發(fā)布時(shí)間：2018-06-07 08:43

  本文選題：結(jié)締組織生長因子 + 骨形態(tài)發(fā)生蛋白��； 參考：《重慶醫(yī)科大學(xué)》2010年博士論文

【摘要】： 本課題的主要研究目的是揭示結(jié)締組織生長因子(CTGF,又稱CCN2)在骨形態(tài)發(fā)生蛋白(BMP)9誘導(dǎo)的成骨分化,以及CTGF在骨腫瘤發(fā)生中的作用。 CCN蛋白家族含有6個(gè)成員,為CCN1到CCN6,它們均為富含半胱氨酸的分泌性小分量蛋白。CCN蛋白是含有四個(gè)功能性結(jié)構(gòu)域的modular蛋白。多數(shù)CCN蛋白家族的成員受生長因子、細(xì)胞因子或細(xì)胞壓力的誘導(dǎo)調(diào)控。CCN蛋白在成體和胚胎組織的表達(dá)模式廣泛且差異很大。CCN蛋白可以通過參與并調(diào)節(jié)多種信號分子而發(fā)揮作用,如整合素、BMPs、血管內(nèi)盤生長因子(VEGF)、Wnts和Notch等。其中,整合素介導(dǎo)的CCN信號傳遞具有組織和細(xì)胞特異性,根據(jù)細(xì)胞外基質(zhì)不同而作用各異。 在本課題中,我們首先檢測了CCN蛋白家族中最重要的4個(gè)成員,CCN1、CCN2、CCN3和CCN4,對BMP9誘導(dǎo)的成骨分化和對骨肉瘤的作用。結(jié)果顯示CCN2(CTGF)抑制了BMP9誘導(dǎo)的成骨分化,促進(jìn)了骨肉瘤細(xì)胞株143B的增殖和遷移,兩個(gè)方面均具有顯著性差異。于是,我們選擇CTGF做進(jìn)一步深入研究。為了方便表述,本課題大致分為兩部分: 在第一節(jié),我們從孕12.5到13.5天的小鼠胚胎中分離得到了小鼠胚胎成纖維細(xì)胞(MEFs)。進(jìn)而,我們用攜帶SV40T基因的逆轉(zhuǎn)錄病毒感染MEFs,通過抗生素篩選,得到了永生化的MEFs(iMEFs)。iMEFs細(xì)胞株的建立為研究CTGF在BMP9誘導(dǎo)的成骨分化和在骨肉瘤細(xì)胞中的作用奠定了基礎(chǔ)。 在第二節(jié)中,我們評價(jià)了4中CCN蛋白,CCN1、CCN2、CCN3和CCN4,對BMP9誘導(dǎo)的成骨分化的影響。處于對數(shù)生長期的iMEFs共感染AdBMP9和AdR-CCN1、Ad-CCN2、Ad-CCN3、Ad-CCN4或AdGFP重組腺病毒。在7天后成骨分化早期標(biāo)志堿性磷酸酶(ALP)用比色發(fā)定量測定。結(jié)果顯示CCN2(CTGF)具有最強(qiáng)的抑制BMP9誘導(dǎo)的骨形成的能力。這一結(jié)果促使我們選擇CTGF做進(jìn)一步深入研究。 由于CTGF是一種含有4個(gè)結(jié)構(gòu)域的mosaic蛋白,為進(jìn)一步研究哪個(gè)或哪幾個(gè)結(jié)構(gòu)域發(fā)揮著抑制作用,在第三節(jié)里我們構(gòu)建了CTGF的中間缺失和C末端缺失突變體。利用分子生物學(xué)技術(shù),我們構(gòu)建了一系列克隆,最終制作了6種重組腺病毒,它們分別表達(dá)CTGF全長、CTGFCD1(無結(jié)構(gòu)域IV),CTGFCD2(無結(jié)構(gòu)域III和IV),CTGFID1(無結(jié)構(gòu)域II),CTGFID2(無結(jié)構(gòu)域III)和CTGFID3(無結(jié)構(gòu)域II和III)。6種腺病毒經(jīng)蛋白酶K裂解,以基因組DNA為模板做PCR,鑒定了CTGF全長及其突變體的存在。以抗Flag抗體為一抗做Western blot,證實(shí)了6種腺病毒可以成功表達(dá)CTGF全長及其突變體(CTGF及其突變體均與3×Flag標(biāo)簽融合表達(dá)。CTGF全長和缺失突變體重組腺病毒的成功構(gòu)建,為進(jìn)一步解析在成骨分化中發(fā)揮功能的結(jié)構(gòu)域奠定了基礎(chǔ)。 在第四節(jié),我們檢測了CTGF全長及其缺失突變體對BMP9誘導(dǎo)的成骨分化的體外影響。用AdBMP9和CTGF腺病毒(AdR-CTGF, CTGFCD1, CTGFCD2, CTGFID1, CTGFID2, CTGFID3)聯(lián)合感染出于對數(shù)生長期的iMEFs細(xì)胞,以AdBMP9 + AdRFP感染作為對照組。感染7天后檢測成骨早期指標(biāo)ALP的活性(組織染色)。結(jié)果顯示CTGF全長、CTGFCD1和CTGFCD2能夠抑制BMP9誘導(dǎo)的成骨分化,而CTGFID1、CTGFID2和CTGFID3的抑制效果更為顯著。 在第五節(jié)中,我們檢測了CTGF全長及其突變體對BMP9誘導(dǎo)的骨形成的體內(nèi)作用。感染BMP9+RFP或BMP9+CTGF突變體腺病毒的iMEFs細(xì)胞種植于裸鼠皮下。皮下成骨包塊在6周后收獲。用Micro-CT、HE染色和Masson’s Trichrome染色分析皮下成骨包塊的大體和細(xì)節(jié)信息結(jié)果顯示,與BMP9+RFP對照組相比,BMP+CTGFCD2和BMP9+ CTGFCD1感染得到的包塊體積明顯增大,而BMP+CTGFID1/2/3組得到的包塊體積明顯小于對照組。BMP9+CTGF全長組似乎對包塊體積無明顯影響。組織學(xué)分析顯示,CTGF缺失突變體通過促進(jìn)或抑制iMEFs細(xì)胞的增殖而影響B(tài)MP9誘導(dǎo)的成骨作用。然而,CTGF及其突變體并沒有影響B(tài)MP9誘導(dǎo)的iMEFs分化。 第二部分:CTGF在腫瘤發(fā)生過程中的作用 在本部分的第一節(jié)中,我們利用結(jié)晶紫染色和劃痕試驗(yàn),研究了CCN1、CCN2、CCN3和CCN4對骨肉瘤細(xì)胞株143B和MG63的生長和遷移作用。結(jié)果顯示CCN1和CCN2可促進(jìn)143B細(xì)胞的增殖和MG63細(xì)胞的遷移,而CCN3和CCN4對這些骨肉瘤細(xì)胞株的增殖和遷移沒有明顯的促進(jìn)作用,甚至有輕微的抑制作用�？紤]到我們在第一部分選擇了CCN2(CTGF)做深入研究,在第二部分,我們同樣選擇CCN2(CTGF)做進(jìn)一步深入研究,即體內(nèi)實(shí)驗(yàn)。 在第二節(jié)中,我們利用逆轉(zhuǎn)錄病毒感染和抗生素篩選的方法,制作了兩種穩(wěn)定整合和表達(dá)熒光素酶基因的骨肉瘤細(xì)胞株143B-Luc和MG63-Luc,并用熒光素活性測定實(shí)驗(yàn)體外測定了兩個(gè)細(xì)胞株的功能。143B-Luc和MG63-Luc細(xì)胞的熒光素酶活性均超過1,000,000,而對照組143B和MG63細(xì)胞的熒光素酶活性均低于100。143B-Luc和MG63-Luc細(xì)胞株的成功構(gòu)建為研究CTGF的體內(nèi)功能奠定了基礎(chǔ)。 在第三節(jié),我們檢測了CTGF腺病毒直接感染對143B-Luc骨肉瘤細(xì)胞體內(nèi)作用。143B-Luc細(xì)胞感染Ad-CTGF或Ad-GFP腺病毒18小時(shí)后,注射到裸鼠背部皮下,用Xenogen活體成像技術(shù)跟蹤腫瘤的生長情況,注射后32天處死裸鼠,收獲腫瘤。結(jié)果顯示,CTGF組的腫瘤包塊體積顯著大于GFP對照組的包塊體積,說明CTGF更夠促進(jìn)骨肉瘤細(xì)胞的體內(nèi)生長。 在第四節(jié),我們利用iMEFs細(xì)胞作為CTGF基因的遞送載體,檢測了CTGF對MG63-Luc細(xì)胞的體內(nèi)作用。iMEFs分別用Ad-CTGF或Ad-GFP感染18小時(shí),感染后的iMEFs細(xì)胞與MG63-Luc細(xì)胞按照1:1的細(xì)胞比例混合,總細(xì)胞數(shù)為2×106,注射到裸鼠背部皮下。不與iMEFS細(xì)胞混合的單獨(dú)的MG63-Luc細(xì)胞作為對照組。用Xenogen活體成像技術(shù)跟蹤腫瘤的生長情況,注射后25天處死裸鼠,收獲腫瘤包塊。結(jié)果顯示,iMEFs+CTGF組的腫瘤包塊體積遠(yuǎn)遠(yuǎn)大于iMEFs+GFP對照組。iMEFs+GFP對照組與不與iMEFS細(xì)胞混合的單獨(dú)的MG63-Luc細(xì)胞作為對照組相比,所得包塊體積無明顯差別。說明CTGF可促進(jìn)骨肉瘤細(xì)胞的生長,且iMEFs細(xì)胞可作為有效的基因遞送載體。 總之,我們的研究結(jié)果顯示,CTGF的結(jié)構(gòu)域IV可對BMP9誘導(dǎo)的體內(nèi)成骨有抑制作用,而結(jié)構(gòu)域I可促進(jìn)這種成骨。我們的研究結(jié)果還顯示CTGF能促進(jìn)骨肉瘤細(xì)胞的體內(nèi)和體外增殖。另外,表達(dá)CTGF的iMEFs細(xì)胞顯著地促進(jìn)了骨肉瘤的體內(nèi)生長,提示CTGF的功能與其所處的細(xì)胞外微環(huán)境密切相關(guān)。 最終,我們的結(jié)果將為BMP9和CTGT缺失突變體促進(jìn)成骨的臨床應(yīng)用,和CTGF作為治療骨肉瘤靶點(diǎn)的臨床應(yīng)用提供參考。我們的結(jié)果也加深了對干細(xì)胞分化與增殖理論的認(rèn)識。
[Abstract]:The aim of this study is to reveal the osteodifferentiation of connective tissue growth factor ( ctgf , also called ccn2 ) in bone morphogenetic protein ( bmp ) 9 , and the role of ctgf in bone tumors .



There are six members , CCN1 to CCN6 , which are secrete small - component proteins , which are rich in cysteine . The majority of the members are regulated by growth factors , cytokines or cell pressure .



The results showed that CCN1 , CCN2 , CCN3 , CCN4 , CCN1 , CCN2 , CCN3 and CCN4 , inhibited BMP9 - induced osteogenic differentiation and promoted the proliferation and migration of osteosarcoma cell line 143B .



In the first section , we isolated mouse embryonic fibroblasts ( MEFs ) from the mouse embryos from 12.5 to 13.5 days of pregnancy . In addition , we used the retroviral infection MEFs carrying the SV40T gene to screen the MEFs by antibiotic . The establishment of iMEFs cell line was to study the osteogenic differentiation induced by BMP9 and its role in osteosarcoma cells .



In the second section , we evaluated the effects of cCN1 , CCN2 , CCN3 , CCN4 and CCN4 on bone differentiation induced by BMP9 . The results showed that CCN2 ( ctgf ) had the strongest ability to inhibit bone formation induced by BMP9 . The results suggested that CCN2 ( ctgf ) had the strongest inhibitory ability to inhibit bone formation induced by BMP9 .



A series of clones were constructed and six recombinant adenovirus were constructed . Six kinds of recombinant adenovirus were constructed by using molecular biology technique . Six kinds of recombinant adenovirus were constructed by using molecular biology technique . The whole length of ctgf and the mutant of CTGFCD 3 ( no domain II ) , CTGFID2 ( no domain III ) and CTGFID3 ( no domain II and III ) were identified .



In the fourth section , we examined the effect of the full length of ctgf and its deletion mutant on bone differentiation induced by bmp9 .



In the fifth section , we examined the effect of the full length of ctgf and its mutants on bone formation induced by bmp9 . The size of imesfs infected with bmp9 + rfp or bmp9 + ctgf mutant adenovirus was significantly increased compared with control group .



The second part : the role of ctgf in the pathogenesis of tumor



The growth and migration of CCN1 , CCN2 , CCN3 and CCN4 to osteosarcoma cell lines 143B and MG63 were investigated in the first section of this section . The results showed that CCN1 and CCN2 could promote the proliferation of 143B cells and the migration of MG63 cells . The results showed that CCN3 and CCN4 had no significant effects on proliferation and migration of these osteosarcoma cell lines .



In section II , we produced two kinds of osteosarcoma cell strains 143B - Luc and MG63 - Luc stably integrated and expressed luciferase gene by means of reverse transcription virus infection and antibiotic screening . The luciferase activity of 143B - Luc and MG63 - Luc cells was more than 1,000,000 , while the luciferase activity of 143B - Luc and MG63 - Luc cells was lower than 100 . 143B - Luc and MG63 - Luc cells were successfully constructed .



In section III , we examined the effect of direct infection on 143B - Luc osteosarcoma cells . 143B - Luc cells were injected into the back of the nude mice after being infected with Ad - ctgf or Ad - GFP adenovirus for 18 hours . After 18 hours of injection into the back of the nude mice , the growth of the tumor was tracked by using a living body imaging technique , the nude mice were sacrificed on 32 days after injection , and the tumor was harvested . The results showed that the volume of tumor masses in the ctgf group was significantly larger than that of the gfp control group , suggesting that ctgf is more effective in promoting the in vivo growth of osteosarcoma cells .



In the fourth section , we used the iMEFs cell as the delivery vector of ctgf gene to detect the in vivo effect of ctgf on mg63 - Luc cells . imeFs were infected with Ad - ctgf or Ad - GFP for 18 hours , the infected iMEFs cells were mixed with MG63 - Luc cells in a ratio of 1 : 1 , the total number of cells was 2 脳 106 , and the tumor mass was harvested 25 days after injection .



In conclusion , the results of our study showed that the structural domain IV could inhibit BMP9 - induced osteogenesis in vivo , and domain I could promote the osteogenesis . Our findings also showed that ctgf can promote in vivo and in vitro proliferation of osteosarcoma cells . In addition , imesfs cells expressing ctgf significantly promote the in vivo growth of osteosarcoma , suggesting that the function of ctgf is closely related to the extracellular microenvironment of the cells .



Finally , our findings will provide a reference for the clinical use of the BMP9 and CTGT deletion mutants for bone formation and the clinical application of ctgf as a target for the treatment of osteosarcoma . Our results also deepen the understanding of stem cell differentiation and proliferation theory .
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 謝亞芹;李秀華;;TGF-β_1和CTGF與慢性心力衰竭心肌纖維化的關(guān)系[J];承德醫(yī)學(xué)院學(xué)報(bào);2011年02期

2 杜業(yè)軍;司維柯;何通川;粟永萍;萬瑩華;潘靜;趙宸;;Cyr61在單純及放創(chuàng)復(fù)合傷愈合過程中的表達(dá)及其臨床意義[J];重慶醫(yī)學(xué);2008年09期

3 鮑艷芳;應(yīng)素芬;何斐;泮瑛瑛;張偉;徐靈芝;;CCN蛋白的功能與機(jī)制[J];當(dāng)代醫(yī)學(xué);2011年35期

4 丁偉;司維柯;孫世俊;潘靜;李招權(quán);;過表達(dá)外源性Cyr61對人肝癌細(xì)胞株HepG2生長和遷移的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年05期

5 孫世俊;司維柯;丁偉;潘靜;李招權(quán);趙宸;;Cyr61與β-catenin在肝癌組織和細(xì)胞株中表達(dá)的相關(guān)性研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年06期

6 顏林楓;南海燕;崔光彬;殷茜;魏經(jīng)國;;肺微血管內(nèi)皮細(xì)胞表型改變與博萊霉素所致大鼠肺纖維化的關(guān)系[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2008年16期

7 呂建發(fā);鄒友成;張純偉;毛志福;;Cyr61和WISP-3在非小細(xì)胞肺癌組織中的表達(dá)及其臨床意義[J];中國肺癌雜志;2010年12期

8 姚芳;閆U，

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