【作者】 祝文涛；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 目的:(1)建立高低不同转移潜能的人骨肉瘤MG63细胞亚株M6,M8肺转移动物模型;(2)建立稳定表达TSLC1基因的人骨肉瘤细胞系;(3)探讨抑癌基因TSLC1对高转移潜能人骨肉瘤MG63细胞亚株侵袭和转移能力的影响。方法:(1)高低不同转移潜能的人骨肉瘤MG63细胞亚株M8、M6,制成2x10~7细胞悬液,通过尾静脉注射于裸鼠体内,4周起分批处死裸鼠,观察皮下成瘤时间,大小,肺转移出现时间,转移灶大小,以及瘤组织病理学和组化检测;(2)采用RT-PCR方法,以人正常肺组织中提取的总RNA为模板扩增TSLC1全长片段,克隆至pCI-neo真核表达载体。限制性酶双酶切及测序鉴定重组质粒pCI-TSLC1。脂质体转染的方法将表达载体稳定转染至高转移潜能人骨肉瘤MG63细胞亚株M8,经克隆化培养以及G418筛选,建立了16株稳定表达TSLC1蛋白的细胞系。对其中一株高表达细胞系M8T的细胞纯度、遗传稳定性、外源基因整合等生物学特性做了鉴定。(3)选用稳定高表达外源性抑癌基因TSLC1的人骨肉瘤MG63细胞亚株M8T细胞株为研究对象。转染空载体的细胞系M8P和未经处理的M8细胞株设为对照,MTT法检测M8T细胞增殖,流式细胞术检测细胞周期变化,细胞凋亡的发生;Transwell检测M8T细胞株的体外侵袭和迁移能力;重悬1×10~7细胞皮下注射裸鼠,一周检测一次肿瘤生长情况。将2×10~7细胞悬液尾静脉注射裸鼠,观察骨肉瘤细胞体内肺转移情况。结果:MTT法检测细胞生长显示高转移潜能M8细胞增殖能力强于细胞亚株M6。尾静脉注射瘤细胞后,M8亚细胞株组裸鼠肺转移发生率为85%,其中60%肉眼即可观察到肺转移灶,转移灶数目平均为1-3/只,25%可以观察到镜下转移灶的形成;而低转移潜能的M6组,20只裸鼠中仅有5只出现肉眼可见的肺转移,有1只可见镜下肺转移灶,其转移率仅为30%。裸鼠发生肺转移的肺组织石蜡切片,免疫组化结果显示转移灶cyclinE1和Bcl-2表达阳性。另外,本研究成功构建了pCI-TSLC1真核表达载体并获得高表达TSLC1的稳定细胞系M8T。生物学性状研究结果表明该转基因细胞系纯度好,遗传性状稳定,抽提细胞RNA进行RT-PCR,扩增到与预期结果大小一致的DNA片段,说明TSLC1基因成功整合到细胞基因组中。稳定转染TSLC1基因的实验组M8T细胞与对照组细胞M8P和M8相比较,其细胞增殖能力下降,生长速度明显受到抑制;M8T细胞的生长周期出现了G0-G1期阻滞,细胞凋亡总数明显增加(P<0.01);体外侵袭和迁移能力变弱;M8T细胞与对照组细胞相比,裸鼠皮下成瘤形成时间晚,体积较小,体内肺转移形成时间晚,转移灶数目少。结论:(1)成功建立了高低不同转移潜能的骨肉瘤裸鼠肺转移模型。(2)成功建立了稳定表达TSLC1的骨肉瘤细胞系。该细胞系遗传性质稳定。(3)TSLC1基因可以明显抑制具有高转移潜能的骨肉瘤MG63细胞亚株的侵袭和转移能力。TSLC1基因有可能成为有效治疗骨肉瘤的候选基因。更多还原

【Abstract】 Objective :1. To establish a nude mice model of human osteosarcoma spontaneous lung metastasis. 2. To establish the osteosarcoma cell sublines which stably expressing TSLC1 protein and to identify its biological characteristics. 3. The effecct of TSLC1 on invasion and metastasis of osteosarcoma cell MG63.Methods: 1. The growth of human osteosarcoma cell sublines M8 and M6 was determined by MTT assay. 2x10~7 cells were injected into the tail vein of nude mice. Mice were sacrificed started on week 4 after injection, and lung metastases were evaluated under both macroscopic and microscopic observation with HE staining and immunohistology. 2. Full length of TSLC1 cDNA was amplified from RNA of normal human lung by RT-PCR, cloned into pCI-neo expression vector. The recombinant plasmid pCI-TSLC1 was identified with restriction enzyme and sequencing, and then was stably transfected into M8 cells with Lipofectamine 2000. The positive clones were developed by selection by G418. Biological characteristics of one of the 16 cell lines, namely, the M8T were studied. 3. Human osteosarcoma cell subline M8T was stably transfected with exogenous gene TSLC1. Cell growth was analyzed with MTT assay. FACSort flow cytometry analysis was performed to assess the cell cycle distribution and apoptosis. Transwell was performed to access the ability of migration and invasion of M8T. 1×10~7cells were injected into the two flanks of nude mice. The tumor growth was monitored once a week. 2×10~7 cells were injected into the tail vein of nude mice. Mice were sacrificed started on week 4 after injection, and lung metastases were evaluated under both macroscopic and microscopic observation with HE staining.Results: 1. The growth of low-metastatic subline M6 was lower than high-metastatic sublines M8. 17 mice after injected M8 had occurred lung metastases while just 5 mice had occurred in M6 group. Moreover, the expression of Bcl-2 and cyclin E1 of lung metastasic tissue were positive. 2. The eukaryotic expression vector pCI-TSLC1 was successfully constructed and the stable cell subline highly expressing TSLC1 protein was obtained. The genetic stability and purity of the cell population were confirmed. The kinetics and genetic of M8T was stable. The DNA product amplified from total RNA of osteosarcoma cell sublines reveals that ectogenous gene TSLC1 has integrated into the genomes of the M8 cell. The cell line M8T was not contaminated by microorganisms. 3. The growth of TSLC1-transfected cells M8T was significantly suppressed in vitro, displaying that the amount of G0-G1 cells increased and the amount of S phase cells decreased significantly, which suggested a G0-G1 cell cycle arresting. The exogenous gene TSLC1 could induce cell apoptosis. The M8T cells proliferation and the ability of invasion or migration which d were significantly reduced in vitro in comparison to the control. Moreover, tumorigenicity of M8T cells was suppressed in vivo, Lung metastases of nude mice occurred later and less than the control.Conclusions: 1. A mouse model for human osteosarcoma cancer spontaneous lung metastasis can be established by injection different ability of metastasis MG63 cells into tail vein. 2. The stable osteosarcoma cell sublines highly expressing TSLC1 has successfully established, which provided a basis for further exploring the roles of TSLC1 in osteosarcoma. 3. The ability of migration and invasion of high-metastatic osteosarcoma cell subline M8 was significantly suppressed by TSLC1 both in vitro and in vivo. TSLC1 would serve as a good candidate molecular target for the treatment of osteosarcoma.更多还原