结缔组织生长因子（CTGF）在BMP9诱导的成骨分化及在肿瘤发生过程中的作用

发布时间：2018-06-07 08:43

本文选题：结缔组织生长因子 + 骨形态发生蛋白　；参考：《重庆医科大学》2010年博士论文

【摘要】： 本课题的主要研究目的是揭示结缔组织生长因子(CTGF,又称CCN2)在骨形态发生蛋白(BMP)9诱导的成骨分化,以及CTGF在骨肿瘤发生中的作用。 CCN蛋白家族含有6个成员,为CCN1到CCN6,它们均为富含半胱氨酸的分泌性小分量蛋白。CCN蛋白是含有四个功能性结构域的modular蛋白。多数CCN蛋白家族的成员受生长因子、细胞因子或细胞压力的诱导调控。CCN蛋白在成体和胚胎组织的表达模式广泛且差异很大。CCN蛋白可以通过参与并调节多种信号分子而发挥作用,如整合素、BMPs、血管内盘生长因子(VEGF)、Wnts和Notch等。其中,整合素介导的CCN信号传递具有组织和细胞特异性,根据细胞外基质不同而作用各异。在本课题中,我们首先检测了CCN蛋白家族中最重要的4个成员,CCN1、CCN2、CCN3和CCN4,对BMP9诱导的成骨分化和对骨肉瘤的作用。结果显示CCN2(CTGF)抑制了BMP9诱导的成骨分化,促进了骨肉瘤细胞株143B的增殖和迁移,两个方面均具有显著性差异。于是,我们选择CTGF做进一步深入研究。为了方便表述,本课题大致分为两部分: 在第一节,我们从孕12.5到13.5天的小鼠胚胎中分离得到了小鼠胚胎成纤维细胞(MEFs)。进而,我们用携带SV40T基因的逆转录病毒感染MEFs,通过抗生素筛选,得到了永生化的MEFs(iMEFs)。iMEFs细胞株的建立为研究CTGF在BMP9诱导的成骨分化和在骨肉瘤细胞中的作用奠定了基础。在第二节中,我们评价了4中CCN蛋白,CCN1、CCN2、CCN3和CCN4,对BMP9诱导的成骨分化的影响。处于对数生长期的iMEFs共感染AdBMP9和AdR-CCN1、Ad-CCN2、Ad-CCN3、Ad-CCN4或AdGFP重组腺病毒。在7天后成骨分化早期标志碱性磷酸酶(ALP)用比色发定量测定。结果显示CCN2(CTGF)具有最强的抑制BMP9诱导的骨形成的能力。这一结果促使我们选择CTGF做进一步深入研究。由于CTGF是一种含有4个结构域的mosaic蛋白,为进一步研究哪个或哪几个结构域发挥着抑制作用,在第三节里我们构建了CTGF的中间缺失和C末端缺失突变体。利用分子生物学技术,我们构建了一系列克隆,最终制作了6种重组腺病毒,它们分别表达CTGF全长、CTGFCD1(无结构域IV),CTGFCD2(无结构域III和IV),CTGFID1(无结构域II),CTGFID2(无结构域III)和CTGFID3(无结构域II和III)。6种腺病毒经蛋白酶K裂解,以基因组DNA为模板做PCR,鉴定了CTGF全长及其突变体的存在。以抗Flag抗体为一抗做Western blot,证实了6种腺病毒可以成功表达CTGF全长及其突变体(CTGF及其突变体均与3×Flag标签融合表达。CTGF全长和缺失突变体重组腺病毒的成功构建,为进一步解析在成骨分化中发挥功能的结构域奠定了基础。在第四节,我们检测了CTGF全长及其缺失突变体对BMP9诱导的成骨分化的体外影响。用AdBMP9和CTGF腺病毒(AdR-CTGF, CTGFCD1, CTGFCD2, CTGFID1, CTGFID2, CTGFID3)联合感染出于对数生长期的iMEFs细胞,以AdBMP9 + AdRFP感染作为对照组。感染7天后检测成骨早期指标ALP的活性(组织染色)。结果显示CTGF全长、CTGFCD1和CTGFCD2能够抑制BMP9诱导的成骨分化,而CTGFID1、CTGFID2和CTGFID3的抑制效果更为显著。在第五节中,我们检测了CTGF全长及其突变体对BMP9诱导的骨形成的体内作用。感染BMP9+RFP或BMP9+CTGF突变体腺病毒的iMEFs细胞种植于裸鼠皮下。皮下成骨包块在6周后收获。用Micro-CT、HE染色和Masson’s Trichrome染色分析皮下成骨包块的大体和细节信息结果显示,与BMP9+RFP对照组相比,BMP+CTGFCD2和BMP9+ CTGFCD1感染得到的包块体积明显增大,而BMP+CTGFID1/2/3组得到的包块体积明显小于对照组。BMP9+CTGF全长组似乎对包块体积无明显影响。组织学分析显示,CTGF缺失突变体通过促进或抑制iMEFs细胞的增殖而影响BMP9诱导的成骨作用。然而,CTGF及其突变体并没有影响BMP9诱导的iMEFs分化。第二部分:CTGF在肿瘤发生过程中的作用在本部分的第一节中,我们利用结晶紫染色和划痕试验,研究了CCN1、CCN2、CCN3和CCN4对骨肉瘤细胞株143B和MG63的生长和迁移作用。结果显示CCN1和CCN2可促进143B细胞的增殖和MG63细胞的迁移,而CCN3和CCN4对这些骨肉瘤细胞株的增殖和迁移没有明显的促进作用,甚至有轻微的抑制作用。考虑到我们在第一部分选择了CCN2(CTGF)做深入研究,在第二部分,我们同样选择CCN2(CTGF)做进一步深入研究,即体内实验。在第二节中,我们利用逆转录病毒感染和抗生素筛选的方法,制作了两种稳定整合和表达荧光素酶基因的骨肉瘤细胞株143B-Luc和MG63-Luc,并用荧光素活性测定实验体外测定了两个细胞株的功能。143B-Luc和MG63-Luc细胞的荧光素酶活性均超过1,000,000,而对照组143B和MG63细胞的荧光素酶活性均低于100。143B-Luc和MG63-Luc细胞株的成功构建为研究CTGF的体内功能奠定了基础。在第三节,我们检测了CTGF腺病毒直接感染对143B-Luc骨肉瘤细胞体内作用。143B-Luc细胞感染Ad-CTGF或Ad-GFP腺病毒18小时后,注射到裸鼠背部皮下,用Xenogen活体成像技术跟踪肿瘤的生长情况,注射后32天处死裸鼠,收获肿瘤。结果显示,CTGF组的肿瘤包块体积显著大于GFP对照组的包块体积,说明CTGF更够促进骨肉瘤细胞的体内生长。在第四节,我们利用iMEFs细胞作为CTGF基因的递送载体,检测了CTGF对MG63-Luc细胞的体内作用。iMEFs分别用Ad-CTGF或Ad-GFP感染18小时,感染后的iMEFs细胞与MG63-Luc细胞按照1:1的细胞比例混合,总细胞数为2×106,注射到裸鼠背部皮下。不与iMEFS细胞混合的单独的MG63-Luc细胞作为对照组。用Xenogen活体成像技术跟踪肿瘤的生长情况,注射后25天处死裸鼠,收获肿瘤包块。结果显示,iMEFs+CTGF组的肿瘤包块体积远远大于iMEFs+GFP对照组。iMEFs+GFP对照组与不与iMEFS细胞混合的单独的MG63-Luc细胞作为对照组相比,所得包块体积无明显差别。说明CTGF可促进骨肉瘤细胞的生长,且iMEFs细胞可作为有效的基因递送载体。总之,我们的研究结果显示,CTGF的结构域IV可对BMP9诱导的体内成骨有抑制作用,而结构域I可促进这种成骨。我们的研究结果还显示CTGF能促进骨肉瘤细胞的体内和体外增殖。另外,表达CTGF的iMEFs细胞显著地促进了骨肉瘤的体内生长,提示CTGF的功能与其所处的细胞外微环境密切相关。最终,我们的结果将为BMP9和CTGT缺失突变体促进成骨的临床应用,和CTGF作为治疗骨肉瘤靶点的临床应用提供参考。我们的结果也加深了对干细胞分化与增殖理论的认识。
[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 .
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

【共引文献】