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目的 研究血管抑素血管他丁 (angiostatin)基因转染人肝癌细胞系HCC772 1,对肿瘤细胞体外生长、周期分布、形态以及体内致瘤力的影响 ,探讨血管抑素的作用机制。方法 应用定向克隆技术构建鼠源性血管抑素血cDNA基因真核表达载体 pcDNA3 .1( + ) angio ,酶切鉴定和测序。采用脂质体基因转染技术将真核表达载体导入人肝癌细胞系HCC772 1,新霉素G4 18筛选抗性克隆 ,设置转染空载体细胞为阴性对照和未转染细胞为空白对照。通过RNA点杂交和流式细胞荧光免疫检测血管抑素的表达 ,测定细胞生长曲线 ,计算细胞倍增时间 ,流式细胞仪检测细胞周期分布。建立动物模型 ,观察转染前后肿瘤细胞致瘤力的改变。免疫组化检测肿瘤组织微血管密度 (MVD)和血管抑素体内表达。结果 成功构建带有碱基序列正确的血管抑素基因片段的重组真核表达载体 pcDNA3 .1( + ) angio。分别转染脂质体 /pcDNA3 .1( + ) angio和脂质体 /pcDNA3 .1( + )的实验组 ,HCC772 1肝癌细胞和阴性对照组经G4 18筛选 ,3 0d后均得到稳定的抗性细胞克隆 ,空白对照组在筛选 1周后全部死亡。实验组HCC772 1细胞在体内和体外均表达目的蛋白 ,而对照组细胞中无表达。与对照组相比 ,虽然实验组细胞在体外的生长速度和细胞周期分布未发生明显改变 ,但在体?
Objective To study the effect of angiostatin gene on human hepatocellular carcinoma cell line HCC772 1 in vitro, and to investigate the effects of angiostatin on tumor growth, cell cycle distribution, morphology and in vivo tumorigenicity in vitro. Methods The eukaryotic expression vector pcDNA3. 1 (+) angio of murine angiostatin blood cDNA was constructed by directional cloning technique. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The eukaryotic expression vector was transfected into human hepatocellular carcinoma cell line HCC772 1 by lipofectamine 2000. The neomycin-resistant G4 18 cells were screened for resistance. The transfected empty vector cells were negative control and untransfected cells were blank control. The expression of angiostatin was detected by RNA dot blot and flow cytometry, the cell growth curve was measured, the cell doubling time was calculated, and the cell cycle distribution was detected by flow cytometry. Animal models were established to observe the tumorigenicity changes of tumor cells before and after transfection. Tumor tissue microvessel density (MVD) and angiostatin in vivo were detected by immunohistochemistry. Results The recombinant eukaryotic expression vector pcDNA3. 1 (+) angio was successfully constructed with the correct angiostatin gene fragment. (+) Angio and liposome / pcDNA3 .1 (+) groups, HCC772-1 hepatocellular carcinoma cells and negative control group were selected by G418, respectively, and all were stable after 30d Cell clones, the blank control group all died after 1 week of screening. The experimental group HCC772 1 cells in vitro and in vivo expression of the target protein, while the control group cells no expression. Compared with the control group, although the experimental group of cells in vitro growth rate and cell cycle distribution did not change significantly, but in vivo?