【作者】 王艳红；

【导师】 王桂琴；

【作者基本信息】 山西医科大学 ， 微生物学， 2005， 硕士

【摘要】 核心蛋白聚糖(decorin,DCN)是一种小分子富含亮氨酸的硫酸皮肤素蛋白聚糖,广泛存在于正常细胞外基质中,其核心蛋白具有抑制细胞增殖、影响细胞外基质形成等多种生物学作用。DCN 基因外显子序列长1080bp,编码395 个氨基酸。目的通过克隆hDCN 基因,构建原核表达体系并表达DCN 蛋白,进一步研究其对胃癌细胞株BGC-823 的抗增殖作用以及对细胞突变P53、血管内皮细胞生长因子(VEGF)表达的影响。方法首先利用DCN 特异引物,通过RT-PCR 方法从人肌肉组织中扩增编码DCN 分子的外显子序列,与pET-21a(+)载体连接,构建原核表达体系pET-21a(+)-DCN 的重组质粒。经双酶切、测序鉴定基因序列,然后将鉴定正确的重组质粒转入表达工程菌E.coli BL21(DE3)中,用IPTG 37℃诱导获得DCN 融合蛋白。SDS-PAGE 凝胶电泳分离、洗脱、透析纯化后,得到纯化的融合蛋白。在胃癌细胞株BGC-823 培养液中加入不同浓度的DCN,作用一定时间后,用活细胞计数、MTT 法测定DCN 对BGC-823细胞生长的抑制作用,通过倒置显微镜、HE 染色观察细胞的形态学变化。利用免疫细胞化学染色法检测胃癌细胞中突变P53、VEGF 在处理前后的变化。结果本研究克隆了hDCN 基因,成功构建了pET-21a(+)-DCN 重组质粒。重组质粒双酶切以及测序结果均证明克隆的DCN 基因完全正确,获得了稳定表达pET-21a(+)-DCN 的原核表达体系,并得到了纯化的融合蛋白。用DCN 对胃癌细胞株BGC-823 进行干预的结果表明,DCN 在5~30μg/ml 对胃癌细胞BGC-823 均有抑制作用,并表现出浓度依赖性关系,各剂量组间均有显著性差异(P<0.05),且随着作用时间延长生长抑制率也增加(P<0.05)。在倒置显微镜下及HE 染色后可以看到处理组细胞体积缩小、变圆、细胞突起回缩,细胞核浓缩、有凋亡小体形成。免疫细胞化学结果表明突变P53、VEGF 在处理组中的表达显著低于未处理的对照组,而且作用呈现时间和剂量效应(P<0.05,P<0.01)。结论本课题制备了pET-21a(+)-DCN 稳定的原核表达体系,表达并纯化了DCN,进一步研究了其生物学活性。DCN 有诱导胃癌细胞凋亡和抗增殖的作用,并能下调胃癌细胞中突变P53 和VEGF 的表达,这种作用可能与其抗增殖、抑制肿瘤细胞浸润有关。更多还原

【Abstract】 Background Decorin is a leucine-rich and ubiquitous proteoglycan that is a normal component of the extracellular matrix and has recently been described as capable of retarding the growth of various tumor cell line, both in vitro and in vivo.The cDNA of decorin extron, was composed of 1080 bp of nucleotides and encoded a protein of 395 amino acids. Objects Our aim is to clone and expresse the human decorin extron gene, to evaluate the anti-proliferation on BGC-823 gastric cancer cell line and influence on mutational P53 and VEGF expression using decorin. Methods Firstly, using decorin specific primer and by RT-PCR, the full length of cDNA of decorin extron was obtained through human muscle, then recombined plasmid -decorin was constructed and sequenced to identify the gene sequence. Furthermore, pET-21a(+)-decorin was transformed into E.coli BL21(DE3)and after higher temperature and IPTG induction, the fusion protein was expressed. After being separated by SDS-PAGE electrophoresis, being eluted and purified through dialysis, the purification of fusion protein pET-21a(+)-decorin was obtained. Secondly, After a period when BGC-823 gastric cancer cell line were treated with decorin in different concentrations, the suppressive effect of decorin on BGC-823 gastric cancer cell line were detected by the MTT assay and counting cell. The morphological changes of gastric cancer cell were observed by inverted microscope and HE stain. The positive expression of mutational P53 and VEGF was determined by immunocytochemistry(ICC). Results The recombinant vector was constructed and identified using genetic cloing technology, and the target peptide was produced in E.coli BL21(DE3)in this experiment. The fusion protein was high expressed. After a period when BGC-823 gastric cancer cell line were treated with decorin in different concentrations, the proliferation of BGC-823 gastric cancer cell line were inhibited by decorin respectively showed by MTT assay and counting cell. These results demonstrated that had a dosage and time dependent relationship. There was a statistically significant decreased in the proliferation rate among the three groups with different concentrations(P<0.05 或P<0.01). The morphological changes of gastric cancer cell that the microvillus disappeared, cytoplasm had vacuolation, chromation were pyknosis and cogulate to mass clustered around the membrane were revealed by invert microscope and HE stain.The expressions of mutational P53 and VEGF were decreased and had a concentration and time dependenent relationship by immunocyto-chemistry(ICC). The positive expression rate was differs significantly(P<0.05 或P<0.01)among the three groups with different concentration. Conclusion The prokaryotic expression vector with pET-21a(+)-decorin can effectively expression decorin fusion protein. The experiment will establish the basis for studying the mechanism of the anti-tumor function of decorin. Our study indicated that decorin can induce the anti-proliferation of BGC-823 gastric cancer cell line and the molecular biological mechanism may be related to the suppressing the expression of mutational P53 and VEGF.更多还原