核受体SHP对BMP9诱导间充质干细胞成骨分化的影响

发布时间：2019-03-03 16:32

【摘要】：目的：研究核受体SHP (small heterodimer partner)在BMP9 (bone morphogenetic protein 9)所诱导的间充质干细胞(mesenchymal stem cells, MSCs)成骨分化中的作用。方法：首先我们构建并鉴定了过表达SHP (Ad-SHP)及沉默SHP(Ad-siSHP)的重组腺病毒。通过RT-PCR及Western Blot检测外源性BMP9对MSCs细胞中SHP表达的影响；然后利用腺病毒Ad-SHP或Ad-siSHP感染细胞,并加以BMP9刺激,通过定量及染色分析其对BMP9诱导的MSCs成骨分化早期指标碱性磷酸酶(Alkaline phosphotase, ALP)的影响；茜素红染色分析成骨晚期指标钙盐沉积的变化；RT-PCR检测成骨晚期指标OCN (Osteocalcin)和OPN(Osteopontin)的变化。接着,通过RT-PCR检测SHP对BMP9所诱导的成骨标志基因Runx2、Id家族和CTGF的影响;Western blot检测SHP对BMP9诱导的Runx2和D1x5蛋白表达的影响；荧光素酶报告基因实验和Western Blot检测SHP对BMP9活化的经典Smad信号通路的影响。结果：RT-PCR和Western Blot均显示Ad-SHP及Ad-siSHP构建成功且有效,接着通过RT-PCR和Western Blot检测发现外源性BMP9可上调C3H10T1/2细胞和C2C12细胞中内源性SHP的表达；过表达SHP能够促进BMP9所诱导的ALP活性的增加,同时促进了晚期成骨指标钙盐沉积以及OCN和OPN的表达,而沉默SHP后BMP9所诱导的早晚期成骨标志均受到了抑制。RT-PCR和Western Blot结果同样显示SHP促进了BMP9成骨关键转录因子Runx2、Dlx5及相关靶基因Idl、Id2、和CTGF的表达,而当SHP被干扰后,这些基因及蛋白的表达均受到抑制；BMP9本身能够激活经典Smad信号通路,但是SHP对于BMP9诱导的经典信号通路的磷酸化并无明显影响；值得注意的是,干扰SHP能够抑制BMP9激活的SBE荧光素酶活性。结论：SHP可调控BMP9所诱导的间充质干细胞的成骨分化,很可能是通过促进BMP9诱导的Smad转录活性而实现。
[Abstract]:Aim: to investigate the role of nuclear receptor SHP (small heterodimer partner) in the osteogenic differentiation of mesenchymal stem cells (mesenchymal stem cells, MSCs) induced by BMP9 (bone morphogenetic protein 9. Methods: first, we constructed and identified recombinant adenovirus expressing SHP (Ad-SHP) and silencing SHP (Ad-siSHP). The effects of exogenous BMP9 on the expression of SHP in MSCs cells were detected by RT-PCR and Western Blot. Then the cells were infected with adenovirus Ad-SHP or Ad-siSHP and stimulated by BMP9. The effects of adenovirus on BMP9-induced alkaline phosphatase (Alkaline phosphotase, ALP) in the early stage of osteogenic differentiation of MSCs were analyzed by quantitative analysis and staining. Alizarin red staining was used to analyze the changes of calcium salt deposition in the late osteogenic stage, and RT-PCR was used to detect the changes of OCN (Osteocalcin) and OPN (Osteopontin) in the late osteogenic stage. Then, the effect of SHP on Runx2,Id family and CTGF induced by BMP9 was detected by RT-PCR.; Western blot was used to detect the effect of SHP on Runx2 and D1x5 protein expression induced by BMP9. Luciferase reporter gene assay and Western Blot were used to detect the effect of SHP on the classical Smad signaling pathway activated by BMP9. Results: both RT-PCR and Western Blot showed that Ad-SHP and Ad-siSHP were constructed successfully and effectively. Then RT-PCR and Western Blot showed that exogenous BMP9 could up-regulate the expression of endogenous SHP in C3H10T1/2 cells and C2C12 cells. Overexpression of SHP could promote the increase of ALP activity induced by BMP9, calcium deposition and expression of OCN and OPN in late osteogenic markers. The results of RT-PCR and Western Blot also showed that SHP promoted the expression of Runx2,Dlx5, the key transcription factor of BMP9 osteogenesis, and the related target genes Idl,Id2, and CTGF, and when SHP was interfered with, the expression of osteogenic markers induced by BMP9 was inhibited by silencing SHP, and the expression of Runx2,Dlx5 and related target genes Idl,Id2, and CTGF were also enhanced by SHP. The expression of these genes and proteins were inhibited. BMP9 itself can activate the classical Smad signaling pathway, but SHP has no obvious effect on the phosphorylation of the classical signaling pathway induced by BMP9. It is worth noting that interference SHP can inhibit the activity of SBE luciferase activated by BMP9. Conclusion: SHP can regulate the osteogenic differentiation of mesenchymal stem cells induced by BMP9, which may be achieved by promoting the transcriptional activity of Smad induced by BMP9.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

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