视黄酸可调控的IFNα表达载体的构建及其对肿瘤细胞凋亡的影响

【作者】 张乾勇；

【导师】 程天民； 糜漫天；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2001， 博士

【摘要】 视黄酸（retinoic acid，RA）是维生素A的活性代谢产物，具有多种生物化学功能。RA的许多生物学效应是通过其核受体介导的，视黄酸受体可分为RAR和RXR两个超家族，每个超家族都有α、β和γ三个成员。RA与其受体的同源或异源二聚体（RXR/RXR、RAR/RXR）结合后，活化的受体能与靶基因上的视黄酸反应元件（retinoic acid responseelement，RARE）结合而调节基因表达。体内外及临床实验研究表明，RA可抑制癌细胞增殖、诱导癌细胞分化和细胞凋亡，研究和应用较多的RA是全反式视黄酸（ATRA）和13-顺式视黄酸（13-cRA）。目前ATRA和13-cRA已用于白血病、肺癌、胃癌、肾癌、黑色素细胞瘤、鳞状上皮细胞癌、神经胶质细胞瘤等多种癌症的临床治疗，并有较好的疗效。但是，单用RA治疗有一定的缺陷：易复发或应用RA治疗一段时间后，患者出现对RA的抵抗性或耐药性。因此，探索克服RA耐药的方法具有重要的临床意义。 癌症患者出现对RA的抵抗性或耐药性可能与机体加速RA的代谢有关。α-干扰素（α-interferon，IFNα）不仅可抑制RA的代谢酶——细胞色素P450的活性而减缓RA的代谢，还能与RA协同调节多种转录因子、抑癌基因、癌基因的表达而发挥其协同抗肿瘤作用。临床研究表明，RA和IFNα联合应用对某些白血病和实体瘤细胞的增殖抑制、诱导分化以及凋亡都有协同效应。但是，临床上联合应用RA和IFNα治疗肿瘤时，RA有较大的毒副作用，长期全身应用大剂量IFNα也会出现严重毒副效应。如何才能发挥RA和IFNα的协同抗肿瘤作用，同时又能减轻它们的毒副效应的研究就显得非常重要。 有研究表明，RA和IFNα单独应用均能提高肿瘤细胞对电离辐射诱导细胞凋亡的敏感性，联合应用时作用更明显，而且 RA和 IFN a对辐射引起的机体损伤还具有保护作用。因此，有学者认为，联合应用RA和 IFN a并结合放疗治疗某些肿瘤是一种有效而可行的治疗策略。但其分子机制仍不清楚，它们是否能协同上调caspase的表达和／或活性，有待子深入研究。 针对以上问题，结合国内外研究进展，本研究首先验证了RA可调控带有RARE的荧光素酶（LUC）报告基因的表达；接着，应用DNA重组技术构建了带有 RARE的 IFN a表达载体中RARE4二FN a人并用 ELISA和 RTICR分析证实了 IFN a在 pM朋4－IFN a转染细胞内的表达受 RA诱导；最后，运用MTT实验、DNA梯形带检测、流式细胞仪分析、核酸原位分子杂交、RTICR兔疫印迹、caspase活性分析以及caspase抑制剂应用等技术手段，探讨了 RA及其诱导的 IFN a联合抗肿瘤作用及其可能分子机制，以及 RA对 pRA朋4IFN a转染肿瘤细胞辐射敏感性的影响及其可能分子机制。该研究的目的意义在于：（）视黄酸可诱导的干扰素表达载体的成功构建，使干扰素在靶细胞的高效定位限时表达受控于RA，体内应用时不仅有利于发挥它们的协同抗肿瘤作用，又能避兔全身应用 IFN a的严重副效应，为 RA和 IFN a联合治疗肿瘤开辟了新的途径。 Q）探讨 RA和 IFN a协同抗肿瘤作用及其提高肿瘤细胞对电离辐射的敏感性的可能分子机制，为它们在临床上的联合治疗策略提供理论依据。 主要研究结果和结论如下： 1．带有 RARE的 LUC报告基因表达载体转染 HL－60细胞后，2 X10‘’mol／L RA处理一定时间，LUC活性升高数十倍。说明 RA可诱导带有RARE的外源基因的表达，构建RA可调控的目的基因表达载体是可行的。 2．运用 DNA重组技术构建了带有 4个 RARE重复序列的 IFN a的真核表达载体（pRARE4－IFN a），其转染 HL60、Beau37和 MCF7细胞，G418筛选稳定表达后，ELISA分析 IFN a表达水平，IFN a分泌量分别为21、16和24ng／ffil／10‘细胞，RA处理不同时间后，IFN a分泌量均显著增高，最高时分别达到184、Zll、24lug／ffil八0‘细胞。而经RA处理24h XI ③的转染细胞，换无 RA的新鲜培养基继续培养 24h，IFN a分泌量又下降到接近处理前的水平。表明 pRA旺4 IFN a转染细胞 IFN a表达受 RA诱导，可限时高效表达，说明 RA可调控的 IFN a表达载体的构建是成功的。 3．pRARE4IFN a转染和未转染的 HL七0和 MCFJ细胞经 RA处理后，生长减慢、细胞周期被阻滞在G；0。期、细胞凋亡率增加、DNA片段化百分率升高、并可检测到DNA梯形带，其中，以pRARE4IFN。转染细胞经 RA处理后变化更显著。说明 RA及其诱导的外源性 IFN a基因产物也具有协同抗肿瘤作用。体内应用时，IFN a局部限时诱导表达有利于发挥 IFN a与 RA的协同抗肿瘤作用，还可避免 IFN a的全身性副效应。 4．pRARE4－IFN a转染和未转染的 HL60和 MCF7细胞经 RA处理后，IRFq和 STATI mRNA水平明显增高，IRF4和 STATI在IFN信号转导通路中发挥中心作用，RA上调IRF－1和 STATI基因表达可能是RA和 IFN a协同抗肿瘤的分子机制之一。RA处理后，H更多还原

【Abstract】 Retinoids are derived from vitamin A, which play an essential role in normal cell growth and differentiation. Retinoic acid (RA), particularly all- trans retinoic acid (ATRA), has been regarded as one of the most suitable agents for differentiative therapy because of its activity on acute promyelocytic leukemia(APL) cells at relatively low concentrations,which can be reached in vivo without major toxicities. Indeed, several clinical trials have reported ATRA efficacy in inducing complete remission in APL patients. Retinoids mediate their antiproliferative action, as well as their ability to induce differentiation and apoptosis through their binding and activation of specific nuclear receptors, ie, RARs or RXRs. These activated nuclear receptors, in turn, bind to specific DNA sequences called as retinoic acid response element (RARE) that are located in the regulatory portions of genes and thus modulate gene activity. Considerable previous studies have demonstrated that retinoids are promising as chemotherapeutic agents for the prevention and treatment of several types of cancer. Nevertheless, the recurrence after RA therapy and/or development of resistance to RA therapy have often been observed in patients. Therefore, it is necessary to take optimal strategies to overcome the clinical resistance to RA. One of the major molecular mechanisms of clinical resistance to RA is up-regulation of metabolic enzyme of RA in cells stimulated with RA. In our previous study, as well as in papers from other groups, evidence was reported that the expression and activity of metabolic enzyme of RA could be inhibited V by interferons (IFNs). Numerous studies have demonstrated differentiation and/or apoptosis induction and growth inhibition in human cancer cells by treatment with retinoids and IFNs. The combination of both compounds, which probably act through different molecular mechanisms, often results in a synergistic amplification. They synergistically inhibited growth and induced differentiation and apoptosis in several types of cancer, including hematological malignancies and solid tumors. IFN a even could restore responsiveness of a subline of HL-60, which was otherwise resistant to the effects of RA. Although the combination of retinoids and lENs has shown promising results in preclinical studies and clinical trials, the major toxicities were not neglectable, and the median duration of response was less than 20 weeks. Thus, it is important and necessary to explore optimal strategies to relieve the side effects and enhance the response. In order to relieve the side effects of combination of RA and LEN a, firstly,the possibility of the expression of exogenous lEN a gene mediated by RA through RARE was investigated in this paper. An IFN a gene eukaryotic expression vector containing four copies of RARE (pRARB4-IFN a ) was constructed with recombinant DNA technique, identificated with restriction endonuclease digest and PCR and DNA sequence analysis, then transfected into the cells of HL-60,Bcap-37 and MCF-7 with liposome DOTAP, the expression of LEN a gene was analyzed in transfected and nontransfected cells stimulated with RA by RT-PCR and ELISA. Secondly, the synergistic effects of RA and the transfection of pRARE4-IFN a on the proliferation and apoptosis of tumor cells and its possible molecular mechanisms were study. After tested that the expression of lEN a in pRARE4-IFN a transfected cells was更多还原