Expression Of NSE, Nestin And NF-H During Neural Cell Differentiation Of Mesenchymal Stem Cells From Human Umbilical Cord Induced By Radix Astragali

It is difficult to cure diseases of the central nervous system. With the development of modern biotechnology, the stem cell transplantation for hope provides strategies in therapy for nervous system diseases. Human umbilical cord considerated a biological wast tissue after delivery, and Wharton’s jelly of human umbilical cord derived mesenchymal stem cells (WJ-MSCs) belong to adult stem cells exhibited self-renewal, multilineage differentiation potential and the unique immunomodulatory properties. Studies have shown that, WJ-MSCs possess the potential to differentiate into neural like cells with proper induction conditions in vivo and in vitro. During this process, the application of traditional Chinese medicine China received the great attention. Radix Astragali with benefiting Qi and supporting healthy energy, is widely used in traditional Chinese medicine formula. According to reports, Radix Astragali has a protective effect on the nervous tissue, and induce WJ-MSCs to differentiate into neural like cells. Our research group has been established technology platform for cultivation of WJ-MSCs, and observed its biological characteristics. The expression of nestin and NF was detected by immunohistochemistry during WJ-MSCs differentiation to neural like cells induced by Radix Astragali. However, their mRNA expression level is unclear.AimrThis study explored WJ-MSCs potential differentiation to neural like cells induced by Radix Astragali, and the intervention effect of calcium channel blocker. Observed the effect of Radix Astragali on the expression of NSE, nestin and NF-H in WJ-MSCs.Methods:WJ-MSCs were prepared by tissue explant culture and pured by passaged using trypsinizing. WJ-MSCs at passage 3-5 was performed in the experiment. WJ-MSCs were induced to differentiate into neural like cell by Radix Astragali in different concentration(5ug/ml,50ug/ml,500ug/ml,5000ug/ml, 25000ug/ml) and time (3h,6h,24h,48h and 72h). The gene expression of NSE, nestin and NF-H was detected by RT-PCR, and observed the its impaction of calcium channel blocker LY294002 and manganese chloride (MnCl2). In addition, the expression of NSE protein was tested by Western blot (WB). It had great significance to clarify the efficacy of Radix Astragali and the differentiation potential of WJ-MSCs, and provided important information for establishment induction system for WJ-MSCs differentiation into neural like cells, as well as laid the theoretical foundation to explore the mechanism in induction of the neural differentiatio by Radix Astragali.Results:WJ-MSCs grew typically in spindle-like or fibroblast-like appearance, grew intensively and arranged in scrol and fascicular shape companied with active proliferation capacity and strong light refraction of cytoplasm. RT-PCR analysis showed that the expression of gene NSE, nestin, and NF-H appearanced in the control group and induction group with Radix Astragali. But in induction group, the expression of NSE gene was increased obviously with dose-response relationship at 48h and 72h. The expression of NF-H gene was increased at the concentration of 5ug/ml、50ug/ml and 25000ug/ml 48h, as well as similar effect at 72h with 5ug/ml、 50ug/ml. It was observed to augmen the nestin gene expression with 5000ug/ml、 25000ug/ml at 24h. Compared with the control group, it was found that there was significant difference(P<0.05) in the expression of NSE, NF-H and nestin. The experiments of adding of calcium channel blockers (LY294002 and MnCl2) showed that the expression of nestin and NF-H gene was significantly decreased(P<0.05) in WJ-MSCs induced by 50 ug/ml Radix Astragali 6h,24h, respectively. But it can’t influence NSE gene expression. The strong protein expression of NSE in WJ-MSCs induced by Radix Astragali at the concentration from 12.5ug/ml to 62.5ug/ml in 24h was detected by Western blot, which showed the best concentration was 50ug/ml. While WJ-MSCs were incubated with 50ug/ml Radix Astragali 6h, the NSE protein expression can be effectively induced.Conclusion:At before and after Radix Astragali induction, the expression of NSE, nestin and NF-H mRNA was appeared in WJ-MSCs. But their expression was upregulated as well as NSE protein. The expression of nestin and NF-H can be down-regulated by LY294002 and MnCl2. Above all, Radix Astragali can induce WJ-MSCs to differentiate into neural like cell, and be affected by the inhibition of calcium ion channel.