【作者】 梅玫；

【导师】 张维铭； 余虹；

【作者基本信息】 天津医科大学 ， 病理学， 2002， 硕士

【摘要】 目的 端粒酶是一种特异性核糖核蛋白酶，它复制细胞染色体端粒重复序列TTAGGG，使细胞分裂后缩短的端粒得到补长。绝大多数永生化细胞和90％以上的恶性肿瘤端粒酶呈高活性，而正常体细胞端粒酶活性极低或缺失。端粒酶具有反转录功能，以内部RNA序列中的11个碱基为模板，识别、结合端粒3’端，并合成、延伸端粒重复序列。端粒酶构成了反义核酸基因治疗肿瘤的新靶点。 癌基因C-myc变异可以引起细胞DNA复制及mRNA转录功能紊乱，使细胞周期调控失衡，可以导致细胞恶性转化。因此，纠正基因变异可以控制肿瘤的发生、发展。 反义核酸可以在介导物作用下，进入细胞，特异性识别、结合靶基因，在复制和转录水平阻断异常基因的调控，抑制癌细胞的代谢生长。 本实验检测八种肺癌细胞的端粒酶活性。采用硫代修饰型特异性端粒酶RNA反义寡核苷酸、端粒DNA反义寡核苷酸和C-myc反义寡核苷酸，导入端粒酶呈高活性表达的人肺癌LTEP-a-2细胞，探讨反义核酸封阻靶基因，抑制癌细胞代谢、生长的作用。 方法 1．培养人肺癌细胞系GLC-82、LTEP-a-2、SPC-a-1、PA-a-1、SCLC、PG-49、801-D和LTEP-78。2．采用Telomerase PCR ELISA方法检测人肺癌细胞系端粒酶活性。3．设计、合成针对端粒酶RNA、端粒DNA和C-myc基因的硫代磷酸型特异性反义寡核苷酸封条和随机序列寡核苷酸片段，用Clonfectin包裹反义寡核苷酸导入LTEP-a-2细胞系，作用72小时。4．检测反义寡核苷酸抑制细胞端粒酶活性；用MTT光吸收法检测反义寡核苷酸对琥珀酸氧化脱氢酶的抑制情况；观察分析反义寡核苷酸抑制细胞代谢和增殖的作用。 结 果 1．8种人肺癌细胞系端粒酶活性均为阳性。2．三种针对端粒酶 RNA反义寡核音酸（hTR PS－ODN卜 hTR PS－ODN小 hTR PS－ODN；／、端 粒DNA反义寡核昔酸和C－p c反义寡核苦酸，spo浓度抑制LTEP－aZ细 胞25．7ds34．87％的端粒酶活性；10pM抑制28．39to2．7％端粒酶活性，其中 hTR PS－ODN。抑制 49M％的端粒酶活性，C－IYlyC PS－ODN＊抑制端粒酶活性 率为 62．7％。3．三种hTR PS－ODNS和端粒PS－ODN。，sliM时抑策细胞氧 化脱氢酶活性（SDH）19．4－28％，C－myc PS－ODN口 对 SDH抑制率为 42．38％。 10pM hTR PS－ODNs和端粒PS－ODN。对 SDH抑末率为 25．71－33．87％。C－myc PS－ODN；。对SDH抑击率达57．78％。4．SPM hTR PS－ODNS、端粒PS－ ODN。抑制细胞增殖为 14．77－－－－28j％，10po hTR PS－ODNS和端粒 PS－ODN。 抑制细胞增殖为 3ds36．92％，C－myc PS0DN；。抑制细胞生长达52．94％。5． 对照随机序列硫代修饰寡核昔酸20pM仅抑制LTEPa习细胞2．37％端粒酶 活性，抑制氧化脱氢酶活性和细胞增殖分别为4％和4．5％。 结 论人不同肺癌细胞系端粒酶活性均呈阳性。针对端粒酶RNA，端粒 DNA和C－myc靶基因的反义硫代磷酸型寡核昔酸，具有特异性抑制人肺腺 癌细胞端粒酶活性和细胞周期代谢，进而抑制肺癌细胞的生长、增殖。反 义寡核苦酸特异性抑制作用随药物浓度升高而增强。更多还原

【Abstract】 Objectives: Telomerase is a ribonucleoprotein that replicates and adds repeating units of TTAGGG to the end of chromosomes. It participates in the maintenance of telomere length after cell division. Almost immortal cells and 90% of tumor cell lines and tissues express more telomerase activity than normal somatic cells and tissues which have no detectable telomerase activity. Telomerase undertakes reverse transcription task. The RNA component of human telomerase, termed hTR, is discribed. the template region of hTR encompasses 11 nucleotides (5’ CUAACCCUAAC 3’), complements to 3’end of the human telomere sequence and lengthens telomere. This suggests that it may be a novel target for gene therapy of tumor using antisense oligodeoxynucleotides.Deregulation C-myc , a proto-oncogen , may induce upset of DNA replication and mRNA transcription, which leads to cell cycle imbalance and cellular oncogenesis.Antisense oligonucleotides are able to penetrate cell membrane, which recognize and complement to cellular target gene by the specificity of Watson-Crick base pairing to inhibit DNA replication, mRNA transcription and cell metabolism.We measured telomerase activity hi 8 Lung tumor cell lines and transfected antisense phosphorothiate oligodeoxynucleotides (anti-PS-ODN) of hTR, anti PS-ODN of Telomere DNA and anti PS-ODN of C-myc as therapeutic agents into target cell LTEP-a-2 which express more tolomerase activity to investigate the effect of inhibition of telomerase activity and tumor cell growth. Methods: 1. Cell culture including GLC-82. LTEP-a-2. SPC-a-1 PA-a-1. SCLC. PG-49. 801- D and LTEP-78. 2. Telomerase activity assay hi human Lung tumor cell lines using Telomerase PCR ELISA .3. Oligonuleotides sythesisincluding Antisense PS-ODNs of hTR , Telomere DNA and C-myc Oligonucleotides were transfected with Clonfectin into LTEP-a-2 cell for 72hr. 4. Measurement of Telomerase activity, SDH. activity and cell growth, usingTelomerase PCR ELISA.. MTT optical absobance test and cell count. Results: 1. Eight cell lines showed high expression of telomerase activity. 2. Concentrations of 5uM anti-PS-ODNs of hTR. Telomere DNA and C-myc specifically reduced the telomerase activity by 25.74-34.87% for 72hr after the initial treatment. lOuM of anti-PS-ODN6 of hTR and Anti C-myc PS-ODNI2 had the ability to inhibit the telomerase activity by 49.44% and 62.7% 3. PS-ODNs of anti-hTR and anti-telomere DNA were able to reduce SDH activity by 19.4-28%.C-myc PS-ODN reduced the SDH activity by 42.38% at 5uM. lOuM of three PS-ODNs of hTR and PS-ODN9 of anti-telomere DNA reduced enzyme activity by 25.71-33.87 %. lOuM of Anti C-myc PS-ODN12 inhibit SDH activity by 57.78%?4. The concentration of 5jaM PS-ODNs of anti-hTR and Telomere DNA had the ability to inhibit cell growth by 14.77-28.13%. the inhibitory rate of 10|^M PS-ODNs of hTR were 34-36.92% and PS-ODNI2 of anti-C-myc can reduce cell growth by 52.94%. 5. The result indicated that 20uM of random sequence of PS-ODN9 failed to inhibit Telomerase, SDH activity and LTEP-a-2 cell growth specially.Conclusions: Human lung tumor cell lines expressed high activity of telomerase. PS-ODNs of anti-hTR and Telomere DNA had the ability to inhibit telomerase activity and reduced LTEP-a-2 cell growth. Anti C-myc PS-ODNI2 resulted in cell growth inhibitory activity by regulating cell cycle and the reduction was concentration dependent.更多还原