【作者】 李建华；

【导师】 许洪伟；

【作者基本信息】 山东大学 ， 内科学， 2009， 硕士

【摘要】 【背景与目的】胃癌是人类常见的恶性肿瘤之一,在我国居恶性肿瘤的发病率和死亡率之首,多数病例在确诊时已属中晚期,手术治疗的5年生存率仅为20%～30%,预后仍然不佳。近年来虽然出现了各种手术方式的改良及化疗、放疗方案的改进,但收效甚微。手术切除无法清除所有的肿瘤细胞,且存在术后复发和转移的弊端。化疗是胃癌除手术外非常重要的辅助治疗手段,但胃癌细胞对单用一种化疗药物的敏感性较差,且易出现耐药现象。同时,化疗作为临床上应用较多的抗肿瘤治疗方法,在杀伤肿瘤细胞的同时,对正常细胞也有较大的损伤,这在一定程度上限制了其疗效。而近年来作为恶性肿瘤第四种治疗模式的基因治疗研究得到了蓬勃发展,取得了一系列可喜的成果,有望成为手术、放疗和化疗的有效辅助疗法。在肿瘤发生过程中,原癌基因激活、抑癌基因失活、误配修复基因改变等起了重要作用。为了彻底征服癌症,人们一直在进行肿瘤基因的研究,这导致许多癌基因、抑癌基因、误配修复基因的发现,脆性组氨酸三联体(fragile histidine triad,FHIT)就是其中之一。FHIT基因是1996年Ohta等人在研究某家族性肾细胞癌时利用外显子捕获的方法发现的,并克隆了相应的cDNA。因包含有主要的脆性位点FRA3B及从属于组氨酸三联体(histidine triad,HIT)蛋白超家族,故命名。FHIT基因自发现后即被拟定为一候选抑癌基因。虽然在早期有学者对它的抑癌作用提出质疑,但越来越多的研究表明,FHIT基因是一个抑癌基因。FHIT基因及其基因产物的抑癌机制目前研究的还不是很清楚,FHIT很可能从多条途径发挥肿瘤抑制作用。研究发现,FHIT基因在胃癌等与环境致癌因素关系密切的恶性肿瘤中表达缺失和异常率较高。有学者将FHIT基因导入肿瘤细胞并有效表达,可以使肿瘤细胞的一些恶性表型得以逆转,还可能诱导肿瘤细胞发生凋亡,从而达到治疗肿瘤或增强传统治疗方法的目的。还有学者将FHIT基因导入肺癌细胞株并有效表达,研究其对一些化疗药物敏感性的影响。化疗药物多西紫杉醇(docetaxel,DOC)作为一种微管稳定剂一直处于胃癌临床研究的领先地位,2期临床试验显示,单药多西紫杉醇一线或二线治疗晚期胃癌的有效率为17%—24%。基于对多西紫杉醇单药的较好活性和FHIT基因在胃癌的发生、发展和转归过程中作用的认识,本研究采用这两种因素干预FHIT基因表达缺失的人胃癌细胞株MGC-803,探讨外源性基因FHIT的表达对多西紫杉醇诱导胃癌细胞凋亡的影响,为胃癌的临床基因与化学药物的联合治疗提供理论基础,国内尚无报道。【方法】采用MTT法检测不同浓度(1.0、5.0、10、20、40μg/ml)及不同时间(24h、48h、72h)下多西紫杉醇对MGC-803细胞的抑制率并选择最佳浓度和作用时间;用脂质体将包含有外源性FHIT基因的重组真核表达质粒稳定转染胃癌细胞株(MGC-803);采用流式细胞术检测FHIT转染和DOC单独及联合作用后的细胞凋亡率;蛋白免疫印迹法检测干预前后cleaved-Caspase3的蛋白表达【结果】1.获得稳定表达FHIT基因的胃癌细胞。2.DOC对胃癌细胞具有抑制作用,且浓度为20μg/ml、时间为48h时最明显。3.DOC+pRcCMV-FHIT组细胞的凋亡水平(65.54%)较阴性对照组(3.27%)、pRcCMV组(3.55%)、pRcCMV-FHIT组(13.94%)、DOC组(44.13%)、DOC+pRcCMV(45.29%)组明显增高,且差异具有统计学意义(P＜0.05);4.DOC+pRcCMV-FHIT组细胞的cleaved-Caspase3蛋白表达较其它各组明显增强。【结论】FHIT基因表达与DOC能够协同促进胃癌细胞的凋亡,这可能与二者能够协同上调Casepase-3蛋白表达相关。更多还原

【Abstract】 【Background and Objective】Gastric cancer is one of the most frequent malignant tumors in human beings.In addition,the morbidity and mortality of gastric cancer is highest in China.Most cases are at midst or advanced stage until recieved a final diagnosis.The 5-year survival rate of gastric cancer is only about 20%-30%after a surgery operational therapy.Its prognosis has not been satisfactoried jet.These years,the therapy of gastric cancer has been developed greatly;however,its treating effect is very poor.Surgical resection can not get rid of all of cancer cells and has the potential possibility of recidivation and transferred.Therefore,gene therapy for gastric cancer is a very important adjunctive therapy method for surgery,radiotherapy and chemotherapy.Some factors,such as proto-oncogene activation,anti-oncogene inactivation and reparation gene mismatching,has been thought to take a key role for the process of tumorigenesis.To conquer cancer thoroughly,many studies about oncogene are being performed,therefore,proto-oncogene activation,anti-oncogene inactivation and reparation gene mismatching have been found.Fragile histidine triad(FHIT) gene, which is located in chromosomal region 3p14.2,has been cloned by Ohta et al in 1996. It spans not only the t(3:8)(p14.2;q24) translocation breakpoint found in familial renal cell carcinoma,but also the most common human fragile site,FRA3B.It is subordinated to histidine triad(HIT) protein superfamily.FHIT has been recently proposed as a tumor suppressor gene.Although there was some doubt about its antitumous effect,more and more studies have revealed that FHIT is an anti-oncogene. The mechanism of this anti-oncogene is not very clear.Several pathways may be associated with its anti-cancer effect.It is found that FHIT has a close correlation with cancers(such as gastric cancer) which are associated with environment carcinogenic agents,and its expression deletion and abnormality rate are high.FHIT has been transfected into tumour cells and expressed well,which can reverse some malignant phenotypes of the tumour cells.In addition,it can also induce tumor apoptosis,which can cure tumor or enhance the traditional therapy.FHIT has also been studied for its effect on chemistry drug sensitivity.As a stabilizer of microtubule,Docetaxel is important in the therapy of gastric cancer.The effective rate of late gastric cancer treated with Docetaxel is about 17%-24%.Based on the good activity of Docetaxel and the effect of FHIT in gastric cancer,we used two factors to treate the human gastric cancer cell line,MGC-803,which has a absence expression of FHIT.And to investigate the effect of exogenous FHIT on the apoptotis of gastric cancer cells induced by Docetaxel,which provide the rationale for the combined treatment of gene and chemical for gastric cancer.It’s not reported in China.【Methods】At different concentrations(1.0、5.0、10、20、40μg/ml) and different time(24h、48h、72h),Methyl thiazolyl tetrazolium(MTT) assay was used to measure the inhibition ratio of gastric cancer cell MGC-803 treated by Docetaxel,and chose the best concentration and time.The recombination eukaryotic expression plasmid with exogenous FHIT was transfected into MGC-803 by liposome.Flow cytometry was employed to detect the apoptosis ratio of gastric cancer cell treated by FHIT and DOC. And the proteinum expression of cleaved-Caspase3 was measured by Western blot.【Results】1.Stable expression of FHIT protein in transfected MGC-803 cells was obtained.2.DOC had inhibitory effect,which is the most obviously at 20μg/mL and 48h, to gastric cancer cell.3.The apoptosis ratio in DOC combined with pRcCMV-FHIT group(65.54%) was significantly higher than that in control group(3.27%),pRcCMV group(3.55%), pRcCMV-FHIT group(13.94%),DOC group(44.13%),DOC and pRcCMV group (45.29%) respectively(P＜0.05).4.Cleaved-Caspase3proteinum expression in DOC+ pRcCMV-FHIT group was significantly higher than any other group.【Conclusions】FHIT expression and DOC could promote the apoptosis of gastric cancer cell synergistically,which may be a result of the up-regulation of Caspase3 proteinum expression induced by FHIT expression and DOC synergistically.更多还原