中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
3期
234-238
,共5页
树突状细胞%T淋巴细胞%CD40%RNAi%慢病毒载体
樹突狀細胞%T淋巴細胞%CD40%RNAi%慢病毒載體
수돌상세포%T림파세포%CD40%RNAi%만병독재체
Dendritic cell%T lymphocyte%CD40 RNAi%Lentiviral vector
目的 构建针对小鼠CD40基因的RNA干扰(RNA interference,RNAi)慢病毒载体,制备低表达CD40的树突状细胞(dendritic cells,DC),探讨其阻断CD40/CD40L共刺激通路诱导异系T淋巴细胞无反应的作用机制.方法 采用培养基选择法体外培养小鼠骨髓源DC,构建针对小鼠CD40 RNAi慢病毒载体,体外感染小鼠骨髓源DC.荧光实时定量PCR及流式细胞术检测感染前后CD40 mRNA及DC表面抗原CD40表达.混合淋巴细胞培养观察CD40 RNAi慢病毒感染DC对异系T淋巴细胞增殖能力的影响.结果 体外培养可获得成熟的小鼠骨髓源DC.成功构建小鼠CD40RNAi慢病毒载体,检测滴度为8×10~9 TU/ml.慢病毒感染DC后与感染前相比,明显抑制CD40mRNA的表达(P<0.05),CD40蛋白表达也明显减少(P<0.05).混合淋巴细胞培养结果显示,CD40RNAi慢病毒感染DC组(1.381±0.442)与未感染组(2.444±0.485)及空慢病毒感染DC组(2.294±0.367)相比,淋巴细胞刺激指数(SI)明显下降(P<0.01).结论 培养基选择法可收获大量骨髓源DC,所获骨髓源DC能激活初始型T细胞免疫应答.CD40 RNAi慢病毒感染小鼠骨髓源DC,可以使DC低表达CD40蛋白.CD40 RNAi慢病毒感染的DC体外刺激异系T淋巴细胞增殖的能力下降.为研究CD40 RNAi在同种器官移植诱导免疫耐受奠定基础.
目的 構建針對小鼠CD40基因的RNA榦擾(RNA interference,RNAi)慢病毒載體,製備低錶達CD40的樹突狀細胞(dendritic cells,DC),探討其阻斷CD40/CD40L共刺激通路誘導異繫T淋巴細胞無反應的作用機製.方法 採用培養基選擇法體外培養小鼠骨髓源DC,構建針對小鼠CD40 RNAi慢病毒載體,體外感染小鼠骨髓源DC.熒光實時定量PCR及流式細胞術檢測感染前後CD40 mRNA及DC錶麵抗原CD40錶達.混閤淋巴細胞培養觀察CD40 RNAi慢病毒感染DC對異繫T淋巴細胞增殖能力的影響.結果 體外培養可穫得成熟的小鼠骨髓源DC.成功構建小鼠CD40RNAi慢病毒載體,檢測滴度為8×10~9 TU/ml.慢病毒感染DC後與感染前相比,明顯抑製CD40mRNA的錶達(P<0.05),CD40蛋白錶達也明顯減少(P<0.05).混閤淋巴細胞培養結果顯示,CD40RNAi慢病毒感染DC組(1.381±0.442)與未感染組(2.444±0.485)及空慢病毒感染DC組(2.294±0.367)相比,淋巴細胞刺激指數(SI)明顯下降(P<0.01).結論 培養基選擇法可收穫大量骨髓源DC,所穫骨髓源DC能激活初始型T細胞免疫應答.CD40 RNAi慢病毒感染小鼠骨髓源DC,可以使DC低錶達CD40蛋白.CD40 RNAi慢病毒感染的DC體外刺激異繫T淋巴細胞增殖的能力下降.為研究CD40 RNAi在同種器官移植誘導免疫耐受奠定基礎.
목적 구건침대소서CD40기인적RNA간우(RNA interference,RNAi)만병독재체,제비저표체CD40적수돌상세포(dendritic cells,DC),탐토기조단CD40/CD40L공자격통로유도이계T림파세포무반응적작용궤제.방법 채용배양기선택법체외배양소서골수원DC,구건침대소서CD40 RNAi만병독재체,체외감염소서골수원DC.형광실시정량PCR급류식세포술검측감염전후CD40 mRNA급DC표면항원CD40표체.혼합림파세포배양관찰CD40 RNAi만병독감염DC대이계T림파세포증식능력적영향.결과 체외배양가획득성숙적소서골수원DC.성공구건소서CD40RNAi만병독재체,검측적도위8×10~9 TU/ml.만병독감염DC후여감염전상비,명현억제CD40mRNA적표체(P<0.05),CD40단백표체야명현감소(P<0.05).혼합림파세포배양결과현시,CD40RNAi만병독감염DC조(1.381±0.442)여미감염조(2.444±0.485)급공만병독감염DC조(2.294±0.367)상비,림파세포자격지수(SI)명현하강(P<0.01).결론 배양기선택법가수획대량골수원DC,소획골수원DC능격활초시형T세포면역응답.CD40 RNAi만병독감염소서골수원DC,가이사DC저표체CD40단백.CD40 RNAi만병독감염적DC체외자격이계T림파세포증식적능력하강.위연구CD40 RNAi재동충기관이식유도면역내수전정기출.
Objective To construct the mouse CD40 RNA interfering(RNAi) lentiviral vector and prepare dendritic cells (DCs) with low expression of CD40. And to explore the mechanism of inducing T lymphocyte incompetence by blocking CD40/CD40L costimulatory pathway. Methods Mouse myeloid DCs were cultured in selective medium containing necessary cytokines for DC growth in vitro. CD40 RNAi gene was transfected into DCs with lentiviral vector. The expression levels of CD40 mRNA and protein were assayed by real-time quantitative PCR and flow cytometry respectively. The influences on DCs stimulating the proliferation ability of T lymphocyte were observed through mixed lymphocyte culture(MLC). Results Myeloid DCs have been harvested from mouse through cell culture in vitro. A mouse CD40 RNAi ientiviral vector was built successfully. The lentiviral titer was 8×10~9 TU/ml. The CD40 mRNA inhibition rate after infection was significantly higher(P<0.05). The CD40 protein expression of DCs was significandy lower(P<0.05). The result of MLC demonstrated that the index of stimulation to T lymphocyte of CD40 RNAi transfected DC significantly decreased compared with non-transfected DC and empty plasmid transfected DC(P< 0.01). Conclusion Large quantity of myeloid DCs with typical histological configuration are obtained through in vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. While DCs were infected by CD40 RNAi lentiviral vector, CD40 protein expression was inhibited significantly. The DCs could hamper the activation of allogeneic T lymphocyte by blocking CD40/CD40L cestimulatory pathway and induce T cell allergy. This will make the good foundation for studying the immune tolerance of CD40 RNAi.
