肝筛、脂质代谢与肝硬化

脂质代谢异常是肝纤维化和肝硬化的发病机理之一。肝筛对脂质代谢具有严格的选择性,肝筛结构和功能的变化可引起脂质代谢障碍导致肝纤维化、肝硬化。本文阐述肝筛理论及在分子生物学水平上的研究,以期为肝纤维化和肝硬化的治疗提供更新、更有价值的方案。

论肝窦内皮窗孔异常是肝脾失调致动脉粥样硬化的微观病机

脾主健运与肝主疏泄在生理上相辅相成,协同运化精微,与现代解剖学的肝脏(在胁之肝)功能密切相关。肝脏脂质代谢紊乱是肝脾不调致动脉粥样硬化(AS)的重要原因,与肝窦内皮窗孔的结构和功能异常相关。因此提出假说:解剖学的肝脏可能是肝脾不调致AS的靶器官,肝窦内皮窗孔形成的肝筛功能异常是脂质代谢紊乱的重要因素,肝窦内皮窗孔异常可能是肝脾不调致AS的超微结构之一。

一氧化氮、内皮素-1在内毒质所致肝脏损伤中的作用机制研究

目的 探讨一氧化氮 (NO)和内皮素 -1(ET-1)在内毒素所致肝脏损伤中的作用机制。方法 Waster大鼠 40只随机分成对照组、内毒素组、内皮素受体拮抗剂组和左旋精氨酸组 ,观察各组大鼠血浆 NO和 ET-1水平、肝脏功能变化 ,在光镜下观察肝细胞损伤程度 ,在电镜下观察肝脏超微结构改变。结果 大鼠投予内毒素 12 h后 ,血浆 NO和 ET-1水平明显下升 ,血浆转氨酶水平增加。肝细胞明显水肿、变性和坏死 ,肝筛的数量和直径明显减少。左旋精氨酸和内皮素受体损坏抗剂均能明显减轻肝脏功能和结构的损伤。结论 在内毒素所致肝损伤中 ,NO起保护性作用 ,而 ET-1则起促进作用 。

一氧化氮和内皮素-1在淤胆性肝纤维化中的作用机制

目的 :探讨一氧化氮 (NO)和内皮素 1(ET 1)在淤胆性肝纤维化中的作用机制。方法 :将Wistar大鼠 4 8只 ,随机分成对照组、胆总管结扎组和胆总管结扎 +内毒素拮抗剂组 ,观察术后 3d、7d、14d和 2 1d血浆内毒素、NO和ET 1水平 ,肝脏结构和功能的变化。结果 :胆总管结扎致胆汁淤积后 ,随着血浆内毒素水平升高 ,NO和ET 1水平相应上升 ,血清AST和ALT也明显升高。肝纤维化程度随梗阻时间逐渐加重 ,电镜下见肝窦变窄 ,Disse间隙增宽 ,内有大量胶原纤维沉积 ,肝筛的数量和直径均明显减少。应用乳果糖能部分逆转肝脏功能和结构的病理改变。结论 :NO和ET 1在淤胆性肝纤维化进程中具有重要作用 ,二者可能是通过肝筛而发挥作用的。

Activity Determination of 8 Chinese Herbs against Hepatoma Cell SMMC-7721 in Vitro by MTT Method

[Objective] The aim was to build up a set of efficient and rapid models for laboratory to screen anti-hepatocellular carcinoma active substance in vitro. [ Method] By using MTT method, the activities of anti-hepatocellular carcinoma SMMC-7721 in vitro from Cymbopogon distans, Lobelia chinensis, Buddleja offlcinalis, Glycyrrhiza uralensis, Sanguisorba officinalis, Bupleurum chinense, Apium graveolen and Curuma zedoaria were tested. The growth curve of hepatoma cell was described, and the growth status in different periods were observed by inverted microscope. [ Result] Cells induced by active substance would be condensing, clear brim, which have significant differences from normal SMMC- 7721 cells. The results suggested that ESCG, ESCC, ESCB could inhibit proliferation of SMMC-7721 cells at the concentration of 1.0 -1.5 mg/ml, and the inhibition rate were 51.6%, 48.5%, 52.9% respectively. With the increasing of concentration, the inhibition strengthened. [ Conclusion] MTT method could be used as a basic model for screening important anti-hepatoma.

Screening in liver disease

A disease is suitable for screening if it is common, if the target population can be identifi ed and reached and if both a good screening test and an effective therapy are available. Of the most common liver diseases only viral hepatitis and genetic hemochromatosis partially satisfy these conditions. Hepatitis C is common, the screening test is good and the therapy eliminates the virus in half of the cases, but problems arise in the definition of the target population. In fact generalized population screening is not endorsed by international guidelines, although some recommend screening immigrants from high prevalence countries. Opportunistic screening (case finding) of individuals with classic risk factors, such as transfusion before 1992 and drug addiction, is the most frequently used strategy, but there is disagreement whether prison inmates, individuals with a history of promiscuous or traumatic sex and health care workers should be screened. In a real practice setting the performance of opportunistic screening by general practitioners is low but can be ameliorated by training programs. Screening targeted to segments of the population or mass campaigns are expensive and therefore interventions should be aimed to improve opportunistic screening and the detection skills of general practitioners. Regarding genetic hemochromatosis there is insufficient evidence for population screening, but individual physicians can decide to screen racial groups with a high prevalence of the disease, such as people in early middle age and of northern European origin. In the other cases opportunistic screening of high risk individu-als should be performed, with a high level of suspicion in case of unexplained liver disease, diabetes, juvenile artropathy, sexual dysfunction and skin pigmentation.

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to this cell substrate to maintain high growth characteristics.By serial passages in Vero cells,the B/Yunnan/2/2005va(B)strain was successfully adapted to Vero cells,with the hemagglutination titer(HAT)of the virus reaching 1:512.The high growth characteristic of this strain is stable up to 21 passages.The strain was identified by hemagglutination inhibition (HAI)test and sequencing respectively;the HA1 gene sequence of the virus was cloned and analyzed.The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.

Screening and cloning for proteins transactivated by the PS1TP5 protein of hepatitis B virus:A suppression subtractive hybridization study

AIM： To clone and identify human genes transactivated by PSITP5 by constructing a cDNA subtractive library with suppression subtractive hybridization （SSH） technique. METHODS： SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1（-）- myc-his（A）-PS1TP5 and pcDNA3.1（-）-myc-his（A） empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa Ⅰ, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subb-active library. Amplification of the library was carried out with E.. coil strain DH5α. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification. RESULTS： The subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown.One novel gene with unknown functions was found and named as PSITP5TP1 after being electronically spliced, and deposited in GenBank （accession number： DQ487761）. CONCLUSION： PSITP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PSlTP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

Objective： Hepatocellular carcinoma （HCC） is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods： Lectin affinity chromatography （LAC） was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography （LC） separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results： A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain （FGG）, FOS-like antigen 2 （FOSL2）, and a-l, 6-mannosylglycoprotein 6-^-N-acetylglucosaminyltransferase B （MGATSB） were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion： A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC.

Perspectives of physicians regarding screening patients at risk of hepatocellular carcinoma

Background and aims:Screening patients at risk for hepatocellular carcinoma(HCC)facilitates early detection of disease,with improved outcome.The most common causes of HCC include chronic viral hepatitis infection—namely hepatitis B,hepatitis C,and cirrhosis.The aim of this study was to assess the awareness of screening among physicians involved in the management of patients at risk for HCC.Methods:Three hundred physicians from three academic centers were invited to participate in a mailed survey questionnaire.The main outcome measure was physicians’knowledge of the current HCC screening guidelines.Demographic and clinical variables were obtained from the survey questionnaire.Results:A total of 177(59.0%)out of the 300 invited physicians responded to the survey questionnaire,including faculty members(n=129),residents(n=46),and fellows(n=2).The specialty areas of the responding physicians were internal medicine(62.1%),family medicine(16.4%),gastroenterology(15.3%),oncology(3.4%)and others(2.8%).The number of physicians who performed HCC screening in patients with cirrhosis secondary to chronic hepatitis B and chronic hepatitis C infection were 163(92.1%)and 167(94.4%),respectively;35.0%of them used alpha-fetoprotein(AFP)every 6 months,while 22.0%used imaging modalities every 6 months to screen for HCC.Further,22 physicians(12.4%)did not check for serum AFP levels and 33(18.6%)never used imaging to screen for HCC.Conclusion:The majority of the participating physicians screen high-risk patients for HCC.However,the most appropriate modality of screening(i.e.imaging)is not employed by most physicians and there is greater reliance on AFP levels.

Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy

A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease,treatment decisions,and assessing drug efficacy.This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging.Pooled plasma from different liver fibrosis stages,which were assessed in advance by the current gold-standard of liver biopsy,was quantitatively analyzed.A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process,and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis.Validation results of fibronectin by Western blotting reconfirmed the mass-based data.Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients.Consequently,quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis.We quantitatively monitored the fibrogenesis process in CHB patients.We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease.These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.

Antigenically dominant proteins within the human liver mitochondrial proteome identified by monoclonal antibodies

Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.