Rehmannia Polysaccharide By Sample Notch Signaling Pathways Induced Bmscs Differentiation Into Neural Cells Of Experimental Research

Purposes:To explore the inductive effect of Rehmannia Glutinosa Polysaccharide (RGP) on bone marrow mesenchymal stem cells (BMSCs) differentiation into neuron-like cells and to screen its induction methods, meanwhile to further study its mechanism on Notch cell signaling pathway during induction.Materials and Methods:Test1:The femoral bone marrow of4-6week old healthy Wistar Rats were used. BMSCs were isolated and cultured using whole bone marrow adherence screening method, meanwhile cellular general states were observed, the proliferation vitality of BMSCs were tested by MTT method, the positive marker CD29. CD44and negative marker CD34were detected by immunocytochemistry method and flow cytometer.Test2:The effects of different concentrations of RGP on rat BMSCs’s vitality were tested by MTT method, then the cultured BMSCs were divided into the control group. BME group, BDNF group, and RGP induction groups with different concentrations and different induction durations, and then they were pre-induced and induced respectively. After7days of regular culture, the NSE protein expressions in different groups were tested by Elisa, then the screening for optimized methods was conducted. The percentages of induced BMSCs with positive neural markers were obtained by immunocytochemistry; the expression of Nestin、 βⅢ-tubuln、NSE、GFAP at different durations time point in RGP group C③were tested by Western blot, and after3days of induction, the expression of neural cells surface markers were tested by RT-PCR.Test3:The P3-P5generations of BMSCs were divided into the control group and RGP group, and they were induced and cultured for7days using the optimized method, the Notchl and Jaggedl protein expression at different durations were tested by immunocytochemical method, the Notchl protein intracellular Domain (NICD) and Jaggedl protein expression at different durations were tested using Western blot, the Notch signaling pathway related molecular mRNA expression and changes were analyzed by Real-time PCR. Results:1. The isolation, culture and identification of rat BMSCs:(1) After10days of adherent culture, the primary cultured BMSCs demonstrated healthy growth; the3rd generation had uniform morphology and maintained favorable reproductive activity.(2) the cell growth profile reveals:P1had irregular growth, from P3, cells demonstrated regular growth, P3and P5BMSCs had similar growth patterns, each generation had a short-term detention period before cell growing, then showed apparent acceleration afterwards, double growth rate occurred, after logarithmic phase, the cells showed slow growth, entered the plateau stage.(3) the BMSCs were identified by immunocytochemistry, which showed the CD29and CD44marker expression was positive, the CD34expression was negative.(4) The flow cytometer testing of BMSCs showed:The CD29and CD44expression positive rates were97.6%and93.8%. The CD34expression was2.3%.2. The screening of optimized method for RGP induced rat BMSCs differentiation into neuron-like cells:(1) In a range of concentrations. RGP can promote the proliferation of BMSCs after passage. At lower concentrations, the proliferation effect was dose dependent. However, at higher concentrations. BMSCs proliferation was not increased accordingly, but OD value decreased.(2) The ELISA results showed:with the increase of induction duration, the OD values of NSE expression in the BME group. BDNF group and RGP all groups become higher, while those of control group. BME group, BDNF group at3d and5d were significantly lower than RGP group C③(P<0.05).(3) General states observation:no neuron-like were observed in control group. In BME group, cellular morphological changes occurred within half an hour after the input of inductor, within6h, neuron-like cells increased significantly, which reached its peak at24h. then large scale of cell death occurred. The cellular changes of BDNF group were similar to those in RGP group, while occurred a little later than those in BME group, as duration increase, more and more neuron-like cells occurred, especially in RGP group, cells demonstrated favorable growth and longer duration till5d.(4) Immunocytochemistry showed:at3d. NSE positive expression rate in RGP group is (76.17±6.27)%. which was higher than those in other groups(P<0.05), while GFAP positive expression rate (10.17±1.27)%is significantly lower than BDNF group (P<0.05).(5) Western blot and RT-PCR testing showed, in line with immunocytochemistry staining outcomes, the expression of nestin, NSE, GFAP, βⅢ-tubulin mRNA after induction were detected in all test groups.3. The effects of RGP on Notch signaling pathway during BMSC differentiation:(1) In RGP group, after completion of induction, the Notch1protein expression of BMSCs decrease with time, positive cell percentage decreased likewise, which was significantly different from normal control group(p<0.01). The Jagged1positive cell rate of RGP group increased from induced end Od to1d after induction, decreased significantly from1d to7d, and there was significant difference between each time point comparison(P<0.05).(2) Western blot testing showed:the NICD expression in normal control group maintained high level, there was no significant difference between different durations(P>0.05). However, in RGP group. NICD expression decreased with time, which of ld.3d,7d was significantly different from0d(P<0.01). the Jaggedl protein relative expression of RGP group went up from Od to1d after induction, and went down significantly from1d to7d. In RGP group, the expression of Jaggedl were higher than those in control group at0d,1d,3d,however lower at7d,the difference was statistically significant (P<0.05).(3) Real-time PCR testing analysis:in RGP group, Notchl mRNA expression decreased with time. Presenilin1expression decreased and then went up mildly afterwards. Hes1expression decreased, Mash1expression increased, Jagged1expression increased then decreased. Which were significantly different from control group at all durations(P<0.01).Conclusions:1. RGP, the effective ingredient of kidney invigorating Chinese medicine named prepared rehmannia root, can successfully induce bone marrow mesenchymal stem cells (BMSCs) to differentiate into neuron-like cells. With basic fibroblast growth factor (bFGF) pre-inducing, BMSCs were induced by200μg/ml concentration of Rehmannia glutinosa polysaccharides. which the induction was mild in action and relatively potent.2. Although the proliferation of BMSCs depends on the hyperexpression of Notch protein, RGP can cause the decrease of notch protein and the expression quantity of its intracellular domain (NICD) during the induction for BMSCs to defferentiate towards neuron-like cells. Furthermore, it can influence the the expression of Notch signaling pathway related molecular mRNA. It showed that Notchl mRNA expression decreased with induction time, Presenilin1expression decreased then went up mildly afterwards, Hes1expression decreased, Mash1expression increased, Jagged1expression increased and then decreased.