CAJ | 학술논문

目的构建小鼠galectin-9重组腺病毒pAd-gal-9,探讨galectin-9的真核表达定位以及galectin-9诱导T细胞凋亡与Tim-3表达的关系.方法常规分子克隆方法结合LR反应构建腺病毒重组质粒pAd/CMV/V5-DEST-gal-9,以Pac I线性化后,用脂质体2000体外转染293A细胞,8 d后反复冻融细胞3次收集含病毒上清,并以此上清感染293A细胞大量扩增病毒pAd-gal-9.CsCI密度梯度离心法纯化病毒,96孔板法梯度感染293A细胞测定病毒滴度,然后感染CHO细胞,用免疫组化、Western blot和流式细胞仪分析galectin-9的表达水平和定位;以新鲜培养的上清或固相化的细胞分别与外周淋巴结细胞进行培养,AnnexinV/PI法检测T细胞凋亡情况,对比凋亡细胞比例与Tim-3~+ T细胞的比例.结果重组腺病毒构建及表达成功,免疫组化表明galectin-9在CHO胞质表达;Westernblot证实galectin-9表达;流式分析表明胞内染色组galectin-9平均荧光强度显著高于表面染色组,病毒感染CHO表面染色组与空白对照组galectin-9平均荧光强度无明显区别;新鲜培养的上清可以明显诱导T细胞发生凋亡,显著高于Tim-3~+ T细胞的比例.结论重组腺病毒pAd-gal-9构建成功,体外感染pad-gal-9的CHO分泌galectin-9诱导T细胞凋亡可以不依赖Tim-3的表达.
목적 구건소서galectin-9중조선병독pAd-gal-9,탐토galectin-9적진핵표체정위이급galectin-9유도T세포조망여Tim-3표체적관계.방법 상규분자극륭방법 결합LR반응구건선병독중조질립pAd/CMV/V5-DEST-gal-9,이Pac I선성화후,용지질체2000체외전염293A세포,8 d후반복동융세포3차수집함병독상청,병이차상청감염293A세포대량확증병독pAd-gal-9.CsCI밀도제도리심법순화병독,96공판법제도감염293A세포측정병독적도,연후감염CHO세포,용면역조화、Western blot화류식세포의분석galectin-9적표체수평화정위;이신선배양적상청혹고상화적세포분별여외주림파결세포진행배양,AnnexinV/PI법검측T세포조망정황,대비조망세포비례여Tim-3~+ T세포적비례.결과 중조선병독구건급표체성공,면역조화표명galectin-9재CHO포질표체;Westernblot증실galectin-9표체;류식분석표명포내염색조galectin-9평균형광강도현저고우표면염색조,병독감염CHO표면염색조여공백대조조galectin-9평균형광강도무명현구별;신선배양적상청가이명현유도T세포발생조망,현저고우Tim-3~+ T세포적비례.결론 중조선병독pAd-gal-9구건성공,체외감염pad-gal-9적CHO분비galectin-9유도T세포조망가이불의뢰Tim-3적표체.
Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9's pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.