| ObjectiveSpinal cord injury is a serious complication of spinal diseases,often leads to severe dysfunction of lower limbs,its incidence increased year by year.The transplantation of exogenous stem cells provides a new idea for the treatment of spinal cord injury.Bone marrow mesenchymal stem cells(BMSCs)have advantages of self renewing and value-added ability,wide source,easy separation and culture,but due to the BMSCs after transplantation in vivo survival rate and low conversion rate,thus limits its wide application.B cell lymphoma/leukemia gene 2(Bcl-2)is essential in regulating cell apoptosis and apoptosis can be caused by many reasons against,is considered to be an anti apoptosis gene can inhibit apoptosis.Through the construction of experimental rat bcl-2 gene expression vector was transfected into BMSCs,We observed the efficacy of Rcl-2 overexpressing BMSCs in the treatment of spinal cord injury。Methods1 、 culture and passage of rat BMSCs were observed using inverted microscope,flow cytometry was used to detect cell surface antigen CD44,CD45 and cd90.construction and identification of Bcl-2 gene expression vector was transfected into BMSCs,in order to obtain the Bcl-2 over expression of BMSCs,RT-PCR and Western-blot were used to detected the expression of Bcl-2.The efficiency of cell proliferation was detected by MTT method.2 、 20 healthy male SD rats were randomly divided into 4 groups,5 rats in each group,using the method of the intramuscular injection,MSCs transplantation in rats with complete spinal cord injury after surgery.Four groups as follow: A: sham operation group(group Sham,n=5);B: injury control group(SCI group,n=5);C: emptyvector BMSCs treatment group(BMSCs group,n=5): in the injury site,empty vector BMSCs was injected;overexpression of D:Bcl-2 BMSCs treatment group(Bcl-2 + BMSCs group,n=5): in the injury site,Bcl-2 over expression of BMSCs was injected.All rats were observed for 6 consecutive weeks,the scores of Basso,Beattie and Bresnahan(BBB)score was record every week,to evaluate the limb function in rats.In sixth weeks after the injury score selection stage or rats and damage corresponding stage slicing,TUNEL method was used to observe the neural apoptosis.At the end of the sixth week score,the rats were injured or injured at the corresponding stage,and TUNEL method was used to observe the neuronal apoptosis.Results1 、 The third generation of cells cultured for Twelfth days,the cell morphology was observed by inverted microscope,cell body to nucleus shrinkage,cells were bipolar or multipolar,smaller cell body,less cytoplasm,high refractive index,slender protrusions and connected with each other for the cross,the typical morphology of neurons.Flow cytometry was used to detect the surface markers of BMSCs showed that CD44 and CD90 positive,the positive rate were 97.8% and 99.9%,CD45 negative expression,the positive rate was0.9%.2 、 Bcl-2 over expression vector group Bcl-2 expression was significantly higher than the other two groups.3 、 The BMSCs amplification efficiency in Bcl-2 over expression vector group was significantly higher than empty carrier control group(P<0.05)4 、 SCI group,BMSCs group,Bcl-2+mbscs group rats BBB score improved gradually in six weeks.in the 5,6 week,BMSCs group and Bcl-2 mbscs group rats were higher than SCI group(P < 0.05),but at 6 week,The score in Bcl-2+BMSCs group was higher than BMSCs group(P < 0.05).5 、 At sixth weeks,the TUNEL positive cells were few in SCI group.the TUNEL positive cells were more in Sham group,BMSCs group,Bcl-2 mbscs group.TUNEL positive cells were found in the spinal cord,the distribution of gray and white matter,mostly in white matter.the number of positive cells in Bcl-2+ MBSCs group was 17.09 + 3.87 /hp,BMSCs group is 8.23 + 1.09 /HP,statistical analysis found that the number of positive cells in the Bcl-2+mMBSCs group were more than BMSCs group,the difference was statistically significant(P < 0.01).6 、 BDA positive nerve fibers in SCI group were less,and most in BMSCs group.The quantitative analysis showed that SCI group,Sham group,BMSCs group,Bcl-2 BMSCs group were 4.37±1.12%,18.76±1.98%,38.65 ±3.01%,61.09±4.08%.Statistical analysis showed that SCI group was lower than Sham group 、BMSCs group or Bcl-2+BMSCs group(P<0.05),Bcl-2 + MBSCs was significantly higher than BMSCs group,the difference were statistically significant(P < 0.01).Conclusion1 、 BMSCs transplantation can treatment of spinal cord injury in rats effectivly.2 、 The efficacy of Bcl-2 over expression of BMSCs transplantation for the treatment of spinal cord injury in rats was better than simple transplant bsmcs,specifically in the lower limbs motor function better recovery,reduce the apoptosis of nerve cells. |
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