【作者】 张冠中；

【导师】 焦顺昌；

【作者基本信息】 中国人民解放军军医进修学院 ， 肿瘤内科， 2011， 博士

【摘要】 目的研究培美曲塞对人结肠癌细胞的生长抑制作用及其与培美曲塞相关基因表达的相关性；研究吉非替尼对人结肠癌细胞的生长抑制作用及其与结肠癌细胞EGFR信号传导通路基因突变的相关性；研究培美曲塞联合吉非替尼对人结肠癌细胞的生长抑制作用；研究培美曲塞、吉非替尼及其联合对于人结肠癌细胞细胞周期及凋亡的影响；研究培美曲塞、吉非替尼及其联合对于人结肠癌细胞培美曲塞相关基因表达的影响；研究培美曲塞、吉非替尼及其联合对于人结肠癌细胞吉非替尼相关蛋白表达的影响。材料和方法1.以体外药敏实验(MTT方法)检测培美曲塞对HT-29、LoVo、SW620、HCT116、SW1116及WiDr六株人结肠癌细胞的生长抑制作用；以Real-time Quantitive RT-PCR方法检测六株结肠癌细胞培美曲塞相关基因RFC、FPGS、TS及DHFR的mRNA表达；分析培美曲塞对六株结肠癌细胞的生长抑制作用与相关基因表达的相关性。2.以体外药敏实验(MTT方法)检测吉非替尼对HT-29、LoVo、SW620、HCT116、SW1116及WiDr六株人结肠癌细胞的生长抑制作用；以基因序列测定方法检测六株结肠癌EGFR及K-RAS基因突变情况；分析吉非替尼对六株结肠癌细胞的生长抑制作用与细胞EGFR及K-RAS基因突变的相关性。3.以体外药敏实验(MTT方法)检测培美曲塞同时联合吉非替尼及培美曲塞序贯联合吉非替尼对于HT-29、LoVo、SW620、及SW1116四株人结肠癌细胞的生长抑制作用。4.以流式细胞仪检测培美曲塞、吉非替尼、培美曲塞同时联合吉非替尼及培美曲塞序贯联合吉非替尼对于人结肠癌细胞HT-29及LoVo的细胞周期及凋亡的影响。5.以Real-time Quantitive RT-PCR方法检测培美曲塞、吉非替尼及培美曲塞同时联合吉非替尼对人结肠癌细胞HT-29及LoVo的培美曲塞相关基因RFC. FPGS、TS及DHFR mRNA表达的影响。6.以Western blot方法检测培美曲塞、吉非替尼及培美曲塞同时联合吉非替尼对人结肠癌细胞HT-29及LoVo的吉非替尼相关蛋白pEGFR. pAKT及pERK表达的影响。结果1.培美曲塞可抑制HT-29、LoVo、SW620、HCT116、SW1116及WiDr六株人结肠癌细胞的生长,培美曲塞对各株结肠癌细胞的生长抑制作用各异(P<0.01)。六株结肠癌细胞RFC、FPGS、TS及DHFR基因mRNA表达各异(P<0.01)。培美曲塞对六株结肠癌细胞的生长抑制作用与细胞TS基因表达呈负相关(r=-0.886,P<0.05)。2.吉非替尼可抑制HT-29、LoVo、SW620、HCT116、SW1116及WiDr六株人结肠癌细胞的生长,吉非替尼对各株结肠癌细胞的生长抑制作用各异(P<0.01)。六株结肠癌细胞中LoVo细胞出现19外显子2236-2250位点缺失突变,HT-29、SW1116及WiDR细胞出现20外显子2361位点G>A点突变。本实验中仅一株细胞出现了有意义的EGFR基因的19外显子突变,相关性分析缺乏统计学意义。3.培美曲塞联合吉非替尼对于HT-29、LoVo、SW620及SW1116四株人结肠癌细胞的药效学作用均表现为协同作用。在对两药相对不敏感的HT-29、SW620及SW1116表现为培美曲塞序贯联合吉非替尼的协同作用强于培美曲塞同时联合吉非替尼,而在对两药相对敏感的LoVo细胞表现为序贯联合的协同作用等于甚至小于同时联合。各组CI值显著不同,P<0.01。4.培美曲塞、吉非替尼、培美曲塞同时联合吉非替尼及培美曲塞序贯联合吉非替尼均可阻滞HT-29和LoVo细胞的细胞周期进程并影响凋亡细胞百分率。差异具有统计学意义(P<0.01)。5.吉非替尼可使HT-29及LoVo细胞的TS及DHFR基因表达降低(P<0.01)。6.培美曲塞可使HT-29细胞及LoVo细胞的pEGFR、pAKT及pERK蛋白的表达升高(P<0.01)。结论1.培美曲塞可抑制人结肠癌细胞的生长,培美曲塞对人结肠癌细胞的生长抑制作用与细胞TS基因表达成负相关。培美曲塞在结肠癌病人治疗中拥有一定的治疗前景。2.吉非替尼可抑制人结肠癌细胞的生长。提高剂量吉非替尼对结肠癌病人的治疗可能也拥有一定的治疗前景。3.培美曲塞联合吉非替尼对于人结肠癌细胞具有协同增效作用。两药联合在结肠癌治疗中前景广阔。4.培美曲塞、吉非替尼及二者联合可阻滞人结肠癌的细胞周期,影响人结肠癌细胞的凋亡百分率。5.吉非替尼可降低人结肠癌细胞培美曲塞相关基因表达,提高人结肠癌细胞对培美曲塞化疗的敏感性,两药联合增效。6.培美曲塞可提高人结肠癌细胞吉非替尼相关蛋白的表达,吉非替尼可显著降低培美曲塞引起的蛋白表达增高,两药联合增效。更多还原

【Abstract】 Objectives1. To study the antitumor effects of pemetrexed on human colon cancr cells and the correlation of antitumor effects of pemetrexed and expressions of genes related with pemetrexed in human colon cancer cells.2. To study the antitumor effects of gefitinib on human colon cancer cells and the correlation of antitumor effects of gefitinib and EGFR signal pathway gene mutations in human colon cancer cells.3. To study the pharmacodynamic effects of pemetrexed combined with gefitinib on human colon cancer cells.4. To study the effects of pemetrexed, gefitinib, and their combination on cell cycles and apoptosis cell percentages in human colon cancer cells.5. To study the effects of pemetrexed, gefitinib, and their combination on expressions of genes related with pemetrexed in human colon cancer cells.6. To study the effects of pemetrexed, gefitinib, and their combination on the expressions of proteins of EGFR signal pathway in human colon cancer cells.Materials and Methods1. Antitumor effects of pemetrexed on human colon cancer cells HT-29, LoVo, SW620, HCT116, SW1116 and WiDr were detected by drug sensitivity test in vitro (MTT method); the mRNA expressions of RFC, FPGS, TS, and DHFR genes related with pemetrexed in six human colon cancer cells were detected by Real-Time Quantitive RT-PCR method; the correlation of antitumor effects of pemetrexed and expressions of genes related with pemetrexed in human colon cancer cells were analyzed.2. Antitumor effects of gefitinib on human colon cancer cells HT-29, LoVo, SW620, HCT116, SW1116 and WiDr were detected by drug sensitivity test in vitro (MTT method); EGFR and K-RAS gene mutations of six human cancer cells were detected by gene sequences detection; the correlation of antitumor effects of gefitinib and EGFR and K-RAS gene mutations in human colon cancer cells were analyzed.3. The pharmacodynamic effects of concurrent pemetrexed and gefitinib and sequential pemetrexed followed by gefitinib in human colon cancer cells HT-29, LoVo, SW620 and SW1116 cells were detected by drug sensitivity test in vitro (MTT method).4. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib and sequential pemetrexed followed by gefitinib on cell cycles and apoptosis cell percentages of HT-29 and LoVo cells were studied by Flow cytometry detection,5. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib on the mRNA expressions of RFC, FPGS, TS, and DHFR genes of HT-29 and LoVo cells were detected by Real-time quantitive RT-PCR method.6. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib on the protein expressions of pEGFR, pAKT and pERK genes of HT-29 and LoVo cells were detected by western blot method.Results1. Pemetrexed inhibited the growth of HT-29, LoVo, SW620, HCT116, SW1116 and WiDr cells (P<0.01). The antitumor effects of pemetrexed were negatively correlated with TS gene expressions in six human colon cells (r=-0.886, P<0.05)2. Gefitinib inhibited the growth of HT-29, LoVo, SW620, HCT116, SW1116 and WiDr cells (P<0.01). Sequence analysis showed that the exon 19 of EGFR gene were deleted in LoVo cells, and exon 20 of EGFR gene in HT-29, SW1116 and WiDr cells were mutated. Because of cells having mutation are less, we did not find correlation between gefitinib’s effect on and EGFR and K-RAS gene mutations of six colon cancer cells (P>0.05).3. Pemetrexed combined with gefitinib showed synergistic effects on human colon cancer cells HT-29, LoVo, SW620 and SW1116 (P<0.01).4. Pemetrexed, gefitinib, concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib affected the cell cycles and apoptosis of HT-29 and LoVo cells(P<0.01).5. Gefitinib decreased TS and DHFR gene expressions of HT-29 and LoVo cells (P<0.01).6. Pemetrexed increased pEGFR, pAKT and pERK protein expressions of HT-29 and LoVo cells (P<0.01).Conclusions1. Pemetrexed can inhibit the growth of human colon cancer cells. The sensitivities to pemetrexed are negatively correlated with TS gene expressions in colon cancer cells.2. Gefitinib can inhibit the growth of human colon cancer cells.3. Pemetrexed combined with gefitinib has synergistic effects on human colon cancer cells.4. Pemetrexed, gefitinib and their combination affect the cell cycles and apoptosis of human colon cancer cells.5. Gefitinib reduces the gene expressions of TS and DHFR, and increases the sensitivities of human colon cancer cells to pemetrexed.6. Pemetrexed increases the protein expressions of pEGFR, pAKT and pERK, and gefitinib can reduce the increase caused by pemetrexed.更多还原