摘要
目的:探讨乳凝集素调节肠相关淋巴组织淋巴细胞分泌表达细胞因子的作用.方法:应用基因重组技术从MCF-7乳腺癌细胞中提取总RNA,通过逆转录PCR方法得到目的基因(乳凝集素)片段,利用酶切,连接等技术,将目的基因构建至PET28载体内,转化入DH5a细胞后,鉴定阳性质粒;将含有目的基因的阳性质粒转化入表达细胞BL-21,IPTG诱导表达目的蛋白乳凝集素,应用特异性镍鏊合的亲和层析柱得到纯化的目的蛋白.利用H3-Tdr法明确乳凝集素剂量与人类肠上皮内淋巴细胞株EEI-10细胞增殖之间的关系.给予适当剂量乳凝集素作用于EEI-10细胞后,利用ELISA方法检测EEI-10分泌IL-2,IL-4及IFN-γ浓度,应用RT-PCR方法检测IL-2,IL-4及IFN-γmRNA表达.结果:酶切阳性的质粒经测序证实与基因文库一致.所得纯化的目的蛋白进行SDS-PAGE电泳和特异性抗体鉴定,明确为目的蛋白乳凝集素.利用H3-Tdr法检测乳凝集素剂量与细胞增殖之间的关系,结果显示乳凝集素作用剂量为250mg/L时淋巴细胞增殖显著.给予乳凝集素处理后细胞培养上清中IL-2(P=0.0394)和IFN-γ(P=0.0082)的含量高于未处理细胞组,而IL-4没有显著的增高;同时处理后IL-2mRNA表达量升高,IL-4mRNA表达量无显著性改变,IFN-γmRNA表达量明显增高并高于PHA刺激组.结论:乳凝集素具有上调EEI-10细胞分泌和表达IL-2和IFN-γ的作用.
AIM： To define the function that lactadherin adjusts lymphocytes of gut-associated lymphoid tissue （GALT） cytokines secretion and expression.
METHODS： We extracted mRNA from human mammary cancer MCF-7 cells, and amplified the target gene from cDNA. The lactadherin was cloned into PET28 vector by digestion, ligation then transformation into DHSa cells to check positive clones. The cDNA encoding the desired polypeptide was transformed into BL-21 cells. The expression of recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside （IPTG）. The protein was purified with a nickel column. We detected the proliferation of EEI-10 cells after treated by different concentration of lactadherin using H3-Tdr assay. Then we checked the secretion of IL-2, IL-4 and IFN~ in treated EEI-10 cells using enzyme-linked im- munosorbent assay （ELISA） and the cytokines mRNA expression using reverse transcription polymerase chain reaction （RT-PCR）.
RESULTS： Lactadherin protein was success- fully obtained and confirmed by SDS-PAGE electrophoresis and specific antibody verifica- tion. When treated with 250 mg/L lactadherin, EEI-10 cells showed a significant increase in proliferation. The levels of IL-2 and IFN-γ secretion enhanced significantly in the cells with lactadherin functional peptide in comparison with those in the untreated cells （P = 0.0394, P = 0.0082, respectively）, but IL-4 secretion had no marked change. The expression of IL-2 and IFN-γ mRNA, especially IFN-γ mRNA, were also higher in the lactadherin-treated cells than those in the untreated cells.
CONCLUSION： Lactadherin can up-regulated the secretion and expression of IL-2 and IFN-γ in EEI-10 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第12期1151-1155,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30271375~~
