【作者】 卢宏达；

【导师】 孔庆志；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2008， 博士

【摘要】 恶性肿瘤,这种由于多种环境因素长期共同作用所产生的多基因疾病,在现代医学注重对单基因、单靶点的对抗性治疗下难以取得良好的效果。然而中医讲究整体、注重变化、注重平衡、辨证施治等基础理论的挖掘,不但可以解决诸如恶性肿瘤等复杂多基因疾病的问题,而且可以促进现代医学向更高境界发展。中医整体观念的治法治则为解决肿瘤分子生物学端粒抗癌靶点的难题提供了良好的思路。端粒是真核生物细胞染色体末端的特殊核酸蛋白结构,能保护染色体末端,以维持染色体结构的稳定。端粒长度的复制性缩短是通过端粒酶的逆转录合成来修复的,而端粒长度的维持对于肿瘤细胞保持无限增殖的永生化倾向至关重要。因此,通过抑制端粒酶来控制肿瘤是近年来国内外学者关注的热点之一。然而,理论推理的逻辑性和实验室工作的初步结果并没有给临床带来满意的效果。因为从整体的角度,除了端粒酶之外,还有众多的端粒相关蛋白因子参与端粒的调控;从平衡的角度,单独的端粒酶抑制是一种不完全的抑制。从理论上看,端粒结合蛋白1(TRF1)通过抑制端粒酶与端粒相互作用而对端粒长度起负向调节作用;而端锚酶能使TRF1发生ADP核糖基化而抑制其与端粒重复片段结合,是端粒长度的正向调节因子。端锚酶、端粒酶同时与TRF1相联系,两者的联合则有可能成为肿瘤基因治疗的一个靶点。为此,我们首先构建端锚酶反义寡核苷酸和反义端粒酶催化亚单位寡核苷酸,以其相应的正义寡核苷酸为对照,采用脂质体法将其导入端粒酶阳性的人肺腺癌细胞A549,观察反义寡核苷酸对A549细胞端粒动力学的影响、与Bcl-2凋亡基因家族的相互联系及反义寡核苷酸作用下A549细胞形态、功能的改变,探讨端锚酶、端粒酶联合作为肿瘤基因治疗的可能性和机理,为日后肿瘤基因治疗提供可靠的实验室数据和充足的理论准备。目的1.观察端锚酶、端粒酶两种反义寡核苷酸联合作用对端粒酶催化亚单位(hTERT)mRNA转录、端粒酶及端锚酶蛋白质表达水平的影响;探讨两种反义寡核苷酸作用对细胞端粒长度的影响。2.探讨两种反义寡核苷酸与Bcl-2凋亡基因家族相互作用的联系,收集其基因学证据。3.观察两种反义寡核苷酸持续作用下A549细胞形态学的改变,细胞传代与端粒缩短之间的相互关系,细胞氚摄取率及X-Gal转染率等功能学改变,探讨其肿瘤基因治疗的潜在价值。方法与结果1.运用逆转录-聚合酶链反应(RT-PCR)对A549细胞端粒酶催化亚单位(hTERT)mRNA表达进行检测。结果发现:端粒酶催化亚单位反义寡核苷酸(ashTERT)下调hTERT mRNA的转录;而端锚酶正、反义寡核苷酸(sTANKS、asTANKS)不影响其转录;端粒酶催化亚单位及端锚酶反义寡核苷酸联合(ashTERT+asTANKS)时hTERT mRNA的转录水平无额外的变化。2.采用多聚合酶链-酶联免疫吸附实验(PCR-ELISA)定量分析端粒酶活性。结果显示:ashTERT明显抑制端粒酶活性;asTANKS、sTANKS作用下端粒酶活性无明显变化;asTANKS与ashTERT联合作用下端粒酶活性亦无明显额外变化。3.采用Western Blot法检测端锚酶活性显示:asTANKS明显抑制端锚酶活性;ashTERT、shTERT作用下端锚酶活性无明显变化;ashTERT与asTANKS联合作用下端锚酶活性亦无明显额外变化。4.通过定量荧光原位杂交法(Q-FISH)上流式细胞仪检测A549细胞端粒长度。结果显示:与sTANKS、shTERT比较,asTANKS、ashTERT均可导致细胞端粒长度的明显缩短;而asTANKS+ashTERT的联合作用使细胞端粒长度的缩短更为明显,两者有协同作用。5.以RT-PCR法分析反义寡核苷酸作用下A549细胞mcl-1、Bcl-2和Bax基因的转录水平发现:ashTERT不影响上述三种基因的转录水平;asTANKS除上调mcl-1基因的转录外,对Bcl-2和Bax基因无影响;并且asTANKS的此种作用不因ashTERT的联合而发生改变。6.Western Blot法检测Mcl-1、Bcl-2及Bax蛋白活性显示:ashTERT对Mcl-1总蛋白活性无影响,但可使蛋白短拼接体的(Mcl-1s)含量下降;ashTERT对Bcl-2、Bax蛋白的表达无影响。asTANKS上调Mcl-1蛋白的表达,并且上调Mcl-1s的比例;对Bcl-2、Bax蛋白的表达无影响。asTANKS+ashTERT的联合作用可逆转Mcl-1s含量的下降,促进Mcl-1s、Mcl-1总蛋白活性的上调。7.普通光学显微镜及荧光显微镜观察,经asTANKS、ashTERT作用的细胞在形态学上渐出现细胞衰老的征象,并且在后期的传代试验中细胞凋亡的比例渐增;而asTANKS+ashTERT联合作用时细胞衰老和凋亡的特征更明显。8.细胞传代实验的结果显示:经asTANKS、ashTERT处理后的细胞传代时间明显延迟,细胞增殖周期明显缩短;而asTANKS+ashTERT联合作用时细胞增殖周期的缩短更为明显,两者有协同作用。9.经asTANKS、ashTERT处理后的细胞氚摄取率随细胞传代的进行逐渐下降,并且asTANKS+ashTERT联合作用组细胞氚摄取率降低更为明显,作为sTANKS、shTERT对照组的细胞氚摄取率无明显改变。10.经asTANKS、ashTERT处理后的细胞细胞X-Gal转染率随细胞传代的进行逐渐升高,并且asTANKS+ashTERT联合作用组细胞X-Gal转染率升高更为明显,作为sTANKS、shTERT对照组的细胞X-Gal转染率无明显改变。结论1.基于中医学整体平衡观念的治法治则对于解决恶性肿瘤端粒抗癌靶点的难题具有明显的指导意义。2.端锚酶反义寡核苷酸通路是完全有别于端粒酶的端粒抑制途径,其缩短细胞端粒长度的作用发挥有赖于减少自身端锚酶的活性。3.端锚酶和端粒酶反义寡核苷酸在加速细胞端粒长度的缩短、促进肿瘤细胞衰老、凋亡方面具有协同作用。4.端锚酶和端粒酶反义寡核苷酸协同作用的机理可能与mcl-1基因在转录及转录后水平的上调有关。总之,在中医学整体平衡观念的治法治则的指导下,端锚酶和端粒酶催化亚单位两种反义寡核苷酸协同作用限制A549恶性肿瘤细胞无限增殖的能力、加速其衰老、凋亡的研究,是肿瘤基因学治疗的新思路,有可能成为抗肿瘤基因治疗的新靶点。更多还原

【Abstract】 Human diseases is related to gene directly or indirectly.Gene treatment,based on changing gene structure,can be used to mono-gene diseases,but nearly all multi-gene diseases,such as tumor result from regulation imbalance of multiple functional genes,namely that of genome and gene network of every layer.So "regulating gene function,not changing gene structure is main strategy to disease no matter now and future."Traditional Chinese Medicine,which core is equilibrium and integration concept(Yin-Yang theory and Zang-Fu theory),believe that disease result from the balance of integration of function.Functional regulation as a whole is just priority of traditional Chinese medicine.It has been proved by experiences of TCM of thousands years.There are many the same features between TCM and modem medicine,with the equilibrium concept and the concept of viewing situation as a whole corresponding to system biology,harmonization between human and nature corresponding to Environment Genomic Project,treat principle based on system and individualization(treat aiming at the pathogenesis ascertained by differentiating and analyzing the clinical presentation)corresponding to pharmaco-genetics,regulate treatment(strengthening vital Qi,eliminating pathogen,monarch,minister,assistant and guide)corresponding to multi-aim and medicine effect in coordination.TCM and Western medicine will reached integration gradually,and the priority of functional regulation as a whole will get more development.At present,there is an examples for telomere to regulate gene function as a whole.BACKGROUND:Telomeres,which contain repeated TTAGGG sequences,are large nucleoprotein complexes that protect the ends of chromosomes against degradation and fusion.The end-replication problem results in progressive shortening of telomeres leading to genome instability,and telomerase provides a means to replace telomere repeats which are lost during replication as a result of the inability of DNA polymerase to replicate to the end of a linear chromosome.Therefore,telomerase activity not only maintains the telomeres of proliferating cells but is implicated in the process of cellular immortalization and oncogenesis.In recent years,inhibition of telomerase used to be discussed as a promising approach for treating a variety of malignant tumors,but failed in clinic. The key is,in addition to telomerase,many other factors play a role in telomere maintenance including tankyrase.Tankyrase,which poly(ADP-ribosyl)ate telomere repeated-binding factor1 and releases it from telomere,allows access of telomerase to telomere and enhances telomere elongated.Conversely,it was possible that combination of both enzyme inhibitors can increase the risk of critically shortened telomere and promote the following crisis and death of cancer cells.OBJECTIVE:This study is to determine the effect and mechanism of antisense tankyrase oligonucleotide (asTANKS)combined antisense human telomerase reverse transcriptase oligonucleotide (ashTERT)on telomere in human lung adenocarcinoma A549 cells:1.To investigate the role of hTERT mRNA expression,telomerase activity,tankyrase activity and telomere length in t A549 cells teeated by asTANKS and/or ashTERT.2.To determine the association between expression of mcl-1,Bcl-2,Bax and action with asTANKS and/or ashTERT and obtain the mechanism.3.To observe the alteration in morphous and function for A549 cells and explore potential target of telomere-based molecular cancer therapeutics.METHODS AND RESULTS:1.The expression of hTERT mRNA by RT-PCR was markedly repressed by ashTERT and alteration was observed for neither asTANKS nor sTANKS.2.PCR-ELISA showed that telomerase activities in A549 cells were strongly suppressed by ashTERT,but not by asTANKS.3.By Western blot,asTANKS significantly inhibited tankyrase activity,while ashTERT not as well.4.Telomere length measured by Q-FISH,became significantly decreased with the treatment of ashTERT or asTANKS and the efficacy was more remarkable with the combination of them.5.Compared with the circumstance that neither asTANKS nor ashTERT was correlated with the regulation in Bcl-2 or Box,mcl-1 mRNA levels increased dramatically in the presence of asTANKS and no alteration with ashTERT.6.Both total Mcl-1 protein levels and the content of Mcl-1s were upregulated by asTANKS, while the content of Mcl-1s was downgraded by ashTERT,although total Mcl-1 protein levels had no change.However,addition of asTANKS reversed downregulation of Mcl-1s induced by ashTERT and result in elevation of them.Consistent with expression of mRNA in mcl-1,Bcl-2,either asTANKS or ashTERT was unassociateds with Bcl-2 or Bax levels.7.Cells was prone to senescence in morphous with asTANKS or ashTERT as passage time was delayed well and the trend combinated between asTANKS and ashTERT was more significant.8.Cells came more rapidly to an end and population double times was shortened more quickly with the combination of asTANKS and ashTERT,although the same effect was observed with single factor.9.Uptake rate in[~3H]-TdR trend to suppression under continuous treatment with ashTERT or asTANKS and combinated effect was more markedly.10.Transfection efficiency in X-Gal was enhanced gradually with ashTERT or asTANKS and increase combinated with them was more significant.CONCLUSION:1.Distinguished from telomerase,tankyrase is an unique negative-regulated route for telomere maintenance,in which antisense oligos diminishs poly(ADP-ribosyl)ation of TRF1,prevents it from release in telomeres,and trun down access of telomerase to telomeres and unconcerned with hTERT mRNA and protein levels as well.2.The combination between ashTERT and asTANKS can enhance the efficacy of telomere shortening induced by single factor,facilitate cancer cell senescence and hasten earlier tumor cellular crisis.3.The synergistic mechanism of them was concerned with ranscriptic and post-ranscriptic regulation in mcl-1.Concomitant with tankyrase activity downragulation,asTANKS might reverse potential resistance induced by ashTERT. In a word,pharmacological targeting of tankyrase enhances telomere shortening by means of a telomerase inhibitor and results in earlier cellular crisis.This study provides insight into strategies for telomere-based molecular cancer therapeutics by means of the iatreusis of integration concept in TCM.更多还原