【作者】 屈艺；

【导师】 王正荣；

【作者基本信息】 四川大学 ， 生物医学工程， 2003， 博士

【摘要】 目的： 为有效切割肿瘤细胞端粒酶RNA组分及逆转录酶组分mRNA，抑制肿瘤细胞端粒酶活性，从而达到肿瘤基因治疗的目的。开展该系列研究： 方法与结果： 1．针对端粒酶RNA组分模板区设计合成锤头状端粒酶核酶（teloRZ），构建了teloRZ基因体外转录质粒p^SPT19-teloRZ和真核表达质粒p^{XJ-neo-teloRZ}，用T₇/SP₆体外转录系统检测了teloRZ对端粒酶RNA组分的体外切割效率，达60％左右。用脂质体介导法，将p^{XJ-neo-teloRZ}导入HeLa细胞和裸鼠移植瘤，结果使HeLa细胞端粒酶活性降至对照细胞的11％～12.5％，细胞生长速度明显变慢，传至19～20代时出现95％凋亡，裸鼠移植瘤注射p^{XJ-neo-teloRZ}14d后，端粒酶活性下降69％，抑瘤率达62％。 2．设计并合成了针对端粒酶逆转录酶mRNA-2239的锤头状核酶基因，构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中，检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明，该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA;在细胞内能明显抑制端粒酶活性，使其降至对照细胞的50%一25%。并使细胞生长变慢，倍增时间延长。3.设计合成了针对端粒酶逆转录酶mRNAS’端区的锤头状核酶hTERT一5’Rz基因，并将该基因重组入peDNA3.1（+），经体外转录得到小片段hTERT一5，RZ。用脂质体介导法，将hTERT一5，RZ导入Hela细胞以检测其对Hela细胞端粒酶活性的抑制效力。同时比较了同法合成的针对端粒酶RNA模板的核酶（t el0RZ）和二种核酶混合物对Hela细胞端粒酶活性的抑制效力。结果，小片段hTERT一5，双能有效抑制Hela细胞端粒酶活性，且其抑制效力较单纯teloRZ为高，二种核酶混合物的端粒酶抑制效力与hTERT一5’RZ相当，也高于单纯tel0RZ。提示，小片段核酶hTERT。5’RZ可望成为优于teloRZ的端粒酶抑制剂，在肿瘤基因治疗中发挥作用。4.为探讨肝癌裸鼠移植瘤细胞DNA合成与端粒酶表达的生物节律。在时辰同步化裸鼠中构建肝癌细胞SMMC一7721裸鼠移植瘤，继续在程控光照条件下饲养，于光照后3，6，9，12，巧，18，21小时取样，用流式细胞仪测定各时期细胞DNA含量，细胞周期分布及端粒酶活性水平。结果，肝癌裸鼠移植瘤细胞DNA合成随昼夜节律变化，且伴随端粒酶活性的同步节律变化，节律周期呈余弦分布。表明，肝癌裸鼠移植瘤的生长与端粒酶活性表达在静止相达高峰，为制定肿瘤时辰化疗方案提供了有价值的参考。5.用hTERT一5’RZ核酶对肝癌裸鼠移植瘤施以时辰治疗。比较在光照后9小时（9 HALO）用药和光照后21小时（21 IIALO）用药的疗效。结果，21HALO组抑瘤率为63%，gHALO组抑瘤率为50%。表明，hTERT一5’核酶择时用药，能更好地抑制肿瘤生长。结论: 我们设计合成的端粒酶核酶Telo一RZ及hTERT一5’Rz均可望成为有效的端粒酶抑制剂，在肿瘤基因治疗中发挥作用。肿瘤细胞端粒酶活性存在近日节律表达，因而端粒酶抑制剂择时用药能有效提高肿瘤治疗疗效。更多还原

【Abstract】 OBJECTIVE:Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. To evaluate the possibility of using ribozyme technology for telomerase inhibition and cancer therapy, to investigate chronobiological characteristic of telomerase and chronotherapy for tumor.Methods and Resultes:1 .A hammer head ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized to serve as a telomerase inhibitor. An in vitro transcription plasmid and a eukaryotic expression plasmid containing teloRZ gene were constructed. In vitro cleavage reaction was carried out by mixing the ribozyme RNA with DIG-labeled-hTR in different reaction conditions. Cleavage bands were detected by digoxin chemiluminescent assay. The eukaryotic expression plasmid was inducted into HeLa cells by lipofectamine; the telomerase activitiesand bio-characteristics of HeLa cells were detected continuously. Through the study we find that, teloRZ showed a specific cleavage activity against the telomerase RNA component used as template. The in vitro cleavage ratio reached about 60%. The telomerase activities of cells expressing teloRZ dropped to eight times; the doubling times became longer and apoptosis ratios became higher with increasing population doublings (PDS); at 19-20 PDS 95% cells showed apoptosis.2. A hammer head ribozyme directed against the hTERT mRNA (hTERT-2239RZ) was designed and synthesized to serve as a telomerase inhibitor. In order to test its in vitro cleavage activity, two in vitro transcription plasmids containing hTERT-2239RZ and hTERT gene respectively were constructed. Ribozyme RNA and DIG-Labeled-hTERT were synthesized by in vitro transcription. In vitro cleavage reactions were carried out by mixing the hTERT-2239RZ with DIG-labeled-hTERT under different reaction conditions, and cleavage bands were detected by digoxin chemiluminescent assay. HTERT-2239RZ showed a specific cleavage activity against the hTERT used as template. To investigate its in vivo effect of telomerase inhibition in tumor cells, a eukaryotic expression plasmid containing the hTERT ribozyme gene was introduced into HeLa cells and hepatoma cells by using LipofectAMINE. In the transfectants, the level of intact hTERT mRNA and the telomerase activity were clearly reduced, and the telomere length of these clones was apparently shortened at the beginning period, then kept a fixed value without further shortening. All the transfectants with ribozymegrew clearly more slowly than the parental cell line. The doubling time of the tansfectants prolonged compared to the negative control, but no apparent apoptosis was shown even at their 37th passage.3. A hammer head ribozyme targeted to hTERT-5’end (hTERT-5’RZ) was synthesized and cloned into pCDNA3.1(+), the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of pSPT19-teloRZ which we constructed before. The ribozymes were transiently transfected into Hela cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed. We found that, the ribozyme targeting the 5’-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combining use of hTERT-5’RZ and teloRZ also showed clear inhibitory activity, but the inhibitory efficiency of teloRZ only was not so strong. These observations suggest that the use of hTERT-5’RZ and the combining use of hTERT-5’RZ and teloRZ are more effective in telomerase inhibition than the use of teloRZ only. They may possess potential for cancer therapy.4. To study the Circadian rhythms of DNA synthesis and telomerase expression in hepatic cancer nude mice transplanted tumor. Sixteen BALB/C mice were synchronized with an alternative lighting regimen with 12 hours for light and 12 hours for dark (12:12 LD) for 4 weeks. Hepatic cancer cells (SMMC-7721) were implanted into b更多还原