中文摘要：

目的研究核糖核酸酶抑制因子（ribonuclease inhibitor,RI）基因表达对小鼠黑色素瘤B16细胞生长和凋亡的影响。方法构建RI真核表达质粒p IRES2-EGFP-RI稳定转染B16细胞,未转染和空质粒作对照组（p IRES2-EGFP组）,经G418筛选出阳性克隆。RT-PCR、免疫印迹和免疫荧光检测RI的表达;MTT检测细胞的增殖;流式细胞术、TUNEL及Hochest33342检测细胞周期及凋亡;Western blot检测凋亡相关的蛋白及ILK/PI3K/AKT信号通路关键分子的表达;分别注射各组B16细胞到C57/BL小鼠建立移植瘤模型,观察肿瘤的生长及肿瘤微血管的变化,进一步分析ILK/PI3K/AKT信号通路蛋白在肿瘤组织中的表达。结果转染RI细胞组的RI mRNA和蛋白表达水平显著升高;MTT结果显示B16-RI细胞较对照组增殖能力明显降低,流式细胞结果显示B16-RI细胞的S期明显增加,表明RI表达上调后细胞生长停滞于S期（P〈0.05）;Hochest 33342、TUNEL及Annexin V和PI双染色流式法细胞凋亡检测结果证实,B16-RI细胞与对照组相比出现大量凋亡细胞（P〈0.01）及典型凋亡形态特征：染色质凝集,核碎裂和明亮的蓝色荧光等;Western blot结果显示,与对照组相比,B16-RI细胞中ILK、p-Akt、p-GSK3β、Bcl-2及β-catenin的表达明显降低,而活化的Caspase-3和Bax蛋白的表达显著增加（P〈0.01）;动物的移植瘤实验结果显示,转RI组的小鼠的瘤重显著降低（P〈0.01）,瘤组织中的血管密度明显减少,同时ILK、p-Akt、p-GSK3β、β-catenin在瘤组织中表达减少。TUNEL肿瘤组织凋亡检测结果与体外细胞一致。结论过表达RI可能通过调节ILK/PI3K/AKT信号通路显著抑制了小鼠黑色素瘤B16细胞的生长并诱导了B16细胞的凋亡。

英文摘要：

Objective To investigate the effects of ribonuclease inhibitor（ RI） on the proliferation and apoptosis in mice melanoma B16 cells. Methods B16 cells was stably transfected with RI eukaryotic expression plasmid p IRES2-EGFP-RI,using the cells transfected with empty vector and untransfected cells as controls,positive clones were screened with G418. The transcription level of RI in B16 cells was determined by RT-PCR,while its protein level was determined by Western blotting and immunofluorescence assay. The cell proliferation was detected by MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry（ FCM）,TUNEL and Hochest33342 staining. The expression of apoptosis-related proteins and key molecules of ILK /PI3 K / AKT signaling pathway were determined by Western blotting. B16-RI cells,B16 vector cells,and B16 cells were used to implant into the C57 BL /6 mice to establish transplantation tumor model. The growth of tumor was observed,tumor microvessel density was surveyed by HE staining and CD31 immunofluorescence assay,and the expression of ILK / PI3 K / AKT signaling pathway proteins in tumor tissues were further analyzed.Results The RI expression at mRNA and protein levels was significantly increased in B16 cells transfected with p IRES2-EGFP-RI plasmid comprised with the other 2 control cells. MTT assay indicated that the proliferation of B16-RI cells was notably decreased compared with those in B16 vector and B16 cells. Compared with B16 cells or B16 vector cells,the percentage of B16-RI cells at the S phase was increased,indicating more cells were arrested at S phase. A large number of apoptotic cells showed typical apoptotic morphological features,such as chromatin,nuclear fragmentation and bright blue fluorescence in B16-RI cells（ P〈0. 01）.Western blotting indicated that the expression of ILK,p-Akt,p-GSK3β,Bcl-2 and β-catenin was distinctly decreased in B16-RI cells,while that of activated Caspase-3 and Bax was significantly increased compared with the control cells（ P〈0. 01）.