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结直癌组织中GP-96肽复合物的提取纯化和体外免疫效应

Purification of gp96-peptide complex from human colorectal cancer tissues and the immune effect in vitro of gp96-peptide complex
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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Westernblot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Westernblot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(r11NF)-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,gp96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。 Objective 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 colorectat cancer tissues. (3) SDS- PAGE 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-α 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 50μg/g tumor tissues ( wet weight ) ; In tumor group the level of TNF-ot 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-α 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-peptide complex obtained by this method have high purity and strong immune effect in vitro. The method is simple and reproducible.
出处 《中华实验外科杂志》 CAS CSCD 北大核心 2009年第12期1648-1651,共4页 Chinese Journal of Experimental Surgery
基金 湖北省自然科学基金资助项目(2004ABA159) 湖北省科技攻关计划资助项目(2005AA304805、2005AA304804) 武汉市科技攻关计划重点项目(200860423230)
关键词 gp-96 肿瘤多肽复合物 免疫 结直肠癌 gp96 Tumor peptide complex Immune Colorectal cancer
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参考文献7

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