【作者】 胡剑峰；

【导师】 张琍；

【作者基本信息】 南华大学 ， 内科学， 2007， 硕士

【摘要】 目的：探讨PPARγ配体罗格列酮（RSG）能否逆转有多药耐药表型的人胃癌SGC7901／VCR细胞对丝裂霉素C（MMC）的耐药性，并进一步探讨RSG逆转人胃癌SGC7901／VCR多药耐药的可能机制。方法：实验分成空白对照组、MMC组、RSG组、CSA+MMC组和RSG+MMC组，以不同浓度的RSG和MMC分别作用于SGC7901／VCR和SGC7901细胞，应用：（1）生长曲线测定RSG对人胃癌多药耐药细胞系SGC7901／VCR及其亲代生长的影响；（2）MTT比色法及集落形成实验研究RSG对MMC抑制上述两株人胃癌细胞细胞增殖的影响；（3）流式细胞仪技术及AO-EB荧光染色研究RSG对MMC诱导人胃癌细胞SGC7901／VCR细胞凋亡的影响；（4）流式细胞仪进行细胞周期分析。（5）RT-PCR和westem-blot法检测多药耐药相关基因MDR1、MRP基因及凋亡相关的Livin基因及P-gp蛋白表达的变化，并进行半定量分析。结果：（1）20μM浓度的RSG对人胃癌SGC7901／VCR细胞及SGC7901细胞的生长无明显抑制作用，与未用药组比较差异无显著性（P＞0.05），而40μM以上浓度的RSG则对SGC7901／VCR及SGC7901细胞生长的具有明显抑制作用，与对照组比较差异有显著性（P＜0.05），且其作用和RSG浓度呈时间-剂量依赖关系；（2）MMC对人胃癌SGC7901／VCR细胞及SGC7901细胞的半数抑制浓度(IC₅₀)分别为9.1mg·L^-1和0.73mg·L^-1，耐药倍数为12.4倍，两组比较差异有显著性（P＜0.01），表明SGC7901／VCR细胞对MMC耐药；RSG对SGC7901／VCR细胞及SGC7901细胞的IC₅₀分别为75.9μM和58.4μM，两组IC₅₀比较差异无显著性（P＞0.05），说明SGC7901／VCR对RSG无耐药性；（3）RSG+MMC组中，RSG的浓度由QμM递增至8QμM，MMC对SGC7901／VCR细胞的IC₅₀由9.1mg·L^-1降至0.87mg·L^-1，20、40、80μM RSG对SGC7901／VCR的逆转倍数分别为2.84、9.6、10.5；40μM RSG逆转SGC7901／VCR对MMC耐药性与40μ·L^-1CSA的作用相当（P＞0.05），表明RSG能逆转SGC7901／VCR细胞株对MMC的耐药性，其作用与RSG浓度呈剂量依赖关系；（4）集落形成实验结果显示：RSG（40μM）+MMC(1mg·L^-1)组的集落形成率与MMC(1mg·L^-1)组、ROS（40μM）组比较，差异有显著性（P＜0.01）；其与CSA(4μg·L^-1)+MMC(1mg·L^-1)组效果相似（P＞0.05），说明RSG与MMC联用减少SGC7901／VCR细胞的集落形成率；（5）AO-EB染色及流式细胞仪检测对SGC7901／VCR细胞凋亡的影响显示相似的结果：RSG+MMC组对诱导SGC7901／VCR细胞株的凋亡率增高，与空白对照组、RSG组、MMC组差异有显著性（P＜0.01），与CSA+MMC组比较差异亦有统计学意义（P＜0.05），说明RSG与MMC联用能增强MMC对诱导SGC7901／VCR细胞凋亡的作用；（6）RT-PCR及Western-blot结果显示：①RSG组、RSG+MMC组SGC7901／VCR细胞内PPARγmRNA表达较空白对照组、MMC组及CSA组明显升高（P＜0.05），提示RSG能诱导SGC7901／VCR细胞PPARγ的表达；②空白对照组（无处理的SGC7901／VCR细胞）与SGC7901细胞比较，MDR1 mRNA、P-gp及Livin mRNA的表达均明显升高（P＜0.01），显示SGC7901／VCR细胞内MDR1、Livin基因的表达较SGC7901细胞内强；③MRP在SGC790／VCR细胞、SGC7901细胞内的均未见明显阳性表达条带；④RSG组及RSG+MMC组对MDR1 mRNA、P-gp及Livin mRNA在SGC7901／vCR细胞内表达下调，与空白对照组和MMC组比较降低，差异有显著性意义（P＜0.05），但和CSA联用MMC比较，差异无显著性（P＞0.05），说明RSG、CSA均能抑制MDR1、Livin基因在SGC7901／VCR细胞内表达。结论：（1）40μM以上浓度的RSG对SGC7901／VCR及SGC7901细胞生长的均有抑制作用，且SGC7901／VCR细胞株对其不具耐药性；（2）RSG能部分逆转SGC7901／VCR对MMC的耐药性；（3）RSG能诱导SGC7901／VCR细胞株的凋亡；（4）多药耐药基因MDR1及凋亡抑制基因Livin在SGC7901／VCR细胞中过度表达，提示可能是SGC7901／VCR产生多药耐药的原因之一；（5）RSG能下调MDR1基因和Livin基因在SGC7901／VCR细胞中的表达，提示这可能是其部分逆转SGC7901／VCR细胞多药耐药的重要机制之一。更多还原

【Abstract】 Objective: To explore whether Rosiglitazone（RSG）, a selective Peroxisome proliferator-activated receptor gamma （PPARγ） ligand, can reverse the resistance to Mitomycin （MMC） of human gastric cancer SGC7901/VCR cell line which has multidrug resistant phenotype, and further to explore the possible mechanism of RSG reserving multidrug resistance.Methods: There are blank control group、MMC group、RSG group, CSA+ MMC group and RSG+MMC group, with different concentrations of RSG and MMC affecting SGC7901/VCR and SGC7901 cell lines, using: （1） The influence of RSG on human gastric cancer SGC7901/VCR cell line which had multidrug resistant phenotype and its parental generation SGC7901 cell line was observed with the analysis of cell growth curve; （2） MTT assay and clone formation rate were used to determine the influence of RSG on MMC-inhibited proliferation of the two human gastric cell lines which mentioned above; （3） The influence of RSG on MMC-induced apoptosis was determined by flow cytometry and AO-EB fluorescent staining; （4） Cell cycle was determined by flow cytometry; （5） The expressions of multidrug resistance related genes MDR1、MRP and apoptotic related gene Livin were measured by RT-PCR and western-blot, and undertaken semi quantitative analysis.Results: （1） RSG at 20、40、80、160μM had inhibitory effects on the growths of human gastric cancer SGC7901/VCR cell line and its parental generation SGC7901 cell line, but 20μmol/L RSG group and blank control group had no statistical significance （P＞0.05）, while 40, 80, 160μM RSG groups had statistical significance with control group（P＜0.05）, and expressing a dose-dependent relationship; （2） The IC₅₀ of MMC to human gastric cancer SGC7901/VCR cell line and its parental generation SGC7901 cell line are 9.1mg·L^-1 and 0.73mg·L^-1 respectively, the multiple of drug resistance is 12.4, the two groups had statistical significance（P＜0.05）, illustrating SGC7901/VCR cell line had drug resistance to MMC; The IC₅₀ of RSG to SGC7901/VCR cell line and SGC7901 cell line are75.9μM and 58.4μM respectively, the IC₅₀ between MMC and RSG had no statistical significance （P＞0.05）; illustrating SGC7901/VCR cell line had no drug resistance to RSG. （3） In RSG+MMC group, when the concentration of RSG increased from 0μM to 80μM progressively, the IC₅₀ of MMC to SGC7901/VCR cell line decreased from 9.1mg·L^-1 to 0.87mg·L^-1, the reserving multiple of RSG at 20、40、80μM to SGC7901/VCR cell line are 2.84、9.6、10.5 respectively, the difference had obvious statistical significance compared with blank control、MMC and RSG group（P＜0.01）. The effect of reversing SGC7901/VCR drug resistance to MMC between RSG at 40μM and CSA at 4μg/L is familiar （P＞0.05）, illustrating that RSG can reverse the resistance to MMC of the human gastric cancer SGC7901/VCR cell line, expressing an obvious dose-dependent relationship. （4） Clone formation rate indicated: the difference of colon formation rate between RSG（40μM）+MMC(1mg·L^-1) and MMC(1mg·L^-1)、RSG（40μM） had statistical significance（P＜0.01）, but it had similar effects with CSA（4mg/L）+ MMC(1mg·L^-1) group （P＞0.05）, illustrating that RSG combined MMC can decrease the colon formation rate of SGC7901/VCR. （5） The results of apoptosis between AO-EB fluorescent staining and flow cytometry is coincident, RSG+MMC group can increase the apoptotic rate of SGC7901/VCR cell line, compared with blank control、RSG and MMC groups, the difference had obvious statistical significance （P＜0.01）, compared with CSA+MMC group the difference had statistical significance（P＜0.05）, illustrating that RSG combined MMC can enhance the apoptosis of MMC induced in SGC7901/VCR cell line; RSG enhanced the G₁-phase percents of gastric cancer SGC7901/VCR cell line and SGC7901 cell line obviously, indicating that RSG arrested cell growth in G1 phase. （6） RT-PCR and Western-blot indicated:①The expression of PPARγ, mRNA and PPARγ, protein of SGC7901/VCR cell line in RSG and RSG+MMC groups is increased than RSG、MMC and CSA groups, the difference had statistical significance（P＜0.05）, RSG can increase the expression of PPARγ, mRNA and PPARγ, protein in SGC7901/VCR cell line;②Compared blank control group to SGC7901 cell line, the expression of MDR1 and Living mRNA all enhanced obviously（P＜0.01）, showing that the expression of MDR1 and Livin mRNA in SGC7901/VCR cell line is stronger than in SGC7901 cell line;③The expression of MRP had no obvious difference between SGC7901/VCR cell line and SGC7901 cell line （P＞0.05）;④The expression of MDR1 mRNA, P-gp and Livin mRNA of SGC7901/VCR cell line in RSG and RSG+MMC groups decreased than blank control and MMC groups, the difference had statistical significance（P＜0.05）, but compared with CSA + MMC group, the difference had no statistical significance （P＞0.05）, illustrating that RSG and CSA all can inhibit the expression of MDR1 and Livin in SGC7901/VCR cell line. （8） RSG at 40μM can down-regulate the expression of MDR1 mRNA, P-gp and Livin mRNA in SGC7901/VCR cell line, compared with blank control and MMC groups, the difference had statistical significance （P＜0.01）, but compared with CSA+MMC group, the difference had no statistical significance（P＞0.05）.Conclusions: （1） RSG inhibits the growth of gastric cancer SGC7901/VCR cell line and SGC7901 cell line at 40μM、80μM、160μM; And has no drug resistance in SGC7901/VCR cell line. （2） RSG can reserve the drug resistance of SGC7901/VCR cell line to MMC partly. （3） RSG can induce the apoptosis of SGC7901/VCR cell line, and has G₁-phase arresting effect. （4） The over expression of multidrug resistance gene MDR1 and apoptotic inhibited gene Livin is a possible reason of multidrug resistance of SGC7901/VCR. cell line. （5） RSG can down-regulate the expression of MDR1 and Livin, this maybe an important mechanism of RSG reversing multidrug resistance of SGC7901/VCR cell line partly.更多还原