中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2014年
10期
777-782
,共6页
粟英%兰亚明%卢义琼%田国红%胡烈献
粟英%蘭亞明%盧義瓊%田國紅%鬍烈獻
속영%란아명%로의경%전국홍%호렬헌
Itch%基因沉默%T淋巴细胞%胃肿瘤%免疫疗法
Itch%基因沉默%T淋巴細胞%胃腫瘤%免疫療法
Itch%기인침묵%T림파세포%위종류%면역요법
Itch%Gene silencing%T-lymphocytes%Stomach neoplasms%Adoptive immunotherapy
背景与目的：Itch蛋白是一种具有调节T细胞免疫应答起始的关键分子，属于E3泛素转移酶家族，广泛参与细胞内多种信号蛋白如ZAP70、P85、VAV、PLC-γ和PKC-θ等的泛素化修饰过程，在肿瘤诱导机体免疫耐受中起着重要作用。Itch通过调节T细胞表面受体活性及转移生长因子-β信号通路来介导T细胞免疫无反应性，诱导外周组织Treg细胞增殖。因此，通过改变Itch蛋白表达活性有望成为一种治疗自体免疫性疾病和肿瘤的有效途径。我们利用特异性小干扰RNA(small interfering RNA，siRNA)沉默T细胞Itch基因的表达，观察转染T细胞对小鼠MFC胃癌细胞的体外免疫杀伤作用。方法：分离615小鼠脾脏T细胞，筛选高效特异性沉默Itch基因的siRNA序列转染T细胞，蛋白质印迹法检测各分组Itch蛋白的表达水平；转染72 h后，利用酶联免疫吸附法(enzyme-linked immunosorbent assay，ELISA)检测细胞因子IL-2、INF-γ分泌情况，比较空白组、空转组及转染组T细胞与小鼠MFC胃癌细胞混合培养肿瘤杀伤率。结果：转染48 h后，与对照组相比，转染T细胞Itch蛋白表达率降低至16%；转染72 h后，检测转染组、空转组、空白组细胞因子IL-2分泌水平分别为(1891.96±141.91)pg/mL，(1241.69±91.67)pg/mL，(1175.03±89.14)pg/mL(P<0.001)，转染组、空转组、空白组细胞因子INF-γ分泌水平分别为(958.33±75.46)pg/mL，(683.33±66.67)pg/mL，(691.72±68.72)pg/mL(P<0.05)。在体外实验中，与空白组及空转组T细胞相比，转染组T细胞能更高效地杀伤小鼠MFC胃癌细胞，最高杀瘤率达到(54.18±2.96)%。结论：利用特异性siRNA技术沉默Itch基因能够促进小鼠T细胞因子IL-2、INF-γ分泌，增强T细胞对小鼠MFC胃癌细胞的体外免疫杀伤作用。
揹景與目的：Itch蛋白是一種具有調節T細胞免疫應答起始的關鍵分子，屬于E3汎素轉移酶傢族，廣汎參與細胞內多種信號蛋白如ZAP70、P85、VAV、PLC-γ和PKC-θ等的汎素化脩飾過程，在腫瘤誘導機體免疫耐受中起著重要作用。Itch通過調節T細胞錶麵受體活性及轉移生長因子-β信號通路來介導T細胞免疫無反應性，誘導外週組織Treg細胞增殖。因此，通過改變Itch蛋白錶達活性有望成為一種治療自體免疫性疾病和腫瘤的有效途徑。我們利用特異性小榦擾RNA(small interfering RNA，siRNA)沉默T細胞Itch基因的錶達，觀察轉染T細胞對小鼠MFC胃癌細胞的體外免疫殺傷作用。方法：分離615小鼠脾髒T細胞，篩選高效特異性沉默Itch基因的siRNA序列轉染T細胞，蛋白質印跡法檢測各分組Itch蛋白的錶達水平；轉染72 h後，利用酶聯免疫吸附法(enzyme-linked immunosorbent assay，ELISA)檢測細胞因子IL-2、INF-γ分泌情況，比較空白組、空轉組及轉染組T細胞與小鼠MFC胃癌細胞混閤培養腫瘤殺傷率。結果：轉染48 h後，與對照組相比，轉染T細胞Itch蛋白錶達率降低至16%；轉染72 h後，檢測轉染組、空轉組、空白組細胞因子IL-2分泌水平分彆為(1891.96±141.91)pg/mL，(1241.69±91.67)pg/mL，(1175.03±89.14)pg/mL(P<0.001)，轉染組、空轉組、空白組細胞因子INF-γ分泌水平分彆為(958.33±75.46)pg/mL，(683.33±66.67)pg/mL，(691.72±68.72)pg/mL(P<0.05)。在體外實驗中，與空白組及空轉組T細胞相比，轉染組T細胞能更高效地殺傷小鼠MFC胃癌細胞，最高殺瘤率達到(54.18±2.96)%。結論：利用特異性siRNA技術沉默Itch基因能夠促進小鼠T細胞因子IL-2、INF-γ分泌，增彊T細胞對小鼠MFC胃癌細胞的體外免疫殺傷作用。
배경여목적：Itch단백시일충구유조절T세포면역응답기시적관건분자，속우E3범소전이매가족，엄범삼여세포내다충신호단백여ZAP70、P85、VAV、PLC-γ화PKC-θ등적범소화수식과정，재종류유도궤체면역내수중기착중요작용。Itch통과조절T세포표면수체활성급전이생장인자-β신호통로래개도T세포면역무반응성，유도외주조직Treg세포증식。인차，통과개변Itch단백표체활성유망성위일충치료자체면역성질병화종류적유효도경。아문이용특이성소간우RNA(small interfering RNA，siRNA)침묵T세포Itch기인적표체，관찰전염T세포대소서MFC위암세포적체외면역살상작용。방법：분리615소서비장T세포，사선고효특이성침묵Itch기인적siRNA서렬전염T세포，단백질인적법검측각분조Itch단백적표체수평；전염72 h후，이용매련면역흡부법(enzyme-linked immunosorbent assay，ELISA)검측세포인자IL-2、INF-γ분비정황，비교공백조、공전조급전염조T세포여소서MFC위암세포혼합배양종류살상솔。결과：전염48 h후，여대조조상비，전염T세포Itch단백표체솔강저지16%；전염72 h후，검측전염조、공전조、공백조세포인자IL-2분비수평분별위(1891.96±141.91)pg/mL，(1241.69±91.67)pg/mL，(1175.03±89.14)pg/mL(P<0.001)，전염조、공전조、공백조세포인자INF-γ분비수평분별위(958.33±75.46)pg/mL，(683.33±66.67)pg/mL，(691.72±68.72)pg/mL(P<0.05)。재체외실험중，여공백조급공전조T세포상비，전염조T세포능경고효지살상소서MFC위암세포，최고살류솔체도(54.18±2.96)%。결론：이용특이성siRNA기술침묵Itch기인능구촉진소서T세포인자IL-2、INF-γ분비，증강T세포대소서MFC위암세포적체외면역살상작용。
Background and purpose: Itch protein is an established regulator of T cell immune response thresholds, belong to a class of E3 ubiquitin-transferring enzymes, widely involve in the ubiquitination of several key signaling molecules, such as ZAP70, P85, VAV, PLC-γ, PKC-θ, etc, plays a critical role in tumor induced immu-nosuppression. Itch ligase activity regulate T-cell anergy and development of regulatory T cells in the periphery by modulating key components of T-cell receptor and transforming growth factor-βsignaling. Therefore, manipulation of Itch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmunity and cancer. speciifc small interfering RNA(siRNA) was utilized to silence the expression of Itch gene of T-lymphocytes and investigate the cytotoxicity activity of transfected T lymphocytes against MFC stomach neoplasms cells in vitro. Methods:T lymphocytes were isolated from the spleen of 615 mice and transfected by speciifc siRNA to silence the expression of Itch gene, The expression of Itch protein were examined by Western bolt in each group;72 hours after transfection, The secretion level of IL-2, INF-γwere measured by enzyme-linked immunosorbent assay (ELISA). At the end, the cytotoxicity activity changes against MFC stomach neoplasms cells was compared between transfected T lym-phocytes, negative control and blank control in vitro. Results:Compared with control group, the expression rate of Itch protein of transfected T-lymphocytes was decreased to 16%after transfection 48 hours;72 hours after transfection, the secretion level of IL-2 in transfection group, negative control and blank control respectively were (1 891.96±141.91)pg/mL, (1 241.69±91.67)pg/mL and (1 175.03±89.14)pg/mL (P<0.001), the secretion level of INF-γin transfection group, negative control and blank control respectively were (958.33±75.46)pg/mL, (683.33±66.67)pg/mL and (691.72±68.72) pg/mL (P<0.05). Transfected T lymphocyte also showed more efifcient killing ability against MFC stomach neoplasms cells than negative control and blank control in vitro, the highest killing rate has reached (54.18±2.96)%. Conclusion:Silencing Itch gene can signiifcantly promoted the secretion level of IL-2, INF-γof mice T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against MFC stomach neoplasms cells in vitro.
