聚丙交酯-乙交酯微球搭载鹿茸多肽联合骨髓间充质干细胞对大鼠坐骨神经损伤的修复作用及其机制

doi:10.13481/j.1671-587x.20180611

摘要/Abstract

摘要： 目的：观察聚丙交酯-乙交酯（PLGA）微球搭载的鹿茸多肽（VAP）联合骨髓间充质干细胞（BMMSCs）对大鼠坐骨神经损伤的修复作用，探讨其相关作用机制。方法：横断法复制大鼠外周神经损伤模型。60只大鼠随机分为坐骨神经横断对照组（对照组）、VAP微球组（VAP组）、BMMSCs移植组（BMC组）和VAP微球联合BMMSCs移植组（VAP-BMC组），每组15只。造模7 d后，对照组大鼠在横断坐骨神经区域注射PBS溶液，VAP组大鼠在相同位置注射含有载药PLGA微球的PBS溶液，BMC组大鼠注射含有BMMSCs的PBS溶液，VAP-BMC组大鼠注射含有载药PLGA微球和BMMSCs的PBS溶液。3个月后采用Basso、Breattie和Bresnahan （BBB）运动评级量表评估各组大鼠的神经功能，HE染色检测各组大鼠坐骨神经组织形态表现，Western blotting法检测各组大鼠坐骨神经组织中二硫键异构酶（PDI）、葡萄糖调节蛋白78（GRP-78）和半胱胺酸蛋白酶12（Caspase-12）蛋白表达水平。结果：BBB运动评级量表评估，与对照组比较，VAP组、BMC组和VAP-BMC组大鼠BBB评分升高（P<0.01），以VAP-BMC组大鼠BBB评分升高最明显。HE染色，与对照组比较，VAP组、BMC组和VAP-BMC组大鼠坐骨神经纤维更粗，轴突直径更长（P<0.01）。Western blotting法检测，与对照组比较，VAP组、BMC组和VAP-BMC组大鼠坐骨神经组织中PDI和Caspase-12蛋白表达水平明显降低（P<0.01），VAP-BMC组大鼠坐骨神经组织中GRP-78蛋白表达水平明显升高（P<0.01）。结论：PLGA微球搭载VAP联合BMMSCs可促进受损坐骨神经的修复，其作用机制可能与抑制内质网应激有关联。

关键词: 聚丙交酯-乙交酯微球, 鹿茸多肽, 坐骨神经, 内质网应激, 骨髓间充质干细胞

Abstract: Objective: To observe the recovery effect of polylactide-glycolic acid (PLGA) microsphere-loaded velvet antler peptide(VAP) combined with bone marrow mesenchymal stem cells(BMMSCs) on the sciatic nerve injury of the rats, and to explore the related mechanisms.Methods: The sciatic nerve injury models were made by the cross-sectional method.A total of 60 rats were randomly divided into sciatic nerve transection control group(control group),VAP group,BMMSCs transplantation (BMC) group and VAP microsphere combined with BMMSCs transplantation(VAP-BMC) group(n=15).Seven days after modeling, the rats in control group were injected with PBS solution in the transecting sciatic nerve region,the rats in VAP group were injected with PBS solution containing PLGA microspheres loading drug, the rats in BMC group were injected with PBS solution containing BMMSCs,and the rats in VAP-BMC group were injected with PBS solution containing PLGA microspheres loading drug and BMMSCs.After three months,the neurological function of the rats in various groups were evaluated by Basso,Beattie,Bresnahan(BBB) functional sports rating scale,and the morphology of sciatic nerves of the rats in various groups were detected by HE staining.The expression levels of protein disulfide isomerase(PDI),glucose regulated protein-78(GRP-78) and Caspase-12 in sciatic nerve of the rats in various groups were detected by Western blotting method.Results: The results of BBB functional sports rating scale showed that compared with control group, the BBB scores of the rats in VAP group,BMC group and VAP-BMC group were increased (P<0.01),especially in VAP-BMC group.The results of HE staining showed that compared with control group, the sciatic nerve fibers of the rats in VAP group,BMC group and VAP-BMC group got thicker and the diameters of axon were longer (P<0.01).The results of Western blotting showed that compared with control group, the expression levels of PDI and Caspase-12 in the sciatic nerve of the rats in VAP group,BMC group and VAP-BMC group were significantly decreased(P<0.01),and the expression level of GRP-78 protein in the sciatic nerve of the rats in VAP-BMC group was significantly increased (P<0.01).Conclusion: PLGA microsphere-loaded VAP combined with BMMSCs can significantly promote the repairment of injured sciatic nerves, and its mechanism may be related to the inhibition of endoplasmic reticulum stress.

Key words: polylactide-glycolide microspheres, velvet antler peptide, sciatic nerve, endoplasmic reticulum stress, bone marrow mesenchymal stem cells

中图分类号:

R651.3

龚庆, 宋秋莹, 邱莉晶, 黄晓巍, 赵文海. 聚丙交酯-乙交酯微球搭载鹿茸多肽联合骨髓间充质干细胞对大鼠坐骨神经损伤的修复作用及其机制[J]. 吉林大学学报(医学版), 2018, 44(06): 1174-1178.

GONG Qing, SONG Qiuying, QIU Lijing, HUANG Xiaowei, ZHAO Wenhai. Recovery effect of PLGA microsphere-loaded VAP combined with BMMSCs on sciatic nerve injury of rats and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2018, 44(06): 1174-1178.

参考文献

[1] 刘颜芬,张雪晶,段晓琴,等.富血小板血浆对外周神经损伤修复的研究现状[J].中国实验诊断学,2018(1):145-148.
[2] Bastami F,Vares P, Khojasteh A.Healing effects of platelet-rich plasma on peripheral nerve injuries[J].J Craniofac Surg,2017,28(1):e49-e57.
[3] Rbia N,Shin AY.The role of nerve graft substitutes in motor and mixed motor/sensory peripheral nerve injuries[J].J Hand SurgAm,2017,42(5):367-377.
[4] 李时珍.本草纲目[M].太原:山西科学技术出版社,2014:1243-1244.
[5] 刘佳,赵海平,李春义.鹿茸成分研究进展[J].特产研究,2016,39(4):50-54.
[6] 苏立宁,宋小青,朱登祥,等.川芎嗪联合脑源性神经营养因子诱导大鼠骨髓间充质干细胞向神经样细胞分化[J].中国老年学杂志,2018,38(1):28-31.
[7] 陈娜子,姜潮,李校堃.内质网应激与疾病[J].中国生物工程杂志,2016,36(1):76-85.
[8] 李锐,李逸阳,李多慧,等.神经生长因子通过抑制内质网应激促进外周神经损伤后髓鞘碎片的清除[J].中国生物化学与分子生物学报,2018,34(2):170-176.
[9] 程兴龙.外膜纵行小切口神经完全横断法构建坐骨神经损伤动物模型[D].承德:承德医学院,2016.
[10] Ma M,Basso DM,Walters P,et al.Behavioral and histological outcomes following graded spinal cord contusion injury in the C57BL/6 mouse[J].Exp Neurol,2001,169(2):239-254.
[11] 李玲.中医对外周神经损伤的认识[J].陕西中医,2011,32(6):768-769.
[12] 王逸文,徐方剑,孙浩,等.鹿茸多肽提取工艺及其药理学作用[J].上海中医药杂志,2016,50(4):94-96.
[13] 王楠,高晓霞,代子彦,等.鹿茸药效物质基础、药理作用、临床应用及质量控制的研究进展[J].中草药,2017,48(22):4784-4790.
[14] 梅兵,杨吉平,常桂娟,等.鹿茸多肽提取分离及药理活性的研究进展[J].中华中医药杂志,2014,29(6):1926-1928.
[15] 王克立,路来金,富旭.鹿茸多肽-PLGA复合膜促进大鼠周围神经再生[J].吉林大学学报:医学版,2006,32(2):199-202.
[16] 路来金,王克利,李立军,等.鹿茸多肽对周围神经再生的影响[J].中国修复重建外科杂志,2008,22(12):1458-1461.
[17] 王煜,闵祥辉,张勤,等.骨髓间充质干细胞与施万细胞联合移植对大鼠周围神经损伤端侧吻合的修复效果研究[J].现代生物医学进展,2016,16(32):6206-6209.
[18] 黄婷婷,刘玉梅,张自强,等.干细胞在周围神经损伤修复中的应用进展[J].中国临床药理学杂志,2016,32(10):945-948.
[19] 朱德志,王伯珉.骨髓间充质干细胞与新型神经导管复合材料修复周围神经缺损[J].中国组织工程研究,2015,19(45):7320-7324.
[20] 尚修超.载GDNF基因的骨髓间充质干细胞促周围神经再生的实验研究[D].扬州:扬州大学,2015.
[21] Chiu HW,Xia T,Lee YH,et al.Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress[J].Nanoscale,2015,7(2):736-746.
[22] 肖斌,刘荣荣,刘炳彤,等.TTF1-NP诱导人肝癌HepG-2细胞凋亡的内质网应激作用[J].吉林大学学报:医学版,2015,41(6):1118-1123.
[23] Zhang HY,Wang ZG,Lu XH,et al.Endoplasmic reticulum stress:relevance and therapeutics in central nervous system diseases[J].Mol Neurobiol,2015,51(3):1343-1352.
[24] Wang Z,Zhang H,Xu X,et al.bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways[J].Toxicol Lett,2012,212(2):137-146.
[25] 麻庆乐,卢德赵.葡萄糖调节蛋白78的研究进展[J].生命科学,2017,29(4):331-335.
[26] 杨方万,穆茂媛,肖娟娟,等.内质网应激诱导细胞凋亡机制的研究进展[J].医学研究杂志,2014,43(10):176-180.
[27] 徐京京,王晓非.间充质干细胞在系统性红斑狼疮发病和治疗中的研究进展[J].中国实用内科杂志,2018,38(9):870-874.