摘要目的 建立从人结直癌组织中提取gp-96肽复合物方法,并检测体外免疫效应.方法 采取三步蛋白纯化法,即硫酸铵沉淀法、ConA亲和层析法、阴离子交换蛋白亲和层析法,从10例结直癌组织提取、纯化gp-96肽复合物;SDS-PAGE和Western blot鉴定gp-96肽复合物;检测GP-96肽复合物的体外免疫效应.结果 该方法提取的蛋白质经SDS-PAGE和Western blot鉴定,证实是gp-96肽复合物,蛋白质得率为50μg/g;体外免疫效应分析:肿瘤组中gp96肽复合物组肿瘤坏死因子(TNF)-α浓度为(801.54±109.25)ng/L,与阳性对照组(509.17±65.34)ng/L、阴性对照组(156.19±32.08)ng/L比较明显升高,差异有统计学意义(P<0.01);健康人对照组与肿瘤组比较,gp96肽复合物组比较差异有统计学意义(P<0.01).肿瘤组中gp96肽复合物组IL-10浓度为(95.71±17.55)ng/L,与阳性对照组(334.55±69.00)ng/L、阴性对照组(205.75±42.04)ng/L比较,差异有统计学意义(P<0.01);健康人对照组和肿瘤组比较,sp96肽复合物组比较差异有统计学意义(P<0.01).结论 使用该方法提取结直癌组织中gp-96肽复合物具有操作简单、重复性好、获得率高、体外免疫效应强等特点.

abstractsObjective To build the method that can purify gp96-peptide complex from human colorectal cancer tissues and research the immune effect in vitro of gp96-peptide complex. Methods (1) After get 10 human colorectal cancer tissues,we homogenate and centrifuge the tissues. (2) Using threestep purification (ammonium sulfate precipitation,ConA-Sepharose affinity chromatography and anion exchange chromatography) ,we purify gp96-peptide complex from human colorectal cancer tissues. (3) SDSPAGE and Western blot was used to certify gp96-peptide complex from human colorectal cancer tissues. (4) ELISA was used to detect level of IL-10 and TNF-a by PMBCs,which were stimulated by gp96-peptide complex from human colorectal cancer tissues. Results We have successfully purified gp96-peptide complex that was confirmed by SDS-PAGE and Western blot. The final protein recovery was 50 μg/g tumor tissues (wet weight); In tumor group the level of TNF-α stimulated by gp96-peptide complex was (801.54 ±109.25) ng/L,followed by the level of TNF-α stimulated by LPS (509. 17 ±65.34) ng/L, buffer (156.19 ±32.08) ng/L and control group (675.71 ±84.63) ng/L. Statistically significant differences were noted between the four groups (P<0.01).In tumor group the level of IL-10 stimulated by gp96-peptide complex was (95. 71 ± 17. 55) ng/L, followed by the level of TNF-a stimulated by LPS (334.55 ±69.00) ng/L,buffer (205.75±42.04) ng/L and control group (133.98 ±24.55) ng/L. Statistically significant differences were noted between the four groups (P<0.01). Conclusion gp96-pep-tide complex obtained by this method have high purity and strong immune effect in vitro. The method is simple and reproducible.