山东医药
山東醫藥
산동의약
SHANDONG MEDICAL JOURNAL
2014年
34期
14-17
,共4页
果蝇zeste基因增强子人类同源物2%小干扰RNA%胶质瘤%药物敏感性%细胞凋亡%卡莫司汀
果蠅zeste基因增彊子人類同源物2%小榦擾RNA%膠質瘤%藥物敏感性%細胞凋亡%卡莫司汀
과승zeste기인증강자인류동원물2%소간우RNA%효질류%약물민감성%세포조망%잡막사정
E2H2 GENE RNA interference%human glioma%chemosensitivity%apoptosis%gemcitabine
目的：观察RNA干扰技术抑制EZH2基因表达对人胶质瘤U251细胞凋亡及其对卡莫司汀敏感性的影响。方法针对EZH2 mRNA序列设计合成小干扰RNA（ siRNA）的DNA模板，构建pRNAT-EZH2重组表达载体，并用其转染人胶质瘤U251细胞。用RT-PCR法检测pRNAT-EZH2转染前后U251细胞中内源性EZH2表达；激光共聚焦显微镜观察转染后及加入卡莫司汀后U251细胞凋亡情况；MTT法检测转染前后U251细胞对化疗药物卡莫司汀敏感性；用分光光度法检测转染前后以及加入卡莫司汀后U251细胞中Caspase-3活性。结果成功构建pR-NAT-EZH2重组质粒，并成功转染U251细胞。 pRNAT-EZH2重组质粒转染后U251细胞中EZH2 mRNA表达量降低（ P＜0．01）；卡莫司汀组及卡莫司汀加pRNAT-EZH2组U251细胞凋亡明显；与卡莫司汀联合时，pRNAT-EZH2组U251细胞生长抑制率明显增高（P＜0．01）；Caspase-3活性明显高于空白对照组和阴性对照组（P均＜0．01）。结论 pRNAT-EZH2可抑制EZH2在人胶质瘤U251细胞中的表达，并增强U251细胞对卡莫司汀敏感性。
目的：觀察RNA榦擾技術抑製EZH2基因錶達對人膠質瘤U251細胞凋亡及其對卡莫司汀敏感性的影響。方法針對EZH2 mRNA序列設計閤成小榦擾RNA（ siRNA）的DNA模闆，構建pRNAT-EZH2重組錶達載體，併用其轉染人膠質瘤U251細胞。用RT-PCR法檢測pRNAT-EZH2轉染前後U251細胞中內源性EZH2錶達；激光共聚焦顯微鏡觀察轉染後及加入卡莫司汀後U251細胞凋亡情況；MTT法檢測轉染前後U251細胞對化療藥物卡莫司汀敏感性；用分光光度法檢測轉染前後以及加入卡莫司汀後U251細胞中Caspase-3活性。結果成功構建pR-NAT-EZH2重組質粒，併成功轉染U251細胞。 pRNAT-EZH2重組質粒轉染後U251細胞中EZH2 mRNA錶達量降低（ P＜0．01）；卡莫司汀組及卡莫司汀加pRNAT-EZH2組U251細胞凋亡明顯；與卡莫司汀聯閤時，pRNAT-EZH2組U251細胞生長抑製率明顯增高（P＜0．01）；Caspase-3活性明顯高于空白對照組和陰性對照組（P均＜0．01）。結論 pRNAT-EZH2可抑製EZH2在人膠質瘤U251細胞中的錶達，併增彊U251細胞對卡莫司汀敏感性。
목적：관찰RNA간우기술억제EZH2기인표체대인효질류U251세포조망급기대잡막사정민감성적영향。방법침대EZH2 mRNA서렬설계합성소간우RNA（ siRNA）적DNA모판，구건pRNAT-EZH2중조표체재체，병용기전염인효질류U251세포。용RT-PCR법검측pRNAT-EZH2전염전후U251세포중내원성EZH2표체；격광공취초현미경관찰전염후급가입잡막사정후U251세포조망정황；MTT법검측전염전후U251세포대화료약물잡막사정민감성；용분광광도법검측전염전후이급가입잡막사정후U251세포중Caspase-3활성。결과성공구건pR-NAT-EZH2중조질립，병성공전염U251세포。 pRNAT-EZH2중조질립전염후U251세포중EZH2 mRNA표체량강저（ P＜0．01）；잡막사정조급잡막사정가pRNAT-EZH2조U251세포조망명현；여잡막사정연합시，pRNAT-EZH2조U251세포생장억제솔명현증고（P＜0．01）；Caspase-3활성명현고우공백대조조화음성대조조（P균＜0．01）。결론 pRNAT-EZH2가억제EZH2재인효질류U251세포중적표체，병증강U251세포대잡막사정민감성。
Objective To explore the relationship between the expression of EZH2 and BCNU Chemosensitivity and Apoptosis of human glioma U251 cells by decreasing EZH2 gene expression by RNA interference.Methods One pair of DNA template coding siRNA was synthesized against U251 to reconstruct pRNA-EZH2, which was transfected into U251 cells.The EZH2 expression in U251 cells were transfected with pRNAT-EZH2, cells were exposed to BCNU and it was de-tected by RT-PCR.MTT was used to observe the growth inhibiting ratio induced by BCNU in U251 cells.Cell apoptosis was analyzed by fluorescence microscopy.Caspase-3 activity was measured by spectrofluorometer.Results RT-PCR ana-lyses demonstrated that pRNAT-EZH2 could significantly inhibit the expression of EZH2 in U251 cells (P<0.01); after U251 cells were exposed to BCNU, the growth inhibiting ratio, Caspase-3 activity and cell apoptosis of pRNAT-EZH2 transfected cells was significantly increased compared to that of control group and negative group (P<0.01).Conclusion pRNAT-EZH2 can significantly inhibit the expression of EZH2, increase chemosensitivity to BCNU and apoptosis of U251 cells.
