关键词: 脂肪源性干细胞, 脱细胞支架, 睫状神经营养因子, Janus激酶2, 信号转导与转录激活因子3信号通路, 脊神经节

Abstract:

Objective To observe the effects of adipose-derived stem cells （ADSC） combined with acellular scaffold （AS） on the ultrastructure of dorsal root ganglion and the protein and mRNA expression levels of ciliary neurotrophic factor （CNTF）， Janus kinase 2 （JAK2）， phosphorylated JAK2 （p-JAK2）， signal transducer and activator of transcription 3（STAT3） and phosphorylated STAT3（p-STAT3） in the rats with sciatic nerve injury （SNI）， and to clarify the protective effect of ADSC combined with AS on dorsal root ganglion in the SNI rats and its possible mechanism. Methods The rat ADSCs were isolated and cultured and their multidirectional differentiation potential was detected. The AS of rats was prepared， and ADSCs were injected into the AS to construct tissue-engineered nerve. A total of 36 rats were randomly divided into control group， model group， AS group， and ADSC+AS group. The rats in control group were routinely fed， and the rats in other groups were used to establish the SNI models by resecting 10 mm of right sciatic nerve. The rats in model group received no further treatment， while the rats in AS group and ADSC+AS group were bridged with AS and the constructed tissue-engineered nerve at the two ends of the injured nerve， respectively. At 6 weeks after surgery， transmission electron microscope was used to observe the ultrastructure of dorsal root ganglion of the rats in various groups； immunofluorescence method was used to detect the protein expression levels of CNTF， p-JAK2， and p-STAT3 in dorsal root ganglion of the rats； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the mRNA expression levels of CNTF， JAK2， and STAT3 in dorsal root ganglion of the rats in various groups. Results After 7 d of primary ADSC culture， a large number of large and long spindle-shaped cells were observed under the inverted microscope， arranged in clusters or whirlpools； red lipid droplets were observed with oil red O staining under microscope， and calcified nodules were observed with Alizarin red staining under microscope， indicating that the isolated and cultured cells had multidirectional differentiation ability.Compared with normal nerve tissue， the level of DNA in AS of rats was significantly decreased （P<0.05）. Compared with control group， the nuclear membrane of dorsal root ganglion cells in model group was uneven and serrated， the number of organelles in the cytoplasm was decreased， mitochondria were swollen with broken or missing cristae and unclear structure； the CNTF protein and mRNA expression levels were significantly decreased （P<0.01）， the p-JAK2 and p-STAT3 protein expression levels were significantly increased （P<0.01）， and the JAK2 and STAT3 mRNA expression levels were significantly increased （P<0.01）. Compared with model group， the serrated change of nuclear membrane of the dorsal root ganglion cells in AS group was significantly alleviated， the number of organelles in the cytoplasm was increased， and mitochondrial swelling was reduced； in ADSC+AS group， the nuclear membrane of dorsal root ganglion cells tended to be intact， the number of organelles was increased， and mitochondrial swelling and vacuolization were significantly reduced； the CNTF protein and mRNA expression levels in the dorsal root ganglion in AS group and ADSC+AS group were significantly increased （P<0.01）， the p-JAK2 and p-STAT3 protein expression levels were significantly decreased （P<0.01）， and the JAK2 and STAT3 mRNA expression levels were significantly decreased （P<0.01）. Compared with AS group， the CNTF protein and mRNA expression levels in ADSC+AS group were significantly increased （P<0.05 or P<0.01）， the p-JAK2 and p-STAT3 protein expression levels were significantly decreased （P<0.01）， and the JAK2 and STAT3 mRNA expression levels were significantly decreased （P<0.01）. Conclusion The application of ADSC combined with AS can improve the ultrastructure of dorsal root ganglion in the SNI rats， and the mechanism may be related to the increased CNTF expression and decreased activation of the JAK2/STAT3 signaling pathway in the dorsal root ganglion by ADSC combined with AS application.

Key words: Adipose-derived stem cells, Acellular scaffold, Ciliary neurotrophic factor, Janus kinase 2, Signal transducer and activator of transcription 3 signaling pathway, Dorsal root ganglion