miR-155在BMP9诱导间充质干细胞成骨分化中的作用及机制研究
发布时间：2018-12-05 13:05
【摘要】：目的:研究miR-155在BMP9诱导成骨分化过程中的表达情况,探究miR-155对BMP9诱导的间充质干细胞成骨分化的调控作用,并对其相关调控机制进行初步研究。方法:用BMP9腺病毒诱导C2C12和MEF细胞成骨分化,q RT-PCR检测miR-155在成骨分化的0d,1d,3d,5d,7d的表达水平,并同时检测成骨相关标志物Runx2和ALP的表达,对BMP9诱导的成骨分化进行验证。miR-155模拟物过表达或miR-155抑制剂抑制miR-155的表达后通过ALP染色及活性检测其对成骨分化早期的影响,茜素红S(Alizarin Red S,ARS)染色观察其对BMP9诱导的成骨分化晚期的影响。q RT-PCR检测成骨相关标志物Runx2,OSX,ALP和OCN的表达,从基因水平检测miR-155对BMP9诱导的成骨分化的作用。Western blot检测Runx2,p-Smad1/5/8的表达,以及晚期成骨标志物OCN和OPN的表达。生物信息学分析miR-155潜在靶基因,在众多靶基因中选择与BMP9诱导成骨分化作用密切相关的Runx2和BMPR2进行分析,用q RT-PCR和Western blot检测miR-155对其潜在靶基因的调控,荧光素酶报告基因实验对miR-155与靶基因的直接靶向作用进行验证。干扰其靶基因后q RT-PCR检测成骨相关标志物的表达,探究靶基因在miR-155对BMP9诱导的成骨分化作用中的影响。裸鼠皮下异位成骨以后取异位骨进行micro CT扫描以及切片后进行HE染色和Masson染色体内验证miR-155对成骨分化的影响。结果:在BMP9诱导C2C12和MEF细胞成骨分化过程中,miR-155的表达呈现出一个先升高后降低的趋势。过表达miR-155后,细胞的ALP染色减弱,活性减低,ARS染色减弱。q RT-PCR检测结果显示在BMP9诱导的成骨分化过程中,过表达miR-155显著降低Runx2,OSX,ALP和OCN的表达,而抑制miR-155以后,其对成骨分化相关基因表达的抑制作用消失。Western blot结果显示miR-155能显著抑制Runx2和p-Smad1/5/8的表达,并能显著降低晚期成骨标志物OCN和OPN的表达。q RT-PCR结果显示miR-155对其潜在靶基因Runx2和BMPR2的m RNA影响不大,但Western blot结果显示miR-155能显著降低Runx2和BMPR2的蛋白表达水平。荧光素酶报告基因结果指出miR-155能直接靶向于Runx2和BMPR2,分别干扰Runx2和BMPR2后,q RT-PCR检测成骨相关标志物,结果显示干扰了靶基因后,成骨相关标志基因表达显著降低,与过表达miR-155作用相似。裸鼠皮下异位成骨后取异位骨组织micro CT扫描,结果表明miR-155能抑制BMP9诱导MEF细胞皮下成骨的体积和密度,HE染色和Masson染色结果显示miR-155能抑制异位骨的骨组织成熟度。结论:体内外实验结果证明miR-155能够抑制BMP9诱导的间充质干细胞成骨分化,其可能是通过抑制Smad/BMP信号通路发挥作用。
[Abstract]:Aim: to investigate the expression of miR-155 in osteogenic differentiation induced by BMP9 and to explore the regulation of miR-155 on the osteogenic differentiation of mesenchymal stem cells induced by BMP9. Methods: BMP9 adenovirus was used to induce osteogenic differentiation of C2C12 and MEF cells. Q RT-PCR was used to detect the expression of miR-155 on the 1st day, 3d, 5d and 7d of osteogenic differentiation, and the expression of Runx2 and ALP, the markers related to osteogenesis. Osteogenic differentiation induced by BMP9 was verified. Overexpression of miR-155 mimics or inhibition of miR-155 expression by miR-155 inhibitor were detected by ALP staining and activity. Alizarin red S (Alizarin Red S, was used to detect the effect of miR-155 on osteogenic differentiation in the early stage of osteogenesis. Q RT-PCR was used to detect the expression of osteoblast-related markers Runx2,OSX,ALP and OCN, and miR-155 to BMP9 induced osteogenic differentiation was detected at gene level. Western blot was used to detect Runx2,. Expression of p-Smad1/5/8 and late osteogenic markers OCN and OPN. The potential target genes of miR-155 were analyzed by bioinformatics, Runx2 and BMPR2, which were closely related to the osteogenic differentiation induced by BMP9, were selected among many target genes. The regulation of miR-155 on the potential target genes was detected by Q RT-PCR and Western blot. Luciferase reporter gene experiment was used to verify the direct targeting effect of miR-155 and target gene. After interfering with its target gene, Q RT-PCR was used to detect the expression of osteoblast-related markers, and to explore the effect of miR-155 on osteogenic differentiation induced by BMP9. After hypodermic ectopic osteogenesis in nude mice, ectopic bone was taken for micro CT scanning, HE staining and Masson chromosome test were performed after section. The effect of miR-155 on osteogenesis differentiation was verified. Results: during the osteogenic differentiation of C2C12 and MEF cells induced by BMP9, the expression of miR-155 increased first and then decreased. After overexpression of miR-155, the ALP staining and the activity of the cells decreased, while the ARS staining decreased. Q RT-PCR showed that the overexpression of miR-155 significantly decreased the expression of Runx2,OSX,ALP and OCN during the osteogenic differentiation induced by BMP9. After inhibiting miR-155, the inhibitory effect of miR-155 on the expression of osteoblastic differentiation related genes disappeared. Western blot results showed that miR-155 could significantly inhibit the expression of Runx2 and p-Smad1/5/8. Q RT-PCR results showed that miR-155 had little effect on the m RNA of Runx2 and BMPR2, but miR-155 could significantly decrease the protein expression of Runx2 and BMPR2. The results of luciferase reporter gene showed that miR-155 could directly target Runx2 and BMPR2, to interfere with Runx2 and BMPR2, and Q RT-PCR could detect osteoblast-related markers. The results showed that the expression of osteoblast-related marker gene decreased significantly after interfering with target gene. Similar to overexpression of miR-155. Micro CT scanning of heterotopic bone tissue was taken from nude mice after subcutaneous ectopic osteogenesis. The results showed that miR-155 could inhibit the volume and density of BMP9 induced subcutaneous osteogenesis of MEF cells. HE staining and Masson staining showed that miR-155 could inhibit the bone maturity of heterotopic bone. Conclusion: in vitro and in vivo, miR-155 can inhibit the osteogenic differentiation of mesenchymal stem cells induced by BMP9, which may play a role by inhibiting the Smad/BMP signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
本文编号：2365240
[Abstract]:Aim: to investigate the expression of miR-155 in osteogenic differentiation induced by BMP9 and to explore the regulation of miR-155 on the osteogenic differentiation of mesenchymal stem cells induced by BMP9. Methods: BMP9 adenovirus was used to induce osteogenic differentiation of C2C12 and MEF cells. Q RT-PCR was used to detect the expression of miR-155 on the 1st day, 3d, 5d and 7d of osteogenic differentiation, and the expression of Runx2 and ALP, the markers related to osteogenesis. Osteogenic differentiation induced by BMP9 was verified. Overexpression of miR-155 mimics or inhibition of miR-155 expression by miR-155 inhibitor were detected by ALP staining and activity. Alizarin red S (Alizarin Red S, was used to detect the effect of miR-155 on osteogenic differentiation in the early stage of osteogenesis. Q RT-PCR was used to detect the expression of osteoblast-related markers Runx2,OSX,ALP and OCN, and miR-155 to BMP9 induced osteogenic differentiation was detected at gene level. Western blot was used to detect Runx2,. Expression of p-Smad1/5/8 and late osteogenic markers OCN and OPN. The potential target genes of miR-155 were analyzed by bioinformatics, Runx2 and BMPR2, which were closely related to the osteogenic differentiation induced by BMP9, were selected among many target genes. The regulation of miR-155 on the potential target genes was detected by Q RT-PCR and Western blot. Luciferase reporter gene experiment was used to verify the direct targeting effect of miR-155 and target gene. After interfering with its target gene, Q RT-PCR was used to detect the expression of osteoblast-related markers, and to explore the effect of miR-155 on osteogenic differentiation induced by BMP9. After hypodermic ectopic osteogenesis in nude mice, ectopic bone was taken for micro CT scanning, HE staining and Masson chromosome test were performed after section. The effect of miR-155 on osteogenesis differentiation was verified. Results: during the osteogenic differentiation of C2C12 and MEF cells induced by BMP9, the expression of miR-155 increased first and then decreased. After overexpression of miR-155, the ALP staining and the activity of the cells decreased, while the ARS staining decreased. Q RT-PCR showed that the overexpression of miR-155 significantly decreased the expression of Runx2,OSX,ALP and OCN during the osteogenic differentiation induced by BMP9. After inhibiting miR-155, the inhibitory effect of miR-155 on the expression of osteoblastic differentiation related genes disappeared. Western blot results showed that miR-155 could significantly inhibit the expression of Runx2 and p-Smad1/5/8. Q RT-PCR results showed that miR-155 had little effect on the m RNA of Runx2 and BMPR2, but miR-155 could significantly decrease the protein expression of Runx2 and BMPR2. The results of luciferase reporter gene showed that miR-155 could directly target Runx2 and BMPR2, to interfere with Runx2 and BMPR2, and Q RT-PCR could detect osteoblast-related markers. The results showed that the expression of osteoblast-related marker gene decreased significantly after interfering with target gene. Similar to overexpression of miR-155. Micro CT scanning of heterotopic bone tissue was taken from nude mice after subcutaneous ectopic osteogenesis. The results showed that miR-155 could inhibit the volume and density of BMP9 induced subcutaneous osteogenesis of MEF cells. HE staining and Masson staining showed that miR-155 could inhibit the bone maturity of heterotopic bone. Conclusion: in vitro and in vivo, miR-155 can inhibit the osteogenic differentiation of mesenchymal stem cells induced by BMP9, which may play a role by inhibiting the Smad/BMP signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
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相关期刊论文 前4条
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