中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2010年
6期
400-404
,共5页
赵占考%姜忠敏%刘晓智%陈彦婷%贾文娟
趙佔攷%薑忠敏%劉曉智%陳彥婷%賈文娟
조점고%강충민%류효지%진언정%가문연
神经胶质瘤%RNA干扰%肿瘤侵润%细胞凋亡%动物实验
神經膠質瘤%RNA榦擾%腫瘤侵潤%細胞凋亡%動物實驗
신경효질류%RNA간우%종류침윤%세포조망%동물실험
Glioma%RNA interference%Neoplasm invasiveness%Apoptosis%Animal experimentation
目的 利用小干扰RNA(siRNA)技术沉默与胶质瘤细胞凋亡和侵袭密切相关的p75神经营养因子受体(p75NTR)基因,观察其逆转胶质瘤恶性表型的治疗效果.方法 设计靶向p75NTR基因的siRNA片段,脂质体转染人U251胶质瘤细胞系,逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法 检测p75NTR的mRNA和蛋白表达;Transwell细胞侵袭实验检测U251细胞的侵袭力;软琼脂集落形成实验检测细胞集落形成能力;建立裸鼠颅内U251胶质瘤荷瘤模型,原位重复注射siRNA-p75NTR/脂质体复合物3次,MRI检测颅内瘤体积,免疫细胞化学SP法检测p75NTR、神经生长因子(NGF)和细胞周期蛋白(cyclin)D2表达,用TUNEL法做移植瘤细胞原位细胞凋亡检测.结果 转染siRNA基因片段的U251细胞p75NTR mRNA和蛋白质表达水平显著下降(P<0.05),细胞侵袭能力及软琼脂集落形成能力均显著降低(P<0.05);体内实验显示p75NTR的表达程度与NGF的表达呈正相关,与cyclin D2表达及原位细胞凋亡数量呈负相关,MRI示瘤体积增长缓慢,边界轮廓清晰,动物生存期明显延长(P<0.05).结论 靶向p75NTR的siRNA技术可有效降低胶质瘤细胞的侵袭及增殖能力,诱导肿瘤细胞凋亡.
目的 利用小榦擾RNA(siRNA)技術沉默與膠質瘤細胞凋亡和侵襲密切相關的p75神經營養因子受體(p75NTR)基因,觀察其逆轉膠質瘤噁性錶型的治療效果.方法 設計靶嚮p75NTR基因的siRNA片段,脂質體轉染人U251膠質瘤細胞繫,逆轉錄聚閤酶鏈反應(RT-PCR)和免疫細胞化學方法 檢測p75NTR的mRNA和蛋白錶達;Transwell細胞侵襲實驗檢測U251細胞的侵襲力;軟瓊脂集落形成實驗檢測細胞集落形成能力;建立裸鼠顱內U251膠質瘤荷瘤模型,原位重複註射siRNA-p75NTR/脂質體複閤物3次,MRI檢測顱內瘤體積,免疫細胞化學SP法檢測p75NTR、神經生長因子(NGF)和細胞週期蛋白(cyclin)D2錶達,用TUNEL法做移植瘤細胞原位細胞凋亡檢測.結果 轉染siRNA基因片段的U251細胞p75NTR mRNA和蛋白質錶達水平顯著下降(P<0.05),細胞侵襲能力及軟瓊脂集落形成能力均顯著降低(P<0.05);體內實驗顯示p75NTR的錶達程度與NGF的錶達呈正相關,與cyclin D2錶達及原位細胞凋亡數量呈負相關,MRI示瘤體積增長緩慢,邊界輪廓清晰,動物生存期明顯延長(P<0.05).結論 靶嚮p75NTR的siRNA技術可有效降低膠質瘤細胞的侵襲及增殖能力,誘導腫瘤細胞凋亡.
목적 이용소간우RNA(siRNA)기술침묵여효질류세포조망화침습밀절상관적p75신경영양인자수체(p75NTR)기인,관찰기역전효질류악성표형적치료효과.방법 설계파향p75NTR기인적siRNA편단,지질체전염인U251효질류세포계,역전록취합매련반응(RT-PCR)화면역세포화학방법 검측p75NTR적mRNA화단백표체;Transwell세포침습실험검측U251세포적침습력;연경지집락형성실험검측세포집락형성능력;건립라서로내U251효질류하류모형,원위중복주사siRNA-p75NTR/지질체복합물3차,MRI검측로내류체적,면역세포화학SP법검측p75NTR、신경생장인자(NGF)화세포주기단백(cyclin)D2표체,용TUNEL법주이식류세포원위세포조망검측.결과 전염siRNA기인편단적U251세포p75NTR mRNA화단백질표체수평현저하강(P<0.05),세포침습능력급연경지집락형성능력균현저강저(P<0.05);체내실험현시p75NTR적표체정도여NGF적표체정정상관,여cyclin D2표체급원위세포조망수량정부상관,MRI시류체적증장완만,변계륜곽청석,동물생존기명현연장(P<0.05).결론 파향p75NTR적siRNA기술가유효강저효질류세포적침습급증식능력,유도종류세포조망.
Objective To study the therapeutic efficacy of siRNA fragments silencing p75 neurotrophin receptor (p75NTR), which may be a key regulator of glioma cell apoptosis and invasion.Methods The siRNA sequence fragments targeting p75NTR were designed and transferred into human glioma cell line U251. RT-PCR and immuocytochemistry method were used to explore the expression of p75NTR mRNA and protein. Cell adhesion assay was employed to detect cellular adhesion ability, and soft agar clone formation assay was adopted to identify oncogenicity, and a U251 glioma model was established in nude mice.The intracranial tumor volume was detected by MRI. The expression of p75NTR, NGF and cyclin D2 were identified using immunohistochemistry. Cell apoptosis was detected by apoptosis kit in situ. Results The siRNA fragments targeting p75NTR were capable of decreasing mRNA and protein expression of p75NTR in U251 glioma cell line. Both the cellular adhesion ability and oncogenicity were weakly relevant. The p75NTR expression level was negatively correlated with cyclin D2 and apoptosis, and positively correlated with NGF expression. The siRNA sequence fragments targeting p75NTR were effective in decreasing the gross volume of tumor;prolonged the survival time of mice, and the edge of tumor was much sharper than that of the control group. Conclusions The gene silencing technique by siRNA targeting p75NTR is capable of decreasing tumor invasion and cell proliferation as well as inducing cell apoptosis. It is expected to be a new choice for glioma gene therapy.
