Effects Of Zuogui Wan On Cartilage Differentiation And Related LncRNA

Objective:Through the separation process,culture and passage of rat bone marrow mesenchymal stem cells（BMSCs） has been studied. one way is to grasp the master of basic techniques of cell culture and observe the intervntion process of rats containing serum about Zuoguiwan deal with P2 generation BMSCs;Predicting and selecting the possible related Long nonco-ding RNA（lnc RNA） of BMSCs during the process of differentiation into cartilage by gene chip technology.Validate the impact of lnc RNA during the process of differrenttiation into cartilage.In order to get a new idea to explore the Epigenetic mechanisms of Zuoguiwan,we need to promote cartilage different-iation and state the theory mechanism about "the kidney being in charge of bone".Methods: Adult SD rats abdominal aorta blood to prepare containing serum,after Zuoguiwan decoction intragastric administration.Take the 4-week-old rats as subjects,using the modified whole bone marrow adherent method to collect the Primary BMSCs,cultured in the condition of dulbecco’s modified eagle medium（DMED） with 10% Fetal Bovine Serum（FBS）and saturated humidity,37℃ and 5% CO2 environment in the Incubator.In liquid according to the growth of cells,passaged when Cells were covered 70%⁹0% of bottom area.Purified the BMSCs through continuous passage and medium ischanged,when the cells were cultured to the second generation,identified by cell morphology and flow cytometry（FCM）.The cytotoxicity test for Zuoguiwan containing serum with the second generation of stem cells;Sellect the fittest containing serum drug concentrations about rat BMSCs growth and proliferation by MTT colorimetry assay;Take P2-generation BMSCs as the research subjects,the experiments are divided into three groups: no-treatment Control Group（containing 10%FBS-H-DMEM complete medium）,control group（serum-free chondrogenic conditions H-DMEM）and experimental group（10% Zuo guiwan serum containing and serum-free chondrogenic conditions H-DMEM complete medium）,intervention at the same environmental conditions for 21 days;Detecting the chondrogenic results by observing changes in cell morphology,the m RNA levels of collagentypeⅡthr-ough realtime quantitative PCR（Real time RT-PCR）,in order to filter the related lnc RNA,we compare the achievement of gene chip technology between two groups.Verifying the effect of Zuoguiwan on cartilage process and the relevant lnc RNA through the lnc RNA spectrums of experimental group.Result:1This extracted and cultured experiment cells were mesenchymal stem cells;2MTT results:5% and 10% Zuoguiwan containing serum can promote BMSCs proliferation,compared with 10% FBS group,the proliferation of 10% Zuoguiwan serum containing even more significant;3Inducted 21 days in different condi-tions,it is found that:group B and experimental group Ⅱcollagen m RNA sione-xpres was higher than the no-treatment Control Group, control group was higher than group C（P<0.05）;4After the intervention, screened lnc RNA differ-ence expression profiles:There were 5 lnc RNAs significantly increased,4lnc RNAs significantly reduced.Conclusion: The modified whole bone marrow adherent method to collect the Primary BMSCs is a practical extractive method and it is simple to operate,easy to master;10% Zuoguiwan contains serum can promote BMSCs growth and proliferation;The related lnc RNA of BMSCs differentiate into cartilage may be:XR₀07242、MRuc008sbh、U50044、XR₀08107、uc.161+、MRAK166199、 BC098827、BC085903 and S74338;Zuoguiwan may regulate above lnc RNA to effect BMSCs to chondrocytes.