Evaluation on expression of the recombinant S gene of human hepatitis B virus in vitro and in vivo

OBJECTIVES: To construct a DNA vaccine capable of expressing the S gene of hepatitis B virus (HBV)and evaluate the expression of the recombinant S gene in vitro and in vivo.METHODS: A cloned S-X gene fragment was inserted into an eukaryote expression vector to construct arecombinant expressing plasmid pCMV-SX. The recombinant plasmid was transcribed in vitro with a T7promoter transcription system and transfected into a human hepatoblastoma cell line HepG2. Theexpression of the S gene was detected by Northern blot hybridization, Western blot hybridization, andenzyme-linked immunosorbent assay (ELISA), respectively. BALB/c mice were inoculated with therecombinant plasmid, and the efficiency of DNA-based immunization in eliciting anti-HBs was evaluatedby ELISA.RESULTS: In vitro transcription of the subcloned HBV S gene was confirmed by Northern blothybridization. The results of Western blot hybridization and ELISA showed that the S gene was expressedexactly in HepG2. In immune experiment, 2 of 10 immunized mice were shown to induce antibody againstHBsAg.CONCLUSION: The recombination and expression of the S gene can be achieved successfully in vitro.And the recombinant plasmid is able to elicit humoral immune response in mice.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

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Host cellular micro RNA involvement in the control of hepatitis B virus gene expression and replication

A large number of studies have demonstrated that the synergistic collaboration of a number of micro RNAs(mi RNAs), their growth factors and their downstream agents is required for the initiation and completion of pathogenesis in the liver. mi RNAs are thought to exert a profound effect on almost every aspect of liver biology and pathology. Accumulating evidence indicates that several mi RNAs are involved in the hepatitis B virus(HBV) life cycle and infectivity, in addition to HBVassociated liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma(HCC). In turn, HBV can modulate the expression of several cellular mi RNAs, thus promoting a favorable environment for its replication and survival. In this review, we focused on the involvement of host cellular mi RNAs that are directly and indirectly associated with HBV RNA or HBV associated transcription factors. Exploring different facets of the interactions among mi RNA, HBV and HCV infections, and the carcinogenesis and progress of HCC, could facilitate the development of novel and effective treatment approaches for liver disease.

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells

Objective: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells. Methods: Polymerase chain reaction (PCR) was per- formed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR prod- uct was cloned into the pGEM-T vector for sequen- cing. After being identified, the HBV preSl gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expres- sive plasmid pGBKT7 to constructe pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl suifate-polyacrylamide gel electro- phoresis(SDS-PAGE) and Western blotting. Results: The HBV preS1 gene was amplified success- fully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blot- ting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecu- lar weight of the expression product was about 30 000 D. Conclusion: The HBV preS1 gene was successfully cloned and expressed in yeast cells.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

瞄准：与丙肝的长期的复制建立一个房间文化系统病毒(HCV ) 染色体和病毒的抗原的表示在试管内。方法：HepG2 房间线被孵化与长期的丙肝从一个病人与浆液为它的危险性测试到 HCV。房间和上层清液在文化期间在各种各样的时间点被收获。文化上层清液为它感染天真的房间的能力被测试。存在减(反感觉) 在房间的核心和 E1 抗原的 RNA 海滨，和察觉被 RT-PCR 和免疫学的技术(流动血细胞计数和西方的污点) 分别地检验。结果：细胞内部的 HCV RNA 首先在 d 上被检测 3 在感染以后然后能一致地在至少三个月的一个时期上在房间和上层清液被检测。新鲜房间能从有教养的感染的房间感染上层清液。流动 cytometric 分析证明表面和在房子里使用的细胞内部的 HCV 抗原表示使 polyclonal 成为了抗体(反核心，和 anti-E1 ) 。西方的污点分析证明在分子量的产生免疫性的肽的簇的表示在一个月内在 31 和 45 kDa 之间延长了感染的房间的旧文化而这簇在 uninfected HepG2 房间是无法发现的。结论：HepG2 房间线产生 HCV 感染而且支持它的复制在试管内不仅。HCV 结构的蛋白质的表示能在感染的 HepG2 房间被检测。这些房间也能够流病毒的粒子进接着对 uninfected 房间变得传染的培养基。

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Expression of core gene cDNA of Chinese hepatitis C virus in HepG2 cell line

Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.

Gene expression of NS5a strong antigenic regions of hepatitis C virus in E. coli and detection of their antigenicity

Objective: To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic re- gion at position 2212-2313 by using recombinant proteins. Methods: Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthe- tic genes encoding for this antigenic region, These genes were assembled by PCR from synthetic olionu- cleotides and expressed in E. coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6. Results: A comparison of sequences derived from dif- ferent HCV genotypes showed that the primary struc- ture of this strong antigenic region is highly variable. Percent homology between different genotype se- quences varied from 40. 4% to 72. 5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype spe- cific antigenic reactivity was detected, only one pro- tein demonstrated a noticeable preference to immu- noreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immuno- reacted with significantly different efficiency with HCV antibodies. Conclusions: Different genotype HCV genes were successfully cloned, expressed and purified. Se- quence variability has a profound effect on the anti- genic properties of the HCV NS5a immunodominant region. This finding should be considered in the de- velopment of diagnostic tests for the efficient detec- tion of anti-HCV activity in serum specimens.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

HBeAg gene expression with baculovirus vector in silk worm cells

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Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B

AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinaseactivity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice.In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2)was detected in 21 of 23 patients (91.30%)with severe AOC hepatitis B, while only 1 of 13 patients(7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1of 10 with mild chronic hepatitis B had detectable hfgl2expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2protein play a pivotal role in initiating severe hepatitis.The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Establishment and primary application of a mouse model with hepatitis B virus replication

瞄准：与肝炎 B (HBV ) 复制建立一个快速、方便的动物模型。方法：HBV-replication-competent 原生质标志的一个裸体 DNA 解决方案经由尾巴静脉被转移到 BALB/C 鼠标，用一个水动力学在活体内 transfection 过程。在注射以后，这些老鼠在 d 上被牺牲 1， 3， 4， 5， 7 和 10。在肝的 HBV DNA 复制中介被南部的污点杂交分析。肝炎 B 核心抗原(HBcAg ) 和在肝的肝炎 B 表面抗原(HBsAg ) 的表示被免疫组织化学检查。浆液 HBsAg 和肝炎 B e 抗原(HBeAg ) 被连接酶的免疫吸着剂试金(ELISA ) 检测。HBV 复制的抑制在与 polyinosinic-polytidylin 酸(polyIC ) intraperitoneally 对待的 HBV 复制模型老鼠被比较或缓冲磷酸盐 saline (PBS ) 。结果：在水动力学在活体内 transfection 以后，在老鼠肝的 HBV DNA 复制中介在 d 上是可检测的 1 并且在 d 上丰富 3 和 4，层次稍微被减少并且在 d 之间仍然保持相对稳定 5 和 7，并且在 d 上是几乎无法发现的 10。HBcAg 和 HBsAg 的表示模式类似于 HBV 复制中介 DNA 的，除了他们在 d 上到达了一座山峰之外 1 在注射以后。在 HBV DNA 复制中介的明显的差别都没在肝的左、正确、中间的脑叶被观察。有 polyIC 的术后疗法，在肝的 HBV 中间的 DNA 的水平在与 PBS 注射的控制老鼠是比那低的。结论：有 HBV 复制的高水平的一个快速、方便的老鼠模型被开发并且过去常在 HBV 复制上调查 polyIC 的禁止的效果，它为未来提供一个有用工具 HBV 染色体的功能的研究。

Inhibition of hepatitis B virus by oxymatrine in vivo

AIM To investigate the anti-HBV effect ofoxymatrine (oxy) in vivo.METHODS HBV transgenic mice were producedby micro-injection of a 4.2kb fragmentcontaining the complete HBV genomes.Expression level of HBsAg and HBcAg in thetransgenic mice liver was determined byimmunohistochemical assay.RESULTS Four groups (6 mice in each group)were injected intraperitoneally with oxy at thedosage of 100,200, and 300 mg/kg or with salineonce a day for 30 days. Both HBsAg and HBcAgwere positive in livers of all the six mice in thecontrol group (injected with saline), and werepositive in livers of two mice in 100 mg/kg groupand 300mg/kg group. In 200mg/kg group,HBsAg and HBcAg were negative in livers of allthe six mice. Based on the results, 200 mg/kg isthe ideal dosage to explore the effect of oxy atdifferent time points. According to the oxytreatment time, mice were divided into fourgroups: 10 d, 20 d, 30 d and 60 d (4 mice in eachgroup). Each mouse underwent liver biopsy twoweeks before the treatment of oxy. Down-regulation of HBsAg and HBcAg appeared aftertreatment of oxymatrine for 10 d and 20 d, Dane-like particles disappeared after the treatment ofoxy for 20d under electron microscopy,however, the expression level of HBsAg andHBcAg returned to normal 60 d later after oxytreatment.CONCLUSION oxymatrine can reduce thecontents of HBsAg and HBcAg in transgenic miceliver, longer treatment time and larger dosagedo not yield better effects.

Changes in gene expression in liver tissue from patients with fulminant hepatitis E

AIM: To study host gene expression and numberof immune cells in liver tissues from patients with fulminant hepatitis E(FH-E).METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_Bead Chip arrays on post-mortem liver tissue from 5 patients with FH-E,and compared with similar tissue from 6 patients with fulminant hepatitis B(FH-B; disease controls) and normal liver tissue from 6 persons.Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E,than healthy liver tissue.For some genes that showed differential expression in FH-E,microarray data were validated using quantitative reverse transcription PCR.Differentially expressed gene lists were then subjected to "Gene Ontology" analysis for biological processes,and pathway analysis using Bio Carta database on the DAVID server.In addition,tissue sections were stained for CD4+,CD8+ and CD56+ cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups.RESULTS: Compared to normal livers,those from patients with FH-E and FH-B showed differential expression of 3377 entities(up-regulated 1703,downregulated 1674) and 2572 entities(up 1164,down 1408),respectively.This included 2142(up 896,down 1246) entities that were common between the two sets; most of these belonged to metabolic,hemostatic and complement pathways,which are active in normal livers.Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways,particularly those involving cytotoxic T cells.The fold-change values of m RNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis.At immunohistochemistry,CD8+ T cells showed an increase in liver biopsies from both FH-E [median 53.4 per arbitrary unit area(range 31.2-99.9)] and FH-B [median 49.3(19.3-51.0); P = 0.005] compared to control liver tissue [median 6.9(3.1-14.9)].CONCLUSION: FH-E patients show CD8+ T cell infiltration and increased gene expression of cytotoxic T cell pathways in liver,suggesting a possible pathogenetic role for these cells.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

瞄准：为了表明使用土拨鼠的可行性，在人的微数组上取样，到提供卓见进包含正电子排放断层摄影术(宠物) 的小径成像 tracers 并且分子的成像为土拨鼠肝细胞癌指向识别能潜在的基因。方法：从土拨鼠织物样品的标记的 cRNA 是到加 2.0 GeneChips 的 Affymetrix U133 的 hybridized。十基因被选择因为用量的 RT-PCR 和文学评论的确认被做。结果：睾丸提高了基因抄本(BAX 禁止者 1 ) ， alpha-fetoprotein， isocitrate 脱氢酶 3 (NAD+) 贝它， acetyl-CoA 合成酶 2，肉毒碱 palmitoyltransferase 2，并且 N-myc2 是起来调整的，亚精胺 / 精胺 N1-acetyltransferase 在土拨鼠 HCC 是下面调整的。我们也发现了以前出版的结果支持 10 很起来调整的基因中的 8 个和 10 很下面调整的基因中的所有 10 个。结论：许多用 RT-PCR 或文学搜索我们的微数组结果被验证。因此，我们相信土拨鼠 HCC 和非癌的肝样品能在人的微数组上被使用产出有意义的结果。