国际免疫学杂志
國際免疫學雜誌
국제면역학잡지
INTERNATIONAL JOURNAL OF IMMUNOLOGY
2013年
2期
149-153
,共5页
梁媛%芦凤亮%宗卫娇%刘晔%高翔%金艾顺
樑媛%蘆鳳亮%宗衛嬌%劉曄%高翔%金艾順
량원%호봉량%종위교%류엽%고상%금애순
B淋巴细胞%浆细胞%体外活化%外周血单个核细胞
B淋巴細胞%漿細胞%體外活化%外週血單箇覈細胞
B림파세포%장세포%체외활화%외주혈단개핵세포
B lymphocytes%plasma cells%in vitro activation%PBMCs
目的 建立体外活化外周血单个核细胞(PBMCs)中B淋巴细胞扩增和分化浆细胞的方法,为高亲和力治疗性人源单克隆抗体的研发提供研究基础.方法 从3名接种过流感疫苗健康志愿者(>10个月)的末梢血中分离PBMCs,加入TLR9激动剂CpG DNA 2006和细胞因子等培养;流式细胞分析、酶联免疫吸附试验(ELISA)、酶联免疫斑点试验(ELISPOT)、细胞计数法评价B淋巴细胞体外活化效果.结果 与对照组相比,刺激组中CD19阳性细胞和CD138阳性细胞的比率明显增高(t=2.625,3.571,P<0.05);培养上清中总lgG和抗原特异性IgG含量增加,且分泌IgG和抗原特异性IgG的浆细胞数量明显增加(t=2.942,3.196,P<0.05),刺激组比未刺激组细胞生存率提高(t=2.182,P<0.05).通过体外活化方法,只需约10 ml外周血即可检测到足量的抗原特异性抗体分泌细胞.结论 本试验所建立的体外活化方法能够有效地诱导外周血B淋巴细胞扩增和分化浆细胞.
目的 建立體外活化外週血單箇覈細胞(PBMCs)中B淋巴細胞擴增和分化漿細胞的方法,為高親和力治療性人源單剋隆抗體的研髮提供研究基礎.方法 從3名接種過流感疫苗健康誌願者(>10箇月)的末梢血中分離PBMCs,加入TLR9激動劑CpG DNA 2006和細胞因子等培養;流式細胞分析、酶聯免疫吸附試驗(ELISA)、酶聯免疫斑點試驗(ELISPOT)、細胞計數法評價B淋巴細胞體外活化效果.結果 與對照組相比,刺激組中CD19暘性細胞和CD138暘性細胞的比率明顯增高(t=2.625,3.571,P<0.05);培養上清中總lgG和抗原特異性IgG含量增加,且分泌IgG和抗原特異性IgG的漿細胞數量明顯增加(t=2.942,3.196,P<0.05),刺激組比未刺激組細胞生存率提高(t=2.182,P<0.05).通過體外活化方法,隻需約10 ml外週血即可檢測到足量的抗原特異性抗體分泌細胞.結論 本試驗所建立的體外活化方法能夠有效地誘導外週血B淋巴細胞擴增和分化漿細胞.
목적 건입체외활화외주혈단개핵세포(PBMCs)중B림파세포확증화분화장세포적방법,위고친화력치료성인원단극륭항체적연발제공연구기출.방법 종3명접충과류감역묘건강지원자(>10개월)적말소혈중분리PBMCs,가입TLR9격동제CpG DNA 2006화세포인자등배양;류식세포분석、매련면역흡부시험(ELISA)、매련면역반점시험(ELISPOT)、세포계수법평개B림파세포체외활화효과.결과 여대조조상비,자격조중CD19양성세포화CD138양성세포적비솔명현증고(t=2.625,3.571,P<0.05);배양상청중총lgG화항원특이성IgG함량증가,차분비IgG화항원특이성IgG적장세포수량명현증가(t=2.942,3.196,P<0.05),자격조비미자격조세포생존솔제고(t=2.182,P<0.05).통과체외활화방법,지수약10 ml외주혈즉가검측도족량적항원특이성항체분비세포.결론 본시험소건립적체외활화방법능구유효지유도외주혈B림파세포확증화분화장세포.
Objective In order to obtain enough antibody secreting cells (ASCs,plasma cells) from peripheral blood mononuclear cells (PBMCs) for the generation of therapeutic human monoclonal antibodies with high affinity,we established the method of expansion and differentiation of B lymphocytes into plasma cells in vitro activation.Methods PBMCs were isolated from peripheral blood of three healthy donors with influenza vaccination (> 10 months) and then activated by TLR9 agonistic CpG DNA 2006 and cytokines in vitro.By flow cytometric analysis,enzyme-linked immunosorbent assay (ELISA),enzyme-linked immunospot test (ELISPOT) and cell viability,we assessed the efficiency of activation.Results The frequencies of CD19 (+) and CD138(+) cells in the viable PBMCs significantly increased 6 days after activation compared with the unstimulated control groups (t =2.625,3.571,P < 0.05).Total IgG titers and antigen-specific IgG titers also increased in the cultural supernanants of PBMCs.IgG ASCs and antigen-specific ASCs could be detected for all three donors and increased obviously (t =2.942,3.196,P < 0.05).The cell viability of stimulated group was higher than the unstimulated control groups(t =2.182,P < 0.05).We could obtain enough antigen-specific ASCs from approximately 10 ml peripheral blood using our established method.Conclusion In vitro activation method we established can effectively induce the expansion and differentiation of B lymphocytes from peripheral blood into plasma cells.