| Objective: 1.To explore the conditions of isolation,culture and purification of human bone marrow mesenchymal stem cells(h BMSCs)in vitro;to study the methods of identifying stem cells;to explore the feasibility of using them as seed cells for the treatment of cartilage related diseases such as osteoarthritis(OA).2.To explore the regulation of mi RNA-21-5p on the expression of related genes in the process of chondrogenesis of h BMSCs,so as to provide a new idea for the treatment of OA and other cartilage related diseases by h BMSCs mediated by mi RNA.Methods: 1.Separating and extracting of h BMSCs through Ficoll density gradient centrifugation.2.To observe the growth of h BMSCs by microscope,and change the culture medium per three days,purify and propagate h BMSCs by adherent method.3.Morphological identification of stem cells under confocal laser microscopy,identification of molecular markers on the surface of stem cells under reverse transcription polymerase chain reaction(RT-PCR)and chondrogenic identification of stem cells by alcian blue staining after chondrogenic induction of h BMSCs by recombinant human transforming growth factor-β 3(TGF-β 3).4.Fam(fluorescent transfection indicator of mirna-21-5p mimic and inhibitor)was used to detect the transfection efficiency of P3 generation h BMSCs.The experiment was divided into five groups: mirna-21-5p mimic group,mirna-21-5p mimic NC group,mirna-21-5p inhibitor group,mirna-21-5p inhibitor NC group and blank group.On the first day after each agent acted on stem cells,the transfection efficiency was observed under fluorescence microscope.5.HBMSCs of P3 generation were transfected with mirna-21-5p,The experiment was divided into five groups: mirna-21-5p mimic group,mirna-21-5p mimic NC group,mirna-21-5p inhibitor group and mirna-21-5p inhibitor group In NC group and blank group,stem cells of each group were cultured for 3,7,14,21 and 28 days respectively.The growth and chondrogenic differentiation of stem cells were observed under microscope.The cells from each group were collected and RNA was extracted.Cartilage related genes were detected by quantitative real-time PCR(q RTPCR): c-myc,Bax,MMP-13,Sox-9,COL-2,beta catenin.Results: 1.HBMSCs were isolated and extracted.After 24 hours of inoculation,P1 generation stem cells began to adhere to the wall,showing a large oval shape.After 48 hours,the number of cells adhered to the wall gradually increased,showing a uniform long fusiform shape,and gradually forming a colony.2.Reverse transcriptase polymerase chain reaction(RT-PCR)showed that CD45(-),CD105(+),CD13(+),CD34(-),confirming the separation and purification of P3 generation h BMSCs;alcian blue staining was shown in Figure 1-4 to Figure 1-7,and the chondrogenesis induction of h BMSCs was successful.3.Observe the transfection efficiency with fluorescence microscope,as shown in Figure 2-5 to figure 2-8.The transfection effect of each group of cells meets the requirements of this experiment.4.In the process of chondrogenic induction,the expression of Sox-9 gene was the highest in mir21-5p inhibitor group on the 21 st day of chondrogenic induction culture,while the expression of Bax and other apoptosis promoting genes was lower and the amount of chondrogenesis was the highest.Conclusion: 1.Ficoll density gradient centrifugation is an effective method for the separation and extraction of h BMSCs.2.The transfection of h BMSCs by mirna-21-5p inhibitor can promote the chondrogenic effect of h BMSCs,that is,inhibit mirna-21-5p gene,and facilitate the differentiation of BMSCs into chondrocytes. |
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