中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
Chinese Journal of Experimental Surgery
2015年
9期
2081-2084
,共4页
付锴%江普查%宫睿%王伟
付鍇%江普查%宮睿%王偉
부개%강보사%궁예%왕위
高半胱氨酸蛋白61%RNA干扰%胶质瘤
高半胱氨痠蛋白61%RNA榦擾%膠質瘤
고반광안산단백61%RNA간우%효질류
Cysteine-rich 61%RNA interference%Glioma
目的 观察运用RNA干扰技术沉默高半胱氨酸蛋白61(Cyr61)基因表达对人脑胶质瘤细胞及其裸鼠移植瘤生长的抑制作用.方法 构建针对Cyr61的小分子RNA干扰(siRNA)重组质粒载体pRNAT-绿色荧光蛋白(GFP)-Cyr61,转染人脑胶质瘤U-87MG细胞;Western blot检测其对U-87MG细胞内源性Cyr61表达的影响;噻唑蓝(MTT)法观察U-87MG细胞体外增殖活性的变化;Western blot检测其对相关调控元件核因子(NF)-κB和相关转录因子丝裂原活化蛋白激酶(MAPK)表达的影响;凝胶电泳迁移率实验(EMSA)分析Cyr61 siRNA转染U-87MG细胞后NF-KB的DNA结合情况;建立人脑胶质瘤裸鼠颅内移植瘤模型,免疫组织化学法观察通过RNA干扰敲除Cyr61基因后脑胶质瘤的微血管密度(MVD)及相关增殖细胞核抗原(PCNA)的表达.结果 Cyr61 siRNA转染U-87MG细胞后在蛋白水平显著抑制Cyr61基因表达,其表达率最低为34.02％;能明显降低U-87MG细胞的增殖活性,抑制率最高达68.15％;NF-κB与MAPK蛋白的表达均明显降低,其表达率最低分别为34.65％、38.25％.其信号通路中相关调控元件NF-κB的DNA结合活性明显降低,抑制率最高为38.26％;种植Cyr61 siRNA转染U-87MG细胞的裸鼠胶质瘤MVD明显下降,抑制率达24.20％;PCNA蛋白含量也明显降低.结论 Cyr61 siRNA可抑制Cyr61在人脑胶质瘤U-87MG细胞及裸鼠胶质瘤中的表达,降低其信号通路中相关调控元件和转录因子的活性,并抑制人脑胶质瘤的生长.
目的 觀察運用RNA榦擾技術沉默高半胱氨痠蛋白61(Cyr61)基因錶達對人腦膠質瘤細胞及其裸鼠移植瘤生長的抑製作用.方法 構建針對Cyr61的小分子RNA榦擾(siRNA)重組質粒載體pRNAT-綠色熒光蛋白(GFP)-Cyr61,轉染人腦膠質瘤U-87MG細胞;Western blot檢測其對U-87MG細胞內源性Cyr61錶達的影響;噻唑藍(MTT)法觀察U-87MG細胞體外增殖活性的變化;Western blot檢測其對相關調控元件覈因子(NF)-κB和相關轉錄因子絲裂原活化蛋白激酶(MAPK)錶達的影響;凝膠電泳遷移率實驗(EMSA)分析Cyr61 siRNA轉染U-87MG細胞後NF-KB的DNA結閤情況;建立人腦膠質瘤裸鼠顱內移植瘤模型,免疫組織化學法觀察通過RNA榦擾敲除Cyr61基因後腦膠質瘤的微血管密度(MVD)及相關增殖細胞覈抗原(PCNA)的錶達.結果 Cyr61 siRNA轉染U-87MG細胞後在蛋白水平顯著抑製Cyr61基因錶達,其錶達率最低為34.02％;能明顯降低U-87MG細胞的增殖活性,抑製率最高達68.15％;NF-κB與MAPK蛋白的錶達均明顯降低,其錶達率最低分彆為34.65％、38.25％.其信號通路中相關調控元件NF-κB的DNA結閤活性明顯降低,抑製率最高為38.26％;種植Cyr61 siRNA轉染U-87MG細胞的裸鼠膠質瘤MVD明顯下降,抑製率達24.20％;PCNA蛋白含量也明顯降低.結論 Cyr61 siRNA可抑製Cyr61在人腦膠質瘤U-87MG細胞及裸鼠膠質瘤中的錶達,降低其信號通路中相關調控元件和轉錄因子的活性,併抑製人腦膠質瘤的生長.
목적 관찰운용RNA간우기술침묵고반광안산단백61(Cyr61)기인표체대인뇌효질류세포급기라서이식류생장적억제작용.방법 구건침대Cyr61적소분자RNA간우(siRNA)중조질립재체pRNAT-록색형광단백(GFP)-Cyr61,전염인뇌효질류U-87MG세포;Western blot검측기대U-87MG세포내원성Cyr61표체적영향;새서람(MTT)법관찰U-87MG세포체외증식활성적변화;Western blot검측기대상관조공원건핵인자(NF)-κB화상관전록인자사렬원활화단백격매(MAPK)표체적영향;응효전영천이솔실험(EMSA)분석Cyr61 siRNA전염U-87MG세포후NF-KB적DNA결합정황;건립인뇌효질류라서로내이식류모형,면역조직화학법관찰통과RNA간우고제Cyr61기인후뇌효질류적미혈관밀도(MVD)급상관증식세포핵항원(PCNA)적표체.결과 Cyr61 siRNA전염U-87MG세포후재단백수평현저억제Cyr61기인표체,기표체솔최저위34.02％;능명현강저U-87MG세포적증식활성,억제솔최고체68.15％;NF-κB여MAPK단백적표체균명현강저,기표체솔최저분별위34.65％、38.25％.기신호통로중상관조공원건NF-κB적DNA결합활성명현강저,억제솔최고위38.26％;충식Cyr61 siRNA전염U-87MG세포적라서효질류MVD명현하강,억제솔체24.20％;PCNA단백함량야명현강저.결론 Cyr61 siRNA가억제Cyr61재인뇌효질류U-87MG세포급라서효질류중적표체,강저기신호통로중상관조공원건화전록인자적활성,병억제인뇌효질류적생장.
Objective To explore the inhibitory effect of the expression of cysteine-rich 61 (Cyr61) on human glioma U-87MG cells and nude mice by silencing Cyr61 gene expression by RNA interference.Methods One pair of DNA template coding was synthesized against U-87MG to reconstruct pRNAT-green fluorescent protein (GFP)-Cyr61,and transfected into U-87MG cells.The Cyr61 expression was tested by Western blotting in U-87MG cells transfected with pRNAT-GFP-Cyr61.MTT assay was used to observe the proliferation behaviors of U-87MG cells.Western blotting was used to detect the expression of nuclear factor-κB (NF-κB),and mitogen-activated protein kinase (MAPK) in U-87MG cells after Cyr61 small interfering RNA (siRNA) transfection.Electrophoretic mobility shift assay (EMSA) was applied to observe the NF-κB-DNA binding.The nude mice model of glioma was established to observe microvessel density (MVD) and the expression of proliferating cell nuclear antigen (PCNA) after silencing Cyr61 gene expression by RNA interference.Results Western blotting analyses demonstrated that pRNAT-GFP-Cyr61 could significantly decrease the expression of Cyr61 to 34.02％ in U-87MG cells;MTT results showed that pRNAT-GFP-Cyr61 could significantly inhibit the proliferation by 68.15％ in U-87MG cells.Western blotting analyses demonstrated that the expression levels of NF-κB and MAPK were decreased to 34.65％ ％ and 38.25％ respectively in U-87MG cells.EMSA results showed that NF-κB-DNA binding was inhibited by 38.26％ in U-87MG cells.MVD and the expression of PCNA were decreased in the nude mice model of glioma in vivo.Conclusion Cyr61 siRNA could significantly inhibit the expression of Cyr61 in U-87MG cells and the nude mice model of glioma.It could suppress the growth of glioma by adjusting the activity of the factors in its signal path.
