Experimental Study Of Panax Notoginseng Saponin On The Proliferation Differentiation And Expression Of TGF-β1in Rabbit Bmscs In Vitro

Objective1. To observe the growth status and biological characteristics of rabbitBone Mesenchymal Stem Cells (BMSCs). To establish the stabilized culturesystem of rabbit BMSCs in vitro.2. To detect the influence of Panax Notoginseng Saponins (PNS) onproliferation and osteoblast differentiation of rabbit BMSCs.3. To detect the effects of PNS on TGF-β1expression of rabbit BMSCs.And to investigate the mechanism of osteogenic.Methods1. By adopting “Whole bone marrow adherent method” and “Densitygradient centrifugation method”, BMSCs were isolated from the leveret lonebone and BMSCs are purified and amplified of cell culture. Through theobservation of cell morphology and biological characteristics, a relatively stablecultivation system was established.2. The method called flow cytometric detection was used to detect therabbit BMSCs surface antigen markers CD29, CD45and HLA-DR., the growth curve was drawn from the results of MTT, which calculated the optical numberof cells for the experiment based on the reproduction rate, total cells number etc.According to the proliferation rate, cell number select the best generation ofrabbit BMSCs. The biological activity effect to rabbit BMSCs was analyzedunder different concentrations of PNS as a way to choose the most suitable drugconcentration.3. After the cultivation of BMCSs with PNS culture medium, toluidine bluestaining was used to measure the alkaline phosphatase (ALP) activity. Calciumnodules and protein expression of TGF-β1were measured by alizarin redstaining and Western blot method respectively. At the same time, Real timefluorescent quantitative PCR (qRT-PCR) was conducted to detect the mRNAexpression of TGF-β1.Results1.“Density gradient centrifugation method” and “whole bone marrowculture method” can successfully isolate and culture rabbit BMSCs. After24hours, the original generation of rabbit BMSCs can be stick adherence at thebottom. Cell fusion reaches about80to90percent of area in10-14days. Thepurity and reproduce rate of passage increased as well.Microscopic observation of typical rabbit BMSCs shows showed that cells withspindle shape fibroblast-like cell at the bottom.2. The positive rate of CD29on the cell surface, detected by flow cytometry,was97.4%.While CD45and HLA-DR were only0.1%. MTT method suggeststhat the P2cells have relatively high reproduce ability. There was nosignificant difference on proliferative ability of rabbit BMSCs between variousconcentrations of PNS medium and common medium. 3. The result of alkaline phosphatase staining demonstrates that ALPactivity of high drug concentrations group was higher than the negative groupand low concentration group (P<0.05), There was no significant differencebetween100mg/L PNS group and200mg/L PNS group. The result of alizarinred staining method showed that different PNS concentration groups hadvarying degrees of positive expression, osteoblasts induced group was stronglypositive. The number of calcium nodule with100mg/L PNS group wasmore than other PNS group (P<0.05).Western blot told us that the TGF-β1protein expression levels of PNS medication group was higher than the negativegroup, and the highest level was100mg/L group (P<0.05). The qRT-PCRresults showed that the TGF-β1mRNA expression of100mg/L PNS group andosteoblasts induced group higher than negative group (P<0.05).Conclusions1.“Density gradient centrifugation method” and “whole bone marrowculture method” both were able to obtain stability and purify rabbit BMSCs,the former could do much better.2. PNS promoted osteoblastic differentiation of rabbit BMSCs and the bestconcentration of PNS was100mg/L, but PNS had no significant effect on rabbitBMSCs proliferation.3. PNS could promote the expression of TGF-β1in rabbit BMSCs, it mightbe associated with BMSCs osteogenetic mechanism.