【作者】 王丽宏；

【导师】 吕申；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:胃癌、大肠癌是我国常见的恶性肿瘤。二者在临床发现时多为晚期阶段,手术后常有转移,因此,化疗成为必要的治疗手段。然而,肿瘤细胞产生对化疗药物的耐药性影响了化疗的治疗效果。尽管近年对肿瘤耐药性逆转的研究很多,但由于肿瘤耐药性发生的机制不清楚,故到目前为止尚无令人振奋的成果出现。目前认为肿瘤耐药性产生的直接机制是肿瘤细胞中一些与抵抗药物有关的蛋白表达增强。这些蛋白或能将化疗药物排出细胞,使细胞内有效药物浓度下降以致不能发挥杀伤作用,或能降低药物毒性。现已发现了十余种这类蛋白,包括:P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药相关蛋白(LRP)、谷胱苷肽-S-转移酶-π(GST-π)等,它们在不同肿瘤及肿瘤对不同化疗药物的耐药产生过程中发挥作用。然而,较少有人研究肿瘤细胞产生及上调这些蛋白表达的机制。同正常细胞特别是不稳定细胞一样,肿瘤细胞也需要基因修复系统,以修复各种原因引起的基因损伤。错配修复(Mismatch Repair,MMR)系统就是其中之一,它可以纠正DNA复制过程中发生的碱基错配,维持基因组稳定。该系统功能丧失时,DNA复制过程中发生的错配得不到修复,基因组突变聚积,易变性增加,致使基因表达紊乱。这是胃癌、大肠癌、子宫内膜癌等一些不稳定细胞来源恶性肿瘤发生的重要原因。研究发现肿瘤耐药性产生过程中也常伴有MMR系统功能异常。那么肿瘤耐药性产生是否与MMR系统功能缺失引起的基因不稳定性所导致的耐药相关基因表达紊乱有关呢?本研究通过检测胃癌细胞株BGC-823、大肠癌细胞株LoVo在顺铂(cDDP)、5-氟尿嘧啶(5-FU)诱导耐药性产生过程中耐药蛋白P-gp、MRP、LRP、GST-π和错配修复蛋白hMSH2、hMLH1的表达变化,分析二者表达的关系,探讨胃肠道癌耐药性产生的机制。方法:1.胃癌耐药细胞株BGC-823/cDDP、BGC-823/5-FU及大肠癌耐药细胞株LoVo/5-FU的建立和它们耐药指数的测定:①采用小剂量、逐步递增cDDP浓度,间歇作用体外诱导法,建立胃癌耐药细胞株BGC-823/cDDP;采用大剂量冲击,逐步递增5-FU浓度,间歇作用体外诱导法,建立胃癌耐药细胞株BGC-823/5-FU;采用5-FU大剂量间断冲击法,建立大肠癌耐药细胞株LoVo/5-FU。②用MTT法检测耐药细胞株BGC-823/cDDP、BGC-823/5-FU、LoVo/5-FU的耐药指数。2.耐药蛋白表达的检测:用免疫细胞化学法和Western·blot法检测胃癌细胞株BGC-823及其耐药细胞株BGC-823/cDDP、BGC-823/5-FU和大肠癌细胞株LoVo及其耐药细胞株LoVo/5-FU的耐药蛋白P-gp、MRP、LRP、GST-π表达情况,比较亲本株与耐药株这些蛋白表达的差异。3.错配修复蛋白表达的检测:用免疫细胞化学法检测胃癌细胞株BGC-823及其耐药细胞株BGC-823/cDDP、BGC-823/5-FU和大肠癌细胞株LoVo及其耐药细胞株LoVo/5-FU的错配修复蛋白hMSH2、hMLH1表达情况,比较亲本株与耐药株这两个蛋白表达的差异。结果:1.耐药细胞株及其耐药指数:①历时约8个月耐药培养,获得胃癌耐药细胞株BGC-823/cDDP,其对cDDP的耐药指数为3.93。②历时约4个月耐药培养,获得胃癌耐药细胞株BGC-823/5-FU,其对5-FU的耐药指数为8.21。③历时约4个月耐药培养,获得大肠癌耐药细胞株LoVo/5-FU,其对5-FU的耐药指数为10.26。2.耐药蛋白表达:①胃癌细胞株BGC-823与其耐药株BGC-823/ cDDP:免疫细胞化学法检测示耐药蛋白LRP、GST-π在亲本株与耐药株均有表达,但在耐药株的表达明显强于亲本株。②胃癌细胞株BGC-823与其耐药株BGC-823/5-FU:免疫细胞化学法检测示耐药蛋白P-gp、MRP、LRP、GST-π在亲本株与耐药株均有表达,但在耐药株的表达明显强于亲本株;Western·blot法检测示耐药蛋白P-gp、MRP、LRP、GST-π在亲本株与耐药株均有表达,但耐药株明显强于亲本株。③大肠癌细胞株LoVo与其耐药株LoVo/5-FU:免疫细胞化学法检测示耐药蛋白P-gp、MRP、LRP、GST-π在亲本株与耐药株均有表达,但在耐药株的表达明显强于亲本株;Western·blot法检测示耐药蛋白P-gp、MRP、LRP、GST-π在亲本株与耐药株均有表达,但耐药株明显强于亲本株。3.错配修复蛋白表达:①胃癌细胞株BGC-823与其耐药株BGC-823/cDDP:免疫细胞化学法检测示错配修复蛋白hMSH2、hMLH1在亲本株与耐药株均有表达,但hMSH2表达在耐药株明显弱于亲本株,而hMLH1表达在亲本株和耐药株间未见明显差异。②胃癌细胞株BGC-823与其耐药株BGC-823/5-FU:免疫细胞化学法检测示错配修复蛋白hMSH2、hMLH1在亲本株与耐药株均有表达,但hMSH2表达在耐药株明显弱于亲本株,而hMLH1表达在亲本株和耐药株间未见明显差异。③大肠癌细胞株LoVo及其耐药株LoVo/5-FU:免疫细胞化学法检测示错配修复蛋白hMSH2、hMLH1在亲本株与耐药株均有表达,但hMSH2表达在耐药株明显弱于亲本株,而hMLH1表达在亲本株和耐药株间未见明显差异。细胞株BGC-823、LoVo耐药产生后,错配修复蛋白表达呈相同模式。结论:1.化疗药物作用前,胃癌、大肠癌细胞株BGC-823、LoVo已有P-gP、MRP、LRP、GST-π的表达,表明P-gP、MRP、LRP、GST-π与消化道肿瘤原发耐药性有关。2.胃癌细胞对cDDP产生耐药的机制可能是LRP、GST-π蛋白的表达。3.胃癌、大肠癌细胞对5-FU耐药的机制可能是P-gp、MRP、LRP、GST-π蛋白的表达。4.耐药细胞株BGC-823/cDDP、BGC-823/5-FU、LoVo/5-FU均有hMSH2表达下调,提示hMSH2蛋白缺失是肿瘤耐药性产生的原因。5.化疗药物对MMR系统功能抑制所导致的细胞基因不稳定可能是肿瘤细胞MDR产生的原因之一。更多还原

【Abstract】 Objective: Gastric and colorectal carcinomas are two of the most common malignant carcinomas in our country. Chemotherapy is one of the important therapies of them. However, frequently drug resistance, particularly multidrug resistance (MDR) of carcinoma cells resulted in failure of chemotherapy. While the mechanism of drug resistance of carcinoma cells have not been known. Now, it is known that the direct mechanism of drug resistance is up- regulation of some drug resistance associated proteins in carcinoma cells. These proteins, including P-glycoprotein(P-gp), multidrug resistance associated protein(MRP), lung resistance protein(LRP), glutathioneS-transFerase- pi(GST-pi) and so on, can eliminate drugs from carcinoma cells or decrease toxicity of drugs. But the causes inducing these proteins to express in carcinoma cells were seldom involved in researches. As normal cells, carcinoma cells also need gene repair systems so as to repair different gene damage. Mismatch repair system that is one of them can maintain the stability in the process of DNA synthesis by repairing mismathed basic groups. The dysfunction of this system might make mismatched basic groups not to be repaired , gene mutations to be collected and the expressions of genes to be disordered, which can cause the development of some carcinomas of instable cells, such as gastric carcinoma, colorectal carcinoma and endometrial cancer. Resently, some reseachers found that the dysfunction of mismatch repair also took part in the drug resistance.Then,is the expressions of drug resistance associated proteins due to the gene instability caused by the dysfunction of mismatch repair? In this study, the expression changes of drug resistance associated proteins P-gp, MRP, LRP, GST-πand mismatch repair proteins hMSH2, hMLH1 with resistance of gastric carcinoma cell line BGC-823 and colon carcinoma cell line LoVo induced by cDDP and 5-FU were detected, then the expression relationship of two kinds of proteins was studied, in order to explore themechanism of the drug resistance in gastrointestinal carcinomas.Methods: 1. To establish resistance cell lines BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU:①Cell culture and gradually increasing dose of cDDP and intermittent administration were used to establish resistance cell line BGC-823/ cDDP; cell culture and large dose, gradually increasing dose of 5-FU and intermittent administration were used to establish resistance cell line BGC-823/5-FU; cell culture and large dose of 5-FU and intermittent administration were used to establish resistance cell line LoVo /5-FU.②MTT assay was used to test RIs of resistance cell line BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU. 2. To detect the expressions of drug resistance associated proteins P-gp, MRP, LRP, GST-π: immunocytochemistry and Western·blot methods were used to detected the expressions of these proteins in cell line BGC-823 and its resistance cell lines BGC-823/cDDP, BGC-823/5-FU, cell line LoVo and its resistance cell line LoVo/5-FU; the expression relationship of these proteins between parent cell lines and their resistance cell lines was studied. 3. To detect the expressions of mismatch repair proteins hMSH2, hMLH1: immunocytochemistry method was used to detected the expressions of these proteins in cell line BGC-823 and its resistance cell lines BGC-823/cDDP, BGC-823/5-FU, cell line LoVo and its resistance cell line LoVo/5-FU; the expression relationship of these proteins between parent cell lines and their resistance cell lines was studied.Results: 1. Resistance cell lines and their RIs:①After cell line BGC-823 was cultured for 8 months, resistance cell line BGC-823/ cDDP was obtained with RI to cDDP being 3.93;②After cell line BGC-823 was cultured for 4 months, resistance cell line BGC-823/ 5-FU was obtained with RI to 5-FU being 8.21;③After cell line LoVo was cultured for 4 months, resistance cell line LoVo / 5-FU was obtained with RI to 5-FU being 10.26. 2. The expressions of drug resistance associated proteins P-gp, MRP, LRP, GST-π:①Cell line BGC-823 and its resistance cell line BGC-823/cDDP:the result of immunocytochemistry showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line.②Cell line BGC-823 and its resistance cell line BGC-823/5-FU: the results of immunocytochemistry and Western·blot showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line.③Cell line LoVo and its resistance cell line LoVo/5-FU: the results of immunocytochemistry and Western·blot showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line. 3. The expressions of mismatch repair proteins hMSH2, hMLH1:①Cell lineBGC-823 and its resistance cell line BGC-823/cDDP: the result of immunocytochemis- try showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.②Cell line BGC-823 and its resistance cell line BGC-823/5-FU: the result of immunocytochemistry showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.③Cell line LoVo and its resistance cell line LoVo/5-FU the result of immunocytoche- mistry showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.Conclusion: 1. The expressions of P-gp, MRP, LRP and GST-πprotein are concerned with the natural resistance in gastrointestinal carcinoma. 2. The expressions of LRP and GST-πprotein are the mechanism of resistance of gastric carcinoma cells to cDDP. 3. The expressions of P-gp, MRP, LRP and GST-πprotein are the mechanism of resistance of gastrointestinal carcinoma to 5-FU. 4. There has down-regulation of hMSH2 protein expression in resistance cell lines BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU. This indicates that down-regulation of hMSH2 protein expression takes part in drug resistance in carcinoma cells. 5. The gene instability that results from the dysfunction of mismatch repair caused by drugs may be one of the reasons of the resistance development of carcinoma cells.更多还原