重组人B淋巴细胞刺激因子（hsBLyS）对B淋巴细胞的免疫调节作用及其机制研究

【作者】 曹萌；

【导师】 陈龙；

【作者基本信息】 南京师范大学 ， 动物学， 2005， 硕士

【摘要】 本文通过离体原代培养的小鼠脾脏B淋巴细胞和小鼠为实验对象,运用细胞培养、流式细胞分析、激光共聚焦显微镜分析等免疫学和细胞学技术,综合研究和观察了hsBLys在离体和在体条件下对B淋巴细胞的增殖、存活和分化的活性作用及其胞内Ca²⁺信号变化,从多个角度探讨了hsBLyS对免疫细胞特别是B淋巴细胞的发育分化乃至整体免疫系统的调控作用及其作用机制。结果如下: 1 hsBLyS对离体脾B淋巴细胞增殖及存活的影响 用带有抗CD19的磁珠分离脾脏的B淋巴细胞,调整细胞浓度至1×10⁵个/ml,适应性培养4h后加入hsBLyS或/和anti-IgM的不同浓度的工作液,继续培养一定时间后MTT法检测光吸收值。结果表明:hsBLys单刺激能够诱导B淋巴细胞增殖,且hsBLyS在0.5-2μg/ml浓度范围内随着浓度升高而加强;hsBLyS+anti-IgM共刺激脾脏B细胞虽没有增殖,但进一步在LPS刺激后表现出增殖明显升高,其刺激指数极显著高于对照组:hsBLyS在体外可以延长B淋巴细胞的存活时间。提示:hsBLyS能够诱导体外培养的脾脏B淋巴细胞的存活和增殖,hsBLyS+anti-IgM共同作用能增强B淋巴细胞免疫活性。 2 hsBLyS单体及三聚体对离体脾B淋巴细胞作用比较 用带有抗CD19的磁珠分离脾脏的B淋巴细胞,调整细胞浓度至1×10⁵个/ml,适应性培养4h后加入hsBLyS单体或三聚体以及anti-IgM的不同浓度的工作液,继续培养一定时间后MTT法检测光吸收值。结果表明:加入hsBLvS三聚体培养的B细胞的增殖活性高于或显著高于对照组,而加入hsBLvS单体的B细胞与对照组相比变化不明显。提示:比较hsBLyS单体和三聚体对B淋巴细胞增殖变化,确认hsBLyS主要是以三聚体形式发挥生物学活性。 3 hsBLyS对离体脾脏B淋巴细胞分化的作用 实验分两个系列。系列一:将分离的脾脏细胞悬液加入24孔细胞培养板,随机分为对照组、2.5μg/ml hsBLyS组、2.5μg/ml anti-IgM组和2.5μg/mlhsBLyS+2.5μg/ml anti-IgM组,每组3个平行孔,分别加入PBS、hsBLyS和/或anti-IgM工作液,培养72h。系列二:分组同系列一,只是在加入工作液培养24h之后,吸去各孔上清再加入LPS继续培养48h。培养结束后,各孔加入anti-CD45R/B220和anti-CD21单克隆荧光抗体标记,流式细胞仪分析hsBLyS对B淋巴细胞分化的影响。结果表明:加入hsBLyS诱导B淋巴细胞总体数量明显高于对照组;B220⁺CD21^loB淋巴细胞（T1 B淋巴细胞）数量虽略高于对照更多还原

【Abstract】 The present study chose mice and splenic B lymphocytes （B cells） of mice, and employed modern biological techniques in order to systematically observe the effect of recombinant human soluble BLyS（hsBLyS） on the proliferation, survival, differentiation and intracellular Ca²⁺ signal mechanism of splenic B cells in vitro and in vivo. The regulation and mechanism of hsBLyS on the B-cell differentiation and immune system was discussed from physiology, immunology and cytology angles. The results were summarized as follows:1 Effect of hsBLyS on function activity of splenic B cells in vitroB cells were isolated from spleen suspensions with anti-CD19 FluoroBeads, then resuspended and diluted to 2× 10⁵ cells/ml. The freshly isolated B cells were cultured with hsBLyS and/or anti-IgM in different dose. After the culture, The absorbance was measured by the MTT cellular proliferation assay. The hsBLyS alone can stimulate B cell proliferation in a dose-dependent manner within 0.5-2 μ g/ml. The results of B cells costimulated with hsBLyS and anti-IgM close to those of untreated B cells, however the proliferation activity of B cells was dramatically enhanced by further stimulation with LPS. The stimulating index was markedly higher than that of untreated cells. hsBLyS can prolong the survival B cells in vitro. It was suggested that hsBLyS can induce B-cell proliferation and survival. The immune activity of B cells can be enhanced by hsBLyS costimulated with anti-IgM.2 Comparison on effect of hsBLyS monomer and trimer on splenic B cells in vitroB cells were isolated from spleen suspensions with anti-CD 19 FluoroBeads, then resuspended and diluted to 2 × 10⁵ cells/ml. The freshly isolated B cells were cultured with hsBLyS monomer or trimer in the absence or presence of anti-IgM in different dose. After the culture, The absorbance was measured by the MTT cellular proliferation assay. The proliferation activity of B cells cultured with hsBLyS trimer was higher or significantly higher than that of untreated B cells, however no obvious differences were observed between B cells cultured with hsBLyS monomer and untreated cells. To compare the changes of B-cell proliferation with hsBLyS monomer or trimer, we confirm that the trimeric molecule is the functional unit for hsBLyS.3. Effect of hsBLyS on differentiation of splenic B cells in vitroThere were two series for the experiment. （1） In the first series, Freshly isolated splenocytes in spleen suspensions were randomly divided into control group, hsBLyS group, anti-IgM group, hsBLyS+anti-IgM group with triplicate of each group, simultaneously incubated for 72 h with hsBLyS and/or anti-IgM. （2） In the second series, the groups were the same as the first series. The splenocytes were pretreated for 24 h in standard medium with hsBLyS and/or 1 anti-IgM, and further cultured for 48h in standard medium with 10μg/ml of LPS. After the cultures, the cells stained with monoclonal antibodies （mAbs） PE-conjugated anti-CD45R/B220 and FITC-conjugated anti-CD21 and analyzed in a fluorescence-activated cell sorter （FACS） Vantage SE flow cytometer with ModFit 2.0 software. The T1 B-cellpopulation account in the absence or presence hsBLyS was not significantly difference, however T2 B-cell population account treated by hsBLyS were markedly higher than that untreated by hsBLyS. After hsBLyS stimulated B cells, the account of Tl and T2 B cell was further enhanced by LPS. It seems that hsBLyS can induce development of immature B-cell and promote the differentiation from Tl to T2 B-cell.4 Effects of hsBLyS on function activities of splenic B cells and NK cells in miceForty-eight healthy ICR mice were chosen and randomly divided into a control group （group C） and five hsBLyS groups of different dosages which were diluted with PBS and injected in the abdominal cavity with 0.0lmg, 0.1 mg, 0.5mg, lmg and 2mg of hsBLyS per kg body weight, respectively, and group C with PBS of the same dosage in group 2mg/kg over eight days. The proliferation and the differentiation of splenic B cells, the activity of NK cells were analyzed on the 4th and 8th days. During the entire experimental period, the proliferation of splenic B cells was higher or significantly higher in animals administrated hsBLyS from 0.1-2mg/kg than that of control group. The account of splenic B cell was markedly higher in hsBLyS groups with 0.5 and 1 mg/kg than in control group on the 8th day, however, the account in other groups was lower than that in group C but no differences were observed. The account of Tl B-cell in each group was obvious differences with group C, except hsBLyS group with 0.1 mg/kg. The account of T2 B-cell in hsBLyS groups with 0.K 0.5 and 1 mg/kg was higher or markedly higher than that in group C, however, the result in hsBLyS group with 2mg/kg was lower than that in group C. The activity of splenic NK cell was higher or significantly higher in hsBLyS groups with 0.01 to 2 mg/kg than in control group on the 4th day. It suggested that suitable dosage of hsBLyS can greatly activate proliferation of splenic B cells in mice, promote its development, and upregulate NK cell activity.5. Discussion on intracellular Ca2+ signal mechanism of promoted role of hsBLyS on B lymphocyteFreshly isolated splenocytes in spleen suspensions from mice were cultured in the absence or presence of hsBLyS for 72 h（5%CC^ 37°C）. After the cultures, the lymphocytes of spleen were collected and marked with monoclonal antibody B220, then loaded with fluorescence probe Fluo-3/AM. A laser scanning confocal microscope was utilized to measure fluorescence intensity of [Ca2+]i in B lymphocyte through fluorescence visualization. B lymphocytes cultured with hsBLyS increased significantly as compared with that of untreated cells. The [Ca2+]i fluorescence intensity in B lymphocytes cultured with hsBLyS maintained relative high level, however, with tendency to reduce in cells untreated with hsBLyS. The [Ca2+]j fluorescence pixels of hsBLyS-treated B cells was significantly higher than that of untreated cells. It was implied that the promoted role of hsBLyS on B cell hsBLyS may play an effective immune regulation through activating [Ca2+]j signal and maintaining its relative high level.更多还原