| Objective: To investigate the molecular mechanism of bone marrow mesenchymal stem cells(BMSCs)transplantation to regulate the expression of NGF in rats with 2,5-hexodione(HD)poisoning.To elucidate the mechanism of BMSCs transplantation to repair the nerve tissues damage in HD poisoning rats.Methods: We choosed 100 four to six weeks male SD rats which were no specific pathogens(specified pathogen free,SPF),then randomly divided them into:Control group(Control),bone marrow mesenchymal stem cells group(BMSCs),infected group(HD)and its repair group(HD-NS),stem cells repaired group(HD-BMSCs),each group has 20 rats.HD was diluted with 0.9% sterile saline,and the rats were treated by intraperitoneal injection.The dose of exposure group was 400 mg / kg and the injection volume was 0.3 ml / 100 g.The control group received the same dose of sterile saline.Once every weekday,the virus is infected one time and the cycle is 5 weeks.After the successful injection of the model,the rats in HD group were injected BMSCs from the tail vein,and the another same group was given to sterile saline.After 5 weeks all rats' stem cells,serum,ischia,spinal cord and other tissues were extracted from each group of animals and stored at-80 ? in deep freezer.The blood NGF was detected by ELISA.The mi RNA Let-7 family was screened by mi RNA microarray.The changes of Let-7 family in the ischial,spinal cord and VSC 4.1 cells were detected by q RT-PCR and Western Blot.The changes of si NGF 2 transfected into VSC 4.1 cells were detected by q RT-PCR and Western Blot.The interaction between let-7e-5p mimic,inhibitor and si NGF was detected by q RT-PCR and Western Blot.Data analysis using SPSS 13.0 statistical software,the results used Graph Pad Prism 5 mapping software.Results:1.ELISA method was used to determine the NGF concentration in the serum of each group,and the concentration of NGF in the Control group was 263 ng/ml;The NGF concentration of HD poisoning group was 136 ng/ml,lower than the Control group(p <0.05).The NGF concentration of BMSCs was 187 ng/ml,higher than HD group(p <0.05).2.The results of ELISA showed that the concentration of NGF in the control group was 397 ng/ml,the concentration of NGF in the HD group was 176 ng/ml,lower than the Control group(p <0.05).The concentration of NGF in the BMSCs group was 368 ng/ml,higher than the HD group(p <0.05).The results of q RT-PCR and Western Blotting showed that the expression of NGF in spinal nerve tissue of HD group was significantly lower than Control group(p <0.05).The expression of NGF in BMSCs group was higher than HD group(p < 0.05).3.ELISA method to determine the concentration of NGF in each group sciatic nerve tissue results show that the concentration of NGF in Control group was 201 ng/ml;the concentration of NGF in HD group was 134 ng/ml,lower than Control group(p <0.05);NGF concentration in BMSCs group was 163 ng/ml,p <0.05).The expression of NGF in sciatic nerve of each group was detected by q RT-PCR and Western Blotting.The results showed that the expression of NGF in HD group was significantly lower than Control group(p <0.05);the expression of NGF in BMSCs group was higher than HD group(p <0.05).4.The results of q RT-PCR showed that the expression of Let-7e-5p in each group was significantly higher than those in other mi RNA Let-7 families(p <0.01).5.The results of q RT-PCR and Western Blotting showed that NGF in VSC 4.1 cells transfected with Let-7e-5p mimic group was decreased.The level of NGF in Let-7e-5p inhibitor group VSC 4.1 cells increased(p <0.05).6.After knocking out of NGF,the amount of NGF in cells was decreased.7.After transfecting Let-7e-5p mimic,inhibitor and NGF si RNA 2 into VSC 4.1 cells,the results of q RT-PCR and Western Blotting showed that the NGF content in VSC 4.1 cells which was transfected Let-7e-5p mimic with NGF si RNA 2 group was decreased;NGF content was increased in VSC 4.1 cells of Let-7e-5p inhibitor with NGF si RNA 2 group.Conclusions: Transplantation of BMSCs significantly increases the NGF concentration in serum of HD rats.Transplantation of BMSCs upregulates the NGF expression in the spinal cord and sciatic nerve of HD rats.Transplantation of BMSCs downregulates parts of the mi RNA Let-7 family in the spinal cord and sciatic nerve of HD rats.Transplantation of BMSCs mediatly subsets the prosession of mi RNA Let-7 family negatively regulate endogenous NGF expression in nerve tissues of HD Rats. |
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