Investigate The Effect Of Salvia Miltiorrhiza For The Proliferation And Ossification Of Human Mesenchymal Stem Cells (MSCs) In Vitro

ObjectiveInvestigate the effect of Salvia miltiorrhiza for the proliferation and ossification of human mesenchymal stem cells(MSCs)in vitro.There are important academic value, practical value and social significance in Combining Chinese medicine research with cell engineering, promoting the process of modernization of Chinese medicine, and developing Chinese medicine and cell engineering research.Methods1.Establishing the method systems of isolation,cultivation and identification of hMSCs in vitro.Single cell was centrifugalized by Percoll gradient separating medium, cultured and amplified in cubator of 37℃,5%CO2 and saturated humidity,used in the experiment after the third passage. Cell of the experimental group was cultured in salvia miltiorrhiza culture medium of 10 ug/ml, Cell of the control group was cultured in ordinary medium.They have same culture condition and the passage method.Then observed by inverted microscope, its growth curve of proliferation was depicted. Cell surface antigens were detected by euzymelinked immunosorbent assay,and identify mesenchymal stem cells from the cells isolated and cultured from the bone marrow. 2. hMSCs were induced directionally to osteoblast differentiation.Took the hMSCs of third generation in good state,diluted it to about 105/ml,took 0.5mL and inoculated to 12-culture plate each time, When the cells fused to of 60%—70, droped mediums. There were three parallel groups (A, B, C), group A was blank without any inductor, the control group was group B with inductor(which consists of common culture medium, 100nmol/L Dex,10mmol/Lβ-GP,50umol/L AA), the experimental group was group C with the inductor just mentioned and salvia miltiorrhiza culture medium of 10 ug/ml.After 24 hours, Group B replaced by fresh medium and Group C into salvia miltiorrhiza culture medium of 10 ug/ml, kept culturing. According to the change of color of the medium, replace the culture medium every 2-3 days. Cell growth and proliferation of changes in the morphology was observed by inverted microscope every day, undertook alkaline phosphatase staining and calcium nodules staining by Von—Kossa assay.ResultsGradient centrifugation with adherent culture may be an effective method for separation and purification of hMSCs. A small amount of primary cell adhere after cultured in vitro 48 hours, then formed into a fiber-like colony units.With the extension of culture, the morphous of the cells gradually in line, they growed and proliferated actively, CD29 and CD71 positive expression, CD34-negative. In the seventh day of inducting cunlture, alkaline phosphatase staining and calcium nodules staining by Von—Kossa assay both positive in the experimental group.The majority of alkaline phosphatase staining negative and a small number weak positive in the control group, no mineralization of deposition and black nodules by Von—Kossa assay. ALP staining of the blank group have shown that the cytoplasm was pink, the cells still retain a clear fibroblast-like appearance.Conclusions1.The process of Obtaining hMSCs is safe and convenient, gradient centrifugation with adherent culture may be an effective method for separation and purification of hMSCs.2. Mesenchymal stem cells can be better induced into osteoblasts by salvia miltiorrhiza culture medium.