Research Of Repair Function Of QDTMT Plus Mesenchymal Stem Cell Toward Homocysteine Induced Apoptosis Umbilical Vein Endothelial Cell

Background:Endothelial cell damage is the basis of many cardiovascular diseases. It is key solution to reverse the EC damage and enhance the repair processes of the damaged EC. High plasma homocysteine (HCY) levels have been associated with many cardiovascular diseases result from the damaged EC. Caspases are the main driving factor in cells which destined to undergo apoptosis and their activities is affected by Inhibitive Apoptosis Proteins (XIAP). How to reverse the damage of EC, restore the EC'function is the hot spot of researching cardiovascular diseases. As we know, bone marrow stem cell is multiple potential ability stem cells which can differentiate into almost all kinds of cell of three embryonic layers. It has been reported that MSCs could promote neovascularization and regenerated damaged ECs. While can Chinese herbs reverse the apoptotic ECs ? Whether the mechanism of repairing has the relation with XIAP? Those entire questions are not clear. So our experiment is based on the co-culture system of ECs and MSCs to investigate the repair function of QDTMP intervention towards apoptotic ECs. Objective: 1,The vivo co-culture system is established to imitate the damage-repair environment of vitro. To investigate whether the Chinese herbs QDTMP can reverse the apoptotic HUVEC.2,Analysis the expression of caspase-3,c-IAP-1,c-IAP-2, primarily detect the repairing mechanism of QDTMT towards the HCY induced apoptotic HUVEC.Method:1,The preparation of QDTMT plasma: choose the adult 24 SD rats (weight 250-300g). The rats were separated randomly into 3 groups. The two groups were feed with QDTMT while the other group was feed with normal saline. Three days later, put the rats to death and extract the peripheral blood mononcleus cell with the lymphocyte separation medium; Then we get the QDTMT plasma and the normal saline plasma. The QDTMT plasma were used the 10% DMEM to compound into 0.15%,1.5%,15% culture medium.2,Cell-culture: Using the whole marrow-adherence way to get the MSCs, using collagen-digest way to get the HUVEC. Then using MTT to analysis the different concentration QDTMT plasma's affection towards MSCs and HUVEC. Immunocytochemistry staining to detect the Fâ…§expression.3,Trough Transwell to establish the co-culture system: damaged HUVEC were inoculated into the upper chamber and the MSCs were inoculated into the lower chamber. This system rightly inmate the vitro damage-repair environment.4,Ultraviolet spectrophotometer to analysis the dosage of SOD,MDA and LDH. Immunocytochemistry fluorescence to observe the differentiate state of MSCs to ECs.5,Hoechst 33258 and flow cytometry to investigate the percentage of HUVEC' apoptosis.6,West-blotting to investigate the expression of caspase-3,c-IAP-1,c-IAP-2 of HUVEC.Result1,We can get adherence cell through marrow-adherence way the next day. The cell is scattering on the ground, three days later we can see a lot of compressed and Fusiform cells, they are generate very rapidly in a circinate style. Our cultured HUVEC can found adherence 36 hour later, in a small Fusiform state, Fâ…§staining is shows positive. MTT tells that the QDTMT has no toxic affection to both the HUNEC and MSCs.2,HCY can effective induces HUVEC apoptosis. Under the electricity microscope, we can find the damaged cell'core is pycnosis. Compare with the damage-control group, the MDA of all other group is obviously higher and the result has statistic meaning. The SOD of damage-control group is significant lower than other groups, while the SOD of MSCs-control group has no significant difference with the 0.15% QDTMT,1.5%QDTMT plasma groups. The LDH of damage-control group has significant defiance with all other groups while the MSCs-control group has no significant difference with all the QDTMT intervene groups.3,The result of Hoechst 33258 and flow cytometry show that the apoptotic percentages of HUVEC are quite different. The apoptotic percentages of MSCs-control group is lower than the damaged group and the the apoptotic percentages of all the QDTMT intervene groups are lower than the MSCs-control group. 4,The result of western-blotting shows that all the caspase-3 of QDTMT intervene groups whose activity is restrained, and the expression of their c-IAP-1 and c-IAP-2 is inhibited.5,Under the HCY environment, MSCs can differentiate into ECs in the affection of HUVEC, who shows the Fâ…§positivity. Conclusion:MSCs can restore the damaged HUVEC, Chinese prescription QDTMT can promote the repair possess. It can be explain by the two following ways: 1,QDTMT can directly enhance the antioxigenation ability. 2,By increasing the expression of c-IAP-1 and c-IAP-2 to inhibit the activation of caspase-3, which eventually inhibit the apoptosis processes and then protect the HUVEC.