CAJ | 학술논문

目的：探讨 Survivin‐shRNA 对骨肉瘤 MG‐63细胞增殖活性、凋亡指数、超微结构变化以及 Survivin 、VEGF 、PCNA 及 CAS‐3蛋白表达水平的影响。方法：骨肉瘤 MG‐63细胞培养成功后，重组腺病毒 Survivin‐shRNA 转染。细胞以1∶5的比例进行稀释传代培养，挑选稳定转染的细胞并扩增培养用于进一步实验，Survivin‐shRNA 病毒载体转染的细胞为 Survivin‐shRNA 组，阴性对照为 GFP 组和 CON 组。采用 MTT法检测细胞增殖活性，流式细胞仪检测细胞凋亡指数，透射电镜观察细胞超微结构改变，Western blot 法检测 SUV 、VEGF 、PCNA 及 CAS‐3蛋白的表达变化。结果：腺病毒介导的 RNA 干扰构建 Survivin‐shRNA 载体，Survivin‐shRNA 组转染细胞的胞质及胞核可见绿色荧光，并伴有少量小颗粒物质。 MTT结果显示：Survivin‐shRNA 对 MG‐63细胞增殖有明显抑制作用；流式细胞仪检测结果发现：Survivin‐shRNA 组与 Control 组和 GFP 组相比，细胞早期凋亡率增加，可见核碎裂、凋亡小体；透射电镜可见 Survivin‐shRNA 对 MG‐63超微结构有明显作用，核固缩，微绒毛消失，染色质浓集靠近于核膜，并可见凋亡小体分离；Westtern blot分析显示：Survivin‐shRNA 抑制 SUV 、VEGF 和 PCNA 蛋白的表达，增强了 CAS‐3蛋白在 MG‐63细胞中的表达水平。结论：Survivin‐shRNA 可抑制骨肉瘤细胞增殖，诱导细胞凋亡，其机制可能是通过抑制肿瘤新生血管形成或通过调控 CAS‐3的表达、下调 SUV 信号通路来完成。
목적：탐토 Survivin‐shRNA 대골육류 MG‐63세포증식활성、조망지수、초미결구변화이급 Survivin 、VEGF 、PCNA 급 CAS‐3단백표체수평적영향。방법：골육류 MG‐63세포배양성공후，중조선병독 Survivin‐shRNA 전염。세포이1∶5적비례진행희석전대배양，도선은정전염적세포병확증배양용우진일보실험，Survivin‐shRNA 병독재체전염적세포위 Survivin‐shRNA 조，음성대조위 GFP 조화 CON 조。채용 MTT법검측세포증식활성，류식세포의검측세포조망지수，투사전경관찰세포초미결구개변，Western blot 법검측 SUV 、VEGF 、PCNA 급 CAS‐3단백적표체변화。결과：선병독개도적 RNA 간우구건 Survivin‐shRNA 재체，Survivin‐shRNA 조전염세포적포질급포핵가견록색형광，병반유소량소과립물질。 MTT결과현시：Survivin‐shRNA 대 MG‐63세포증식유명현억제작용；류식세포의검측결과발현：Survivin‐shRNA 조여 Control 조화 GFP 조상비，세포조기조망솔증가，가견핵쇄렬、조망소체；투사전경가견 Survivin‐shRNA 대 MG‐63초미결구유명현작용，핵고축，미융모소실，염색질농집고근우핵막，병가견조망소체분리；Westtern blot분석현시：Survivin‐shRNA 억제 SUV 、VEGF 화 PCNA 단백적표체，증강료 CAS‐3단백재 MG‐63세포중적표체수평。결론：Survivin‐shRNA 가억제골육류세포증식，유도세포조망，기궤제가능시통과억제종류신생혈관형성혹통과조공 CAS‐3적표체、하조 SUV 신호통로래완성。
Objective :To explore the effects and molecular mechanisms of baicalin on tumor proliferation and apoptosis of MG‐63 cell .Methods :MG‐63 cells were cultured in DMEM medium and recombinant adenovirus vector Survivin‐shRNA and negative control rAd5‐GFP were transfected into MG‐63 cells .Cells were subcultured at a 1 ∶ 5 dilution .Positive stable transfectants were selected and expanded for further study .The clone in which the rAd5‐Survivin‐shRNA virus vectors transfected was named as Survivin‐shRNA group ,the negative control vectors transfected was named as GFP group and MG‐63 cells were named as CON group .M TT assay was used to detect cell proliferation inhibition rate ;flow cytometry detected apoptosis index ;TEM detected microscopic morphological changes ;Western Blot assay determine the protein expression level of SUV ,VEGF ,PCNA and CAS‐3 .Results :After Survivin‐shRNA was transfected into MG‐63 cells ,it was found that knockdown of SUV could significantly re‐duce the proliferative activities of MG‐63 cells compared with GFP group and CON group .knockdown of Survivin markedly increased the apoptotic index of MG‐63 cells compared with GFP group and CON group indicated by flow cytometry ;real‐time PCR and Western blot assays were performed that the mRNA and protein expression level of SUV and PCNA was decreased ,while CAS‐3 expression was increased in rAd5‐SUV group compared with the GFP group and CON group .Conclusion :Survivin‐shRNA could inhibited proliferation ,induced apoptosis of MG‐63 cells in a time‐dependent manner .The molecular mechanisms may through VEGF‐mediated regulation or regulate the ex‐pression of SUV and CAS‐3 .