HIF-1α小干擾RNA對骨髓間充質(zhì)干細(xì)胞HIF-1α、SDF-1α和VEGF基因表達(dá)的影響
[Abstract]:Part one: culture of MSCs and screening of HIF-1 偽 siRNA objective: to screen the most effective RNA interference sequence of bone marrow mesenchymal stem cells (MSCs / mesenchymal stem cells), RT-PCR) cultured by adherent method. Methods: MSCs were cultured by adherent method. The cells of the 3-5 passage were used in the experiment. The morphology of the cells was observed under inverted microscope, and the surface marker CD11b / cpCD34, CD444 and CD90 were detected by flow cytometry. MSCs was divided into five groups: hypoxia group (without any intervention), liposome group (transfection of empty liposome) siRNA609 group (transfected siRNA609), siRNA658 group (transfected siRNA658) and siRNA2070 group (transfected siRNA2070). The gene expression of HIF-1 偽 was detected by RT-PCR in the above five groups cultured in anoxic condition for 24 h. The result is 1: 1. Flow cytometry showed that the positive cells of CD44 and CD44 were 84.2% 鹵0.2% and 97.91% 鹵0.710% respectively by flow cytometry. The number of CD44 / CD34- cells was 99.4% 鹵0.40.41% 鹵0.97% 鹵0.97%. The results of RT-PCR showed that the gene expression of HIF-1 偽 in siRNA609 siRNA658siRNA2070 group was significantly lower than that in hypoxic group (P 0.05), and the inhibitory effect of siRNA658 group was the highest (P 0.05). Conclusion 1. The cultured cells expressed the corresponding surface marker of MSCs. 2. MSCs could transfect HIF-1 偽 siRNA sequence. 3. SiRNA658 interference sequence had the strongest inhibitory effect. The second part is the effect of HIF-1 偽 small interfering RNA on the expression of HIF-1 偽 -SDF-1 偽 and VEGF gene in MSCs. Objective to investigate the effects of small interfering RNA on the expression of MSCs HIF-1 偽, SDF-1 偽 -stromal derived factor 1 偽 and vascular endothelial growth factor) in MSCs. Methods: four groups of MSCs, Normoxic control group (no intervention factor), hypoxia group (hypoxia 24 h), liposome control group (hypoxia 24 h after transfection of no-loaded liposome) RNA interference group (hypoxia 24 h after transfection of liposome mediated RNA interference sequence) .RT-PCR method to detect HIF-1 偽 -SDF-1 偽 and VEGF mRNA expression water in MSCs The expression levels of HIF-1 偽 -SDF-1 偽 and VEGF protein in the supernatant of MSCs culture were detected by ELISA and the effect of MSCs supernatant on the proliferation of rat smooth muscle cells was detected by CCK8. Results 1. RT-PCR showed that the expression of HIF-1 偽 -SDF-1 偽 mRNA in hypoxia group was higher than that in normoxic control group (P0. 05), and that in RNA interference group was lower than that in liposome control group. Compared with the normoxic control group, the content of HIF-1 偽 -SDF-1 偽 -VEGF in conditioned medium of hypoxia group was increased (P 0.05), and the content of HIF-1 偽 -SDF-1 偽 -VEGF in RNA interference group was lower than that in liposome control group (p0.05). Compared with the normoxic control group, the conditioned supernatant of hypoxia group could stimulate the proliferation of smooth muscle cells (P0. 05), and the supernatant of hypoxia culture supernatant of RNA interference group was weaker than that of liposome control group (P 0. 05). Conclusion 1. Inhibiting the expression of HIF-1 偽 could decrease the expression of SDF-1 偽 and VEGF genes in MSCs. Hypoxia can increase the expression of HIF-1 偽 -SDF-1 偽 and VEGF genes. 3.HIF-1 偽 is one of the main factors of MSC regulating the expression of SDF-1 偽 and VEGF genes. 4. RNA interference can reduce the proliferation of rat smooth muscle cells induced by hypoxia culture supernatant.
【學(xué)位授予單位】:福建醫(yī)科大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2010
【分類號】:R329
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