【作者】 庞建新；

【导师】 吴曙光；

【作者基本信息】 第一军医大学 ， 药理学， 2002， 博士

【摘要】 目的：人端粒酶逆转录酶（human telomerase reverse transcriptase,hTERT）是端粒酶活性调节的限速亚单位，是正常细胞和肿瘤细胞端粒酶活性明显不同的原因。hTERT是通过转录调控的，涉及到转录因子c-Myc,Sp1的转录激活作用；PHA可以诱导Jurkat T细胞端粒酶激活。本文目的在于：探讨转录因子c-Myc，Sp1对端粒酶的转录作用及PHA激活端粒酶的信号传导途径，为更好地理解肿瘤发生的机理及寻找端粒酶特异抑制剂提供理论依据。 方法：用脂质体将Sp1、c-Myc反义寡核甘酸（Oligodeoxynucleotide,ODN）或Sp1、Sp3表达载体转入培养至对数生长期的Jurkat T细胞（2-3×10⁶细胞/ml），预孵育30min后（表达载体预孵育时间为1h），5％CO2、37℃恒温箱中继续培养36h。用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。用逆转录PCR方法检测Sp1、c-Myc和hTERT mRNA水平，用Westernblot检测蛋白水平，用MTT法检测细胞增殖作用；采用脂质体将显性负突变ras、raf表达载体及反义H-ras ODN、反义c-raf ODN转入Jurkat T细胞，或应用信号传导特异抑制剂抑制Jurkat T细胞后，用PHA诱导细胞端粒酶表达，用逆转录PCR方法检测转染效果，并用端粒酶PCR—ELISA方法检测端粒酶活性，PCR产物用15％非变性聚丙烯酰胺凝胶分离，并通过银染显示DNA梯度。 结果：（1）转染反义Sp1 ODN(1μmol·L^-1)明显抑制Sp1 mRNA和蛋白表达，抑制率分别为44.8％（P＜0.05）和57％（P＜0.01）；转染反义c-MycODN(1μmol·L^-1)对c-Myc mRNA表达抑制率为36.2％（P＜0.01）；转染1μmol·L^-1反义Sp1 ODN、反义c-Myc ODN后，端粒酶活性抑制率 第一军医大学博士学位论文分另为43．1％（P＜0刀1）和27．2％（P＜0刀＊；在0二5、o．5、1刀、2刀（n mol ＊”‘）时，反义 SP ODN表现为明显的剂量依赖性端粒酶抑制作用，而反义O＊c0＊在该剂量范围内，剂量依赖性抑制作用不明显：（2转染1n mol·L”‘反义＊ ODN、反义 c－Myc ODN后，hTERTmRNA抑制率分别为 43＊％（P＜0＊1）和 23．4％（P＜0＊5）；（3）Spl、Sp3载体转化 36小时，Spl、Sp3蛋白水平分别升高59＊％（P＜0＊1）和36．8％（P＜0＊5）；转染 SpJp3表达载体后，随着即 l表达水平的增加，端粒酶活性和 hTERT＜ RNRNA水平明显增加，增加率分别为38．5WP＜0刀5）和25．4％0＜0刀5L而即3表达载体对端粒酶活性和hTERT m洲A水平无明显影响。O）浓。度从 0二52刀 u mol·L”l时，反义 Spl对细胞的增殖抑制作用随剂量增加而增强，与反义 C－MyC ODN比较，有较好的量效关系，但各浓度组的抑制作用比反义 c－Myc ODN低；而 2刀 u m＊·LI浓度的反义C－MyC、Spl ODN转染 Jurkat T细胞 24h、48h和 72h后，对细胞增殖抑制作用的时效关系表明，反义C－MyC ODN比反义Spl ODN起效快，作用强。归)2．5 n g砌l的 PHA刺激 Jurkat T细胞时，端粒酶活性增加了5080％o叱刀匀；ras负突变表达载体 pZIPrasl7N和 raf负突变表达载体pCGNraf（l）转染使PHA刺激的端粒酶活性分别降低了38．7％o刃刀5)和 34．3％（P＜0刀匀。（6）转染 1刀 n mol·L”‘反义 H－rs ODN和反义 c一rafODN有效抑制 H－rs mRNA和 c－raf mRNA表达，抑制率分别为52．l％（P＜0＊1）和 42．8％（P＜0＊1）；不同浓度的反义H－ras ODN和反义c－rafODN明显抑制 PHA诱导的端粒酶激活，浓度从 0．5 u mol·L“l至 2刀 umol 工’时，抑制率分别从 19＊％升高至68．6％和从20刀％升高至67刀％， 表现出明显的剂量依赖性＜7）用 10 u mol“’的 PD98059J刀卜 mol”U0126和 5 n mol ．L’l的 Bisindolylmaleimide分别抑制 MEKI、MEKllZ 和 PKC，使 2．5 P g／mlPHA刺激的 JUrkat T细胞端粒酶活性分别降低了 37．2WP＜0＊5)、64．7WP＜0＊1)禾SO．5地P＜0＊1)。 结论：＊)Spl和C－MyC转录因子通过促进hTERT表达水平共同调节 Jurkat T细胞端粒酶活性，但Spl在hTERT转录调控中更为重要八2)SP3 对hTERTInRNA的转录水平虽有一定的抑制作用，但不影响端粒酶活性， 表明即3不是端粒酶活性的负调控因子叶)反义 c－Myc对 Jurkat T细胞 4 第一军医大学博士学位论文增殖抑制作用不是通过抑制端粒酶活性引起的。O)PHA主要通过RasKafMEKERK信号途径激活 Jurka T细胞端粒酶／5)PKC对端粒酶活性的调节作用是双重的，一方面促进端粒酶生成，另一方面增强其功能。…)反义SPI ODN抑制端粒酶活性，进而抑制肿瘤生长，预示其可作为一种潜在的抗肿瘤新策略。更多还原

【Abstract】 Objective Human telomerase reverse transcriptase (hTERT) has been generally acknowledged as a rate-limiting determinant of the telomerase only expressed in immortal or tumor cells, and this may be one of the reason that tumor cells differ from somatic cells. Many observations indicate that transcriptional regulation of the hTERT gene plays a key role in the activation of telomerase, and transcriptional factors of c-Myc and Spl are obviously involved in telomerase expression. In Jurkat T cells, telomerase could be induced by PHA. The purpose of this dissertation is: (1) to investigate the effect of c-Myc and Spl on expression of telomerase and hTERT; (2) to study the signal transduction pathway of telomerase induced by PHA in Jurkat T. Methods Antisense Spl oligodeoxynucleotide (ODN) and antisense c-Myc (ODN) or Spl and Sp3 expression vector(pCMV-Spl, pCMV-Sp3) were delivered to Jurkat T cells by lipofectamin. Telomerase PCR ELISA was used to detect telomerase activity; RT-PCR analysis was used to assess the mRNA expression of Spl ,c-Myc and hTERT; Western blot was used to analyze the levels of Spl and Sp3 protein and the cells proliferation was measured by MTT. The expression vector of dominant negative p21ras (pZIPras17N)and raf-l(pCGNraf(l-130)) or antisense H-ras ODN and antisense c-raf ODN were transferred to Jurkat T cells by lipofectamin, or using selective inhibitors of kinases blocked different signal cascades such as PKC and MEK, and then cells were stimulated with PHA. Telomerase PCR ELISA was used to detect telomerase activity. Results (1) Treatment of Jurkat T with Spl antisense inhibitorC 1 μ mol ·L-1)resulted in dramatically reduced Spl mRNA and protein levels. The inhibition rate were 44.8%(P<0.05) and 57% (P<0.01) respectively. Treatment of Jurkat T with c-Myc antisense inhibitor (1 u mol -L-1) also resulted in significantly reduced c-Myc mRNA with an inhibition rate of 36.2%(P<0.01). Following the transcriptional factor Spl and c-Myc functionally altering, the telomerase activity were significantly suppressed with a 43.1% (P<0.01) and a 27.2% (P<0.01) inhibition rate respectively. From 0.25-2.0 n mol -L-l, a dose-dependent inhibition of telomerase activity by antisense Spl ODN was discovered, but the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN. (2) Treatment of Jurkat T with 1 μmol·L-1 Spl antisense ODN or c-Myc ODN, hTERT mRNA expression were significantly decreased by 43.6% (P<0.01) and a 23.4% (P<0.05) respectively. (3) Treatment of Jurkat T cells with Spl or Sp3 expressing vectors for 36h result in significantly increase of Spl and Sp3 protein levels by 59.6%( P<0.01) and 36.8% (P<0.05) respectively. Enhancement of Spl expression obviously increased telomerase activity and hTERT mRNA levels with a rate of 38.5%(P<0.05) and 25.4% (P<0.05) respectively, whereas Sp3 had no significant effect on both telomerase activity and hTERT mRNA levels. (4) Dose-effect relationship of cells survival rate indicated a dose-dependent survival decreasing after treated with 0.25, 0.5, 1.0, 2.0(μmol·L-l) antisense Sp1 ODN for 48h, the similar dose-dependent inhibition did not occurred by antisense c-Myc ODN, but antisense c-Myc ODN of different concentrations inhibited cells proliferation more significantly than antisense Spl ODN. Time-effect relationship of cells survival rate indicated that antisense c-Myc ODN had an earlier and more powerful effect on cells proliferation than antisense Spl ODN. (5) Telomerase activity was greatly stimulated after exposure to PHA, and treatment of Jurkat T cells with dominant negative p21ras and raf-1 for 36h result in significantly decrease of telomerase by 38.7% (PO.05) and 34.3% (P<0.05) respectively. (6) Transferring to Jurkat T with 1 v mol -L-l H-ras or c-raf antisense inhibitor resulted in dramaticallyreduced H-ras mRNA and c-raf mRNA by 52.1%(P<0.05) and 42.8% (P<0.01) respectively. From 0.5-2.0 μmol·L-1, a dose-dependent inhibition of telomerase activity by antisense H-ras ODN and antisense c-raf ODN. (7) The increase of telomerase activit更多还原