Serum Of Diabetes Affects Human Umbilical Cord Blood Stem Cell Proliferation And Apoptosis By Complement Dependent Pathways

ObjectStudying the influence of proliferation, apoptosis, as well as translation of Human umbilical cord mesenchymal stem cells that stimulated by the diabetic serum, and to investigate the association of diabetes with stem cells.Material and Methods1. MaterialHuman umbilical cord blood (Chongqing Daping Hospital); The diabetic serum (Hospital Secretion Branch); Complement inhibitor (GLBiochem shanghai Ltd); Annexin V-FITC/PI Apoptosis Assay System (BD, America); Steady-Glo(?) Luciferase Assay System (Promega, America).2. MethodsThe separation and purification of hUCB-MSCs was performed by adherent culture method. The hUCB-MSCs after the identification were incubated with glucose and leu, with the serum heated or unheated from diabetes and normal humen, the serum of diabetes and normal humen administrated with complement inhibitor respectively, the proliferation of hUCB-MSCs was detected by MTT assay, the apoptosis was detected by Annexin V/PI double staining and cell cycle was detected by pI staining, the apoptosis related proteins puma, bcl-2, bax, caspase 3 and caspase 9 and the cycle related proteins CyclinA、CyclinB、CDK1and CDK2 were detected by western blotting. Then, the hUCB-MSCs was thansfered with luciferase reporter gene before administrated with unheated serum from diabetes and normal humen with or without complement inhibitor, then chemiluminescence of the hUCB-MSCs were detected.Result1. There was no significantly different between proliferation and apoptosis ratio of hUCB-MSCs when they were treated by the high glucose or leu or glucose and leu.2. There was no significantly different between proliferation and apoptosis ratio of hUCB-MSCs when they were treated with the serum of heated of diabete or normal humen.3. There was no significantly different between proliferation and the apoptosis ratio of hUCB-MSCs when they were treated with the serum of heated or unheated of normal humen.4. Comparing with hUCB-MSCs that were treated with the serum of unheated from normal humen, the unheated serum from diabete significantly inhibited the proliferation of them.5. Comparing with hUCB-MSCs treated with the serum of unheated from normal humen, the unheated serum from diabete significantly inhibited the proliferation and cell cycle and cyclinA、cyclinB、CDK1 and CDK2 and translation of them, at the same time, promoted the apoptosis ratio and the expression of apoptosis protein Puma、Bcl、 Bax、Caspase3 and Caspase9.6.In order to remove the complement influention, the serum which was came from normal and diabete humen was incubated with complement inhibitor pmx-53.To our surprised, the complement inhibitor reversed the effect of diabete serum to hUCB-MSCs.In contrast, the complement inhibitor couldn’t reversed the effect of normal human serum to hUCB-MSCs.Conclusions:1. There was no significantly different of the proliferation of hUCB-MSCs,when they were treated with the high glucose and leu.2. The unheated serum from diabete significantly prohibited the proliferation and cell cycle and cyclinA、cyclinB、CDK1 and CDK2 and translation of them, at the same time, promoted the apoptosis ratio and the expression of apoptosis related protein Puma、 Bcl-2、Bax、Caspase3 and Caspase9 of them.3. The complement inhibitor reversed the proliferation, cell cycle, translation and apoptosis of the hUCB-MSCs treated with diabete serum.