The Early Stage Of Experimental Research Of Fixing The Apoptosis Of Neural Cell By BDNF Over-expressed BMSCs

Objective(s):After isolation,purification,culture,identification of rabbit neural cells,rabbit neural cellular apoptotic model can be constructed by serum-deprivation to explore the onset and progress of the apoptosis of neural cells and the optimal interventional time,and to search the characteristics of apoptotic neural cells in each time spot.Constructing the BDNF over-expressed BMSC and coculturing with apoptotic neural cells had been done to explore the preventive and recovery effects of BDNF over-expressed BMSC to apoptotic neural cells in vitro.Methods:1.Cell isolation,culture,identification of rabbit BMSCs(1)Primary culture and subculture of BMSCs after isolating and purifying;(2)Flow cytometry was used to identify BMSCs(CD29,CD34,CD45,CD90 staining)subculturing to the third generation.2.Isolation,primary culture,identification of rabbit apinal neural cells(1)Primary culture of rabbit apinal neural cells after isolating and purifying;(2)Immunohistochemical method was used to identify the rabbit apinal neural cells(Nestin,NSE staining);3.To construct the rabbit neural cellular apoptotic model by serum-deprivation(1)remove the culture medium in primary culture of rabbit apinal neural cells(2)resuspense of neural cells by using trypsase and culturing rabbit neural cell by serum-deprivation in 72 hours at the density of 1×105cells/spot(3)Identification of apoptotic ratio and status of neural cells by Annexin V-PE and 7-AAD Kit in the time of 24h,48h and 72h4.To construct and identify rabbit BDNF over-expressed BMSCs(1)To construct rabbit BDNF over-expressed BMSCs secreting BDNF constantly(2)Identification of the expressing of BDNF gene and BDNF by RT-PCR and Western Blotting.5.Co-cultivation of BDNF over-expressed BMSCs and apoptotic neural cells and Identification of apoptotic ratio and proliferation condition after then(1)Co-cultivation of BDNF over-expressed BMSCs and apoptotic neural cells using Trans well double petri dishes and identification of apoptotic ratio.(2)Co-cultivation of BDNF over-expressed BMSCs and apoptotic neural cells using CCK-8 Kit and identification of neurogenic differentiations and proliferation condition.6.Results testSPSS 17.0 statistical software was used to analyze,all the data with mean ? standard deviation(x±s)said,the difference of data between groups were analyzed by independent sample t-test,the difference of data within group was analyzed by single factor analysis of variance,and P<0.05 had statistical significance.Results:1.rabbit neural cellular apoptotic model can be constructed by serum-deprivation,then using PE Annexin V Apoptosis Kit to identify the apoptosis of neural cells:Apoptosis Group:In the time of 24h the early apoptosis rate:15.98%,the late apoptosis rate:2.40%,the total rate of apoptosis:18.38%;In the time of 48h the early apoptosis rate:18.13%,the late apoptosis rate:1.12%,the total rate of apoptosis:19.25%;In the time of 72h the early apoptosis rate:39.52%,the late apoptosis rate:15.45%,the total rate of apoptosis:54.97%;Normal Contral Group:the early apoptosis rate:7.92%,the late apoptosis rate:2.22%,the total rate of apoptosis:10.14%.From 48h to 72h the late apoptosis rate was elevated significantly.The difference of data among groups were analyzed by single factor analysis of variance,and P<0.05 had statistical significance.2.Co-cultivation of BDNF over-expressed BMSCs and apoptotic neural cells using Transwell double petri dishes and identification of apoptotic ratio(OD value represent the amount of cells):Apoptosis Group:the early apoptosis rate:6.15%,the late apoptosis rate:2.64%,the total rate of apoptosis:8.79%;Normal Contral Group:the early apoptosis rate:0.8%,the late apoptosis rate:2.41%,the total rate of apoptosis:3.21%;BDNF over-expressed Grroup:the early apoptosis rate:0.78%,the late apoptosis rate:2.05%,the total rate of apoptosis:2.83%;BDNF inhibited Group:the early apoptosis rate:2.49%,the late apoptosis rate:2.22%,the total rate of apoptosis:4.71%;BMSC Group:the early apoptosis rate:1.45%,the late apoptosis rate:2.90%,the total rate of apoptosis:4.35%;eGFP-BMSC Group:the early apoptosis rate:1.81%,the late apoptosis rate:2.56%,the total rate of apoptosis:4.37%;BDNF over-expressed Group have a lower apoptosis rate than other groups.the difference of data among groups were analyzed by single factor analysis of variance,and P<0.05 had statistical significance.3.Co-cultivation of BDNF over-expressed BMSCs and apoptotic neural cells in 72 hours,using CCK-8 Kit and identification of proliferation condition(OD value represent the amount of cells):Apoptosis Group:OD 450:0.512.0.909.1.036;Normal Contral Group:OD 450:1.244.1.045?1.141;BDNF over-expressed Grroup:OD 450:1.902?1.952?1.640;BDNF inhibited Group:OD 450:0.821?0.802?0.736;BMSC Group:OD 450::1.244?1.045?1.141;the difference of data among groups were analyzed by single factor analysis of variance,and P<0.05 had statistical significance.4?Mixed culture of BDNF over-expressed BMSCs and apoptotic neural cells in 72 hours,using CCK-8 Kit and identification of proliferation condition:Apoptosis Group:OD 450:0.512?0.909?1.036;Normal Contral Group:OD 450::1.209?0.373?0.765;BDNF over-expressed Grroup:OD 450:1.461?1.533?1.124;BDNF inhibited Group:OD 450:1.85?0.934?1.024;BMSC Group:OD 450::1.61?1.214?1.74;the difference of data among groups were analyzed by single factor analysis of variance,and P<0.05 had statistical significance.Conclusion(s):1.BDNF over-expressed BMSC shows a significantly protective effect to the early stage of apoptotic neural cells and shows little effect to the late stage of apoptotic neural cells.2.Rabbit BMSC shows a significantly protective effect to the early stage of apoptotic neural cells and shows little effect to the late stage of apoptotic neural cells.4.Rabbit BDNF could accelerate the proliferation of apoptotic neural cells.5.Rabbit BMSC could reduce the proliferation of apoptotic neural cells.6.Rabbit neural cellular apoptotic model can be constructed by serum-deprivation;the apoptosis rate is associated with duration in this model.The optimal interventional time is at the time of 48 hours.