CAJ | 학술논문

目的：构建包含不同Alpha基因片段的重组报告质粒和Alpha1-TFEB融合基因表达质粒，并评价Alpha基因启动子活性。方法：利用软件对Alpha基因进行启动子预测；采用PCR技术扩增出5种不同长度的Alpha基因片段和正常TFEB基因启动子序列pTFEB，构建含Alpha基因片段和pTFEB的重组报告质粒；采用脂质体转染法将其分别转染至人胚肾293T细胞；通过荧光素酶活性检测确定Alpha基因的启动子活性。同时构建含Alpha1-TFEB融合基因表达质粒，转染293T细胞后，采用Western blot法检测转染前后TFEB蛋白的表达水平。结果：成功构建含不同Alpha基因片段和pTFEB的重组报告质粒。与pGL3-Basic质粒转染组进行比较，Alpha1、Alpha2、Alpha3、Alpha4、Alpha5质粒转染组的荧光素酶活性显著增高（P<0.01）；与正常TFEB基因启动子进行比较，Alpha1、Alpha2、Alpha5的荧光素酶活性明显增高（P<0.01）；与pGL3-Enhancer质粒转染组进行比较，pGL3-Enhanc-er-Alpha1-TFEB表达质粒组TFEB蛋白的表达明显升高。结论：t（6；11）染色体易位肾细胞癌中Alpha基因具有启动子活性，可促进TFEB表达，Alpha5片段最强活性区域位于643~693位碱基序列。
목적：구건포함불동Alpha기인편단적중조보고질립화Alpha1-TFEB융합기인표체질립，병평개Alpha기인계동자활성。방법：이용연건대Alpha기인진행계동자예측；채용PCR기술확증출5충불동장도적Alpha기인편단화정상TFEB기인계동자서렬pTFEB，구건함Alpha기인편단화pTFEB적중조보고질립；채용지질체전염법장기분별전염지인배신293T세포；통과형광소매활성검측학정Alpha기인적계동자활성。동시구건함Alpha1-TFEB융합기인표체질립，전염293T세포후，채용Western blot법검측전염전후TFEB단백적표체수평。결과：성공구건함불동Alpha기인편단화pTFEB적중조보고질립。여pGL3-Basic질립전염조진행비교，Alpha1、Alpha2、Alpha3、Alpha4、Alpha5질립전염조적형광소매활성현저증고（P<0.01）；여정상TFEB기인계동자진행비교，Alpha1、Alpha2、Alpha5적형광소매활성명현증고（P<0.01）；여pGL3-Enhancer질립전염조진행비교，pGL3-Enhanc-er-Alpha1-TFEB표체질립조TFEB단백적표체명현승고。결론：t（6；11）염색체역위신세포암중Alpha기인구유계동자활성，가촉진TFEB표체，Alpha5편단최강활성구역위우643~693위감기서렬。
To construct recombinant reporter plasmids containing different Alpha gene segments and Alpha1-TFEB fusion gene and to evaluate the promoter activity of the Alpha gene. Methods:Promoter regions of the Alpha gene were predicted using a software Primer 0.5. Five Alpha gene segments with different lengths and a normal TFEB gene promoter (pTFEB) were amplified via polymerase chain reaction, and recombinant reporter plasmids containing different Alpha gene segments and a normal TFEB gene pro-moter were constructed. Liposome transfection was used to transfect these vectors into the human embryo kidney 293T cells. The pro-moter activity of the Alpha gene was evaluated via luciferase assay. Meanwhile, the recombinant Alpha1-TFEB plasmid was construct-ed and transfected into the 293T cells. The TFEB expression of the recombinant Alpha1-TFEB plasmid was then detected via Western blot. Results: Recombinant reporter plasmids containing different Alpha gene segments and pTFEB were constructed successfully. Compared with the luciferase activity of pGL3-Basic, that of the groups with Alpha1, Alpha2, Alpha3, Alpha4 and Alpha5 significantly increased (P<0.01). The luciferase activity also increased significantly in the groups with Alpha1, Alpha2 and Alpha5 compared with that of the pTFEB group (P<0.01). The TFEB expression of the pGL3-Enhancer-Alpha1-TFEB was significantly higher compared with that of the pGL3-Enhancer group. Conclusion:In t(6;11) translocation RCC, the Alpha gene has a strong promoter activity and it en-hances TFEB expression. The strongest promoter activity region is in Alpha5 with a sequence from 643 bp to 693 bp.