CAJ | 학술논문

背景：CTLA4Ig作为免疫耐受诱导剂是目前预防移植物抗宿主病很有潜力的策略之一。目的：体外研究腺病毒介导CTLA4Ig基因修饰的骨髓基质细胞诱导HLA单倍型供者T细胞免疫耐受的有效性。方法：自HLA单倍型相合供者骨髓分离培养骨髓基质细胞，用CTLA4Ig-重组腺病毒按感染复数50转染骨髓基质细胞72 h，免疫荧光法检测CTLA4Ig在骨髓基质细胞中的表达、定位。分别将2×104，4×104及8×104 CTLA4Ig基因修饰的骨髓基质细胞与105个HLA单倍型相合的供者外周血T细胞及105个受者外周血单个核细胞行混合淋巴细胞培养，MTT法测定T细胞增殖抑制率，收集培养上清以ELISA法检测白细胞介素2水平。构建CTLA4Ig基因修饰的骨髓基质细胞层于6孔板，每孔接种骨髓单个核细胞1×105，于培养第5天计数扩增后的单个核细胞数及CFU-GM集落数。结果与结论：按感染复数50转染的骨髓基质细胞中CTLA4Ig表达阳性率为85%，荧光信号在细胞浆中呈不均匀分布。2×104，4×104及8×104 CTLA4Ig基因修饰的骨髓基质细胞对供者T细胞增殖的抑制率高于未转染基质细胞组，而白细胞介素2水平低于未转染基质细胞组，差异均有显著性意义(P <0.05)。培养第5天， CTLA4Ig 基因修饰的骨髓基质细胞组单个核细胞数及 CFU-GM 集落数与未转染骨髓基质细胞组比较差异均无显著性意义(P >0.05)。结果表明腺病毒介导CTLA4Ig基因修饰的骨髓基质细胞在体外能诱导HLA单倍型相合供者T细胞的免疫耐受。
배경：CTLA4Ig작위면역내수유도제시목전예방이식물항숙주병흔유잠력적책략지일。목적：체외연구선병독개도CTLA4Ig기인수식적골수기질세포유도HLA단배형공자T세포면역내수적유효성。방법：자HLA단배형상합공자골수분리배양골수기질세포，용CTLA4Ig-중조선병독안감염복수50전염골수기질세포72 h，면역형광법검측CTLA4Ig재골수기질세포중적표체、정위。분별장2×104，4×104급8×104 CTLA4Ig기인수식적골수기질세포여105개HLA단배형상합적공자외주혈T세포급105개수자외주혈단개핵세포행혼합림파세포배양，MTT법측정T세포증식억제솔，수집배양상청이ELISA법검측백세포개소2수평。구건CTLA4Ig기인수식적골수기질세포층우6공판，매공접충골수단개핵세포1×105，우배양제5천계수확증후적단개핵세포수급CFU-GM집락수。결과여결론：안감염복수50전염적골수기질세포중CTLA4Ig표체양성솔위85%，형광신호재세포장중정불균균분포。2×104，4×104급8×104 CTLA4Ig기인수식적골수기질세포대공자T세포증식적억제솔고우미전염기질세포조，이백세포개소2수평저우미전염기질세포조，차이균유현저성의의(P <0.05)。배양제5천， CTLA4Ig 기인수식적골수기질세포조단개핵세포수급 CFU-GM 집락수여미전염골수기질세포조비교차이균무현저성의의(P >0.05)。결과표명선병독개도CTLA4Ig기인수식적골수기질세포재체외능유도HLA단배형상합공자T세포적면역내수。
BACKGROUND:CTLA-4Ig as a tolerance-induction agent is a potential strategy in graft-versus-host disease prevention. OBJECTIVE:To investigate the efficacy of CTLA4Ig-gene-modified bone marrow stromal cels mediated by adenovirus to induce T-cel tolerance of haploidentical donors. METHODS: The bone marrow stromal cels isolated culture from the bone marrow of HLA haploidentical donors were transfected by recombinant adenovirus encoding CTLA4IgcDNA (AdCTLA4Ig) at a multiplicity of infection=50 for 72 hours. The expression rate and the location of CTLA4Ig in the transfected cels were detected by fluorescence microscope after immunofluorescence staining. CTLA4Ig-modified bone marrow stromal cels (2×104, 4×104and 8×104) were respectively co-cultured with 105 T cels from the peripheral blood of HLA haploidentical donors and 105 peripheral blood mononuclear cels from recipients. The proliferative inhibition rate was determined by MTT assay, and the level of interleukin-2 in the supernatant was detected by ELISA. The bone marrow mononuclear cels (1×105/wel) were co-cultured with CTLA4Ig-modified bone marrow stromal cel layers constructed in 6-wel plates. The number of bone marrow mononuclear cels and colony-forming unit-granulocyte macrophages were calculated after 5-day culture. RESULTS AND CONCLUSION: The expression rate of CTLA4Ig at the multiplicity of infection=50 was as high as 85%, and the immunofluorescence signals of CTLA4Ig were distributed unevenly in the cytoplasm. The inhibition rates of 2×104, 4×104, and 8×104 CTLA4Ig-modified bone marrow stromal cels on proliferation of T cels were higher than that of untransfected cels. The levels of interluekin-2 in the corresponding cel groups were significantly lower than that in the untransfected cels (P < 0.05). At 5 days of culture, there was no significant difference in the number of bone marrow mononuclear cels and colony-forming unit-granulocyte macrophages between the transfected and untransfected cel groups (P > 0.05). These findings indicate that CTLA4Ig-modified bone marrow stromal cels mediated by adenovirus can induce immune tolerance of T-lymphocyte from HLA haploidentical donors in vitro.