微重力條件下CKIP-1基因?qū)π∈蠊撬栝g充質(zhì)干細(xì)胞增殖及向成骨分化影響研究
發(fā)布時(shí)間：2018-08-05 20:30
【摘要】：目的:隨著我國(guó)太空事業(yè)的發(fā)展,宇航員太空活動(dòng)增多,駐留時(shí)間延長(zhǎng),因失重導(dǎo)致的骨質(zhì)疏松現(xiàn)象加重。同時(shí)隨著我國(guó)社會(huì)老齡化的加劇,骨質(zhì)疏松患者逐漸增多,給患者帶來(lái)嚴(yán)重的健康問(wèn)題和經(jīng)濟(jì)負(fù)擔(dān)。CKIP-1(酪氨酸激酶相關(guān)蛋白酶1)基因是近年來(lái)新發(fā)現(xiàn)的骨分化、形成的負(fù)調(diào)控因子,能夠有效對(duì)抗失重性及廢用性骨質(zhì)疏松。本文主要探索微重力環(huán)境下,CKIP-1基因?qū)π∈驜MSCs細(xì)胞增殖、凋亡及成骨分化的影響及分子機(jī)制探討研究,為治療骨質(zhì)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(xì)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(xì)胞,傳代培養(yǎng),分離,純化。觀察細(xì)形態(tài),胞流式細(xì)胞學(xué)鑒定純度。并用細(xì)胞計(jì)數(shù)法及MTT法檢測(cè)正常重力下CKIP-1對(duì)細(xì)胞增殖影響。2將BMSCs細(xì)胞(KO-BMSCs及WT-BMSCs)分別在不同重力環(huán)境中培養(yǎng)。細(xì)胞分為4組分為:(1)野生型BMSCs細(xì)胞正常重力組(wild type normal gravity,WT-NG);(2)敲除CKIP-1型BMSCs細(xì)胞正常重力組(knockout normal gravity,KO-NG);(3)敲除CKIP-1型BMSCs細(xì)胞微重力組(knockout microgravity,KO-MG)(4)野生型BMSCs細(xì)胞微重力組(wild type microgravity,WT-MG)。微重力環(huán)境用體外細(xì)胞旋轉(zhuǎn)培養(yǎng)系統(tǒng)(RCCS)模擬,均在體外行成骨誘導(dǎo)培養(yǎng)5天后行相關(guān)檢測(cè)。3細(xì)胞增殖及成骨分化相關(guān)指標(biāo)檢測(cè)3.1 MTT測(cè)量細(xì)胞0、3、5天細(xì)胞的增殖3.2流式細(xì)胞學(xué)技術(shù)及RT-PCR測(cè)定Bcl-2基因表達(dá)分析各組細(xì)胞的凋亡3.3茜素紅染色檢測(cè)各組細(xì)胞的礦化程度3.4細(xì)胞微絲染色3.5細(xì)胞堿性磷酸酶(ALP)濃度測(cè)定及染色3.6 RT-PCR檢測(cè)成骨相關(guān)基因的表達(dá)3.7 Western blot檢測(cè)成骨相關(guān)蛋白的表達(dá)結(jié)果:1敲除CKIP-1基因后,小鼠的生命體征及生長(zhǎng)發(fā)育未受明顯影響。KO-BMSCs細(xì)胞和WT-BMSCs在體外可以正常增殖生長(zhǎng)。通過(guò)傳代培養(yǎng)4代后BMSCs純度可達(dá)90%以上。細(xì)胞計(jì)數(shù)顯示KO-BM SCs增殖率低于WT-BMSCs組,MTT試驗(yàn)結(jié)果顯示二者之間未有明顯統(tǒng)計(jì)學(xué)意義;2.微重力條件抑制細(xì)胞增殖及BMSCs向成骨細(xì)胞的分化;微重力環(huán)境促進(jìn)細(xì)胞凋亡,敲除CKIP-1基因促進(jìn)了細(xì)胞的凋亡;3.微重力環(huán)境中BMSCs細(xì)胞由梭形變?yōu)閳A形,WT-BMSCs形態(tài)變化較KO-BMSCs組弱。同時(shí)在微重力條件下兩組BMSCs細(xì)胞的微絲變細(xì),排列紊亂,但敲除CKIP-1基因能有效對(duì)抗微絲變化,保持微絲形態(tài);4.敲除CKIP-1基因后BMSCs細(xì)胞成骨分化能力增強(qiáng),BMP/Smad信號(hào)通道表達(dá)增強(qiáng),成骨相關(guān)基因及蛋白的表達(dá)增強(qiáng),而微重力環(huán)境中該趨勢(shì)受到抑制。結(jié)論:1敲除CKIP-1后小鼠的生命體征及生長(zhǎng)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(xì)胞增值率降低,但敲除CKIP-1基因能有效對(duì)抗微重力阻礙BMSCs向成骨細(xì)胞分化的效應(yīng),促進(jìn)細(xì)胞的成骨分化,可以為治療骨質(zhì)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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R580
本文編號(hào)：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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R580
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 黃懿文;楊銳;陳思;孫嘉;陳容平;黃震;;PPARγ通路對(duì)模擬微重力條件下大鼠骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化的影響[J];南方醫(yī)科大學(xué)學(xué)報(bào);2013年04期
2 毛新建;宋關(guān)斌;羅慶;張晨;;微重力效應(yīng)對(duì)骨髓間充質(zhì)干細(xì)胞增殖分化影響的研究進(jìn)展[J];醫(yī)用生物力學(xué);2013年01期
3 李曉峰;趙勁民;蘇偉;崔向榮;羅世興;馬愛(ài)國(guó);;大鼠骨髓間充質(zhì)干細(xì)胞的培養(yǎng)與鑒定[J];中國(guó)組織工程研究與臨床康復(fù);2011年10期
4 麥燕興;黃震;簡(jiǎn)煉;秦佳升;杜江;鄧偉民;;模擬失重與骨組織的細(xì)胞凋亡[J];中國(guó)組織工程研究與臨床康復(fù);2010年46期
5 郭春;張西正;閆玉仙;郭勇;李瑞欣;王亮;;力學(xué)拉伸強(qiáng)度對(duì)破骨細(xì)胞形成和分化的影響[J];中華創(chuàng)傷雜志;2009年09期
6 黃國(guó)平;鄭強(qiáng);楊金鳳;郭春娟;沈丹;石東燕;徐玉林;潘志軍;王金福;;模擬微重力對(duì)人骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化中細(xì)胞信號(hào)通路影響的分析[J];空間科學(xué)學(xué)報(bào);2008年01期
7 費(fèi)琴明;陳統(tǒng)一;張光健;Boden Scott D;Titu Lsouisa;;Smurf1拮抗BMP-2對(duì)C2C12細(xì)胞的誘導(dǎo)成骨作用[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2006年05期
8 孫怡寧;商澎;梅其炳;;微重力引起骨丟失的細(xì)胞機(jī)制[J];國(guó)際骨科學(xué)雜志;2006年02期
9 張鈺鵬,李非,孫家邦,孫海晨,崔葉青;模擬微重力培養(yǎng)肝細(xì)胞的形態(tài)特點(diǎn)[J];中華實(shí)驗(yàn)外科雜志;2003年10期
10 劉霞,王常勇,郭希民,歐陽(yáng)五慶;生物反應(yīng)器內(nèi)再造組織工程化心肌的實(shí)驗(yàn)研究[J];中國(guó)醫(yī)學(xué)科學(xué)院學(xué)報(bào);2003年01期
相關(guān)碩士學(xué)位論文 前2條
1 梁超;CKIP-1的PH結(jié)構(gòu)域與泛素相互作用的研究[D];安徽醫(yī)科大學(xué);2013年
2 鄒超;大鼠骨髓間充質(zhì)干細(xì)胞的提取與鑒定[D];長(zhǎng)春中醫(yī)藥大學(xué);2013年
，本文編號(hào)：2166929
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2166929.html
最近更新
教材專著
