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
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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。
The overall objective of this research is to study the effects of connective tissue growth factor (CTGF, also named as CCN2) on BMP9- induced osteogenesis and on osteosarcomas.
The CCN proteins contain six members, namely CCN1 to CCN6, which are small secreted cysteine-rich proteins. The CCN proteins are modular proteins, containing up to four functional domains. Many of the CCN members are induced by growth factors, cytokines, or cellular stress. The CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues. The CCN proteins can integrate and modulate the signals of integrins, BMPs, VEGF, Wnts, and Notch. The involvement of integrins in mediating CCN signaling may provide diverse context-dependent responses in distinct cell types.
In this project, the most important CCN family members, CCN1, CCN2, CCN3 and CCN4, were evaluated in BMP9-induced osteogenesis and osteosarcomas. Of the two aspects, CCN2 (CTGF) showed significant effects both in the inhibition of the BMP9-induced bone formation and in the proliferation and migration of osteosarcoma cell line 143B. Thus, CTGF was chosen for more extensive study. To simplify the depiction, this study was divided into two parts.
Part 1: The effect of CTGF on BMP9-induced osteogenesis.
In Chapter 1, we isolated MEFs from mouse embryos, 12.5 to 13.5 days postcoitum. We then established immortalized MEFs (iMEFs) through the immortalization of these MEFs by integration of SV40 T gene mediated by retrovirus vector. The establishment of iMEFs greatly facilitated this study both on BMP9-induced osteogenesis and on osteosarcoma cell lines.
In Chapter 2, we evaluated the effects of four CCN proteins, CCN1, 2, 3 and 4, on the BMP9-induced bone formation. Subconfluent iMEFs were co-infected with AdBMP9 and AdR-CCN1, Ad-CCN2, Ad-CCN3, Ad-CCN4, or, AdGFP adenoviruses and osteogeinc differentiation early marker alkaline phosphatase (ALP) activity was measured by quantitative colorimetric analysis at day 7. Results showed that CCN2 (CTGF) had the strongest inhibitory effect on BMP9-induced bone formation. These results promoted us to focus on CTGF for further study.
Since CTGF is a mosaic protein that contains four modules, to further elucidate which module or modules contribute to this inhibitory function, terminal and internal deletions of CTGF were made in Chapter 3. Using molecular biology techniques, we did a series of cloning and finally generated six adenoviruses which could express CTGF full length, CTGFCD1 (no module IV), CTGFCD2 (no module III and IV), CTGFID1 (no module II), CTGFID2 (no module III), CTGFID3 (no module II and III). The presence of CTGF full length or CTGF deletions in these recombinated adenoviruses was confirmed by PCR, and the expression of these CTGF constructs was confirmed by Western blot with anti-Flag antibody, due to the fusion 3×Flag tag in these constructs. These CTGF full length and deletion mutant adenoviruses provided useful tools for dissecting the functional domains of CTGF in bone formation.
In Chapter 4, we evaluated the effects of CTGF full length and its deletion mutants on the BMP9-induced bone formation in vitro. Subconfluent iMEFs were co-infected with AdBMP9 and AdR-CTGF, CTGFCD1, CTGFCD2, CTGFID1, CTGFID2, CTGFID3, or AdRFP adenoviruses and osteogeinc differentiation early marker alkaline phosphatase (ALP) activity was measured by ALP (histochemical) staining at day 7. Results showed that CTGF full length, CTGFCD1 and CTGFCD2 could inhibit BMP9-induced bone formation, while CTGFID1, CTGFID2, and CTGFID3 had a much more significant inhibitory effect.
In Chapter 5, we evaluated the effects of CTGF full length and its deletion mutants on the BMP9-induced bone formation in vivo. BMP9+RFP or BMP9+CTGF mutants expressing iMEFs were implanted subcutaneously in nude mice. Ectopic osseous masses were retrieved at 6 weeks. Micro-CT, H & E staining, and Masson’s Trichrome staining were employed to inspect the general and detailed information of the retrieved bone masses. Results showed that compared with BMP9+RFP control, BMP+CTGFCD2 and BMP9+CTGFCD1 yielded much larger bone masses, while the bone masses of BMP+CTGFID1/2/3 are much smaller. BMP9+CTGF full length didn’t change the bone mass volume. Histological studies showed that CTGF deletions affect the BMP9-induced bone formation by the promotion or inhibition of iMEFs proliferation. However, CTGF and its deletions didn’t affect the BMP9-induced iMEFs differentiation. These results suggest that CTGF module IV inhibited the BMP9-induced bone formation, while module I may promote this effect.
Part 2: The effect of CTGF on osteosarcoma. In Chapter 1, using crystal violet staining and wound healing assay, we studied the effects of CCN1, 2, 3, and 4 on osteosarcoma cell line 143B and MG63. Results showed that CCN1 and CCN2 promoted the proliferation of 143B cells and promoted the migration of MG63 cells, while CCN3 and CCN4 had no effect or only had slightly inhibitory effect on the proliferation and migration of these osteosarcoma cell lines. Considering we had focused on CCN2 (CTGF) for detailed study in Part One, we also chose CCN2 (CTGF) for further study in Part Two.
In Chapter 2, we generated two osteosarcoma cell lines, 143B-Luc and MG63-Luc by retrovirus-mediated firefly luciferase gene integration. The constitutively express of luciferase of the two cell lines were confirmed by luciferase assay in vitro. The luciferase activities of 143B-Luc and MG63-Luc cells were more than 1,000,000, while the values of the controls, 143B and MG63 cells were lower than 100. The establishment of 143B-Luc and MG63-Luc provided a useful tool for our in vivo study of CTGF.
In Chapter 3, we tested the effect of CTGF on 143B-Luc cells in vivo by direct CTGF adenovirus infection. 143B-Luc cells were infected with Ad-CTGF and Ad-GFP for 18 h, and injected subcutaneously into the back of nude mice. Injected mice were tracked by Xenogen imaging, and tumors were harvested at day 32. Results showed that the volumes of tumors of CTGF group were much larger than those of the GFP group, suggested that CTGF could promote the growth of osteosarcoma cells.
In chapter 4, we tested the effect of CTGF on MG63-Luc cells in vivo, using iMEFs as CTGF gene delivery vehicle. iMEFs were infected with either Ad-CTGF or Ad-GFP for 18 h. Infected iMEFs were mixed with MG63-Luc (1:1 ratio), to a total number of 2×106, and injected subcutaneously into the back of nude mice. MG63-Luc cells, without mix with iMEFs, were injected as control. Injected mice were tracked by Xenogen imaging, and tumors were harvested at day 25. Results showed that the volumes of tumors of iMEFs+CTGF group were much larger than those of the iMEFs+GFP group. There is no significant difference between the iMEFs+GFP group and the MG63-Luc only group. It suggested CTGF promoted osteosarcoma cell growth, and iMEFs was a good gene delivery vector.
In summary, our studies demonstrated that Module IV could exert an inhibitory effect on the BMP9-induced osteogenesis in vivo, while Module I could promote the bone formation. Our study also demonstrated that CTGF could promote the proliferation of osteosarcoma cell line both in vitro and in vivo. In addition, iMEFs expressing CTGF dramatically increased tumor volume in vivo, indicated that CTGF function was closely related to the context or microenviroment.
Ultimately, our results could offer the reference for the clinical use of BMP9 combined with CTGF deletions in bone formation, and the use of CTGF as a therapeutic target for osteosarcomas. Our results also enhanced the theory of stem cell differentiation v.s, proliferation.
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。
The overall objective of this research is to study the effects of connective tissue growth factor (CTGF, also named as CCN2) on BMP9- induced osteogenesis and on osteosarcomas.
The CCN proteins contain six members, namely CCN1 to CCN6, which are small secreted cysteine-rich proteins. The CCN proteins are modular proteins, containing up to four functional domains. Many of the CCN members are induced by growth factors, cytokines, or cellular stress. The CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues. The CCN proteins can integrate and modulate the signals of integrins, BMPs, VEGF, Wnts, and Notch. The involvement of integrins in mediating CCN signaling may provide diverse context-dependent responses in distinct cell types.
In this project, the most important CCN family members, CCN1, CCN2, CCN3 and CCN4, were evaluated in BMP9-induced osteogenesis and osteosarcomas. Of the two aspects, CCN2 (CTGF) showed significant effects both in the inhibition of the BMP9-induced bone formation and in the proliferation and migration of osteosarcoma cell line 143B. Thus, CTGF was chosen for more extensive study. To simplify the depiction, this study was divided into two parts.
Part 1: The effect of CTGF on BMP9-induced osteogenesis.
In Chapter 1, we isolated MEFs from mouse embryos, 12.5 to 13.5 days postcoitum. We then established immortalized MEFs (iMEFs) through the immortalization of these MEFs by integration of SV40 T gene mediated by retrovirus vector. The establishment of iMEFs greatly facilitated this study both on BMP9-induced osteogenesis and on osteosarcoma cell lines.
In Chapter 2, we evaluated the effects of four CCN proteins, CCN1, 2, 3 and 4, on the BMP9-induced bone formation. Subconfluent iMEFs were co-infected with AdBMP9 and AdR-CCN1, Ad-CCN2, Ad-CCN3, Ad-CCN4, or, AdGFP adenoviruses and osteogeinc differentiation early marker alkaline phosphatase (ALP) activity was measured by quantitative colorimetric analysis at day 7. Results showed that CCN2 (CTGF) had the strongest inhibitory effect on BMP9-induced bone formation. These results promoted us to focus on CTGF for further study.
Since CTGF is a mosaic protein that contains four modules, to further elucidate which module or modules contribute to this inhibitory function, terminal and internal deletions of CTGF were made in Chapter 3. Using molecular biology techniques, we did a series of cloning and finally generated six adenoviruses which could express CTGF full length, CTGFCD1 (no module IV), CTGFCD2 (no module III and IV), CTGFID1 (no module II), CTGFID2 (no module III), CTGFID3 (no module II and III). The presence of CTGF full length or CTGF deletions in these recombinated adenoviruses was confirmed by PCR, and the expression of these CTGF constructs was confirmed by Western blot with anti-Flag antibody, due to the fusion 3×Flag tag in these constructs. These CTGF full length and deletion mutant adenoviruses provided useful tools for dissecting the functional domains of CTGF in bone formation.
In Chapter 4, we evaluated the effects of CTGF full length and its deletion mutants on the BMP9-induced bone formation in vitro. Subconfluent iMEFs were co-infected with AdBMP9 and AdR-CTGF, CTGFCD1, CTGFCD2, CTGFID1, CTGFID2, CTGFID3, or AdRFP adenoviruses and osteogeinc differentiation early marker alkaline phosphatase (ALP) activity was measured by ALP (histochemical) staining at day 7. Results showed that CTGF full length, CTGFCD1 and CTGFCD2 could inhibit BMP9-induced bone formation, while CTGFID1, CTGFID2, and CTGFID3 had a much more significant inhibitory effect.
In Chapter 5, we evaluated the effects of CTGF full length and its deletion mutants on the BMP9-induced bone formation in vivo. BMP9+RFP or BMP9+CTGF mutants expressing iMEFs were implanted subcutaneously in nude mice. Ectopic osseous masses were retrieved at 6 weeks. Micro-CT, H & E staining, and Masson’s Trichrome staining were employed to inspect the general and detailed information of the retrieved bone masses. Results showed that compared with BMP9+RFP control, BMP+CTGFCD2 and BMP9+CTGFCD1 yielded much larger bone masses, while the bone masses of BMP+CTGFID1/2/3 are much smaller. BMP9+CTGF full length didn’t change the bone mass volume. Histological studies showed that CTGF deletions affect the BMP9-induced bone formation by the promotion or inhibition of iMEFs proliferation. However, CTGF and its deletions didn’t affect the BMP9-induced iMEFs differentiation. These results suggest that CTGF module IV inhibited the BMP9-induced bone formation, while module I may promote this effect.
Part 2: The effect of CTGF on osteosarcoma. In Chapter 1, using crystal violet staining and wound healing assay, we studied the effects of CCN1, 2, 3, and 4 on osteosarcoma cell line 143B and MG63. Results showed that CCN1 and CCN2 promoted the proliferation of 143B cells and promoted the migration of MG63 cells, while CCN3 and CCN4 had no effect or only had slightly inhibitory effect on the proliferation and migration of these osteosarcoma cell lines. Considering we had focused on CCN2 (CTGF) for detailed study in Part One, we also chose CCN2 (CTGF) for further study in Part Two.
In Chapter 2, we generated two osteosarcoma cell lines, 143B-Luc and MG63-Luc by retrovirus-mediated firefly luciferase gene integration. The constitutively express of luciferase of the two cell lines were confirmed by luciferase assay in vitro. The luciferase activities of 143B-Luc and MG63-Luc cells were more than 1,000,000, while the values of the controls, 143B and MG63 cells were lower than 100. The establishment of 143B-Luc and MG63-Luc provided a useful tool for our in vivo study of CTGF.
In Chapter 3, we tested the effect of CTGF on 143B-Luc cells in vivo by direct CTGF adenovirus infection. 143B-Luc cells were infected with Ad-CTGF and Ad-GFP for 18 h, and injected subcutaneously into the back of nude mice. Injected mice were tracked by Xenogen imaging, and tumors were harvested at day 32. Results showed that the volumes of tumors of CTGF group were much larger than those of the GFP group, suggested that CTGF could promote the growth of osteosarcoma cells.
In chapter 4, we tested the effect of CTGF on MG63-Luc cells in vivo, using iMEFs as CTGF gene delivery vehicle. iMEFs were infected with either Ad-CTGF or Ad-GFP for 18 h. Infected iMEFs were mixed with MG63-Luc (1:1 ratio), to a total number of 2×106, and injected subcutaneously into the back of nude mice. MG63-Luc cells, without mix with iMEFs, were injected as control. Injected mice were tracked by Xenogen imaging, and tumors were harvested at day 25. Results showed that the volumes of tumors of iMEFs+CTGF group were much larger than those of the iMEFs+GFP group. There is no significant difference between the iMEFs+GFP group and the MG63-Luc only group. It suggested CTGF promoted osteosarcoma cell growth, and iMEFs was a good gene delivery vector.
In summary, our studies demonstrated that Module IV could exert an inhibitory effect on the BMP9-induced osteogenesis in vivo, while Module I could promote the bone formation. Our study also demonstrated that CTGF could promote the proliferation of osteosarcoma cell line both in vitro and in vivo. In addition, iMEFs expressing CTGF dramatically increased tumor volume in vivo, indicated that CTGF function was closely related to the context or microenviroment.
Ultimately, our results could offer the reference for the clinical use of BMP9 combined with CTGF deletions in bone formation, and the use of CTGF as a therapeutic target for osteosarcomas. Our results also enhanced the theory of stem cell differentiation v.s, proliferation.
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