【作者】 陈学军；

【导师】 郑伟；

【作者基本信息】 浙江大学 ， 妇科肿瘤学， 2003， 硕士

【摘要】 子宫内膜癌是女性最常见的恶性肿瘤之一，约占女性生殖道癌瘤的7％，占女性生殖道恶性肿瘤的20％～30％。近20年来发病率呈逐年上升趋势。子宫内膜癌的主要治疗方法为手术、放射及药物治疗，由于癌变位于宫腔进展较缓慢，诊断时多为临床早期，手术切除或放射治疗后早期子宫内膜癌的5年生存率达85％以上。如同其它所有恶性肿瘤，晚期和复发性子宫内膜癌是治疗的难题，约有内膜癌10％～13％病灶已扩散至子宫体外，此外早期内膜癌治疗后有1／4会出现复发；对于晚期或复发性癌，目前主张结合手术、放疗及药物的综合治疗，但由于晚期病变的广泛性和复杂性，以及病人相对较差的身体条件，病人往往不能够耐受具极大破坏性的手术或放疗。孕激素以及现有的细胞毒性药物的虽然有一定的治疗效果，但不能达到治愈，并且疗效的持续时间短，所以目前药物治疗在子宫内膜癌治疗的地位仍然处于辅助治疗或者姑一息治疗的地位。几十年来对晚期癌的治疗方式进行了大量的研究和尝试，但生存率仍然徘徊在原来的水平。 端粒酶作为一种逆转录酶，以其RNA亚单位为模板在自身的催化亚单位作用下合成染色体末端的特殊结构——端粒，这对于维持染色体稳定以及保证DNA复制的完整性有重要的作用。端粒酶的异常激活和肿瘤的形成和发展密切相关，绝大多数恶性肿瘤表达端粒酶活性，而大部分正常组织除胚胎组织生殖细胞、造血干细胞以及部分的粘膜上皮以外都没有端粒酶活性的表达。内膜癌组织的端粒酶活性则持续激活，内膜癌组织端粒酶阳性在95％以上。端粒酶活性高度表达与淋巴转移、手术分期显著相关。单纯分析绝经后妇女的内膜癌，淋巴转移和端粒酶活性高度相关，端粒酶活性水平与FIGO分期和FIGO分级显著相关；此外，还与内膜癌的早期复发和术后快速进展有关，端粒酶活性可作为一个独立的因素预后疾病的进展，有研究表明，抑制端粒酶能够影响内膜癌的生长，提示以端粒酶为靶点治疗子宫内膜癌的可能性。 反义技术是近几十年来兴起的生物高技术，是反义技术封闭和抑制特定基因的表达，是基因治疗的重要组成部分。由于AODN能与特定基因按碱基配对原理结合KNA，抑制基因的表达，因此被认为是理想的，具有精确选择性的靶向治疗药物，可广泛应用于多种疾病的治疗。反义治疗与常规的基因治疗有所不同，AOND不改变细胞基因的结构，不需要载体和病毒的介导作用。在动物模型和有选择的人类临床上取得成功，目前己有十儿种反义药物进入临床前及临床实验，证实反义基因治疗潜在的临床应用价值。反义治疗应用范围广，涉及到遗传病、恶性肿瘤、自身免疫性疾病、感染性疾病和兔疫排斥等诸多病种。多项体外及动物实验表明，端粒酶反义核酸能够抑制肿瘤细胞的端粒酶活性，进 3一而影响细胞的生长，针对端粒酶RNA设计的AODN能够延长肿瘤细胞的细胞倍增时间，诱导细胞发生细胞凋亡。当把AODN直接注入移植性肿瘤中，使肿瘤体积缩小，且具有剂量依赖性，提示端粒酶抑制剂作为新型抗癌药物的可能性。 本研究以端粒酶催化亚单位基因hTERT为靶点设计合成硫代反义寡核昔酸，评价反义核酸对于宫内膜癌 HECl A细胞株的抑制作用。采用四甲基偶氮哇蓝（M’IT）法并绘制生长曲线检测AODN对细胞增殖能力及细胞生长的影响；逆转录聚合酶链反应技术（RT－PCR）、定量端粒酶重复扩增法（TRAP）、Western Blot蛋白免疫印迹法、凋亡相关CASPASE酶活性测定检测反义核酸对hTERT基因转录、蛋白表达、细胞端粒酶活性以及CASPASE活性，探讨AODN抑制子宫内膜癌细胞的作用机理。 体外实验结果表明：不同浓度的AODN转染细胞后，均不同程度的影响细胞的生长，药物的抑制作用有明显的剂量依赖性；在5天内药物的作用随着时间的延长而增加，0．in mol／L、0．4 n mol／L、0．8 u mol／L。1石 v mol／L浓度 AODN经脂质体包裹转染至 HEC刁 A细胞，在转染 2天后抑制率分别为6．8％、26．8％、45．9％、60％；5天后的抑制率分别为 0．069O、46．35O、，65．22O、79．5O。实验证实，AODN抑制细胞的生长作用是通过抑制端粒酶活性进行的。分别测定AODN作用后细胞的端粒酶活性、以及 hTE盯的表达情况，结果 AODN转染后的 HEC1细胞端粒酶活性显著降低，hTERT mRNA及蛋白的表达明显下降，且这种抑制作用具有明显的时间和剂量依赖性，证明AODN抑制靶基因表达、抑制细胞生长的有效性。此外，HEC1 A被 AODN作用抑制端粒酶后，细胞的生长的活力的降低也从反面证明hTERT基因表达调节的 4一异常在子宫内膜癌增殖中的重要地位。我们采用CASPASE化学荧光法测定AODN转染前后细胞CASPASE酶，转染后CAPASE－l，CASPASE－3的活性增加，提示AODN的细胞抑制作用与凋亡信号转导途径激活有关。 为进一步评价AODN的对子宫内膜癌的治疗效果，我们建立了子宫内膜癌裸鼠皮?更多还原

【Abstract】 Endometrial cancer is the second most common gynecologic malignancy, accounting for 20%~30% of all gynecologic cancers. Endometrial carcinoma usually presents as early-stage disease and can generally be managed with surgery or radiotherapy, and survival of early stage disease was about 85% at 5 years. But advanced or recurrent disease still have a poor survival, about 10%~13% of all endometrial cancers have clinical evidence of disease spread outside of uterine body, and about one fourth of patients treated for early endometrial cancer develop recurrent disease. Treatment of advanced or recurrent disease involves surgery, radiation therapy and systemic hormonal or chemotherapy. As the complicated complications and poor performance status, many patientscannot be tolerant with surgery or radiation therapy. Hormonal therapy or cytotic agents are capable of inducing objective response, but response and survival times are short, so far systemic agents therapy just provide adjuvant or relief therapy of disease. Although there are many studies about advanced endometrial cancer, the survival of diseases has not improved significantly for decades.Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the end of eukaryotic chromosomes, called telomeres, which appear to be associated with senescence, replication, and cell cycle clock. Telomerase is composed of an RNA molecule and protein. The RNA component of human telomerase (hTER) functions as a template the extension reaction of the telomere repeats. Human telomerase reverse transcriptase (hTERT), the key protein component of the telomerase, is a catalytic subunit of telomerase, and studies have shown that hTERT is a rate-limiting determinant of telomerase activity. Thus, it has proposed that the specific inhibition of telomerase activity in tumors might have significant and beneficial therapeutic effects.In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase , resulting in stabilized telomere length. These observations indicate that telomerase maintenance is essential to the proliferation of tumor cells, and telomerase activation is therefore a crucial step in cellar immortalization and carcinogenesis. Previous studies have reported that approximately 95% of denometrial cancer telomerase positive.High telomerase activity was significantly correlated with advanced surgical stage and with pelvic lymph node metastasis. In recently reported studies, the treatment of various tumor derived cell lines with telomerase inhibitorresulted in cellular senescence.Antisense technology is a new strategy, and plays an important role in gene therapy. A synthetic antisense molecule recognizes complementary mRNA or DNA by sequence-specific base paring, and hence prevents translation or transcription, resulting in a selective inhibition of protein synthesis. Antisense oligonucleotides offer the potetial for blocking the expression of targeted gene and acting as new therapeutic agents in human disease, such as malignant tumor, vascular disease, inflammatory, viral infection. Designed to specifically inhibit the expression of disease -related genes, antisense compounds have shown efficacy in numerous preclinical studies.Studies have shown that inhibition of telomerase activity leads to inhibition of cell growth, and therefore, telomerase can be considered as an attractive target for therapy of cancer or other pathogenically proliferating cells. Recent data have demonstrated inhibition of cell division in cancer cells and inducing apoptosis in presence of telomerase antisense oligodeoxyonucleotides (AODN), and also AODN was demonstrated to be effective in animal tumor model, all of which provided a proof of concept for the use of telomerase inhibitors as candidate cancer drugs.In this study, we synthesized a 20 bases antisense oligonucleotide against human telomerase reverse transcriptase and investigated itsantit更多还原