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

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.