中文摘要
本研究从遗传学和表观遗传学的角度研究新近发现的候选抑癌基因BLU、FUS1 和TUSC4 在食管鳞癌中的作用。应用PCR、MSP-PCR方法，研究三个基因在食管鳞癌的细胞系EC109、SHEE、SHEEC中基因组、表达水平的改变，发现：BLU基因在DNA水平上存在缺失，且启动子存在甲基化，导致mRNA水平的表达下调；FUS1和TUSC4基因在基因组水平上无缺失，但在细胞中表达下调。从临床患者石蜡切片提取DNA，发现FUS1、BLU基因均存在缺失现象，而TUSC4基因缺失较少。成功获得三个基因克隆，构建于慢病毒载体中，转染EC109细胞，采用MTT法、平板克隆形成实验、流式细胞术检测等方法，发现FUS1具有强的肿瘤抑制作用，MTT降低40%，与之相应的平板克隆形成也减少40%；但通过荧光克隆数校正抑制达80%；细胞周期 G1期增加。而BLU、TUSC4基因对食管癌细胞株EC109不存在抑制作用。应用组织芯片的方法研究发现：在食管癌病例中E2F1高表达，而正常组织粘膜检测不到；并且E2F1表达强度与组织分期具有相关性。通过本项研究为进一步深入研究FUS1在食管癌中的作用打下了基础。
英文摘要
In this project, the function of BLU, FUS1 and TUSC4 gene was investigated in the esophageal cancer in the field of genetics and epigenetics. The genetic and expression changes of these genes were studied in the cell model of EC109, SHEEC and SHEE by methods of PCR and MSP-PCR. The results showed that:BLU gene was found deletion in genome, and its promoter was methylated, which might resulted in it's down-regulation in the cell lines; however there was no deletion in the FUS1 and TUSC4 genes, FUS1 gene was significantly down regulated. The deletion of BLU and FUS1 also were found in the clinic patients, but TUSC4 deletion was seldom. The three gene cDNA clones were constructed in the lentivirus vector, then tranfected into EC109 cells. The effect of three genes were observed by methods of MTT, plate colony forming and flow cytometry. The results showed that the cell growth was inhibited by about 40% compared with the control group by methods of MTT. At same time, the plate colony formation results concord with that of MTT. The G1 cycle of cells apparently increased. These indicated that FUS1 gene strongly inhibit the EC109 cell growth, while BLU and TUSC4 gene did not have this effect of inhibitory. The other finding was that E2F1 was highly expressed in the tissue of esophageal cancer, and associated with the p
结题摘要
本研究从遗传学和表观遗传学的角度研究新近发现的候选抑癌基因BLU、FUS1 和TUSC4 在食管鳞癌中的作用。应用PCR、MSP-PCR方法，研究三个基因在食管鳞癌的细胞系EC109、SHEE、SHEEC中基因组、表达水平的改变，发现：BLU基因在DNA水平上存在缺失，且启动子存在甲基化，导致mRNA水平的表达下调；FUS1和TUSC4基因在基因组水平上无缺失，但在细胞中表达下调。从临床患者石蜡切片提取DNA，发现FUS1、BLU基因均存在缺失现象，而TUSC4基因缺失较少。成功获得三个基因克隆，构建于慢病毒载体中，转染EC109细胞，采用MTT法、平板克隆形成实验、流式细胞术检测等方法，发现FUS1具有强的肿瘤抑制作用，MTT降低40%，与之相应的平板克隆形成也减少40%；但通过荧光克隆数校正抑制达80%；细胞周期 G1期增加。而BLU、TUSC4基因对食管癌细胞株EC109不存在抑制作用。应用组织芯片的方法研究发现：在食管癌病例中E2F1高表达，而正常组织粘膜检测不到；并且E2F1表达强度与组织分期具有相关性。通过本项研究为进一步深入研究FUS1在食管癌中的作用打下了基础。