miR-155在BMP9誘導(dǎo)間充質(zhì)干細(xì)胞成骨分化中的作用及機(jī)制研究
發(fā)布時間：2018-12-05 13:05
【摘要】：目的:研究miR-155在BMP9誘導(dǎo)成骨分化過程中的表達(dá)情況,探究miR-155對BMP9誘導(dǎo)的間充質(zhì)干細(xì)胞成骨分化的調(diào)控作用,并對其相關(guān)調(diào)控機(jī)制進(jìn)行初步研究。方法:用BMP9腺病毒誘導(dǎo)C2C12和MEF細(xì)胞成骨分化,q RT-PCR檢測miR-155在成骨分化的0d,1d,3d,5d,7d的表達(dá)水平,并同時檢測成骨相關(guān)標(biāo)志物Runx2和ALP的表達(dá),對BMP9誘導(dǎo)的成骨分化進(jìn)行驗證。miR-155模擬物過表達(dá)或miR-155抑制劑抑制miR-155的表達(dá)后通過ALP染色及活性檢測其對成骨分化早期的影響,茜素紅S(Alizarin Red S,ARS)染色觀察其對BMP9誘導(dǎo)的成骨分化晚期的影響。q RT-PCR檢測成骨相關(guān)標(biāo)志物Runx2,OSX,ALP和OCN的表達(dá),從基因水平檢測miR-155對BMP9誘導(dǎo)的成骨分化的作用。Western blot檢測Runx2,p-Smad1/5/8的表達(dá),以及晚期成骨標(biāo)志物OCN和OPN的表達(dá)。生物信息學(xué)分析miR-155潛在靶基因,在眾多靶基因中選擇與BMP9誘導(dǎo)成骨分化作用密切相關(guān)的Runx2和BMPR2進(jìn)行分析,用q RT-PCR和Western blot檢測miR-155對其潛在靶基因的調(diào)控,熒光素酶報告基因?qū)嶒瀸iR-155與靶基因的直接靶向作用進(jìn)行驗證。干擾其靶基因后q RT-PCR檢測成骨相關(guān)標(biāo)志物的表達(dá),探究靶基因在miR-155對BMP9誘導(dǎo)的成骨分化作用中的影響。裸鼠皮下異位成骨以后取異位骨進(jìn)行micro CT掃描以及切片后進(jìn)行HE染色和Masson染色體內(nèi)驗證miR-155對成骨分化的影響。結(jié)果:在BMP9誘導(dǎo)C2C12和MEF細(xì)胞成骨分化過程中,miR-155的表達(dá)呈現(xiàn)出一個先升高后降低的趨勢。過表達(dá)miR-155后,細(xì)胞的ALP染色減弱,活性減低,ARS染色減弱。q RT-PCR檢測結(jié)果顯示在BMP9誘導(dǎo)的成骨分化過程中,過表達(dá)miR-155顯著降低Runx2,OSX,ALP和OCN的表達(dá),而抑制miR-155以后,其對成骨分化相關(guān)基因表達(dá)的抑制作用消失。Western blot結(jié)果顯示miR-155能顯著抑制Runx2和p-Smad1/5/8的表達(dá),并能顯著降低晚期成骨標(biāo)志物OCN和OPN的表達(dá)。q RT-PCR結(jié)果顯示miR-155對其潛在靶基因Runx2和BMPR2的m RNA影響不大,但Western blot結(jié)果顯示miR-155能顯著降低Runx2和BMPR2的蛋白表達(dá)水平。熒光素酶報告基因結(jié)果指出miR-155能直接靶向于Runx2和BMPR2,分別干擾Runx2和BMPR2后,q RT-PCR檢測成骨相關(guān)標(biāo)志物,結(jié)果顯示干擾了靶基因后,成骨相關(guān)標(biāo)志基因表達(dá)顯著降低,與過表達(dá)miR-155作用相似。裸鼠皮下異位成骨后取異位骨組織micro CT掃描,結(jié)果表明miR-155能抑制BMP9誘導(dǎo)MEF細(xì)胞皮下成骨的體積和密度,HE染色和Masson染色結(jié)果顯示miR-155能抑制異位骨的骨組織成熟度。結(jié)論:體內(nèi)外實驗結(jié)果證明miR-155能夠抑制BMP9誘導(dǎo)的間充質(zhì)干細(xì)胞成骨分化,其可能是通過抑制Smad/BMP信號通路發(fā)揮作用。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
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相關(guān)期刊論文 前4條
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2 Maya Fakhry;René Buchet;David Magne;Eva Hamade;Bassam Badran;;Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts[J];World Journal of Stem Cells;2013年04期
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4 Wen Luo,;Qinghua Nie;Xiquan Zhang;;MicroRNAs Involved in Skeletal Muscle Differentiation[J];遺傳學(xué)報;2013年03期
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