HLA classⅠexpression in primary hepatocellular carcinoma

AIM：To investigate whetherCTL vaccine therapy is suitable for primary hepatocellular carcinoma(HCC)from the viewpoint of HLA classIantigens expression.METHODS:The immunocytochemistry,image analysis,flow cytometry,and labeled streptavidin bioti(LSAB)method of immunohistochemistry were applied respectively to study 4HCCcell lines(e.g.Alexander,HepG2,SMMC-7721,andQGY-7703)cultured in vitro and 6frozen tissue specimens of HCC.RESULTS:The positive control cell line Raji had very strong positive staining,Most mitotic and nonmitotic cells of the 4HCCcell lines had various intensity of HLAclassⅠantigens expression.The negative control cell K562 and the control slides of all the cell lines had no positive staining,In the 6HCCspecimens immunohistochemically studied,histological normal hepatocytes had no or very weak positive staining and the liver sinus had very strong positive staining.Most HCCcells in the sections from the 6HCCspecimens had strong positive HLAclassⅠantigens staining.The positive staining was located in the cytoplasm,the perinuclear area,and at the cell membrane of the liver cancer cells.Flow cytometry also revealed that Raji and those 4HCCcell lines had strong HLAclassⅠantigens expression.which was confirmed quantitatively by the image analysis.It showed that the objective grayscale values of Raji and those 4HCCcell lines were significantly different from that of K562(Raji114.04±10.94,Alexander165.97±5.35,HepG2167.02±12.60,QGY-7703161.46±7.13,SMMC-7721 165.93±5.21,K562244.89±4.60,P<0.01).Significant differences were also found ebtween Raji and the 4HCCcell lines.CONCLUSION:HCC cells express HLA classI antigens strongly,Fromthis point of view.the active specific immunotherapy of CTL vaccine is suitable and practicable for HCC.     

Treatment of human hepatocellular carcinoma by fibroblast-mediated human interferon α gene therapy in combination with adoptive chemoimmunotherapy

The therapeutic effect of the fibroblast-mediated human interferon (IFN) gene therapy in combination with interleukin-2 (IL-2) activated killer cells (AK)/doxorubicin (i.e., adoptive chemoimmunotherapy) on nude mice bearing the human hepatocellular carcinoma (HCC) was investigated. A fibroblast cell clone (NIH3T3-IFN⁺) secreting 1024 U/ml human IFN was obtained from 14 positive clones by BMGNeo-INF DNA transfection, G418-resistant selection, limiting dilution and assay of IFN activity. After i.p. implantation of NIH3T3-IFN⁺ encapsulated into collagen, serum human IFN activity could be detected from 12 h to day 15 with a peak at 72 h. AK were prepared from human peripheral mononuclear cells costimulated in vitro by IL-2 and inactivated human SMMC 7721 HCC cells. When the NIH3T3-IFN⁺ cells were i.p. implanted into the HCC-bearing nude mice, the grown of HCC was inhibited and the survival time of the mice was extended. The growth of HCC was inhibited more obviously when AK was i.v. injected and IL-2 was i.p. injected after the NIH3T3-IFN⁺ cells had been implanted. The best therapeutic effect was achieved when NIH3T3-IFN⁺ cells were used in combination with IL-2/AK/doxorubicin. All these results suggested that the fibroblast-mediated human IFN gene therapy could be used to treat the human hepatocellular carcinoma effectively and that when used in combination with IL-2-based adoptive chemoimmunotherapy, the therapeutic effect would be better.Abbreviations IFN interferon - HCC hepatocellular carcinoma - IL-2 interleukin-2 - AK activated killer cells - Dox doxorubicin This research was supported by the National Natural Sciences Foundation of China (grant 39421009)     

Alpha-fetoprotein stimulated the expression of some oncogenes in human hepatocellular carcinoma Bel 7402 cells

AIM:To investigate the molecular mechanism of alpha-fetoprotein (AFP) on regulating the proliferation of humanhepatocellular carcinoma cells.METHODS:Alpha-fetoprotein purified from human umbilicalblood was added to cultured human hepatocellular carcinomaBel 7402 cells in vitro for various treatment periods.Theexpression of c-fos,c-jun,and N-ras mRNA involved inproliferation and differentiation of cells was analyzed byNorthern blot,and the expression of mutative p53 and p21~(ras)proteins was determined by Western blot.RESULTS:The results showed that AFP (20 mg/L) stimulatedmRNA expression of these oncogenes in Bel 7402 cells.Theexpression of c-fos mRNA increased by 51.1%,60.9%,96.0%,and 25.5% at 2,6,12,and 24 h,respectively.Theexpression of c-junand N-ras mRNA reached to the maximumwhich increased by 81.3% and 59.9% as compared withthe control after 6 h and 24 h incubation with AFP,respectively.Western blot assay also demonstrated that AFP promotedthe expression of mutative p53 and p21~(ras) proteins,and theincreased rate of those proteins was 13.0%,39.9%,and70.9%,as well as 35.2%,102.6%,and 46.8% at 6,12,and24 h,respectively,as compared with the control.Both humanserum albumin (the same dosage as AFP) and monoclonalanti-AFP antibody failed to stimulate the expression of theseoncogenes,but anti-AFP antibody could block the functionsof AFP.CONCLUSION:The data indicate that AFP can stimulate theexpression of some oncogenes to enhance the proliferationof human hepatocellular carcinoma Bel 7402 cells.     

Construction of retroviral vector containing HSV-tk gene for colorectal carcinoma tissue-specific gene therapy

ＩＮＴＲＯＤＵＣＴＩＯＮＨｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ（ＨＳＶｔｋ）ｇｅｎｅｉｓｔｈｅｅｎｃｏｄｉｎｇｇｅｎｅｏｆｖｉｒｕｓｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ．Ｃａｔａｌｙｚｅｄｂｙｈｅｒｐｅｓｖｉｒｕｓｔｈｙｍｉｄ...     

Significance of cyclooxygenase—2 expression in human primary hepatocellular carcinoma

AIM：To clarife the significance of cyclooxygenase-2(COX-2)expression in human primary hepatcellular carcinoma(HCC)and adjacent nontumorous tissues.METHODS;TheCOX-2protein and mRNA were investigated in 27HCC tissues with adjacent nontumorous tissues,and 5histologically normal liver tissues,using immunohistochemistry and in situ hybridization.RESULTS:The well-differentiated HCC expressed COX-2protein(5.68±1.19)more strongly than moderated HCC(3.43±1.98)and poor differentiated HCC(3.33±1.50)(P<0.05 respectively),adjacent nontumorous tissues(4.93±1.05)and normal live tissues(3.20±1.92)(P<0.01 respectively);More intensive staining of COX-2in adjacent nontumorous tissues was observed than that in normal liver tissues(P<0.05).There was no significant difference among adjacent nontumorous tissues,moderately differentiated HCC and poorly differentiated HCC(P>0.05).The expression of COX-2mRNA was observed in the cytoplasm of the cells of HCC and of gtthe hepatocytes in adjacent nontumorous tissues in which COX-2 protein was positive.CONCLUSION:The overexpression of COX-2 in well-differentiated HCsuggets that COX-2 may play a role in the early stages of hepatocarcinogensis.     

Construction of human colorectal carcinoma-specific retroviral vector containing cytosine deaminase gene

ＩＮＴＲＯＤＵＣＴＩＯＮＣｙｔｏｓｉｎｅｄｅａｍｉｎａｓｅ（ＣＤ），ａｋｅｙｅｎｚｙｍｅｏｆｔｈｅＤＮＡｐｙｒｉｍｉｄｉｎｅｒｅｍｅｄｉａｌｐａｔｈｗａｙｉｎｆｕｎｇｉａｎｄｓｏｍｅＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ，ｃａｎｔｒａｎｓｆｏｒｍ５ｆｌｕｏ...     

Effect of blocking IGF-Ⅰ receptor on growth of human hepatocellular carcinoma cells

AIM: To study the expression level and localization of insulin-like growth factor -I receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells. METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of aIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1μg/mLαIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P<0.01). However, the aIR3 for 24 h at final concentration of 4.0μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P < 0.01). Compared with control, treated with aIR3 for 48 h at final concentrations ranging from 1.0μg/mL to 4.0μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%, 92.53% vs 100%, P < 0.05 or P < 0.01), treated withαIR3 for 72 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P<0.01), and treated with aIR3 for 96 h at final concentrations ranging from 0.5μg/mL to 4.0μg/mL made GIs of HepG2 cells lower significantly (88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P<0.05 or P<0.01). Moreover, treated withαIR3 from 24 h to 96 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5μg/mL to 2.0μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P<0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P<0.01), in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P< 0.01). CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF-IR. The blockage of IGF-IR with aaaaaIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.     

YKL-40 expression in human hepatocellular carcinoma:a potential biomarker?

BACKGROUND:YKL-40 is a new biomarker with diagnostic value in many different cancers.Whether it may serve as a biomarker for hepatocellular carcinoma (HCC) is still unclear.This study aimed to examine the expression of YKL-40 in the serum and liver tissues of HCC patients and in HCC cell lines,in comparison with that in non-HCC liver disease patients and non-tumor hepatic cell lines,respectively.METHODS:Immunohistochemical staining was used to detect YKL-40 protein expression in liver biopsy specimens from ...     

Transduction of human hepatocellular carcinoma cells with human alpha-interferon gene via retroviral vector

AIM:To investigate the therapeutic potential of gamma interferon (IFN-alpha) genemodified human hepatocellular carcinoma (HCC) cells.METHODS:The IFN-alpha gene was introduced retrovirally into four HCC cell lines.Secreted IFN-alpha activity was assessed using bioassay. The expression of MHC molecules was detected by FACS.Tumorigenicity was analysed by tumor formation in nude mice.RESULTS:Four IFN-alpha gene transduced HCC cell lines secreted different amounts of IFN-alpha, as in the same case of five clones derived from one HCC cell line. Transduction with IFN-alpha caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class I was increased by 2-3 times in terms of mean fluorescence intensities, while for class II expression, the percentage of positive cells augmented from < 10% to &lg 50%. When equal amount of tumor cells were injected into nude mice, the tumor igenicity some transduced cells decreased dramantically.CONCLUSION:IFN-alpha gene transduction can convert weakly imunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.     

肝细胞癌mdm2基因表达及其与p53基因突变的关系

目的研究ｍｄｍ２基因在原发性肝细胞癌（ＨＣＣ）中的表达并探讨其与ｐ５３基因突变的关系．方法用银染ＰＣＲＳＳＣＰ法检测ｐ５３基因第５～８外显子的突变,原位杂交检测ｍｄｍ２基因ｍＲＮＡ的表达,ＳＡＢＣ法检测ｍｄｍ２蛋白的表达．结果３９３％（１１／２８）的病例有异常的电泳迁移率．ｐ５３基因突变与肿瘤的大小、分化及转移无关．原位杂交显示９例ＨＣＣ出现ｍｄｍ２基因ｍＲＮＡ增加,７例ＨＣＣ可检测到ｍｄｍ２蛋白表达,ｍｄｍ２基因表达与ＨＣＣ的大小、分化及是否转移无关．Ⅰ～Ⅱ级ＨＣＣ中ｍｄｍ２阳性表达率（１３３％）明显低于Ⅲ～Ⅳ级ＨＣＣ中的阳性表达率（５３８％）．１１例有ｐ５３基因突变的ＨＣＣ中,只有３例出现ｍｄｍ２基因表达,另外６例有ｍｄｍ２过表达的ＨＣＣ未见ｐ５３基因突变．ｐ５３基因突变的ＨＣＣ与ｐ５３基因无突变的ＨＣＣ相比,ｍｄｍ２基因表达阳性率无显著差别．结论ｐ５３基因突变和ｍｄｍ２基因表达在原发性ＨＣＣ的发病中起重要作用．ｍｄｍ２基因表达与ＨＣＣ的恶性程度相关．ｍｄｍ２基因表达与ｐ５３基因是否突变无关．     

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

AIM:To study the relationship between N-ras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS:The N-ras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry.RESULTS:Thirteen cases of HCCs were p53 positive (44.8%), which showed a rather high Cpercen-tage of p53 gene mutation in Guangxi. The aberrations at N-ras codon 2-37 were found in 79.31% of HCCs and 80.77% of adjacent non-tumorous liver tissues. More than 2 point mutations of N-ras gene were observed in 22 cases (75.86%). Twelve cases (41.37%) of HCCs showed both N-ras gene mutation and p53 gene expression.CONCLUSION:N-ras gene and p53 gene may be involved in the carcinogenesis and the development of HCC.That 38% of HCCs with N-ras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.     

肝细胞癌中耐药基因的表达及意义

目的通过对肝细胞癌（HCC）中3种耐药基因MDR1、MRP、GST-π的检测,探讨肝细胞癌中耐药基因的表达特点及临床意义。方法应用逆转录-聚合酶链反应（RT-PCR）技术检测3种耐药基因在51例肝细胞癌组织和10例正常肝组织中的表达。结果 （1）MDR1、MRP、GST-π在肝细胞癌中的表达分别为0.55±0.27、0.62±0.29、0.64±0.31,正常肝组织中的表达分别为0.23±0.10、0.25±0.07、0.26±0.12,耐药基因在肝细胞癌中的表达高于正常肝组织,差异具有统计学意义（P〈0.05）;（2）耐药基因的表达与肿瘤Edmondson分级呈正相关（P〈0.05）;（3）MRP与GST-π的表达相关。结论肝细胞癌中存在有原发性耐药的现象,且多种机制并存。MDR1、MRP、GST-π在肝细胞癌中有较高的表达。联合检测对制定科学有效的治疗方案有一定价值。     

Construction of eukaryotic expression vector of HBV x gene

ＩＮＴＲＯＤＵＣＴＩＯＮＣｈｒｏｎｉｃｉｎｆｅｃｔｉｏｎｗｉｔｈｈｅｐａｔｉｔｉｓＢｖｉｒｕｓｉｓｃｌｏｓｅｌｙｒｅｌａｔｅｄｔｏｌｉｖｅｒｄｉｓｅａｓｅｓ,ｉｎｃｌｕｄｉｎｇｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａ．ＨｅｐａｔｉｔｉｓＢｖｉｒｕ...     

肝细胞癌组织中PTEN蛋白表达

探讨抑癌基因PTEN在肝细胞癌 (HCC)组织及癌旁组织的表达、临床意义。采用免疫组织化学SP法检测PTEN。 4例正常肝组织均呈PTEN蛋白阳性 ;HCC及其癌旁肝组织中的阳性率分别为 5 8 8%(2 0 / 34)和 10 0 %(34/ 34) ,两者比较差异有显著性 (P <0 0 5 )。中分化癌阳性率为 77 8%(14 / 18) ,低分化阳性率为 2 5 %(3/ 12 ) ,两者比较差异有显著性 (P <0 0 0 1)。PTEN蛋白表达与年龄、性别、肿瘤大小、有无包膜及门脉癌栓均无明显关系 (P >0 0 5 ) ,但与HCC分化程度明显相关 ,HCC分化愈差 ,PTEN蛋白表达愈弱。PTEN蛋白表达与HCC分化程度明显相关。