人端粒酶催化亚单位(hTERT)特异性核酶对乳腺癌细胞MCF-7活性的作用及影响

Effect and Influence of hTERT Specificity Ribozyme on Breast Cancer MCF-7 Activity

目的 探讨端粒酶特异性核酶对乳腺癌细胞MCF-7端粒酶活性、增殖和凋亡的影响．方法 设计合成RZ基因，构建人端粒酶锤头状核酶真核表达质粒pcDNA3．1（＋）．hTERT—RZ，测序鉴定．提取MCF-7细胞总RNA，RT-PCR扩增靶基因的cDNA片段，插入载体pMD18-T并测序鉴定．将核酶重组体及hTERT载体体外转录，琼脂糖凝胶电泳检测RZ的切割活性．核酶载体转染乳腺癌细胞MCF-7，RT—PCR法检测核酶重组子转染效率及核酶转染细胞中hTERTmRNA的表达量，TRAP法测定细胞端粒酶活性，MTT法、流式细胞仪测定细胞增殖及凋亡．结果 测序表明pcDNA3．1（＋）-hTERT-RZ和pMD18-T-hTERT构建成功．5％琼脂糖凝胶电泳表明体外转录出RZ和靶基因hTERT，RZ对靶基因hTERT具有切割活性．构建好的核酶真核表达载体转染乳腺癌MCF-7细胞，发现其潜伏期延长，对数生长期减缓，S期细胞比例降低，G1期细胞比例增高（P〈0．01）．端粒酶活性检测发现端粒酶活性明显降低．结论 端粒酶催化亚单位特异性核酶对靶基因hTERT在体外和体内均有催化切割活性，该核酶在乳腺癌MCF-7细胞中抑制hTERT的表达和细胞增殖，为核酶应用于恶性肿瘤的基因治疗提供了实验依据和新型工具酶．

Objective To study the effect of telomerase specificity ribozyme on the telomerase activity, proliferation and apoptosis of MCF-7 breast cancer cells. Methods The vector pcDNA3.1 （ ＋ ） -hTERT-RZ was constructed and then were analyzed by agarose gel electrophoresis and DNA sequencing, the conservative hTERT gene sequence amplified by polymerase chain reaction （PCR） was inserted into pMD18-T and then were analyzed by PCR and DNA sequencing. Then, the ribozyme gene and hTERT gene were transcribed in vitro respectively. The recombinants containing ribozyme DNA were transiently transfected into MCF-7 cells by GenEscort^TM Ⅱ. The amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT;telomerase activity by TRAP;cell apoptosis by FCM. Results Effects of telomerase hTERT ribozyme on MCF-7 cell is tested, pcDNA3.1 （ ＋ ） -RZ had been successful transfected into MCF-7 cell by GenEscort ^TM Ⅱ. We observed the propagation of the cells and detected their telomerase activities by TRAP-Hyb Kit. After transfection, the propagation of the cells was inhibited and the telomerase activity was descended. Cells in G1 phase significantly increased , and cells in S phase decreased （ P 〈 0.01 ）. Conclusion Telomerase hTERT ribozyme could effectively digest the template element of the human telomerase hTERT and inhibit the telomerase activity and proliferation of breast carcinoma cell, which can provide experimental basis and new therapeutic agents for ribozyme applicated in gene treatment of cancer.