CAJ | 학술논문

目的：构建4个新靶点的人CyclinE干扰RNA真核表达载体，转染人脑胶质细胞瘤U251细胞，经反转录-聚合酶链反应（RT-PCR）检测mRNA表达，获得干扰效果最好的真核表达载体，为CyclinE成为人脑肿瘤标志物提供有价值的资料。方法（1）构建4个新靶点CyclinE干扰RNA的真核表达载体后，用双酶切和碱基序列测定的方法鉴定载体构建成功与否；（2）用Lipofectamine 2000转染4个成功构建的载体到胶质瘤 U251细胞株；（3）通过 RT-PCR的结果检测转染后CyclinEmRNA表达量，选出干扰效果最好的1个。结果（1）成功构建了4个新靶点的CyclinE干扰RNA真核表达载体即PCyclinE-1、PCyclinE-2、PCyclinE-3、PCyclinE-4。（2）CyclinEmRNA 表达明显受到抑制，并获得效果最好的CyclinE干扰RNA真核表达载体。结论成功构建并筛选出效果最好的新靶点Cy-clinE干扰RNA真核表达载体，使U251细胞的CyclinE mRNA表达明显降低。
목적：구건4개신파점적인CyclinE간우RNA진핵표체재체，전염인뇌효질세포류U251세포，경반전록-취합매련반응（RT-PCR）검측mRNA표체，획득간우효과최호적진핵표체재체，위CyclinE성위인뇌종류표지물제공유개치적자료。방법（1）구건4개신파점CyclinE간우RNA적진핵표체재체후，용쌍매절화감기서렬측정적방법감정재체구건성공여부；（2）용Lipofectamine 2000전염4개성공구건적재체도효질류 U251세포주；（3）통과 RT-PCR적결과검측전염후CyclinEmRNA표체량，선출간우효과최호적1개。결과（1）성공구건료4개신파점적CyclinE간우RNA진핵표체재체즉PCyclinE-1、PCyclinE-2、PCyclinE-3、PCyclinE-4。（2）CyclinEmRNA 표체명현수도억제，병획득효과최호적CyclinE간우RNA진핵표체재체。결론성공구건병사선출효과최호적신파점Cy-clinE간우RNA진핵표체재체，사U251세포적CyclinE mRNA표체명현강저。
Objective To construct the 4 new targets eukaryotic expression vectors of human CyclinE interfer-ence specific RNA to transfect into glioma U 251 cells ,the mRNA expression was detected by RT-PCR ,for obtaining the ukaryotic expression vectors with the best interference effect to provide the valuable data for Cyclin E becoming the human tumor marker .Methods (1)Four new targets eukaryotic expression vectors of RNA interference specific CyclinE were constructed ,named as PCyclinE-1 ,PCyclinE-2 ,PCyclinE-3 and PCyclinE-4 .The double digestion method and the base sequence measuring were adopted to detect whether the vectors were successfully constructed .(2)The four newly constructed vectors were transfected into U 251 cell line by Lipofectamine 2000 .(3)The RT-PCR results was adopted to detect the expression quantity of CyclinE mRNA for selecting a vector with best interference .Results (1)4 new targets eukaryotic expression vectors of RNA interference specific for CyclinE were constructed .(2)CyclinEmRNA was obviously suppressed ,the eukaryotic expression vector with best interference effect for human CyclinE interfer-ence specific RNA was obtained .Conclusion The new eukaryotic expression vector of CyclinE siRNA with best effect is successfully constructed ,which significantly suppress the CyclinE mRNA expression of U 251 cells .