致小鼠脾脏T细胞凋亡日本血吸虫虫卵抗原蛋白的筛选

Study on the screening of molecule(s) performing apoptotic effect on T lymphocytes from schistosoma japonicum egg antigen

目的 观察日本血吸虫虫卵致小鼠脾脏淋巴细胞凋亡情况,筛选SEA中致脾脏T细胞凋亡的蛋白组分。方法 采用TUNEL(Terminal Deoxynucleotidyl Transferase-mediated dUTP nick end labeling, TdT介导的dUTP缺口末端标记法)检测日本血吸虫感染鼠脾脏虫卵肉芽肿细胞凋亡情况。制备日本血吸虫可溶性虫卵抗原(soluble egg antigen, SEA)并免疫小鼠。将小鼠处死,取脾,制备脾细胞悬液,分别以15、30、60μg/mlSEA刺激脾脏细胞,流式观察脾脏淋巴细胞凋亡情况。用层析柱及超滤管按分子质量将SEA初步分离为4个分子区段,蛋白分别富集于Fr1,>55 ku;Fr2,30~70 ku;Fr3,5~30 ku;Fr4,<5 ku。体外分离培养免疫小鼠脾脏细胞,分别加入60μg/ml的不同SEA区段分子,培养48 h后以Annexin V标记进行流式检测,采用DNA ladder法和RT-PCR检测T细胞中Fas, FasL mRNA水平。将致凋亡作用显著的SEA区段进行SDS-PAGE,电洗脱富集不同亚区段蛋白,再与T细胞共同培养,观察T细胞凋亡比例,进一步缩小蛋白范围区间。结果 TUNEL法检测日本血吸虫感染后8周小鼠脾脏虫卵肉芽肿虫卵周围有细胞凋亡,免疫小鼠T淋巴细胞在60μg/ml浓度下凋亡比例显著高于其他浓度组,流式检测、DNA ladder法及定量PCR结果均表明Fr1蛋白组分致T细胞凋亡作用最强。将SDS-PAGE电泳后的Fr1分子进行电洗脱,富集不同亚区段蛋白(Fr1-1,Fr1-2,Fr1-3),以60μg/ml的浓度刺激T细胞48 h, Annexin V法显示Fr1-2(55~72 ku亚蛋白区段)致T细胞凋亡率达57.04%,致凋亡效果最为显著(P<0.01)。结论 虫卵周围高浓度SEA有致小鼠脾脏T细胞凋亡作用,以55~72 ku蛋白区段致凋亡作用最强,可进一步从中筛选疫苗靶蛋白。

Objective To observe the apoptosis of spleen lymphocytes in mice caused by eggs, and assess the pro-apoptotic activity effect of soluble egg antigen(SEA) from Schistosoma japonicumv to T lymphocytes. Methods Apoptic cells around the eggs in the splenic granulomas were measured by TUNEL staining in situ,SEA was prepared and used for immunization. Mice were sacrificed and the splenocytes suspension was prepared then stimulated with 15,30,60 μg/ml SEA,respectively. Apoptosis of lymphocytes from spleens of immunized mice cultured in different SEA concentrations were detected by flowcytometry. Proteins in the SEA were initially size-separated into 4 different fractions by superdex 200 gel filtration and 5 ku hyperfiltration tube, proteins were respectively enriched in Fr1,>55 ku;Fr2,30-70 ku;Fr3,5-30 ku;Fr4,<5 ku.Each fraction(60 μg/ml) was then cultured with lymphocytes from spleens of immunized mice, tested for the presence of pro-apoptotic activity through annexin V and propidium iodide staining、DNA fragmentation and RT-PCR that aimed to determine the mRNA expression level of Fas, FasL. These studies would narrowed the activity to a specific fraction and then divided it into sub-fractions and electroeluted from the gel slices by electroelution tube, pooled fractions were concentrated and used for next stimulation(60 μg/ml),the pro-apoptotic activity of the sub-fractions was tested again. Results Cells around the eggs in the splenic granulomas were apoptic when measured by TUNEL staining, a significant increase of Annexin V positive T cells were revealed in splenic T cells when cultured with 60 μg/ml SEA,the results of DNA fragmentation, annexin V/propidium iodide staining, and RT-PCR all showed significant pro-apoptotic activity associated with fraction 1(>55 ku). Fraction 1 was then divided into sub-fractions by molecule mass( Fr1-1,Fr1-2,Fr1-3),then cultured with lymphocytes from spleens of immunized mice in 60 μg/ml for 48 h, annexin V/propidium iodide staining cells were detected by flowcytometry, result showed that the pro-apoptotic activity was correlated with Fr1-2,molecules between 55 to 72 ku, it induced 57.04% apoptosis in T cells in vitro,with the most significant apoptotic effect(P<0.01). Conclusion High concentration of SEA around the eggs can cause significant apoptosis of spleen T cells, The fraction of SEA molecules between 55 to 72 ku caused more apoptosis in T cells in vitro,further study of identify the precise protein will provide candidate molecules for vaccine research.