Effect Of Total Panax Notoginseng Saponins On Inducing Human Bone Mesenchymal Stem Cells Differenting Into Neuron-like Cells And Apoptosis Of Neuron-like Cells In Vitro

Abstract Objective: Human bone mesenchymal stem cells can be differentiated into neuron-likecells(NLCs) by total Panax notoginseng saponins (tPNS) and bFGF in vitro.To detect survival ofdifferentiated NLCs and probe the optimum concent ration of inducing mesenchymal stem cellsinto neurons and resist apoptosis.Methods: Human BMSCs harvested from adult normal human marrow, then were passaged andpurified in culture medium and cultured for 3 passages. Cell surface markers CD29,CD44,CD105,CD34 and NLCs cycle were tested by flow cytometer. Culture medium containing 0.2volume fraction of fe tal bovine serum and 1 mmol/Lβ-mercapto-ethanol (β-ME) were used for24-hour pre-induction. The study was as signed into six groups: tPNS group containing 0.5mg/mL tPNS+20ng/mL bFGF, 1.0 mg/mL tPNS+20ng/mL bFGF, 2.0 mg/mL tPNS+20ng/mLbFGF, Standard control group containing 20ng/mL bFGF. Drugs group containing 1.0 mg/mLtPNS and blank control group without the addition of any induction agent. The expression ofneuron-specific enolase (NSE) and microtubule-associated protein-2(MAP-2) and glia l fibrillaryacidic protein (GFAP) was measured by immunocytochemical method. After treatments,TUNEL method and flow cytometer technique was used to calculate the percentages ofneuron-like cells apoptosis.The effects of tPNS on the proliferation of NLCs were tested byMTT.Morphological changes and survival time of induced MSCs were observed during celldifferentiation and survival.Results:1. The human bone marrow stromal cells can be induced to differentiate into NLCseffectively with tPNS and bFGF in vitro. Flow cytometer studies presented that the culturedhBMSCs were CD29,CD44,CD105 positive and CD34 negative,which displaying features ofhBMSCs. NLCs cycle tested by flow cytometer showed: a high percentage of G0/G1 phasecells suggested hBMSCs high differentiation potential.2. Immunocy tochchemistry staining showed that the cells after induced NSE and MAP-2positive. After the tPNS and bFGF induced the mature neurocyte with high concentration wasNSE,MAP-2 positive (P<0.05),while MAP-2 and NSE positive cells increased markedly inhBMSCs with the increase in tPNS dose(P<0.05). GFAP were negative in control groups(P<0.05).3. The effects of tPNS and bFGF on the activity and cell proliferation of neuron-like cellswere observed by MTT. After the tPNS and bFGF induced, The activity of neuron - like cellssignificantly increased(P<0.05),while the activity of induced neuron-like cells increased with theincrease in tPNS dose(P<0.05). Cell death occurred slowly in the cells that were induced todifferentiate into NLCs by tPNS and bFGF.4. TUNEL method and flow cytometer technique was used to calculate the percentages ofneuron-like cells apoptosis respectively.Compared with control group, apoptosis index ofdifferentiated NLCs were significantly descend (P<0.05), while apoptosis index with the increasein tPNS dose were descend(P<0.05).ConclusiConclusion:1. The human bone marrow stromal cells can be induced to differentiate into NLCseffectively with tPNS and bFGF and expression of neural cell surface antigen in vitro.AftertPNS and bFGF of co-culture we achieved better results, maintaining hBMSCs better activity.Effect was positively correlated with the dose.2. The hBMSCs induced by tPNS and bFGF in vitro obationed more optimummultiplication capacity,After tPNS and bFGF of co-culture we achieved better results,andextend cell survival time ,and they are not to be aged. Effect was positively correlated with thedose.3. The apoptosis index of hBMSCs induced by tPNS and bFGF were descend.While tPNSand bFGF of co-culture the result is more effective. Effect was a negative correlation with drugdose.