Preliminary Experimental Study On The Treatment Of Sciatic Nerve Injury In Rats By VEGF₁₆₅ Secretion Of NSCs Cooperate With Rat Fetal Membrane

Objective: Though to culture SD rat embryonic NSCs(Neural Stem Cells, NSCs) by suspension in vitro, and gene transfection pc DNA-EGFP and pc DNA-EGFP-VEGF165; To explore the effects of cryopreservation on the related indexes of fetal membrane biological activity; To construct sciatic nerve V-grade injury of SD rats model and to find the best optimal timing, and provide experimental basis and theoretical basis for subsequent transplantation to the treatment of sciatic nerve injury in rats. Methods:(1)The brain of 12.5d embryonic SD rat were used as the tissue source, and the application of serum free suspension culture technique was used to isolate and culture NSCs; Nestin immunofluorescence to detect NSCs characteristics; Brd U labeling method to detect the proliferation ability of NSCs; After induction of differentiation of NSCs were NSE, GFAP immunofluorescence technical appraisal; With HE staining, scanning electron microscopy to observe the phenotype of NSCs neurosphere and single NSCs; After identification of the cells and divided into three groups: control group, empty plasmid group, plasmid group, then used So-Fast transfection kit, the empty plasmid group be transfect pc DNA-EGFP and the plasmid group be transfect pc DNA-EGFP-VEGF165, after 24 h screening ampicillin, while the control group without transfection and selected; After transfection 5 days, plasmid group be extracted VEGF165 gene and then to detect gene sequencing; Each extraction of VEGF m RNA in agarose gel electrophoresis was used to detect the integrity of RNA, RT-PCR to detect the expression of VEGF m RNA relative expression; And Nestin expression of plasmid group NSCs was detected by flow cytometry.(2)Fetal membrane tissue obtained from pregnant 12.5d SD rat were divided randomly into untreated group, cryopreservation group, non-cryopreservation group; Untreated conventional treatment immediately backup, cryopreservation group was be storage quickly in liquid nitrogen tank, treatment after 30 ds, non-cryopreservation group placed on ice 1h before handling; Each group of membranes with HE staining observed the change of histological, trypan blue staining to count the cell mortality, in situ hybridization detection of VEGF m RNA and BDNF m RNA expression; Western blot to detect expression of VEGF protein.(3)42 adult SD rats were divided into 7 groups randomly, 6 rats in each group; Sciatic nerve of rats in the model group were completely cut off on the right side of the femur, then were stiched at different time, the control group was only exposed sciatic nerve; HE staining, transmission electron microscopy, immunohistochemistry, Western Blot and TUNEL were used to detect the relative indexes of sciatic nerve injury in each group. Results:(1)Cultivated a large number of Neurospheres in suspension, Nestin and Brd U expression positive; Neurospheres expressed NSE and GFAP after induction differentiationin two ways; HE staining can be seen in the neurospheres gathered by the large number of cells, single NSCs has huge nucleus of, less cytoplasm; Scanning electron microscope can be observed between NSCs neurospheres surface are connected to evacuate, single NSC somata were round, the surface is smooth and no obvious axonal growth; After transfection of 24 hs, under fluorescence microscopy can be a small amount of NSCs expressing green fluorescence was observed, after the 5 days and 7 days screening by ampicillin can be observed in numerous neurospheres expressed green fluorescence; Plasmid group extracted VEGF165 after sequencing and Gen Bank included the VEGF165 gene coding region of nucleic acid sequence(AF486837.1) is exactly same; Each group of VEGF m RNA with good integrity, RT-PCR results showed that no primer dimers with a specific product, use 2-ΔΔCT method to analyze the data, compared plasmid group and empty plasmid group, NSCs group, P < 0.05; Flow cytometry was detected by plasmid transfection of NSCs, Nestin expression was positive.(2)HE staining and trypan blue staining: Cryopreserved group and untreated group, mortality of cells in the fetal tissue morphological changes and fetal membranes was not significantly different; In situ hybridization and Western Blot: untreated group and cryopreservation group, the number of VEGF m RNA and BDNF m RNA positive cells and VEGF protein relative expression compared to the amount, P < 0.05.(3)HE staining, transmission electron microscope and TUNEL detection: compared 6h sutured group with model groups, the variation of sciatic nerve to a lesser extent and fewer apoptotic cells; immunohistochemical and Western Blot: VEGF protain in no-sutured group and 6h sutured group in injury early period(7th day) expression was higher than that other model groups(P < 0.05). Conclusion:(1)Application of serum free culture technology, the in vitro culture of the amplified embryos NSCs with self regeneration and multi differentiation capacity; After transfection, plasmid group can be highly efficient expression of gene products, and no significant impact on NSCs biological characteristics.(2) The cryopreserved tissue stored with freeze-stored liquid have good biological activity.(3)The healing effect of 6h sutured group after sciatic nerve injury is better than other sutrued time points, the mechanism may be related to the improved expression of VEGF in the injured area.