微重力條件下CKIP-1基因對小鼠骨髓間充質干細胞增殖及向成骨分化影響研究
發(fā)布時間：2018-08-05 20:30
【摘要】：目的:隨著我國太空事業(yè)的發(fā)展,宇航員太空活動增多,駐留時間延長,因失重導致的骨質疏松現象加重。同時隨著我國社會老齡化的加劇,骨質疏松患者逐漸增多,給患者帶來嚴重的健康問題和經濟負擔。CKIP-1(酪氨酸激酶相關蛋白酶1)基因是近年來新發(fā)現的骨分化、形成的負調控因子,能夠有效對抗失重性及廢用性骨質疏松。本文主要探索微重力環(huán)境下,CKIP-1基因對小鼠BMSCs細胞增殖、凋亡及成骨分化的影響及分子機制探討研究,為治療骨質疏松找到新的方法。方法:1敲除CKIP-1基因的BMSCs(knockout,KO-BMSCs)及正常BMSCs細胞提取:CKIP-1基因小鼠和野生型小鼠雜交繁育后,PCR法鑒定4周齡敲除CKIP-1基因小鼠(Knockout,KO型)與野生型C57BL/6小鼠(wild type,WT型),股骨離斷用全骨髓法提取兩組BMSCs細胞,傳代培養(yǎng),分離,純化。觀察細形態(tài),胞流式細胞學鑒定純度。并用細胞計數法及MTT法檢測正常重力下CKIP-1對細胞增殖影響。2將BMSCs細胞(KO-BMSCs及WT-BMSCs)分別在不同重力環(huán)境中培養(yǎng)。細胞分為4組分為:(1)野生型BMSCs細胞正常重力組(wild type normal gravity,WT-NG);(2)敲除CKIP-1型BMSCs細胞正常重力組(knockout normal gravity,KO-NG);(3)敲除CKIP-1型BMSCs細胞微重力組(knockout microgravity,KO-MG)(4)野生型BMSCs細胞微重力組(wild type microgravity,WT-MG)。微重力環(huán)境用體外細胞旋轉培養(yǎng)系統(tǒng)(RCCS)模擬,均在體外行成骨誘導培養(yǎng)5天后行相關檢測。3細胞增殖及成骨分化相關指標檢測3.1 MTT測量細胞0、3、5天細胞的增殖3.2流式細胞學技術及RT-PCR測定Bcl-2基因表達分析各組細胞的凋亡3.3茜素紅染色檢測各組細胞的礦化程度3.4細胞微絲染色3.5細胞堿性磷酸酶(ALP)濃度測定及染色3.6 RT-PCR檢測成骨相關基因的表達3.7 Western blot檢測成骨相關蛋白的表達結果:1敲除CKIP-1基因后,小鼠的生命體征及生長發(fā)育未受明顯影響。KO-BMSCs細胞和WT-BMSCs在體外可以正常增殖生長。通過傳代培養(yǎng)4代后BMSCs純度可達90%以上。細胞計數顯示KO-BM SCs增殖率低于WT-BMSCs組,MTT試驗結果顯示二者之間未有明顯統(tǒng)計學意義;2.微重力條件抑制細胞增殖及BMSCs向成骨細胞的分化;微重力環(huán)境促進細胞凋亡,敲除CKIP-1基因促進了細胞的凋亡;3.微重力環(huán)境中BMSCs細胞由梭形變?yōu)閳A形,WT-BMSCs形態(tài)變化較KO-BMSCs組弱。同時在微重力條件下兩組BMSCs細胞的微絲變細,排列紊亂,但敲除CKIP-1基因能有效對抗微絲變化,保持微絲形態(tài);4.敲除CKIP-1基因后BMSCs細胞成骨分化能力增強,BMP/Smad信號通道表達增強,成骨相關基因及蛋白的表達增強,而微重力環(huán)境中該趨勢受到抑制。結論:1敲除CKIP-1后小鼠的生命體征及生長發(fā)育未受明顯影響,敲除CKIP-1基因后小鼠BMSCs增殖率降低;2敲除CKIP-1后BMSCs細胞增值率降低,但敲除CKIP-1基因能有效對抗微重力阻礙BMSCs向成骨細胞分化的效應,促進細胞的成骨分化,可以為治療骨質疏松治療提供新的思路。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R580
本文編號：2166929
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R580
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