Abstract:ObjectiveTo study the geneticrelationship between the isolated Keriyan population and ethnics in Xinjiang. Methods Three autosomal shorttandemrepeat (STR) polymorphisms (D5S818, D7S820 and D13S317) were analyzed by Genescan software in the isolated population in the center of Taklamakan desert of Xinjiang. Sheriver’s Dsw of the isolated population with others were estimated, and phylogenetic tree was constructed by using NeighborJoining method based on Dsw. Results Allelic frequencies of three autosomal STR were calculated, and the genetic distance based on allelic frequencies showed that the isolated population had the closest genetic distant with Uzbek of Xinjiang. ConclusionThe isolated population has the closest relationship with Uzbek, and they may be derived from the same ancestor.

Abstract:ObjectiveTo assess the genetic affinities of the population from the Zhukaigou archaeological site in Inner Mongolia. MethodsThe mitochondrial DNA hypervariable region Ⅰ (HVRⅠ) in teeth of 9 individuals were amplified and sequenced. Different phylogenetic analyses were performed using mtDNA sequences among the ancient population，the Yinniugou ancient population and several modern Asian populations. The sequences were primarily assigned into each mtDNA haplogroups, and compared with modern populations. Results Fragments of mtDNA HVRⅠ, 364 bp in length, were recovered from 7 individuals from the Zhukaigou archaeological site. 7 sequence types and 15 polymorphism sites were found. Haplogroup distributions of the ancient individuals were investigated according to variations in the HVRⅠ sequence. ConclusionThe matrilineal genetic structure of the Zhukaigou site population is close to that of the later Yinniugou population and modern populations in this area. This aDNA study shows the continuity of the matrilineal genetic structure in the populations of the south central Inner Mongolia.

Abstract:ObjectiveTo develop a fluorogenic probe for measuring the concentration of DNA extracted from ancient samples，and evaluate its detection effects . Methods A novel fluorogenic probe, TaqMan-Molecular Beacon (TaqMan-MB), which combined the hairpin structure of molecular beacons and the cleavage mechanism of TaqMan probes，was developed. Combined with a real-time PCR instrument, TagMan-MB was used for quantification of templates of ancient DNA. ResultsIn all five ancient samples, three samples were gotten the copy numbers, one sample had no template, and one sample cannot be detected. Conclusion TaqMan-MB has much better detection effects than TaqMan or molecular beacons alone, and presents a promising approach for quantification of target genes.

To find a simple, rapid, reliable and effective method for ancient DNA extraction. Methods Four methods based on silica particles and silica-based spin columns(A:Modified Silica Particles Method;B:Geneclean Method;C:QIAamp Mothod;D:Modified QIAquick Method) were respectively used to extract mitochondrial DNA (mtDNA) from 5 ancient sheep dated to 4 000 years ago. PCR amplification of mtDNA was performed, and its results were evaluated by agarose gel electrophoresis. The effects of 4 kinds of methods mentioned above were determined by examining amplification success rates of DNA from 5 ancient sheep. Results The amplification success rates of 4 methods were different from 20% to 100%, and the effects of extraction based on silica-based spin columns (60%, 100%) were distinctly better than those based on silica particles (20%, 40%). The effect of extraction based on modified silica particles was the worst, and based on modified QIAquick was the best among 4 methods. Conclusion Modified QIAquick Method is a highly effective method, with a 100% success rate, in which Centricon○[KG-*4/5]〖WT7”〗R ultrafiltration devices are used to concentrate teeth digest solution and purify DNA through silica-based spin columns. It can effectively remove the inhibitor as well as obtain preferable DNA template.

To explore the method of secretory expression of the natural N-terminal rBPTI with high-level in Pichia pastoris. MethodsHuman serum albumin signal peptide (hsasp) and bpti genes were ligated and the eukaryon expression plasmid pPICZ/hsasp-bpti was constructed. The recombinant plasmid was transformed into the Pichia pastoris(X-33)via electroporation. The transforming positive strains were screened by PCR,SDS-PAGE and trypsin inhibition experiment. Results The rBPTI was expressed and secreted in X-33. SDS-PAGE and MS showed that the relative molecular mass of rBPTI were respectively 6 500 and 6 508. Sequence analysis of amino acid proved that 15 amino acids at amido-rBPTI were identical with that of natural BPTI. Trypsin inhibition experiment showed that Ki value of rBPTI［(2.6±0.1)×10^-9］was identi cal with that of natural material. ConclusionrBPTI with natural N-terminal sequence is successfully expressed in Pichia pastoris with hsasp, and the expression level of rBPTI reaches at 200 mg•L^-1.

To observe the alterations of urotensinⅡ (UⅡ) contents in blood and urine in Caulis Aristolochiae Manshuriensis(CAM)-induced renal lesions in rats. Methods Male Wistar rats were divided into two groups: control group(n=5) and administration group(n=25). After CAM were administered by gavage to rats for 0, 3, 7, 15, 25 d, and 10 d after withdrawal of CAM, the blood and 24 hour-urine samples were collected, 24 hour-urinary protein excretion, urinary retinol-binding protein (RBP), N-acetyl-D glucosaminidase (NAG), serum creatinine (Scr) and blood urea nitrgen (BUN) were examined, and the plasma and urinary UⅡ contents were detected. The pathological changes in kidney were observed at these various time. Results ① At various time after administration of CAM, the Scr , BUN and 24 hour-urinary protein excretion levels were not increased than those in control group. Compared with control group, after administration of CAM for 15 d, urinary NAG content was significantly higher( P<0.05). After administration of CAM for 25 d,urinary NAG and RBP contents were significantly higher( P<0.01). After CAM was withdrawn for 10 d, the urinary RBP and NAG contents were not decreased compared with those at 25th day after administration. ② Compared with control group,after administration of CAM for 15 d, the plasma UⅡ content was significantly increased（P<0.05);and after administration of CAM for 25 d, the plasma UⅡ content was peak value (P<0.01); 10 d after withdrawal of CAM, plasma UⅡ content was higher (P<0.01). After CAM-treated for 25 d, the urinary UⅡ content was increased compared with controls (P<0.05);and 10 d after withdrawal of CAM, the urinary UⅡ content was increased still (P<0.05).③ The degeneration and necrosis in tubules was the major pathological changes in rats after administration of CAM for 3 d，and the pathological changes were aggravated as treatment time increased. After CAM was withdrawn for 10 d, the pathological changes were alleviated than 25 d. ConclusionThe plasma and urinary UII contents are significantly elevated in CAM-induced renal lesions in rats, suggesting that UⅡ may play a pathological role in the development of renal lesions.

To construct rat calcineurin catalytic subunit α(CnA)/enhanced green fluorescent protein EGFP gene coexpression plasmid for exploring the effect of calcineurin on the myocardium apoptosis induced by ischemia-reperfusion . Methods Total RNA was isolated from the heart of the adult Wistar rat, and CnA CDS segment of approximate 1 590 bp size was amplified by reverse transcription PCR method. CnA cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-CnA. CnA cDNA segment excised from plasmid T-CnA was ligated with pShuttle2 which had inserted IRES-EGFP segment into before. The DNA of the recombinant plasmid was extracted and was identified by double digesting with Nhe Ⅰ and Not Ⅰ. The right clone was named pShuttle2-CnA-IRES-EGFP. Results Sequencing result verified that the PCR product of CnA gene was identical to GenBank(NM_017041). 1% agarose electrophoresis showed the bands of recombinant plasmid pShuttle2-CnA-IRES-EGFP digested by Nhe Ⅰ and Not Ⅰ were in the right range corresponding with expectation (1 590 bp and 5 240 bp). Conclusion Recombinant eukaryotic expression plasmid carrying rat CnA cDNA as well as a report gene-EGFP gene is successfully constructed in this experiment.

To investigate the protective effect of Ginkgo biloba (Gb) tablet on rat kidney following unilateral ureteral obstruction (UUO). MethodsWistar rats were divided into four groups: sham group, UUO model group, Gb tablet treatment group and benazapril treatment group (n=12). After Gb tablet was given to UUO rats for 2 and 4 weeks, all rats were sacrificed, the index of kidney, ratio of left and right kidney weight, the activity of urinary N-acetyl-beta-D-glucosaminidase (NAG), urea nitrogen in urine and serum, creatinine in urine and serum and the 24 h urinary protein excretion were detected, respectively. ResultsThe NAG activity ［(18.0±5.4) U•L^-1］, serum urea nitrogen ［(7.3±1.3) mmol•L^-1］, serum creatinine ［(35.2±8.4) μmol•L^-1］ and the 24 h urinary protein excretion ［(9.82±4.42) mg］ in Gb tablet treatment group were decreased significantly compared with UUO model group (P<0.05) 2 weeks after administration. The NAG activity ［(10.3±4.1) U•L^-1］, serum urea nitrogen ［(8.7±0.9) mmol•L^-1］, serum creatinine ［(44.2±6.7) μmol•L^-1］ and the 24 h urinary protein excretion ［(10.17±8.42) mg］ in Gb tablet treatment group were obviously lowered than those in UUO model group after treated with Gb for 4 weeks (P<0.05). Gb tablet could lighten the lesion of kidney in UUO rats. Conclusion Gb tablet has obviously protective effect on renal function and structure in UUO rats.

To study the absorption of quercetin and rutin in human intestinal epithelial. MethodsBy using Caco-2 cell line as an intestinal epithelial cell model, the transportatrions of quercetin and rutin were studied from apical side to basolateral side or from basolateral side to apical side. ResultsThe apparent permeabilities (Papp) of quercetin and rutin from the apical side to basolateral side were( 5.15±0.65)×10^-6cm•s^-1 and (0.05±0.01)×10^-6cm•s^-1, respectively. The Papp of quercetin and rutin from basolateral side to apical side were (10.54±1.35)×10^-6cm•s^-1 and ( 0.07±0.01)×10^-6cm•s^-1, respectively. Conclusion Quercetin and rutin can be absorbed across intestinal epithelial cells, moreover qercetin is easier to be absorbed than rutin.

To clone the gene of a novel activin receptor-interacting protein 4 (ActRIP4) which interacts with type Ⅱ activin receptor (ActRⅡA). Methods The ActRIP4 gene from a mouse cDNA library was cloned by using the ActRIP2 gene fragment as the probe. By mammalian cell two-hybrid, its ability to combine with ActRⅡA was confirmed. The expressions of ActRIP4 mRNA in tissues were analyzed by RT-PCR. Results The cloned full-length cDNA for ActRIP4 was 691bp, which ORF was encoding 118 amino acids. The specific mammalian cell two-hybrid analysis revealed that ActRIP4 could specifically combine with ActRⅡA. The results of RT-PCR showed that ActRIP4 mRNA expressed in many tissues. Conclusion ActRIP4 is a novel member of ActRIP family, and has the ability to specifically combine with ActRⅡA, which may mediate signaling transduction of activin receptor in various tissues.

To explore the inhibitory effects of fraction Ⅰ from scorpion venom (SV-Ⅰ) on the growth of SKOV3 ovarian carcinoma cells. MethodsSKOV3 ovarian carcinoma cells were divided into two groups: control group and treatment group with SV-Ⅰ（final concentration as 200，400 and 800 mg•L^-1,respectively）. MTT method was used to detect the inhibitory rate of tumor cells treated with SV-Ⅰ in vitro and colony forming assay was applied to measure the proliferation of tumor cells. Moreover, flow cytometry was further adopted to observe the changes of cell cycle and apoptosis of SKOV3 cells. Results SV-Ⅰ could inhibit the growth of SKOV3 ovarian carcinoma cells significantly at the concentrations of 200, 400 and 800 mg•L^-1 and t he inhibitory rates were 29.87 %，48.11% and 67.77 %， respectively (P<0.01). Furthermore, SV-Ⅰ could inhibit colony forming capacity of SKOV3 ovarian carcinoma cells at 400 and 800 mg•L^-1 and the numbers of colony were 57.00±6.16 and 48.00±4.11, respectively (P<0.01). Compared with control, cells in S stage were decreased and cells in G₂+M stage were increased obviously (P<0.01) after SV-Ⅰ was given at 400 mg•L^-1, while cells in G₂+M stage were decreased (P<0.01) and cells in G₁ stage had a ascending tread after treated with 800 mg•L^-1 SV-Ⅰ. Furthermore, apoptosis could be induced obviously by SV-Ⅰ at 400 and 800 mg•L^-1 compared with control(P<0.01). Conclusion SV-Ⅰ could inhibit the proliferation and growth of SKOV3 ovarian carcinoma cells and the changes of cell cycle and apoptosis may be important mechanism of inhibitory effects of SV-Ⅰ.

To construct an eukaryotic expression vector of the hepatitis C virus（HCV）NS5A gene. and obtain a stable transfected Huh-7 cell line which provides a basis for further investigation of the hepatitis virus C NS5A protein. Methods The HCV NS5A gene from the pcDNA3.1(+)/HCV NS345 plasmid with HCV NS5A gene was amplified by PCR， and cloned to pGEM-T vector，and transformed into E. coli JM109.The positive colonies were first confirmed by restriction enzyme digestion and sequencing and then were inserted to eukaryotic expression vector pCI-neo，and verified by enzyme digestion and sequencing. Results After TA colon of the HCV NS5A was amplified by PCR，the positive colonies were finally verified by sequencing, which were totally in line with the designed coding sequence of HCV NS5A gene.Conclusion Eukaryotic expression vector of HCV NS5A gene has been successfully constructed.

To investigate the relationship between the expression changes of NMDAR subunits in hippocampus and temporal lobe epilepsy. Methods Male Wistar rats were divided into three groups, five were in normal group; the other two groups, sham and epilepsy groups, were divided into 6 subgroups according to the time points of 6, 24 and 72 h, 7, 14, and 21 d after kindling, respectively, each subgroup had 5 rats. Typical kindled model of temporal lobe epilepsy was established by kainic acid(KA) microinjected into right nucleus amygdalae in male Wistar rats. In situ hybridization was used to detect the expression changes of NR₂A mRNA and NR₂B mRNA in hippocampus after KA kindling. Results In general, NR₂A mRNA significantly increased in regions of hippocampus in epilepsy group than that in sham group (P<0.05), and gradually returned to normal level in epileptic rats after kindled for 21 d (P<0.05). NR₂B mRNA was significantly increased in DG and CA₁ regions in epilepsy group than that in sham group (P<0.05), while without difference in CA₃ region between them. Stepwise regression analysis showed that the expressions of NR₂A mRNA and NR₂B mRNA in hippocampus were positively correlated with temporal lobe epilepsy (P= 0.0001). Conclusion NR₂A mRNA and NR₂B mRNA expressions in hippocampus present temporal and spatial changes in epileptic rats, which may be involved in the mechanisms of temporal epilepsy and the brain injury.

To observe the protective effects of rhKD/APP on carbon tetrachloride-induced acute hepatic injury and find possible mechanisms of hepatic injury. Methods Sixty mice were randomly divided into 6 groups（n=10）:control,model,pos itive control and rhKD/APP groups with different doses (4.5,9.0,18.0 mg•kg ^-1). Pathologic models of acute hepatic injury in mice caused by carbon tetrachloride were set up and rhKD/APP with different dosage were administered (ip) to mice.Effects of rhKD/APP on hepatic injury were observed by measuring enzyme level (AST and ALT)in serum,hepatic malondialdehyde and morphology of hepatic tissue section . Results Compared with model group， serum ALT and AST concentrations in rhKD/APP groups with different doses were decreased significantly (P<0.01, P<0.05), MAD in hepatic tissue in rhKD/APP groups with different doses were decreased (P<0.01, P<0.05). The pathological results showed that there were inflammatory cells and hepatocellular necrosis in model group， the protective effects of rhKD/APP on hepatic injury showed linear relation to the drug dose. Conclusion rhKD/APP has markedly protective effects on acute hepatic injury induced by carbon tetrachloride.

To investigate the effects of advanced glycation end products (AGEs) on insulin-like growth factor-1（IGF-1） expression in rat mesangial cells and its relationship with extracellular matrix(ECM) accumulation. Methods Rat mesangial cells were treated with AGEs-modified bovine serum albumin with different concentrations（0, 12.5 , 25.0 , 50.0 , 100.0 and 200.0 mg•L^-1）, corresponding concentration native bovine serum albumin (BSA) as control. Fibrinectin, collagen Ⅳ and IGF-1 protein contents were detected by ELISA. Results Compared with those of corresponding concentration BSA group, FN contents in 12.5-200.0 mg•L^-1 AGEs group increased significantly（P<0.01）; collagen IV contents in 100.0-200.0 mg•L^-1 AGEs group increased significantly（P<0.01）; IGF-1 contents in 25.0-100.0 mg•L^-1 AGEs group increased significantly（P<0.01）; and 200.0 mg•L^-1 AGEs showed no promotion on IGF-1 expression. ConclusionECM accumulation induced by AGEs is relevant to IGF-1 in a certain range

To investigate the biocompatibility of poly(L-lactide)/poly(L-lactide) surface grafted hydroxyapatite composite(PLLA/PLLA-gHAP) as a new biodegradable material, and provide a biological evidence for the application of the material. Methods　The L929 cell bioactivities in PLLA/PLLA-gHAP group, PLLA group, negative control group(DMEM culture media),positive control group（DMEM contain phenol solution）were determined by MTT assay after coculture for 24,48 and 72 h. L929 cells were cultivated on cover glide coated with PLLA/PLLA-gHAP composite for 1, 2 and 3 d, phase contrast microscope was used to observe the morphological changes of the cells. The leaching liquor of the material was injected into mice to test the acute toxicity and genetic toxicity of it. The PLLA/PLLA-gHAP plates were implanted subcutaneously into rabbit, and the anatomical, pathological and hematological changes were examined after 2 weeks, 3 months and 6 months. Results There was no significant difference in cytotoxicity between the PLLA/PLL　A-gHAP goup and negative control group, and the L929 cells grew well on the cover glide which goated with PLLA/PLLA-gHAP composite. No any acute toxicity or genetic toxicity was found in mice. There was only mild inflammatory reaction and thin fibrosis membrane in the early days after PLLA/PLLA-gHAP plate　 Implantation, the fibrosis membrane was nearly disappeared and most of the imflammatory reaction recovered 6 months later. Conclusion　The new bioabsorbable material, PLLA/PLLA-gHAP composite, has good biocompatibility .

To study the healing effect of recombinant human acidic fibroblast growth factor (rhaFGF) expressed in E.coli on dermal chronic ulcers in diabetic rats. Methods　Ten male Wistar rats were used to set up diabetic dermal chronic ulcers models. The wounds were sprinkled with rhaFGF and physiological saline, respectively. The wound area, wound cavity volume and healing time were recorded, granulation tissue growth and epithelization in wound were observed, and wound healing status was evaluated. Results　Compared with control group, the wound area and wound volume at different day in rhaFGF group were significantly diminuted(P＜0.05）, the healing rate was predominantly eleva Ted　(P＜0.05）, the healing time was significantly shortened(P＜0.05）. RhaFGF resulted in a remarkable enhancement of granulation tissue growth and epithelization in wound. Conclusion　The topical application of rhaFGF can significantly accelerate the healing of dermal chronic ulcers in diabetic rats.

To investigate the inhibitory effects of polyI∶C on growth of PC-3M cell lines. Methods　he prostate cancer PC-3M cells were observed by contrast phase microscope after being treated with different doses of PolyI∶C（100,200,400 μg•L^-1）.The cell growth inhibitory rate was detected by 　TT assay, and content of DNA was detected by flow cytometry, as well as the cell cycle and the apoptosis. Results　he cells in control group were fusiform adherence and abundant, the state of cells in Poly I∶C group was worse than that in control group.The inhibitory rates of growth in Poly I∶C with different doses(400,200,100 μg•L^-1)groups were 67.16%,52.84% and 42.00%,respectively,there were significant differences between three groups(P<0.05).The inhibitory rate in Poly I∶C group with the same dose at 72 h was higher than those at 48 and 24 h,there were significant differences between three groups(P<0.05). The result of flow cytometry showed that 　oly I∶C could induce the apoptosis with dose-dependent manner,and the apoptotic rates were 3.9%，27.1%，42.9%，respectively,there were significant differences between three groups(P<0.05) The G₁ phase cell proportion in PolyI∶C group(61.4%) was higher than that in control group（33.9%）. Conclusion　olyI∶C has significant effect on PC-3M cell inhibition and the inhibitory rate is dose and time dependent.The mechanism may be concerned with the cell cycle arrest and inducing apoptosis upon the PC-3M cells

To study the levels of Th1 and Th2 type cytokines mRNA in TNBS colitis without or with Trichinella spiralis (T. spiralis) infection in mice. Methods　emale BALB/C mice were randomly divided into 4 groups: 50% Ethanol, T. spiralis only, TNBS, T. spiralis +TNBS(at least 6 in each group when mice were killed). The levels of IFN-γ, IL-4, IL-10 mRNA in spleen and IL-10 mRNA in colon in mice with colitis induced by TNBS without or with T. spiralis infection 3 and 7 d post-induction were assessed using RT-PCR method. Results　ompared with TNBS group, IFN-γ mRNA production in spleen were reduced in T. spiralis +TNBS group 7 d post-induction of colitis（P<0.05）, IL-4 mRNA levels were increased in T. spiralis +TNBS group both 3 and 7 d post-induction of colitis（P<0.05）. A statistically significant alteration was not detected in spleen IFN-γ mRNA level 3 d post-induction of colitis（P>0.05）. IL-10 mRNA production in colon and spleen were increased in T. spiralis +TNBS group 3 and 7 d post-induction of colitis（P<0.05）. Conclusion　T. spiralis infection significantly attenuates TNBS-induced colitis in mice. The peripheral immunologic mechanism is that T. spiralis can down-regulate strongly Th1-type immune response of colitis and up-regulate Th2 response. At the same time, IL-10 as Th2 and Tr1 type cytokine has an important effect on colonic immunity and general immunity of TNBS-induced mice.

To study the assembly and release of adriamycin in vitro using magnetic thermosensitive liposomes treated with magnetic field and pyrogenation. Methods ll samples were divided into six groups : 21, 33, 39, 42, 45℃ and control groups according to the temperature of water bath. Lucifer yellow iodoacetamide (LY) was used as a fluorescence marker to adriamycin which was encapsulated by magntic thermosensitive liposomes. Liposomes was localized under the action of magnetic field, and adriamycin was released through the use of water baths with LY used as a fluorescence marker to observe the assembly and release. Temperature-treated liposomes were mixed with CT-26 cells to measure the binding of the relea sed LY-marked adriamycin to cell surface. Results he magnetic thermosensitive adriamycin liposomes was assembled directionally under magnetic field. The onset of LY release occurred near 33℃, and reached plateau above 42℃ when 90% of the LY was released. The binding with the HT-29 had positive correlation with the release of the LY-marked adriamycin. Conclusion agnetic field can make the mag netic thermosensitive liposomes drug to assemble directionally, hyperthermia can control the release of drug from magnetic thermosensitive liposomes.

To analyze the changes of different neuropeptide-containning nerve fibres in iodoacetamine-induced gastritis, and the relationship between the nerve fibres and immune cells. Methods The animal models with gastritis were induced by iodoacetamide. Controls received water.Gastric specimens from the control and the model groups were examined by immunohistochemistry and immunocytochemistry and the changes of these fibres containing-Substance P (SP）, vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) and immunocompetent cells were analyzed. Results The nerve fibres showing immunoreactivity for SP, VIP and NPY were seen in all layers of the stomach. Nerve fibres containing-SP and VIP increased significantly in model group（P<0.05）. Immunocompetent cells could be found in mucosa in model group, which were lymphocytes, plasma cells, and mast cells. Immunocompetent cells were very near the fibres. Conclusion The model with mild iodoacetamide-induced gastritis is built successfully. Neuropeptides participate in the processes of gastritis and might involve in neuroimmunomodulation in gastritis.

To observe the effects of lysophosphatidic acid（LPA）on the action potential and contraction force in guinea pig papillary muscles. Methods Eighteen healthy adult guinea-pigs were randomly divided into LPA 0.1,1.0, 10.0 μmol•L^-1groups, with six guinea pigs in every group compared with themselves before administration to observe the effects of LPA on the papillary muscles. The pigs in 10.0 μmol•L^-1 group were perfused with TEA 20 mmol•L^-1+ LPA 10 μmol•L^-1 after desorption. Results LPA 1.0,10.0 μmol•L^-1 could increase contraction force in guinea pigs compared with themselves before administration from 100% to (122.6±7.9)% and (139.48±8.2)%（P<0.05，P<0.01）. The LPA at different doses (0.1, 1.0, and 10.0 μmol•L^-1) could increase the action potential amplitude(APA) compared with themselves before administration from (106.79±11.80 ),(96.86±9.81),(100.22±7.55)mV to (113.49±12.66),(112.54±10.07),(118.91±10.62)mV（P<0.05 or P<0.01）；the action potential durations measured at the 50% repolarization levels and the 90% repolarization levels were prolonged compared with before administation（all P<0.05）.TEA(20 mmol•L^-1) shortened the APD₅₀ compared with LPA 10 μmol•L^-1 group（P<0.05）. Conclusion LPA could increase APA, prolong action potential duration and increase contractionforce .

To detect the expression of TrkA in the brain of the Wistar rats during the embryonic phase. MethodsAdult Wistar rats were fed in one cage.It was E0 day when sepermia were found in vaginal suppository and vaginal smear.The expressions of TrkA in the brain of Wistar rats during various brain regions were observed by immunohistochemistry on E13,E15,E17,E19 and E21 day. Results On E13 day no obvious expression of TrkA was observed in the embryonic brain. The expression of TrkA had an obviously increasing tendency in the cortex of telencephalon from E15 day to E17 day. And on E17 day, the positive rate of the expression of TrkA reached the second peak. While on E19 day, the expression of TrkA had a temporary decreasing. Then on E21 day the expression of TrkA reached the highest peak. The striatum and hippocampus developed later than the telencephalon till E17 day, and the expressions of TrkA during these stages were same as the cerebrum. Conclusion From E15 to E17 day, obvious expressions of TrkA are observed in the embryonic telencephalon, and it accords with embryonic induction.

To investigate the invasion ability and collagenolytic activity of human glioma cells in vitro. Methods Boyden chamber invasion assay was employed to evaluate the cell migration ability of human malignant glioma cell line U87MG in vitro and the effect of conditioned medium of U87MG cells on matrix collagenolytic activity was tested by agar-gelatin gel. Results The number of U87MG glioma cells migrating through the Matrigel-coated membrane was more than that of addition of EDTA or anti-MMP-2 antibody in U87MG glioma cells (P<0.01), and the difference between the numbers of the migrated cells in the latter two was not significant (P>0.05). EDTA or anti-MMP-2 antibody markedly inhibited the number of the migrated cells, the inhibitory rates were 79.2% and 77.1%, respectively. The conditioned medium collected from the U87MG cells showed an increase in the transparent ring area on agar-gelatin gel. Inhibition of MMP-2 enzymatic activity by EDTA or anti-MMP-2 antibody reduced the transparent ring area on agar-gelatin gel. Conclusion Both invasion ability and collagenolytic activity of U87MG cells are depended on MMP-2, suggesting that MMP-2 plays an important role in glioma invasiveness.

To study the time-effect relationship of hypoglycemic effect of fenugreek seed extracts on experimental diabetic rats. Methods The diabetic models were established by using streptozotocin (STZ) in rats. All the rats were randomly divided into three groups：diabetic control group(D)(n=8), fenugreek seed extracts treatment group(F)(n=10), and normal control group(C)(n=6). The time-effect of the blood glucose level was observed. Results Compared with group D, on the 9th day the level of the blood glucose decreased significantly in diabetic rats treated with fenugreek seed extracts (P<0.05), on the 21st day it decreased significantly (P<0.01). On the 27th day approximately it decreased to normal level (P>0.05) and kept this low level to the 12th week. In the first 4 weeks the level of blood glucose in diabetic rats was negatively related with treatment times of fenugreek seed extracts (r=-0.97， P<0.05), and hypoglycemic effect increased correspondingly with the time of treatment. After treatment for 4 weeks and withdrawal for 2 weeks, the level of blood glucose increased lowly. Conclusion Fenugreek seed extracts can reduce significantly the level of blood glucose in diabetic rats and recover it to the normal level with the relations of time-effect.

To explore the effects of 5-Fu on apoptosis of colon cancer cells (Hct/8) and related gene expressions. Methods Hct/8 were divided into 5-Fu group and control group.The apoptosis of Hct/8 treated by 5-Fu was observed using cell counting, morphology ,DNA segmentation and flow cytometry, and the changes of gene expression was analyzed by microarray chip. Results The number of apoptotic cells was greatly increased in 5-Fu group compared with control group(P<0.01),and further comfirmed by aradine staining and genomic electrophoresis. Microarray chip analysis showed that treatment of 5-Fu on Hct/8 cells resulted in changes of various gene expressions,especially down-regulated of DNA injury-repair related genes. Conclusion 5-Fu could induce the apoptosis of Hct/8 due to interaction of various gene expression changes, particularly DNA injury-repair related genes could not repair the damaged gene in time.

To investigate the effects of advanced glycation end products (AGEs) on expression of peroxisome proliferator-activated receptor-γ(PPAR-γ) mRNA in rat renal cortex. Methods Normal rats were given tail vein injection with either AGE-modified rat serum protein（AGEs）or AGE-RSP followed by intraperitoneal injection of AG（AGEs+AG）or native rat serum protein（native RSP）. Normal rats without any treatment were as controls (Control). PPAR-γ mRNA expression was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results PPAR-γ mRNA expressed in all rat kidney cortex. There was a decrease for PPAR-γ in mRNA levels in the renal cortex of AGEs- treated rats(P<0.01). AG treatment prevented partly mRNA changes. ConclusionPPAR-γ mRNA signaling pathways are in rat kidney cortex. AGEs reduce the expression of PPAR-γ in renal cortex and AG inhibits the effect of AGEs. These results support the notion that modulation of PPAR-γ by AGEs may be relevant to the development of diabetic nephropathy (DN).

To explore the mechanism of cytotoxity induced by rotenone in PC12 cells in order to provide the theoretical basis of neuroprotective drugs. Methods he cultivated PC12 cells were divided into control and 0.5　μmol•L^-1 rotenone-treated groups. Cell viability was estimated by MTT 72 h after treatment. Cell apoptosis was examined by Hoechst 33342. Proteins were extracted from two cell groups, respectively. The maps of proteins were set up by DIGE system. The altered protein spots were identified with MALDI-TOF MS and database searching. Results TT showed cell viability decreased significantly in 0.5 μmol•L^-1rotenone-treated group compared with control group（P<0.01）. Hoechst 33342 fluorescence staining showed that more apoptotic cells were found in rotenone-treated group, the ratio of apoptosis increased significantly compared with control group（P<0.01）. Cells showed condensed, fragmented and nuclei demonstrated by fluorescent dye, which were the characteristics of apoptosis. DIGE revealed two protein spots were significantly changed in rotenone-treated cells compared with control（P<0.01）. MALDI-TOF MS analysis and NCBI database searching demonstrated them as vinculin and mitochondrial aconitase, respectively. Conclusion Vinculin and mitochondrial aconitase may be involved in the cytotoxicity，which may be the target of neuroprotective drugs.

To set up an effective and low-price way for enriching dendritic cells precursor from chronic myelogeous leukemia by Percoll density gradients. Methods Peripheral blood mononuclear cells(PBMCs) were collected by separa tion through Ficoll-Hypaque, then PBMCs were separated by 55% Percoll gradients. The cell type and DCs expressing CD1a, CD86 were detected in the high-density group(C) ,low-density group(B) and non-Percoll separation group(A). Results After separation of 55% Percoll, the percentage of promyelocytes and myelocytes in group B was obviously higher than those in group A and group C(P<0.01). Moreover the percentages of lymphocytes, eosinophilic granulocytes and basophilic granulocytes in group B were lower than those in group C and group A. The percentages of CD1a and CD86 cells detected by FACS on 14th day in group B were higher than those in group A and group C(P<0.01). Conclusion The induced rate of dendritic cells is improved after the separation of 55% Percoll. This method is no-toxic, cheap , simple and can handle a large number of cells. It’s useful for clinical application.

To study the effects E.coli purine nucleoside phosphorylase suicide gene on human gastric cancer MKN-45 cells as well as the bystander effect. Methods The ePNP gene was subcloned to pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1-ePNP. The recombinant plasmid was transfected into MKN-45 cells by lipofectamin method and positive cell clones were screened with G418. Then MePdR was added to these gene-modified cells and the sensitivity of the cells to MePdR was studied as well as the bystander effect. Results MePdR treatment of MKN-45/ePNP cells induced cytotoxic and antiproliferative effects in a concentration-dependent manner with 100% cell death since 1 mg•L^-1. Bystander effect was strong in vitro as 100% of tumor cells were killed by MePdR with only 10% MKN-45/ePNP cells. The gene-modified gastric cancer cells MKN-45/ePNP were sensitive to the treatment of MePdR compared with unmodified tumor cells, and also a remarkable bystander effect was observed. Conclusion The efficiency of ePNP/MePdR system and the potential feasibility of suicide gene strategy for clinical therapy of human gastric cancer are demonstrated.

To study the effect of integrin α_v of tumor cells on adenovirus vector transfection. Methods Tumor cells were transfected by vector AdCMVLacZ. The number of transfected cells was assessed by X-gal staining. The sensitivities of M21 melanona cells and M21-L lacking integrin to adenovirus infection were compared. The blocking effect of GRGDSP -a peptide containing RGD sequence on adenovirus transfection after using GRGDSP to treat tumor cells was observed. Results The transfection efficiencyies of adenovirus to different human tumor cells were different. When multiplicity of infection(MOI) of adenovirus was 50, A549 cells had the highest transfection efficiency (90.2%), the transfection efficiency of Hela cells was the lowest, only 7.6%. On the other hand, different tumor cells needed different MOI of AdCMVLacZ to get 100% transfection efficiency. A549 cells needed 75 MOI. The transfection efficiency of AdCMVLacZ in M21 cells was higher than that in M21-L at same MOI. M21 needed 100 MOI for 100% transfection efficiency, however M21-L needed 400 MOI. After M21 was treated with GRGDSP, the transfection efficiency decreased. Conclusion The sensitivity of tumors to adenovirus transfection is related to integrin of tumor cells.

To compare the effects of enteral nutrition(EN) and parenteral nutrition(PN) on bowel mucosal barrier function in rats with obstructive jaundice. Methods By common biliary duct ligating, the obstructive jaundice rat models were set up.Sixty SD rats were randomly divided into group A:sham operation（SHAM）,group B:common biliary duct ligating（CBDL）,group C:CBDL+TPN,group D:CBDL+EN,group E:CBDL+ freedom drinking water with antibiotic,12 rats in each group.After the rats were bred for 1 week,the caval vein blood was got and the serum was separated to wait for the measuring of endotoxin. The mesenteric lymph node,liver and spleen were obtained, cultivated, observed and the condition of bacterium growth were recorded. The small intestine was gained to make microtome section and mucous membrane of small intestine morphology, trophonema altitude, thickness and crypt shade were observed under light microscope.Results Compared with group A, jejunal mucous intestinal membrane depths in groups B and D were shallow(P<0.05), rat jejunal mucous intestinal membrane in group C and E became thinner and atrophy,and had shortened villus and shallower cave.There was no lymphnode mesenterici bacteria transposal in group A, the lymph node mesenterici bacteria transposal rates in groups B,C,D and E were higher than that in group A(P<0.01 or P<0.05).The contents of endotoxin in group B,C,D and E were higher than that in group A(P<0.01 or P<0.05),it was lower in group E than those in groups B,C and D(P<0.05). Conclusion ①When intestinal barrier is injuried, bacteria transposal and endotoxemia exist in obstructive jaundice;②Both EN and standard PN can not maintain intestinal membrane barrier and hold up the occurrence of intestinal bacteria transposal in obstructive jaundice rats,but the effect of EN is better than PN；③Intestinal tract antibiotics can step down incidence rate of endotoxemia and profit to hold up the occurrence of intestinal bacteria transposal.

To explore the effect of lead acetate on the growth of murine mesenchymal stem cells in vitro. Methods 40.00,60.00 and 100.00 μmol•L^-1 of lead acetate were used in the culture of colony-forming unit-fibroblast(CFU-F),the effect on the rate of colony-forming and the rule of variation were observed. Results The rates of colony-forming were(3.30±0.20), (2.40±0.10) and(1.57±0.21)/10⁵ ,when the doses of lead acetate were 40.00 ,60.00 and 100.00 μmol•L^-1 and there were significant differences compared with control group(4.20±0.20)/10⁵, P<0.05). The rates of colony-forming had significant differences between lead acetate with different doses groups (P<0.05). Dose-effect relationship was analyzed with linear correlation and correlation coefficient（r=-0.960,P<0.01）. Colony cells of control group presented fusiform shape while cells of 40.0 μmol•L^-1 of lead acetate group presented irregularity or round shape cultivated in vitro after 6 d. Conclusion The lead acetate suppresses the rate of colony-forming of CFU-F significantly in vitro and the inhibitory effect increases with the increasing of doses of lead acetate.

To investigate the therapeutic effect of ginsenoside Rg3 on herpes simplex encephalitis (HSE) mice and its immunoregulative activity. Methods HSV-1 suspension was diluted into 10^-1,10^-2,10^-3,10^-4,10^-5 and 10^-6 with PBS and injected intraperitoneally to establish HSE mouse model. NO in the brain homogenate was examined by nitrate reductase method. Rg3 with doses of 0.3, 1.0 and 3.0 mg•kg^-1 were administered intraperitoneally and the influence of Rg3 on the mouse survival time and pathological changes of the brain tissue were observed. MTT colorimetry was used to detect the influence of Rg3 on lymphocyte transformation function and NK cytotoxicity. The activity of IL-2 was detected by lymphoblast proliferation assay and IFN-γ by cytopathic effect inhibition. Results HSE mouse model was successfully established. The content of NO in the brain homogenate was higher in the infected group than that in the control group (P<0.05). 0.3 mg•kg^-1 Rg3 could extend survival time and ameliorate pathological changes in HSE mice. 0.4 mg•kg^-1 Rg3 could significantly enhance the lymphocyte transformation function and improve the secretion of IL-2 and IFN-γ (P<0.05). NK cytotoxicity was also strengthened. Conclusion Rg3 has protective effect on HSE mice. It also has immunoregulative activity. Rg3 is a potent antiviral drug which deserves further development.

To observe the effect of YiZhikoufuye(YZ) on apoptosis in the different cerebral regions of experimental Alzheimer’s disease(AD) rats. Methods　The animal model of AD was set up by stereotaxic damage of CA₁ area of rat hippocampus using aggressive Aβ₁-40. After operation thirty male Wistar rats were randomly divided into YZ, melatonin and model control groups,10 in each group.The rats were fed with YZ, melatonin and saline, respectively.On the 41th day,the rats were killed. The percentage of apoptotic cells, the changes of mitochondrial membrane potential and the expression level of cytochrome C protein were observed with immunohistochemical methods. Results　Compared with control group, YZ could inhibit the apoptotic changes caused by Aβ（P<0.05),inhibit the decrease of mitochondrial membrane potential （P<0.05）and the increase of cytochrome C caused by Aβ. ConclusionYZ has protective function on the Aβ damage by inhibiting the decrease of mitochondrial membrane potential and the increase of cytochrome C .

To explore the mechanism of therapeutic effect of Donepezil hydrochloride on Alzheimer’s disease (AD) rats. Methods　According to weight,36 rats were divided into normal group, model group and Donepezil hydrochloride group. AD rat model was set up by injecting D-galactose into abdominal cavity for seven weeks, learning and memory function of rats was determined by using Morris water maze and Step-down test. The section of rat cerebral cortex and hippocampus were stained with haematoxylin eosin(HE),and the effect of Donepezil hydrochloride was observed by detecting the MDA content and SOD activity in cerebral tissue. Results　Compared with model group,latency and distance of Donepez　il hydrochloride rats shortened on the fourth day and the fifth day, starting angle of Donepezil hydrochloride rats shortened on the fourth day and the fifth day in the Morris water maze test, error times of Donepezil hydrochloride rats decreased on the first day and the second day in Step-down test; MDA content in cerebral tissue of Donepezil hydrochloride rat was deceased（P<0.05）, SOD activity in cerebral tissue of Donepezil hydrochloride rats was increased（P<0.05），Donepezil hydrochloride significantly reduced the pathological changes of 　AD rats. Conclusion　Donepezil hydrochloride has theraputic effect on AD rats by decreasing free radical injury.

To explore the distribution and characterization of class 1 integrons in E.coli from healthy feces, and to elucidate the status of gene-cassettes. Methods　Routine method was used to isolate E.coli, antibiotics suscept　ibility was tested by the disk diffusion method; class 1 integron was detected by PCR assay; PCR products were sequenced and analyzed. Results　Of 97 samples, 76 isolates were identified, and 25 isolates were multiple-drug resistant. The ant　ibiogram was sulfamethoxazole-trimethoprim, ampicillin, streptomycin, tetracycline, erythromycin. 14 of 25 isolates carried class 1 integrons, and the size of integrons differed from 1 800 bp (10 strains) to 750 bp (4 strains). The sequenced PCR product demonstrated that the 1 800 bp integron laboured aadA1-dfrA14-orf gene cassette conferred the resistance to sulfamethoxazole-timethoprim, streptomycin and aminoglycoside; the 750 bp integron laboured dfrA14 gene cassette conferred the resistance to sulfamethoxazole-trimethoprim. Conclusion　The different kinds of class 1 integrons exist in E.coli from the healthy students, and determinee the multiple- resistant antibiotics.

To investigate the expressions of ClC-3 and NF-κB in transitional cell carcinoma of the bladder(TCCB) specimen and their significances. MethodsThe expressions of ClC-3 and NF-κB were detected in 31 cases of TCCB and 6 cases of normal bladder tissue as control by immunohistochemical staining.Results In the tissues of TCCB, ClC-3 was mainly located in the cell membrane，the positive rate of ClC-3 was 74% (23 /31);the posivive rates of Ⅰ,Ⅱ and Ⅲ grades were 40%(4/10), 81%(9/11), and 100%(10/10), respectively. NF-κB was mainly distributed in the cytoplasm, the positive rate of NF-κB was 83% (26/31); the positive rates of Ⅰ, Ⅱ and Ⅲ grades were 60%(6/10), 90%(10/11) and 100%(10/10), respectively. Only one specimen showed weak positive staining, and negative staining of NF-κB was found in 6 cases of normal bladder tissues.The positive rates of ClC-3 and NF-κB in tissue of TCCB were higher than those in normal bladder tissue（P<0.01）.Conclusion ClC-3 and NF-κB may be related to the pathologic al grade and proliferation of TCCB.

Abstract:ObjectiveTo investigate the expression of tumor-associated carbohydrate antigen sTn in breast neoplasm, and analyzed the association between the expression of sTn and size of tumor, age, menopause, axillary lymph node metastasis, clinical stage, pathologic grade and the level of estrogen receptor in breast carcinoma. MethodsThe monoclonal antibody TKH-2 was used to detect the immunohistochemical expression of sTn in 46 cases of breast carcinoma, 4 breast adenoma and 2 gynecomastia. The level of estrogen receptor was detected by S-P method in 46 breast carcinoma. ResultsImmunohistochemical expression of sTn was not detected in breast adenoma and gynecomastia. The expression of sTn was found in 28 of 46 breast carcinoma (60.9%). The expression of sTn was significantly associated with axillary lymph node metastasis (P<0.05), clinical stage (P<0.05) and the level of estrogen receptor (P<0.05) in breast carcinoma. But there was no apparent relationship between the expression of sTn and size of tumor(≥2cm), age, menopause and pathologic grade (P>0.05). In 46 breast carcinoma, the positive quantity of estrogen receptor was 25 (54.3%). ConclusionSTn immunostaining appears to be a poor prognostic factor in patients with breast carcinoma. It is more useful in detecting the expression of sTn together with the level of estrogen receptor in breast carcinoma to investigate the prognosis of the patients.

To investigate the realationship between resting heart rate(RHR) and carotid arterial intima-media thickness (IMT)in senile patients with essential hypertension .MethodsAll 164 elderly essential hypertensive patients were divided into three groups according to the levels of blood pressure (BP): the first group, 140/90 mmHg≤SBP<160 mmHg/100 mmHg;the second group, 160/100 mmHg≤BP<180/110 mmHg; the third group, BP≥180/110 mmHg. Each of the groups mentioned above were devided into four groups according to the levels of RHR. RHR₁ group :RHR <60 beats/ min; RHR₂ group :60 beats/min ≤RHR<70 beats/min ;RHR₃ group :70 beats /min ≤RHR <80 beats/min ;RHR₄　group : RHR≥80 beats /min.Blood lipids ,blood glucose, carotid ultrasonography were performed .Carotid arterial IMT and carotid arterial diameter (CAD)were measured. ResultsThe blood glucose,blood triglyeride,blood total cholesterol ,systolic blood pressure in RHR₄ groups were higher than those of RHR₁-RHR₃ groups (P<0.01 or P<0.05). The number of carotid arterial IMT and CAD in RHR₄, RHR₃ groups were higher than those in RHR₁，RHR₂ groups （P<0.01 or P<0.05）.ConclusionWith the increasing of RHR ,the numerical value of carotid arterial IMT and CAD also increase significantly. The increasing of RHR is one of the reasons of the carotid arterial IMT increasing in senile essential hypertensive patients.

To provide a detailed anatomic data of temporal region and evaluate the clinical application of intermedial temporal space in re-time bone graft operation. MethodsStepwise dissection was performed in the temporal region in 15 pre-fixed adult cadavers(30 sides) with the aid of surgical microscope，and the anatomic layers，the distribution of nerves and blood vessels in temporal region were observed. Results① Temporal region was rich in fascia layers，fatty tissue layers, and contained 3 latent anatomic spaces；② The distance between point A,B and dorsal root of ygomatic arch was 0.3-0.85 cm［（0.5±0.1）cm］,2.2-3.5 cm ［（2.6±0.3）cm］, respectively. ③ All of the temporal branches of facial nerve were located in front of line CD，75.0% in subcutaneous tissue, 21.4% in superficial temporal space, 3.6% in intermedial temporal space. Conclusion ① The intermediate fat layer is an important landmark of intermediate temporal space;② The skin flap before dorsal root of ygomatic arch 1 cm is safety to temporal artery;③ The temporal space after the line CD is a safety operation area for the protection of temporal branch of facial nerve.

To investigate the changes and clinical significances of the chemokines of interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1(MCP-1) and growth-related oncogene-α(Gro-α) involved in pathagenesis of Kawasaki disease(KD) and Henoch-Schonlein purpura(HSP). MethodsThe chemokines production of IP-10,MCP-1 and Gro-α were assayed by ELISA in 15 patients with KD, 12 patients with HSP and 10 healthy children. ResultsThe plasma levels of IP-10 and MCP-1 were markedly elevated in KD group ［（394.2±176.4 ）and（420.5±163.4）ng•L^-1］compared with HSP group［（94.8±66.4）and（109.2±76.6）ng•L^-1］ and the control group ［（76.4±46.5）and （87.7±47.8）ng•L^-1］(all P<0.05). There were no differences of the levels of IP-10 and MCP-1 between HSP and control groups ( P>0.05),as well as Gro-α between the three groups. Conclusion Monocyte may enhance the immune damage in KD pathogenesis, and the levels of IP-10, MCP-1 may be important indexes for KD. Neutrophil may be not involved in pathogenesis of HSP and KD.

To study the levels of expression, coexpression and clinical significance of multidrug resistance factors in lung cancer. MethodsThe expressions of p-glutathione(P-gp), multidrug resistance-associated protein(MRP),glutathione s-transferase Ⅱ(GST-Ⅱ) in tumor tissues of 52 lung cancer patients were detected by using immunohistochemical method. ResultsThe positive rates of P-gp,MRP,GST-Ⅱ were 51.9%(27/52), 61.5%(32/52), 78.8%(41/52), respectively. No relationship was observed between the expression of drug resistance factors and TNM stage and cell differentiation. The coexpression rates were as follows: P-gp+MRP，40.1%；P-gp+GST-Ⅱ，44.2%；MRP+GST-Ⅱ，50.0%；P-gp+MRP+GST-Ⅱ，21.2%;correlations were found between P-gp and MRP(r_s=0.632,P<0.01）,P-gp and GST-Ⅱ (r_s=0.521,P<0.01）, MRP and GST-Ⅱ (r_s=0.532, P<0.01）. ConclusionThe multidrug resistance in lung cancer patients is affected by various multidrug resistance factors. The drug resistance factors’〖KG-*3〗 expressions are not related to histological subtypes, TNM stage and cell differentiation.