通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。     

细胞间粘附分子-1和血管细胞间粘附分子-1的结构与功能

细胞粘附分子（ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌｅ，　ＣＡＭ）是一类调节细胞与细胞、细胞与细胞外基质（ｅｘｔｒａｃｅｌｌｕｌａｒ　ｍａｔｒｉｘ，　ＥＣＭ）间相互结合、起粘附作用的膜表面糖蛋白。细胞间粘附分子－１（ｉｎｔｅｒｃｅｌｌｕｌａｒ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌａｒ－１，　ＩＣＡＭ－１）和血管细胞间粘附分子－１（ｖａｓｃｕｌａｒ　ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌａｒ－１，　ＶＣＡＭ－１）均属于ＣＡＭ中免疫球蛋白超家族（ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ　ｓｕｐｅｒｆａｍｉｌ…     

丙型肝炎病毒RNA和蛋白在肝组织中的定位研究

肝组织中丙型肝炎病毒（ｈｅｐａｔｉｔｉｓＣｖｉｒｕｓ，ＨＣＶ）ＲＮＡ和蛋白的定位研究表明，ＨＣＶ具有泛嗜性，不仅能在肝细胞、单个核细胞、枯否氏细胞、窦壁上皮细胞和胆管上皮细胞的胞浆和胞核中贮存与复制，而且可在肝细胞中表达与成熟。肝细胞的损害可能与ＨＣＶ的直接细胞毒作用和宿主介导的免疫病理反应有关。     

关于肝星状细胞的命名

肝星状细胞（ｈｅｐａｔｉｃｓｔｅｌａｔｅｃｅｌ，ＨＳＣ）位于肝窦周间隙（Ｄｉｓｅ间隙）曾被称为星状细胞、Ｉｔｏ细胞、窦周细胞、脂细胞（ｌｉｐｏｃｙｔｅ）和贮脂细胞（ｆａｔｓｔｏｒｉｎｇｃｅｌ）。１８７６年德国Ｋｕｐｆｅｒ首先用氯化金染色确认出ＨＳＣ，...     

HBsAg,AFP,CEA在肝细胞癌中表达的临床病理学意义

ＨＢｓＡｇ、ＡＦＰ、ＣＥＡ在肝细胞癌中表达的临床病理学意义１王兰２刘弋作者单位：１浙江省温州市第二人民医院病理科３２５０００２安徽医科大学附属医院普外科，合肥２３００２２对肝细胞癌（Ｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａ，ＨＣＣ）的临床流行病学...     

丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核 …

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶ Ｃ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ ｂｌｏｔ检测表达的核心抗原     

E-选择素及其配体在人卵巢癌细胞与血管内皮细胞粘附中的作用

目的和方法：为进一步阐明肿瘤血路转移的分子机制,采用活性染料玫瑰红摄入法,研究人卵巢癌细胞系ＨＯ－８９１０与激活的人脐静脉内皮细胞（ＨＵＶＥＣｓ）的粘附性以及抗粘附分子Ｅ－选择素及其配体ｓＬｅ＾ｘ单抗对ＨＯ－８９１０细胞与激活的ＨＵＶＥＣｓ粘附的影响。     

Bcl-2 和Bax蛋白在慢性病毒性肝炎肝组织中的表达

目的研究细胞凋亡相关的Ｂｃｌ－２和Ｂａｘ蛋白在慢性病毒性肝炎（ＣＨ）肝组织中表达的情况与该病发生发展的关系。方法对３５例不同病变程度的ＣＨ肝组织用免疫组化法检测Ｂｃｌ－２和Ｂａｘ的表达及定位。结果Ｂｃ１－２和Ｂａｘ分布基本一致，轻、中度ＣＨ主要见于碎屑样坏死（ＰＮ）区中的单个核细胞（ＭＮ），邻近ＰＮ区边缘的肝细胞浆中也有强阳性表达。ＨＥ染色观察，这些Ｂｃｌ－２和Ｂａｘ阳性肝细胞多呈重度水样变性或嗜酸性变。在重度ＣＨ肝组织中Ｂｃｌ－２和Ｂａｘ阳性的ＭＮ主要分布于重度ＰＮ区和桥接坏死区，周围部分肝细胞Ｂａｘ阳性，而Ｂｃｌ－２为阴性。在慢性重型肝炎，Ｂｃｌ－２和Ｂａｘ除在亚大块肝细胞坏死区浸润的ＭＮ胞浆内表达外，残存肝细胞亦呈阳性表达。结论Ｂｃｌ－２和Ｂａｘ在肝组织中的表达可能不仅与ＣＨ的细胞凋亡有密切关系，而且与慢性重型肝炎的发生发展有关     

免疫老化与肝脏受损易感性

免疫老化与肝脏受损易感性孙文兵１韩本立１许多因素与老化宿主肝脏受损易感性有关。本文就免疫老化（Ｉｍｍｕｎｏｓｅｎｅｓｃｅｎｃｅ）与其之间的关系作一综述。１枯否细胞与肝细胞损伤枯否细胞（ＫｕｐｆｅｒＣｅｌＫＣ）对肝细胞的调节是肝细胞维持正常生理过程的重...     

丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核细胞中的表达

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶＣ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的核心抗原。结果：用限制性内切酶酶切后，片段大小与计算值相符。Ｗｅｓｔｅｒｎｂｌｏｔ证实，表达抗原的Ｍｒ约为２２０００。结论：ＨＣＶＣ基因能够插入ｐｃＤＮＡ３真核表达载体，并使其在真核细胞中表达，为进一步ＨＣＶ基因疫苗的研制和探讨抗ＨＣＶ感染打下了基础。     

大鼠肝癌细胞与I型胶原的黏附特性及其与细胞周期的关系

肿瘤细胞和胞外基质之间的黏附特性与肿瘤的侵袭转移有密切关系。作者从细胞周期的角度,采用微管吸吮技术和细胞同步技术研究了不同细胞周期肝癌细胞与I型胶原裱衬表面的黏附力学特性。结果表明:胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法可分别获得G 1期和S期肝癌细胞,平均同步率分别为74.09%和90.39%;在研究剂量和时间范围内,肝癌细胞与I型胶原的黏附力具有浓度和时间依赖性;S期肝癌细胞和I型胶原的黏附力值与G 1期和未同步组(对照组)相应值比较明显降低。结果提示:肝癌细胞经血道转移的侵蚀细胞间质阶段,G 1期细胞可能起更重要的作用。这一研究对全面认识肝癌的转移机理有重要意义。     

肺癌细胞与胞外基质选择裱衬表面粘附力学的研究

肿瘤细胞与细胞外基质的粘附特性与肿瘤的侵袭转移密切相关,作者力图揭示人肺癌细胞相应的生物力学和生物流变学特征,采用微管吸吮技术定量测定体外培养的低转移人肺腺癌（PAa_)细胞和高转移人肺巨细胞癌（PG）细胞与细胞外基质重要组份胶原蛋白IV和层粘连蛋白（LN）的粘附力学特性。结果表明：与胶原蛋白IV裱衬在的粘附力,在PA,aPG细胞均较裱衬前增加,但在较低胶原浓度（1．00ug/ml,2.00ug/ml)时PAa细胞的粘附力增加幅度大于PG细胞,与LN和固定浓度（2．00ug/ml)胶原蛋白IV的复合裱衬面的粘附力为PAa细胞大于PG细胞,在较低LN浓度,PAa(0.625ug/ml),PG(0.625ug/ml,1.25ug/ml)细胞粘附力相对降低,尤以PG细胞中的降低程度和幅度为大,因此,癌细胞与细胞外基质的粘附和去粘附行为主要通过膜受体介导,从而影响癌细胞生物学特性及侵润转移能力。     

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.     

Mechanism of fibrous capsule formation surrounding hepatocellular carcinoma. Immunohistochemical study.

In 14 cases of hepatocellular carcinoma with capsule, we studied the mechanism of capsule formation by the immunoperoxidase technique using antibodies to types I, III, and IV collagen, antilaminin antibody, and anti-prolyl hydroxylase antibody. Marked round cell infiltration was observed in the noncancerous side of the capsule and around compressed hepatocytes near the capsule. Thin capsules were composed primarily of type III collagen produced by an increased number of fibroblasts, transitional Ito cells, and hepatocytes near the capsule. In thickened capsules, the noncancerous side consisted primarily of type III collagen and the cancerous side of types I and III collagen. Type I as well as type III collagen was produced by fibroblasts, transitional Ito cells, and hepatocytes. The capsule thus formed is suggested to be part of the defense mechanisms against the growth of hepatocellular carcinoma.     

长春花碱和细胞松驰素D对肝癌细胞粘弹性的影响

用微管吸吮技术测定了正常肝细胞和肝实质细胞癌细胞的粘弹性，以三参数标准线性固体模型拟实验结果，进一步研究了两种细胞骨架干扰剂处理后肝细胞和肝癌细胞粘弹性系数的变化。     

Morphometric study of hepatocytes containing hepatitis B surface antigen

The development of hepatocellular carcinoma (HCC) is probably related to infection with hepatitis B virus (HBV). Hepatocytes in livers of patients with HCC have been reported to show putative preneoplastic changes such as hyperplasia, dysplasia, or adenomatous regeneration. To determine quantitatively whether these morphologic changes are associated with HBV-infected cells, the authors performed morphometry of hepatitis B surface antigen (HBsAg)-positive hepatocytes in the nontumorous portion of 10 livers with HCC and in 10 livers without HCC. The diameter of nuclei and cytoplasm of HBsAg-positive hepatocytes was measured after demonstration of HBsAg by the peroxidase-antiperoxidase method. As controls, HBsAg-negative hepatocytes in the same liver sections were measured as well as hepatocytes of 20 age-matched HBsAg-negative patients with normal liver or alcoholic cirrhosis. HBsAg-positive hepatocytes exhibited significantly larger nuclei and a higher nucleocytoplasmic ratio than control hepatocytes. In addition, HBsAg-positive cells were often arranged in foci that consisted of two cell populations: hypertrophic (enlarged nuclei and nucleocytoplasmic ratio) and hyperplastic (two-cell-thick plates of small cells with a high nucleocytoplasmic ratio). While precancerous cells have been difficult to identify, these morphologic changes are frequently associated with the development of malignant neoplasia.     

Alteration of adhesion molecule expression and cellular polarity in hepatocellular carcinoma

AIMS: To study the expression of adhesion molecules in human liver and their possible roles during hepatocarcinogenesis. METHODS AND RESULTS: The expression of adhesion molecules in normal liver tissues, benign including probable premalignant lesions and malignant lesions was systematically investigated by immunohistochemistry and Western blotting. In normal liver, both hepatocytes and bile duct cells expressed symplekin, desmoglein 1/2, desmocollin 2, desmoplakin and plakophilin 2, but did not express desmocollin 1/3 or plakophilin 1. In benign hepatocyte lesions, expression of the adherens junctions and desmosomes was uniform and slightly increased, but symplekin appeared to show reduced expression in dysplastic lesions. In hepatocellular carcinoma (HCC), the expression of adhesion molecules was often heterogeneous and of abnormal location. Tumour cells with an abnormal distribution or loss of adhesion molecules showed an apolar arrangement of tissue architecture. The expression levels of the adhesion molecules correlated with the differentiation grades of HCC cells. CONCLUSIONS: The decreased expression of symplekin may be an early step in the transformation of hepatocytes, whereas alteration of the expression of adherens junctions and desmosomes may indicate more serious changes.     

Differential expression of beta-galactoside alpha2,6 sialyltransferase and sialoglycans in normal and cirrhotic liver and hepatocellular carcinoma

SUMMARY: Sialyltransferases sialylate plasma glycoproteins in hepatocytes and may (as hepatic key enzymes) constitute markers for liver diseases. We examined expression of the prevalent alpha2,6 sialyltransferase (ST6Gal I) and sialoglycans in normal liver, cirrhotic liver, and hepatocellular carcinoma (HCC) using a new ST6Gal I-specific mAb and recombinant fusion proteins of CD22 and sialoadhesin recognizing alpha2,6- or alpha2,3-sialylated glycans in immunohistology and flow cytometry. In normal and cirrhotic liver, ST6Gal I and sialoglycans were localized in the Golgi region of hepatocytes surrounding the bile canaliculi and along the bile canaliculi, respectively. Sialoglycans were additionally recognized in Kupffer cells, bile ducts, endothelial cells, and oval cells. Well-differentiated and moderately differentiated HCC showed Golgi and diffuse cytoplasmic staining of ST6Gal I and sialoglycans, whereas the cytoplasmic staining for ST6Gal I and sialoglycans was decreased or even absent in poorly differentiated HCC. Detection of sialoglycans by the recombinant fusion proteins in Western blots of cell lysates derived from cell lines revealed two major double bands of sialoglycoproteins at 65 and 120 kDa for hepatocytes, three major bands at 54, 49, and 44 kDa for colonic epithelial cells, and one band at 60 kDa for endothelial cells. Our results describe the expression patterns of ST6Gal I and sialoglycans in various liver tissues and demonstrate an altered expression of these structures between benign and malignant hepatocellular lesions.     

Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells

The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell-substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC.     

Nuclear translocation of survivin in hepatocellular carcinoma: a key to cancer cell growth?

Survivin is a recently described anti-apoptosis protein and regulator of cell division. Its expression and localization in hepatocellular carcinoma (HCC) and in normal liver tissue has not been fully elucidated. We examined the expression of survivin, Fas, proliferating cell nuclear antigen (PCNA), and apoptosis in 47 specimens of hepatocellular carcinoma (HCC) and surrounding nonmalignant hepatic tissues. To further determine the relationship between survivin expression and cell proliferation and apoptosis, we performed double immunostaining for survivin and PCNA TUNEL staining in the same HCC specimens. Positive immunostaining for survivin was present in 35 of 47 (74%) HCCs. Twenty-two of 35 survivin-positive HCCs (63%) showed punctate nuclear staining in HCC cells, and the remaining 13 showed predominant cytoplasmic staining. In contrast, nonmalignant hepatocytes showed only cytoplasmic staining. HCC cells had significantly higher PCNA-labeling and apoptotic indices compared with the case of nonmalignant hepatic tissue (P<0.001). Furthermore, nucleus-positive HCC specimens for survivin showed the highest PCNA labeling index. The nuclear localization of survivin in HCC cells correlated with tumor cell de-differentiation with the exception of the HepG2 cell line. Survivin expression was inversely associated with apoptosis and was strongly associated with Fas expression (P=0.01). All 4 HCC cell lines examined showed survivin expression and punctate nuclear localization. Our results indicate that survivin is localized to the cytoplasm in quiescent nonmalignant liver cells to suppress apoptosis and translocates into the nucleus in HCC cells. In conclusion, translocation of survivin from the cytoplasm to the nucleus may constitute an important regulatory mechanism for cell proliferation and differentiation in HCC.