Influence Of TMPZ On The Proliferation, OGD Cell Viability And Paracrine Functions Of BMSCs In Vitro

Objective To observe the influence of TMPZ on the proliferation, OGD cell viability and paracrine functions of BMSCs in vitro and explore the mechanism of TMPZ induced protective effect against OGD, in order to provide cell-based evidence for its further research, and to provide new strategy for in vitro drug screening in the combination therapy of stem cells and complementary medications.Methods①Whole bone marrow adherent culture method combined with differential digestion was applied for the separation and purification of BMSCs. BMSCs from P3to P6perform microscopic examination for cells morphology identification while CD29, CD45and CD90were determined by flow cytometry for cell surface marker identification.②The influence of TMPZ (0.001～100μM·L-1) on the proliferation of BMSCs was determined by MTT assay at time point24h and48h.③The influence of TMPZ (0.001～10μM·L-1) on the cell viability of BMSCs cultured under OGD condition for6h was determined by MTT assay.④The influence of TMPZ (0.01μM·L-1) on the mRNA expression of Bcl-2, Bax, p53and Caspase-3after cultured under OGD condition for6h was determined by quantitative real-time PCR.⑤The influence of TMPZ (0.01uM·L-1) on the protein expression of Bax, p53and Caspase-3after cultured under OGD condition for6h was determined by Western Blot.⑥The release of VEGF and IGF-1and the influence of TMPZ (0.001～10μM·L-1) on the cytokine secretion of BMSCs cultured for48h was determined by ELISA.Results①BMSCs from P3to P6exhibit homogeneous, fusiform or spindle-shaped and had whorled arrangement. Flow cytometry showed that cells were negative for CD45(4.0%) and positive for CD29(86.1%)&CD90(98.9%). These results were consistent with biological characteristics of BMSCs.②TMPZ (0.001～100μM·L-1) has no significant influence on the proliferation of BMSCs in the time scale of24h and48h (P>0.05).③Cultured under OGD for6h significantly reduce BMSCs cell viability (P<0.01);0.01μM·L-1TMPZ could significantly increase BMSCs cell viability from68.5%to85.4%(P<0.05).④Cultured under OGD for6h significantly increase Bax and p53mRNA expression levels (P<0.01) while Bax and p53mRNA expression levels were significantly suppressed in0.01μM·L-1TMPZ treated groups (P<0.01).⑤Cultured under OGD for6h increase Bax and p53protein expression levels and they were suppressed in0.01μM·L-1TMPZ treated groups.⑥The VEGF concentration was significantly higher in BMSCs-conditioned medium versus control (P<0.01), TMPZ (0.001～10μM·L-1) has no significant influence on the cytokines secretion of BMSCs in the time scale of48h (P>0.05).Conclusion The results of cells morphology and surface markers identification by flow cytometry were consistent with biological characteristics of BMSCs.0.01μM·L-1TMPZ could significantly increase BMSCs cell viability cultured under OGD condition and this might be related with its significant suppression to high Bax and p53mRNA and protein expression levels induced by OGD. No significant influence of TMPZ on the proliferation, VEGF and IGF-1secretion of BMSCs were observed in the present study.