Effect Of Extract Of Qiang Ji Jian Li Liquid On Nuclear Receptor In The Proliferation Of Rat Mesenchymal Stem Cell In Vitro

Qiang ji jian li liquid is a major experience prescription of Deng TieTao to treat myasthenia gravis. The prescription is based on Bu Zhong Yi Qi liquid from dynasty of Yuan. Researches indicate that Qiang ji jian li liquid can reduce Skelaxin of benzcurine iodide and take two-ways regulation on gastrointestinal motility. It can even accelerate AChR of Nicotine by the speed of infiltrating diaphragmatic muscle and step down acceptor's degradation to treat myasthenia gravis. whether the therapeutic action of Qiang ji jian li liquid on myasthenia gravis by adjusting stem cells or not still need to be approached.Objective:The topic's objective is approaching the proliferation mechanism of extractives of Qiang ji jian li liquid by definiting the active site and effective target.Methods:1. After segregating MSCs from femoral bone and tibia of rat, adopted adherent culture method in vitro.2. Accrediting CD44 on MSCs's surface by flow cytometrics.3. Detect whether extractives of Qiang ji jian li liquid can proliferate MSCs or not by the method of MTT; authenticate Qiang ji jian li liquid's active site by flow cytometrics.4. Bolting nuclear receptors of MSCs after adherent culture in vitro.5. Bolting growth factor of MSCs after adherent culture in vitro. 6. Detect Desmin and Myosin on MSCs after adherent culture in vitro by immunity class.Result:1. The cells which had been segregated attached partly in 24 hours. In order to reduce suspension cells and some incompact adherent cells, we exchanged half of the medium and washed cells. After 2 days, we could see some adherent cells and Fusiform shape cells distributed powder. After 4-6 days, every 10 or 20 cells buildup a cell clone. After 7-9 days, cells proliferated and extended, connected to slices partly. After exchanging medium several times, we digested and went down cells to the future generation when they connected to 80% fusion.2. After analysis marks on the cells' surface, we found the cells we had cultured expressed CD44, but CD34/CD45 which are marks of HSCs or WBCs. We can conclude the cells are MSCs, because CD44 is the mark of MSCs.3. The result of MTT displayed that the acetic ether extractive of Qiang ji jian li liquid had proliferation activity, other parts of Qiang ji jian li liquid didn't had such activities.In advanced verification, we used flow cytometrics to identify. We found that the acetic ether extractive of Qiang ji jian li liquid really had proliferation activity, and the activity relied on the does of the extractive. MTT and flow cytometrics certificated the acetic ether extractive of Qiang ji jian li liquid had proliferation activity in common.4. The results of immunohistochemistry and western blotting indicated that MSCs which had been cultured by the acetic ether extractive of Qiang ji jian li liquid expressed VDR and RARαof nuclear receptor greatly.5. The results of immunohistochemistry and western blotting indicated that MSCs which had been cultured by the acetic ether extractive of Qiang ji jian li liquid expressed CDK2,PDGF and RAS of growth factors greatly.6. The results of immunohistochemistry and western blotting indicated that MSCs which had been cultured by the acetic ether extractive of Qiang ji jian li liquid expressed Desmin and Myosin greatly. Desmin and Myosin are marks of muscle cell on the surface. We concluded that MSCs are differentiate to muscle cells when Desmin and Myosin expressed masculine.Conclusions:1. The acetic ether extractive of Qiang ji jian li liquid had proliferation activity, other parts of Qiang ji jian li liquid didn't had such activities.2. The acetic ether extractive of Qiang ji jian li liquid can activised nuclear receptors VDR and RARα, which activised PDGF. PDGF associated to cell itself by autocrine, and then activised RAS and CDK2. CDK2 can activised MSCs proliferation.3. The acetic ether extractive of Qiang ji jian li liquid can makes MSCs expresses Desmin and Myosin greatly.