IGFBP5在骨肉瘤生长与转移作用机理的研究

【作者】 宿玉玺；

【导师】 金先庆； Tongchuan-He；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分荧光素酶标记骨肉瘤细胞MG63及转移亚型细胞株的建立目的:荧光素酶标记肿瘤细胞株MG63,通过肺转移灶原代培养的方法建立MG63细胞株转移亚型细胞株MG63-1和MG63-2。方法:体外培养肿瘤细胞株MG63细胞株,荧光素酶标记细胞,通过膝关节注射肿瘤细胞建立肿瘤模型,5周左右,当荧光成像仪扫描发现肺脏广泛转移时,取肺脏转移肿瘤,原代培养得细胞株MG63-1,重复上述操作得细胞株MG63-2。结果:(1)利用BSD筛选方法成功筛选到含荧光素酶的细胞株MG63-luc(2)成功将筛选到的细胞株MG63-luc注射到4周龄裸鼠成瘤,并得到肿瘤转移细胞亚型MG63-1和MG63-2。结论:成功得到含有荧光素酶标记的细胞株MG63-luc及其转移亚型MG63-1和MG63-2,为进一步研究骨肉瘤转移打下了良好的基础。第二部分MG63转移亚型细胞株体外实验的研究目的:通过一系列的肿瘤细胞经典转移能力方法,体外体内实验比较MG63和MG63-2细胞株之间肿瘤转移能力,证实MG62-2较MG63的转移能力强。方法:绘制细胞生长曲线,划痕实验,细胞吸附实验,细胞Matrigel迁移实验,与转移相关的主要5个基因MMP2、MMP9、TIMP1、TIMP2和Ezrin的RT-PCR及western-blotting。结果:(1)MG63-2的生长较MG63生长缓慢,细胞的周期长。(2)MG63-2划痕实验细胞的融合较MG63慢,所需时间长。(3)细胞吸附实验证实MG63-2的细胞吸附能力较MG63强。(4)Matrigel迁移实验证实MG63-2的迁移能力较MG63强(。5)RT-PCR以及western-blotting均证实MMP2和MMP9表达降低,TIMP2和Ezrin表达增高。结论:通过绘制细胞生长曲线,划痕实验,细胞吸附实验、Matrigel迁移实验,与转移相关的主要七个基因MMP2、MMP9、TIMP1、TIMP2和Ezrin的RT-PCR及其western-blotting,有差异表达,证实MG63-2较MG63在体外有着较强的转移能力,为下一步研究打下坚实之基础。第三部分MG63转移亚型细胞株体内实验的研究目的:本研究旨在通过膝关节注射动物肿瘤模型观察MG63和MG63-2之间肿瘤生长及转移能力的区别。方法:将MG63细胞和MG63-2细胞株的PBS细胞悬液注射入裸鼠膝关节内。荧光素酶成像仪器观察裸鼠原位肿瘤生长情况和肺转移情况。石蜡组织切片证实MG63-2在肺部有广泛的转移灶。结果:(1)采用膝关节注射法成原位肿瘤法稳定可靠;(2)原位肿瘤的生长曲线证实MG63肿瘤组成瘤性较MG63-2生长慢。(3)荧光素酶成像仪观察结果显示MG63-2的成瘤性较强,且其肺脏内转移多而广泛,有明显的统计学意义。(4)组织学HE染色,光镜下计数肿瘤转移数量,MG63-2有较强的转移能力。结论:体内实验证实MG63-2较MG63细胞株有较强的细胞成瘤性和转移能力。第四部分IGFBP5对肿瘤生长和转移影响的体内外实验的研究目的:通过构建IGFBP5基因高表达和IGFBP5 RNA干扰腺病毒,重点研究IGFBP5在肿瘤生长其转移的作用。方法:构建IGFBP5的腺病毒载体和IGFBP5的干扰病毒,分别感染Mefs、C3H10和C2C12三类细胞株,观测ALP读数,分析研究IGFBP5对其分化的影响。病毒感染143B、MG63-P、MG63-1、MG63-2细胞株绘制生长曲线,采用matrigel迁移实验分析IGFBP5对骨肉瘤细胞株的迁移能力的影响。游标卡尺及Xenogen image成像技术分析IGFBP5高表达和低表达后,肿瘤在动物体内生长与转移情况,分析其对肿瘤生长与转移的影响,石蜡组织切片,光镜下看转移灶的情况,研究IGFBP5对肿瘤转移的影响。结果:(1)ALP读数证实IGFBP5能促进C3H10、mefs、C2C12的分化,但是抑制其增长。而对SiIGFBP5则促进增长影响分化。(2)IGFBP5能抑制骨肉瘤143B-luc、MG63、MG63-1、Mg63-2的增长,SiIGFBP5则促进其增长。(3)划痕实验、matrigel肿瘤迁移实验,证实IGFBP5抑制肿瘤的迁移能力。(4)体内肿瘤生长的测量及其成像均证实IGFBP5有促进肿瘤分化,抑制增长,抑制转移的特性。结论:IGFBP5能够一定程度的抑制骨肉瘤细胞的增长,促进其分化。其在骨肉瘤的生长和转移中有重要的抑制作用。更多还原

【Abstract】 PART ONE LUCIFERASE TAGGED MG63 CELL LINE AND ESTABLISHMENT OF SUBTYPE OF THE CELL LINEObjective: using the luciferase to tag the MG63 cell line by retro-virus, culture the cells which got from the lung metastasis sites, then got the cell lines of MG63.1 and MG63.2.Methods: culture the MG63 cell lines in vivo, using the cells which was tagged by luciferase, the cells were injected in the intra-tibia, about 5weeks, when we found the signal from the lungs if we made the image by Xengon machine, the lung tumors which were metastasized from the legs were taken and cultured in the incubator, this is the cell line which we definite as MG63.1, repeated the procedure and then we got the cell line MG63.2.Results: (1)Using the classifying method by antibiotic BSD, we successfully got the cell line MG63-luc. (2)Successfully established the cell lines MG63.1 and MG63.2 by injecting the cell line MG63-luc to the intra-tibia of the 4 weeks old nude mice.Conclusion: Successfully established the cell line MG63-luc which tagged luciferase and the subtype cell lines MG63.1 and MG63.2, and this made us easier further research on the metastasis of the osteosarcoma. PART TWO THE IN VITRO STUDY OF THE SUBTYPE CELL LINE OF MG63Objective: through series methods of studying the metastasis ability, compare the metastasis ability between MG63 and MG63.2 and prove the MG63.2 has a stronger ability to metastasis than MG63.Methods: Draw the proliferation curve of MG63 and MG63.2, wound healing test, cell adhesion test, matrigel invasive test, cell migration test, and the seven genes MMP2,MMP9,TIMP1,TIMP2 and Ezrin which had relationship with metastasis run the RT-PCR and western-blotting to test the expression of RNA and protein.Results: (1) Comparing with the MG63, MG63.2 grew slower and the cell cycle was a little longer. (2)Cell adhesion test showed MG63.2 had a stronger ability than MG63. (3)Matrigel migration test showed MG63.2 had a stronger ability than MG63. (4) Both the RT-PCR and western-blotting test showed that the expression of MMP2 and MMP9 decreased while the TIMP2 and ezrin increased in these two cell lines.Conclusions: Through the test of cell proliferation curve, wound healing test, cell adhesion test, matrigel invasive test and RT-PCR and western-blotting of 5 genes MMP2, MMP9, TIMP1, TIMP2, ezrin showed that MG63.2 definitely had the ability to metastasis than MG63 in vitro. PART THREE THE IN VIVO STUDY ON THE SUBTYPE CELL LINE OF MG63Objective: To study the tumor growth and the ability to metastasis by the animal model which were established by injecting the cells to the intratibia between the MG63 and MG63.2.Methods:Inject the suspended cells MG63 and MG63.2 diluted by PBS to the intra-tibia of the nude mice. Observe the tumor grow and lung metastasis. HE staining proved that the MG63.2 cans metastasis to the lungs.Results:(1) The model which can be formed by injecting the cells to the intra-tibia was stable and reliable. (2) The tumor growth in the primary site showed the MG63.2 grew a little faster than MG63. (3)Xengon image showed MG63.2 had the stronger ability to form the tumor metastasis. And there was significant difference between these two groups. (4)HE staining of the histology showed the quantity of the tumor metastasis was more in the MG63.2.Conclusions: MG63.2 had the stronger ability of tumor forming and metastasis than MG63. PART FOUR THE RESEARCH ON THE EFFECT OF TUMOR GROWTH AND METASTASIS IN VITRO AND IN VIVO STUDY FOR THE GENE IGFBP5Objective: To construct the ad-easy virus of IGFBP5 and RNA interference virus, do the research on effect of the tumor growth and metastasis for the gene IGFBP5.Methods:Construct the ad-virus of IGFBP5 and its RNA interference virus, infected the mefs,C3H10 and C2C12 three cell lines, read the ALP reading, analysis the effect on the differentiation of IGFBP5. Draw the proliferation curve for the 143B,MG63-P,MG63.1 and MG63.2 after the IGFBP5 virus infecting. Study the matrigel migration ability for the IGFBP5 virus infected cell lines. Measured the tumour growth and Xengon image analysis the signal express in vivo. Counted the quantity of the metastasis in the lungs and study the effect of IGFBP5 in the metastasis.Results:(1)ALP reading proved that IGFBP5 can promote C3H10, mefs and C2C12 differentiation and inhibit the proliferation. For the SiIGFBP5, it is opposite. (2) IGFBP5 can inhibit the osteosarcoma cell lines 143B-luc, MG63, MG63.1and MG63.2 growth while the SiIGFBP5 was the opposite. (3)Wound healing test, matrigel invasive test proved that IGFBP5 can inhibit the tumour invasive while the SiIGFBP5 was the opposite. (4)Tumour measurement and Xengon image showed that IGFBP5 can inhibit the tumour growth, while the SiIGFBP5 is the opposite. Conclusions: IGFBP5 can inhibit the tumour growth, promote the tumour differentiation. It played an important role in the tumour growth and metastasis.更多还原