【作者】 张璐；

【导师】 王士勇；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的建立小鼠H22肝癌细胞肺高转移细胞株并测定其相关生物学特性和RhoC基因的表达,为研究转移相关分子机制提供经济实用的模型。方法将小鼠H22肝癌细胞(简称为M0)接种于小鼠腹腔形成腹水瘤,取腹水瘤细胞经尾静脉注射,待其肺转移结节形成后,取其肺转移结节处肝癌细胞再次腹腔接种、形成腹水瘤,再尾静脉注射,反复进行此操作后获得第四代肺高转移肝癌细胞(简称为M4)。检测M4在昆明小鼠体内转移能力、体外细胞增殖能力、计算细胞倍增时间、计数细胞分裂指数、Giemsa染色观察其染色体形态、用流式细胞仪检测细胞周期分布,并与M0比较。应用逆转录聚合酶链反应(RT-PCR)方法测定M4细胞RhoC基因mRNA的表达水平,并应用Imagequant软件进行比较分析。结果肺高转移H22细胞M4与M0相比较,尾静脉注射后在第30天和40天,M4肺转移结节总体积明显大于M0；M0与M4细胞体外培养24h、36h、48h、60h及72h,P值均小于0.05,范围从3×10-9至7.2×10-26,M4细胞浓度明显高于M0；M0细胞倍增时间为71.98h,而M4细胞倍增时间缩短为44.10h；M4细胞分裂指数大于M0(P=0.014)；M4细胞周期G0/G1期比例较M0减少(P=0.024),S期比例增多(P=0.022)；染色体数目无差异,但高转移H22细胞染色体存在明显异型性。高转移细胞RhoC基因mRNA的表达高于M0细胞,M0细胞RhoC基因与GAPDH基因表达比值为0.486±0.045,M4细胞RhoC基因与GAPDH基因表达比值为1.011±0.163,存在统计学差异(P=0.0029)。结论建立了小鼠H22肝癌细胞肺高转移细胞株M4,M4转移能力明显增高、体外细胞生长速度快、倍增时间短、分裂期细胞比例多、细胞周期G0/G1期比例较减少、S期比例增多、染色体异型性明显。高转移细胞的RhoC基因mRNA的表达明显增高；RhoC基因的过量表达与小鼠H22肝癌细胞的高转移能力相关。更多还原

【Abstract】 ObjectiveTo establish a mice high-metastasis H22 hepatocellular carcinoma cell line, and measure its biologic characters and RhoC gene expression to provide a suitable model for the study of metastasis-related molecular mechanisms.MethodsH22 hepatocellular carcinoma cells (MO for short) were inoculated into the abdominal cavity of mice, collect the abdominal caicinoma cells, inject them to the tail vein of another healthy mice and the metastatic lesions were harvested in the lung, then re-implanted into mice for the second time of in vivo selection. The same procedure was repeated until get the fourth cells(M4 shorted for). Then measure the metastatic ability of M4 cell in vivo, cell proliferation ability in vitro, calculate the cell doubling time and cell division index, observe the chromatosome after Giemsa stain and test the cell cycle by flow cytometry. At last measure the M4 andMO cell RhoC gene mRNA expression with the method of RT-PCR, then analyse it by the Imagequant software.ResultsComparing the MO cell with the high-metastasis cell M4,30 and 40 days after injecting carcinoma cell by tail vein, it is obviously easy to found that the velume of metastatic nodes of M4 cell in vivo is larger than M0. The concentration of M4 cells is larger than MO when they are cultered in vitro at the time point of 24h、36h、48h、60h 及72h. P<0.05, from3×10-9to 7.2×10-26. The cell doubling time of M0 is 71.98h, but M4 is 44.1 Oh, M4 is obviously shorter than MO.and cell division index of M4 is larger than M0(P=0.014). The high-metastasis cell proportion in G0/G1 stage is smaller than M0(P=0.024), in S stage is larger than M0(P=0.022). For the number of chromatosome, they are similar to each other, but there is obvious heteromorphism difference. The RhoC gene mRNA expression of high-metastatic cell is higher than M0; the relative expressive ratio of MO and M4 are 0.486±0.045and 1.011±0.163, (P=0.0029)ConclusionA mice high-lung metastasis hepatocellular carcinoma cell M4 is established. Comparing the MO cell, The metastatic ability of high-lung metastasis hepatocellular carcinoma cell is higher, growing speed is faster,the ratio of division cell is larger, there is obvious heteromorphism difference, proportion in G0/G1 stage is smaller but S stage is larger. The RhoC gene mRNA expression of high-metastatic cell line is higher, RhoC gene excessive expression is related to H22 hepatocellular carcinoma cell Line high-metastatic ability.更多还原