摘要
背景与目的
肝细胞癌(hepatocellular carcinoma, HCC)是世界第二大致死性癌症,其在全世界范围内的发病率也一直呈上升趋势,全球每年约有75万新发肝癌患者见于报道。由于起病隐匿,早期症状不明显,而且进展迅速,在我国大多数患者确诊时已达晚期,失去手术治疗机会,治疗困难,预后很差。化疗作为肝癌综合治疗手段之一,疗效欠佳,耐药是其主要原因。从不同角度探讨肝癌耐药机制的研究成为国内外学者研究的热点,但仍未取得突破性进展。近年来,内质网应激(endoplasmic reticulum stress, ERs)与细胞生存之间的关系逐渐被认识,内质网(endoplasmic reticulum, ER)的功能对大多数细胞的活动和生存是必需的。ERs途径是独立于死亡受体途径和线粒体途径的第3条凋亡信号途径。GRP78/BiP (glucose-regulated protein78, GRP78; the immunoglobulin heavy chain-binding protein, BiP)表达上调常被视为ERs的标志。大量研究表明：发生ERs的细胞能调节促凋亡分子如caspase-12等和促进细胞存活分子GRP78等的表达、活化,最终决定细胞是生存还是凋亡。GRP78作为一种分子伴侣在蛋白质的折叠和转运过程及内质网应激反应中发挥重要作用,其在多种肿瘤组织中呈高表达。ERs在肝癌发展过程中是否被启动及GRP78在肝癌组织中的表达情况甚少述及。本文拟检测GRP78在原发性肝细胞癌组织中的表达,探讨人类原发性肝细胞癌发生发展过程中是否启动内质网应激,推测其可能的临床意义。
方法
采用免疫组织化学(immunohistochemistry)、Real-time PCR和Western Blot检测53例肝细胞癌组织(17例高分化型癌,23例中分化型癌,13例低分化型癌,均经术后病理证实。)及19例正常肝组织中葡萄糖调节蛋白78的1nRNA及其蛋白的表达。
结果
1.免疫组织化学结果显示正常肝组织及肝癌组织内GRP78蛋白的表达情况
GRP78蛋白阳性染色主要定位于细胞质内,弥漫点状大小不一的棕黄色细颗粒状,核仁不着色。肝癌组织中的表达水平均显著高于正常组织；在肝癌组织中GRP78表达水平与细胞分化程度相关。分化程度越低,表达越高。
2.Real-time PCR结果显示正常肝组织及肝癌组织内GRP78mRNA的表达情况
GRP78及β-actin的扩增动力学曲线为典型的S形,平台期汇于一条直线上,指数区明显；熔解曲线呈单峰型,表明引物的特异性良好。通过内参β-actin校正之后,比较各组GRP78mRNA的相对表达,结果显示,肝癌组织组GRP78mRNA的相对含量显著高于正常肝组织组,且与肝癌组织分化程度成反比。(P<0.05)
3.Western blot结果显示正常肝组织及肝癌组织内GRP78的表达情况
正常肝组织的GRP78蛋白条带较弱,而肝细胞癌组织的GRP78蛋白条带较强。与正常肝组织相比,肝癌组织内GRP78表达量明显提高(P<0.05),且与分化程度成反比,即：分化程度越高,表达量越低；反之,则越高。
结论
GRP78从基因水平和蛋白水平在肝细胞癌组织中的表达明显高于其正常组织,其可能在肿瘤的发生、发展中起着重要的作用,并与细胞的分化程度相关；肝细胞癌组织内存在内质网应激。
Objective
Hepatocellular carcinoma(HCC) is the second most deadly cancer in the world, its incidence has been on the rise in worldwide. It is reported that about750,000new patients with liver cancer found each year. Because the onset of HCC conceals, early symptoms is not obvious, and may be quickly severe, most of the patients in our country has reached late stage when confirmed. So surgery opportunity is lost, treatment is difficult, the prognosis is poor. The study of domestic and foreign scholars discuss liver cancer drug resistance mechanism from different angles which become the hot topics, but still no breakthrough. In recent years, the endoplasmic reticulum stress (ERs) is gradually understanding the relationship related with cell survival. The endoplasmic reticulum(ER) function is essential for most cell activity and survival. ERs pathway is the third apoptosis signaling pathways independent of the mitochondrial pathway and the death receptor pathway. The upregulated expression level of glucose-regulated protein78(GRP78)/the immunoglobulin heavy chain-binding protein(BiP)often has been seen as ERs marker. Numerous studies show that ERs cells can regulate the activation/expression of promoting apoptosis molecules such as CHOP and caspase-12and promoting cell survival molecules such as GRP78,the balance of which is cells dicides the survival or apoptosis finally. Glucose regulating protein78plays an important role in protein folding and transfer process and endoplasmic reticulum stress response as a molecular chaperone, it is highly expressed in many kinds of tumor tissue. It is seldom reported that whether ERs was initiated in the development of cancer of the liver and GRP78expression level is in liver cancer tissue.To detect the expression of glucose-regulated protein78mRNA and protein in the tissue of hepatocellular carcinoma, demonstrating the activation of endoplasmic reticulum stress(ERs) and the unfolded protein response(UPR) in process of development of hepatocellular carcinoma and conjecturing its possible clinical significance.
Methods
To detect GRP78mRNA and protein expression of53case examples of hepatocellular carcinoma(17cases Well-differentiated carcinoma,23cases middle-differentiated carcinoma,13case poorly differentiated carcinoma,which were all confirmed by postoperative pathology) and19case normal liver tissues by immunohistochemistry, Real-time PCR and Western Blot.
Results
1. Immunohistochemistry results showed that the expression of GRP78in normal liver tissue and HCC tissue
The positive staining of GRP78protein is mainly located in cytoplasm, also observed in the cell membrane, not in nucleolus. Expression levels of GRP78in HCC tissues were significantly higher than normal liver tissues, which were associated with cellular differentiation degree. The lower the degree of differentiation, the higher the expression of GRP78protein.
2. Real-time PCR results showed that the expression of GRP78in normal liver tissue and HCC tissue
The amplification dynamics curves of GRP78and beta actin is a typical s-type, the platform stage is gathered into a straight line. Melting curve shows unimodal type, which indicates that the specificity of the primers is good. After the correction of internal reference beta actin, to compare the relative content of GRP78mRNA in each group, the results shows that expression of GRP78mRNA of HCC tissue group is significantly higher than normal liver tissue, and is inversely proportional to the degree of hepatocarcinoma tissue differentiation.(P<0.05)
3. Western blot results showed that the expression GRP78protein in normal liver tissue and HCC tissue
The GRP78protein bands are lighter in normal liver tissue, and the GRP78protein bands is brighter in hepatocellular carcinoma tissues. Compared with normal liver tissue, the expression quantity of GRP78increased significantly in HCC tissues (P<0.05). And it is inversely proportional to the degree of differentiation, namely: the differentiation degree is higher, the expression of quantity is the lower; on the other hand, it is the higher.
Conclusion
It exists activation of ERs and UPR in process of growth of hepatocellular carcinoma. The expression of GRP78protein and its mRNA was accordant. it was higher in hepatoma tissue than in normal hepatic tissue. GRP78was speculated to be closely related to the biological characteristics of human hepatocellular carcinoma.
引文
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