【作者】 罗政强；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 骨外科， 2006， 博士

【摘要】 目的筛选高低不同转移特性并稳定表达绿色荧光蛋白的人乳腺癌MCF7细胞亚株，检测细胞亚株之间基因表达的差异，以筛选乳腺癌骨转移相关基因。方法利用脂质体转染GFP入人乳腺癌MCF7细胞，通过有限稀释法、细胞电泳，获得高低不同转移特性的细胞亚株，测定细胞亚株的增殖率，通过组织形态学观察、软琼脂克隆形成率、transwell小室体外侵袭能力测定、裸鼠体内成瘤试验等研究细胞亚株的生物学特性；将C6作为实验组，A2为对照组，用cDNA微阵列的方法比较两个细胞亚株之间基因表达的差异；用实时定量PCR法检测、验证cDNA微阵列中显著差异表达的整合素aV基因和微阵列中未涉及的整合素a4基因。结果获得稳定表达绿色荧光蛋白不同转移特性的两个细胞亚株A2(高转移特性)和C6(低转移特性)，其中A2的群体倍增时间为25.6h，软琼脂形成率为30.7％；C6群体倍增时间为41.8h，软琼脂形成率为14％；transwell小室体外侵袭能力测定发现，A2细胞亚株的平均侵袭细胞数为136.80±12.36个，明显高于C6的平均侵袭细胞数73.62±9.76个；裸小鼠皮下成瘤试验发现A2成瘤时间短，瘤体直径大，细胞增殖快。A2、C6细胞亚株的基因芯片结果发现，2个细胞亚株之间发生显著性表达变化的基因有767个，其中与A2相比，在C6中上调表达的基因有480个，下调表达的基因有287个。在发生明显表达变化的基因中具有比较明显的功能类别特征的包括与细胞骨架结构相关的基因、与细胞生长及增殖调控相关的基因、以及与细胞氧化张力应激反应相关的基因，提示与A2细胞相比，C6细胞表现出细胞增殖抑制，细胞结构变化以及应激反应活化等特征。实时定量PCR检测A2、C6细胞亚株的ITGA-V基因发现，A2细胞亚株样本的ITGA-V基因平均拷贝数为6574.56，C6细胞亚株ITGA-V基因的平均拷贝数为318.84，A2细胞亚株ITGA-V基因的拷贝数大于C6细胞亚株的ITGA-V基因的拷贝数；实时定量PCR检测A2、C6细胞亚株的ITGA4基因发现，A2细胞亚株样本的ITGA4基因平均拷贝数为19.98，C6细胞亚株的平均拷贝数为49.26，A2细胞亚株ITGA4基因的拷贝数小于C6细胞亚株ITGA-V基因的拷贝数。结论筛选出的两个细胞亚株有不同的转移特性，GFP的整合及表达未对MCF7细胞的生长状态、生物学性状未造成明显影响，可作为报告基因进一步研究乳腺癌骨转移的机制和治疗；cDNA微阵列法对于筛选乳腺癌骨转移相关基因有独特的优势，获得的差异表达基因具有较强的代表性，为寻找乳腺癌骨转移相关基因提供了重要线索；在高低不同转移特性的细胞亚株中，整合素aV的表达随着肿瘤细胞侵袭力的增高而增高，整合素aV的高表达和和乳腺癌的侵袭性相关；整合素a4的表达随着肿瘤侵袭程度的增高而降低，整合素a4的失表达和和乳腺癌的侵袭性相关。更多还原

【Abstract】 [Objective] To establish variant cell sublines expressing green fluorescent protein with high and low metastatic potential and to investigate gene expression profile in breast cancer cell sublines with different metastatic potentialities, in order to find new candidate genes related to metastasis of breast cancer to bone.[Methods] MCF7 cells were transfected by GFP gene, two cloned cell sublines(A2、 C6) were isolated by limiting dilution technique and electrophoretic technique. They were selected for further research on growth rate, colony formation, cell migration assay, subcutaneous transplantation in nude mice. The gene expression of A2 and C6 cell sublines were investigated by using cDNA microarray analysis. The alterations in ITGA-V and ITGA4 gene expression of these two sublines were confirmed by realtime quantitative RT—PCR.[Results] Two clones expressing Green fluorescent protein with high (A2) and low (C6) metastatic potential were isolated from the parent cell line. Compared with C6, A2 was faster in vitro growth rate (tumor cell doubling time was 25.6 h vs 41.8 h). The clone formation rates were 30.7% for A2 and 14% for C6. Invasion assay in vitro demonstrated that the number of penetrating cells was 136.80±12.36 cells/field for A2 vs. 73.62±9.76/field for C6. After subcutaneous transplantation of A2 and C6, A2 showed a stronger tumorigenicity than C6 after transplantation.There were significant differences in gene expression between high and low metastatic potential cell sublines. Among the target genes, 767 differentially expressed genes were identified in these two cell sublines. Compared to A2, 480 of 767 differentially expressed genes were up-regulated and the other 287 were down-regulated. Realtime quantitative RT—PCR revealed the mean ITGA-V gene copies of A2 cell line were 6574.56 while C6 were 318.84, copies in A2 cell line were significantly higher than those in the C6; the mean ITGA4 gene copies of A2 cell line were 19.98 while C6 were 49.26, copies in A2 cell line were lower than those in the C6.[Conclusion] Two clones expressing Green fluorescent protein with different metastatic potential were established, which could be valuable for further study on the molecular mechanisms and therapeutics of breast carcinoma metastasis to bone. There were different gene expression profiles between high and low metastatic potential cell sublines.These genes may provide important clues for finding genes related to metastasis of breast cancer to bone. The overexpression of integrin aV and loss expression of integrin a4 were related to the invasive behaviour of breast cancer cells which metastasis to bone.更多还原