微重力条件下CKIP-1基因对小鼠骨髓间充质干细胞增殖及向成骨分化影响研究

发布时间：2018-08-05 20:30

【摘要】：目的:随着我国太空事业的发展,宇航员太空活动增多,驻留时间延长,因失重导致的骨质疏松现象加重。同时随着我国社会老龄化的加剧,骨质疏松患者逐渐增多,给患者带来严重的健康问题和经济负担。CKIP-1(酪氨酸激酶相关蛋白酶1)基因是近年来新发现的骨分化、形成的负调控因子,能够有效对抗失重性及废用性骨质疏松。本文主要探索微重力环境下,CKIP-1基因对小鼠BMSCs细胞增殖、凋亡及成骨分化的影响及分子机制探讨研究,为治疗骨质疏松找到新的方法。方法:1敲除CKIP-1基因的BMSCs(knockout,KO-BMSCs)及正常BMSCs细胞提取:CKIP-1基因小鼠和野生型小鼠杂交繁育后,PCR法鉴定4周龄敲除CKIP-1基因小鼠(Knockout,KO型)与野生型C57BL/6小鼠(wild type,WT型),股骨离断用全骨髓法提取两组BMSCs细胞,传代培养,分离,纯化。观察细形态,胞流式细胞学鉴定纯度。并用细胞计数法及MTT法检测正常重力下CKIP-1对细胞增殖影响。2将BMSCs细胞(KO-BMSCs及WT-BMSCs)分别在不同重力环境中培养。细胞分为4组分为:(1)野生型BMSCs细胞正常重力组(wild type normal gravity,WT-NG);(2)敲除CKIP-1型BMSCs细胞正常重力组(knockout normal gravity,KO-NG);(3)敲除CKIP-1型BMSCs细胞微重力组(knockout microgravity,KO-MG)(4)野生型BMSCs细胞微重力组(wild type microgravity,WT-MG)。微重力环境用体外细胞旋转培养系统(RCCS)模拟,均在体外行成骨诱导培养5天后行相关检测。3细胞增殖及成骨分化相关指标检测3.1 MTT测量细胞0、3、5天细胞的增殖3.2流式细胞学技术及RT-PCR测定Bcl-2基因表达分析各组细胞的凋亡3.3茜素红染色检测各组细胞的矿化程度3.4细胞微丝染色3.5细胞碱性磷酸酶(ALP)浓度测定及染色3.6 RT-PCR检测成骨相关基因的表达3.7 Western blot检测成骨相关蛋白的表达结果:1敲除CKIP-1基因后,小鼠的生命体征及生长发育未受明显影响。KO-BMSCs细胞和WT-BMSCs在体外可以正常增殖生长。通过传代培养4代后BMSCs纯度可达90%以上。细胞计数显示KO-BM SCs增殖率低于WT-BMSCs组,MTT试验结果显示二者之间未有明显统计学意义;2.微重力条件抑制细胞增殖及BMSCs向成骨细胞的分化;微重力环境促进细胞凋亡,敲除CKIP-1基因促进了细胞的凋亡;3.微重力环境中BMSCs细胞由梭形变为圆形,WT-BMSCs形态变化较KO-BMSCs组弱。同时在微重力条件下两组BMSCs细胞的微丝变细,排列紊乱,但敲除CKIP-1基因能有效对抗微丝变化,保持微丝形态;4.敲除CKIP-1基因后BMSCs细胞成骨分化能力增强,BMP/Smad信号通道表达增强,成骨相关基因及蛋白的表达增强,而微重力环境中该趋势受到抑制。结论:1敲除CKIP-1后小鼠的生命体征及生长发育未受明显影响,敲除CKIP-1基因后小鼠BMSCs增殖率降低;2敲除CKIP-1后BMSCs细胞增值率降低,但敲除CKIP-1基因能有效对抗微重力阻碍BMSCs向成骨细胞分化的效应,促进细胞的成骨分化,可以为治疗骨质疏松治疗提供新的思路。
[Abstract]:Objective: with the development of space in China, the space activities of astronauts increase, the stay time is prolonged, and the osteoporosis caused by weightlessness is aggravated. At the same time, with the aggravation of aging society in China, the number of osteoporosis patients is increasing gradually, which brings serious health problems and economic burden to patients. CKIP-1 (tyrosine kinase-associated protease 1) gene is a newly discovered bone differentiation in recent years. The formation of negative regulatory factors, can effectively combat weightlessness and waste osteoporosis. In this paper, the effects of CKIP-1 gene on the proliferation, apoptosis and osteogenic differentiation of mouse BMSCs cells under microgravity environment and its molecular mechanism were studied in order to find a new method for the treatment of osteoporosis. Methods BMSCs (knockout KO-BMSCs) with CKIP-1 gene knockout 1 and normal BMSCs cells were used to identify 4-week-old knockout CKIP-1 gene mice (KnockoutoutKO type) and wild type C57BL/6 mice (wild type C57BL/6 type), and femur dissected with whole bone marrow. Method to extract two groups of BMSCs cells, Passage culture, separation, purification. The fine morphology was observed and the purity was identified by cell flow cytology. The effects of CKIP-1 on cell proliferation under normal gravity were detected by cell counting and MTT methods. 2 BMSCs cells (KO-BMSCs and WT-BMSCs) were cultured in different gravity environments respectively. The cells were divided into four groups: (1) (wild type normal gravimetric WT-NG); (2) knockout CKIP-1 BMSCs cell KO-NG); (3 (knockout microgravimetric KO-MG) (4) wild-type BMSCs cell microgravity group (wild type microgravimetric WT-MG). The microgravity environment was simulated by in vitro cell rotation culture system (RCCS). After 5 days of osteogenic induction and culture in vitro, the proliferation of 3. 3 cells and the correlation index of osteogenic differentiation were detected by 3. 1 MTT. 3. 2 flow cytology and 3. 2 flow cytometric analysis of cell proliferation and Bcl-2 gene expression analysis by RT-PCR 3. 3. 3 apoptosis: 3. 3 alizarin red staining to detect mineralization degree of 3. 4 cells microfilament staining 3. 5 cell alkaline phosphatase (ALP) concentration and staining 3. 6 RT-PCR to detect the expression of osteoblast-associated gene. 3. 7 Western blot to detect the surface of osteoblast-associated protein. As a result, the CKIP-1 gene was knocked out by 1: 1. The vital signs, growth and development of mice were not significantly affected. KO-BMSCs and WT-BMSCs could proliferate and grow normally in vitro. The purity of BMSCs was over 90% after 4 generations. Cell count showed that the proliferation rate of KO-BM SCs was lower than that of WT-BMSCs group. Microgravity condition inhibited cell proliferation and differentiation of BMSCs into osteoblast, microgravity promoted cell apoptosis and knockout of CKIP-1 gene promoted cell apoptosis. In microgravity environment, the morphological changes of BMSCs cells from fusiform to circular WT-BMSCs were weaker than those in KO-BMSCs group. At the same time, the microfilaments of the two groups of BMSCs cells became thin and disordered under microgravity, but knockout of CKIP-1 gene could effectively resist the changes of microfilaments and maintain the morphology of microfilaments. The osteogenic differentiation ability of BMSCs cells was enhanced after knockout of CKIP-1 gene. The expression of BMP / Smad signal channel and osteoblast-associated genes and proteins were enhanced, but the trend was inhibited in microgravity environment. Conclusion the vital signs, growth and development of CKIP-1 knockout mice were not significantly affected, but the proliferation rate of BMSCs was decreased after knockout of CKIP-1 gene, and the proliferation rate of BMSCs cells was decreased after CKIP-1 knockout. But knockout of CKIP-1 gene can effectively antagonize the effect of microgravity blocking the differentiation of BMSCs into osteoblasts and promote the osteogenic differentiation of cells. It can provide a new idea for the treatment of osteoporosis.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R580

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