Background and Aims: Continuous release and transmission of hepatitis B virus (HBV) is one of the main factors leading to chronic hepatitis B (CHB) infection.However, the mechanism of HBV-host interaction for optimal viral transport is unclear.Hence, we aimed to explore how HBV manipulates microtubule-associated protein 1S (MAP1S) and microtubule (MT) to facilitate its transport and release.Methods: The expression of MAP1S or acetylated MT was investigated by immunofluorescence, RT-PCR, immunoblotting, and plasmid transfection.MAP1S overexpression or knockdown was performed by lentiviral infection or sh-RNA transfection, respectively.HBV DNA was quantified using q-PCR.Results: Significantly higher level of MAP1S in HepG2215 cells compared with HepG2 cells was detected using RT-PCR (p<0.01) and immunoblotting (p<0.001).Notably, stronger MAP1S expression was observed in the liver tissues of patients with CHB than in healthy controls.MAP1S overexpression or knockdown demonstrated that MAP1S promoted MT acetylation and reduced the ratio of HBV DNA copies inside to outside cells.Further, transfection with the hepatitis B virus X protein (HBx)-expressing plasmids induced significantly higher level of MAP1S than that in controls (p<0.0001),whereas HBVX -mutant-encoding HBV proteins (surface antigen, core protein, and viral DNA polymerase) hardly affected its expression.Conclusions: These results demonstrate that HBx induces the forma-tion of stable MTs to promote the release of HBV particles through upregulating MAP1S.Thus, our studies delineate a unique molecular pathway through which HBV manipulates the cytoskeleton to facilitate its own transportation, and indicate the possibility of targeting MAP1S pathway for treatment of patients with CHB.

为了使研究生能更深刻地认识和理解聚合酶链反应技术,熟练掌握并灵活应用于自己今后的科研课题中,本文从实验原理、教学目标、教学过程、衍生实验及用途、实验相关注意事项、教学中发现的问题等方面进行详细阐述,并对研究生实验课带教工作进行了教学总结,对通过实验教学来提高医学研究生的科研能力进行了探索分析.

Integrins are transmembrane glycoproteins expressed on the surface of various cells. They can conduct bidirectional signal transduction across cell membranes, exchange information between extracellular matrix proteins and intracellular molecules, and regulate cell adhesion and activation. During cancer development, integrins mediate crucial regulatory functions in anti-tumor response including tumor antigen uptake, activation of tumor-specific T cells, leukocyte trafficking into the tumor site and tumor cell killing. We provided a comprehensive overview of the structure of integrins, immune regulation, effects of integrins on tumor immunity and covered in vivo and in vitro studies of tissue culture, animal models of human diseases and gene knockout animals as well as the progress in clinical diagnosis and therapy of tumors.

目的 构建血管内皮生长因子(VEGF)原核表达载体并诱导其表达蛋白,为后续迸行VEGF相关研究奠定基础.方法 通过逆转录聚合酶链式反应(RT-PCR)扩增得到目的片段,将DNA片段克隆至带组氨酸标签的pET-30a(+)载体上;构建的重组质粒经过菌液聚合酶链式反应(PCR)及测序方法鉴定;利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组质粒表达,通过蛋白质印迹法(Western blot)迸行鉴定.结果 以HepG2细胞为模板扩增出大小正确的片段;构建的pET-30a-VEGF重组质粒经菌液PCR及DNA测序证实序列正确;考马斯亮蓝染胶和Western blot结果显示IPTG诱导表达的融合蛋白分子量正确且条带单一.结论 成功构建pET-30a-VEGF原核表达质粒,后续可迸行大量诱导表达用于肝癌的诊疗相关研究.

Objective: To study the effect of apoptosis-stimulating protein 2 of p53 (ASPP2) on the activation and apoptosis of hepatic stellate cells induced by transforming growth factor-β1 (TGF - β1), and to explore the role of autophagy in this process. Methods: Mouse hepatic stellate cells were primarily isolated and cultured with green fluorescent protein (GFP) expressing empty vector adenovirus (Ad-GFP) and ASPP2 expressing adenovirus (Ad-ASPP2) for 12 h by transfection kit, and then treated with TGF-β1 (10ng/ml) for 24 h. The experiments were grouped as follows: control group: green fluorescent protein (GFP) expressing empty vector adeno (Ad-GFP); experimental group 1: transfected with Ad-GFP and added with TGF-β1; experimental group 2: transfected with Ad-ASPP2 and induced by TGF-β1. Western blot and quantitative fluorescence PCR were used to detect the expression of ASPP2, α-smooth muscle actin (SMA). At the same time, autophagy was determined by microtubule-associated protein 1 light chain 3-β (LC3). Autophagy and apoptosis of MHSc were observed by immunocytochemistry and RNA interference (RNAi). Multiple pairwise-comparisons between the mean of groups was performed by one-way ANOVA. Results: The relative expression of α-SMA mRNA in mHSC of TGF-β1 + Ad-GFP group (16.83 ± 2.41) was significantly higher than Ad-GFP group (3.62 ± 0.56) (P < 0.05), while the relative expression of α-SMA mRNA (4.22 ± 0.48) in TGF-β1 + Ad-GFP group was significantly lower than TGF-β1 + Ad-GFP group (P < 0.05). The expression of α-SMA protein in each group was consistent with mRNA expression. The proportion of mHSC autophagy in TGF-β1 + Ad-GFP group (80%) was significantly higher than Ad-GFP group (35%); however, there was no statistically significant difference between the two groups. The proportion of mHSC autophagy in TGF-β1 + Ad-ASPP2 group was 42%, which was significantly lower than TGF-β1 + Ad-GFP group, but the apoptotic rate was significantly increased. Cells were simultaneously treated with autophagy inhibitors 3-MA and TGF-β1. The level of autophagy was not statistically significantly different from that of TGF-β1 + Ad-ASPP2 group, but the apoptotic rate was increased. In addition, the RNAi group added with ASPP2 had increased autophagy (LC3-II/LC3-I) than control RNAi group, and the rate of apoptosis was significantly decreased. Conclusion: Overexpression of ASPP2 can alleviate the activation of mHSC and promote the apoptosis of HSC by inhibiting autophagy, so as to alleviate liver fibrosis.

Objective To evaluate the effect of tacrolimus combined with Bailing capsules on fasting blood glucose and renal function in patients after liver transplantation.Methods Clinical data of 73 patients who had liver transplantation from January 2007 to December 2011 in Beijing YouAn Hospital,Capital Medical University were analyzed.All patients were treated with tacrolimus and hormones in the first 3 months after surgery.Three months later,they were divided into control group(13 cases) treated with tacrolimus and observation group (60 cases) treated with tacrolimus plus Bailing capsules.Blood glucose,liver and kidney function indexes were analyzed 1,2,3 years after surgery.Results Levels of fasting blood glucose,urea nitrogen and creatinine in observation group were significantly lower than those in control group at 2 and 3 years after surgery(all P ＜ 0.05).In observation group,there was no significant changes of fasting blood glucose,blood urea nitrogen and creatinine levels in 1,2,3 years after surgery(all P ＞ 0.05);blood uric acid level at 3 years was significantly higher than that at 1 year after surgery [(389 ± 120) μmol/L vs (316 ± 118) μmol/L] (P ＜ 0.01).The total incidence of hyperuricemia and the incidence of hyperuricemia in males at 2,3 years were significantly higher than those at 1 year after surgery in observation group(all P ＜ 0.05).Conclusions Tacrolimus combined with Bailing capsules treating patients after liver transplantation can effectively increase blood urea nitrogen and creatinine levels,delay the progression of chronic renal failure,but it shows no significant effect on blood glucose and uric acid.The combined medication shows no obvious adverse reaction.

在高速发展的知识经济时代,成人继续教育成为我国教育体系中不可或缺的一个部分,而成人学生群体有自身的特殊性,如何根据学生的特点更加合理地设置课程内容、教学方法并增加互动式学习,直接影响学生的学习效率和学习效果.通过两年夜大学生的教学工作,结合学生的特点、学习目的及学习方式等方面总结个人的一些带教心得,在内容安排、授课方法、结课考试、课堂考勤几个方面浅谈了个人的一些体会,旨在提高本人夜大教学的带教质量,使学生能够学以致用,为今后的工作打下更夯实的基础.

Apoptosis-stimulating protein of p53-2 (ASPP2) is a damage-inducible P53-binding protein that enhances damage-induced apoptosis. Fibrosis is a wound-healing response, and hepatic stellate cells (HSCs) are key players in liver fibrogenesis. However, little is known about the relationship between ASPP2 and hepatic fibrosis.

Objectives: There is growing concern about mitochondrial DNA (mtDNA) mutations with long-term NRTI exposure in HIV-1 infected children. Methods: Twenty-four HIV-1 infected children who started ART more than 2 years earlier who had an excellent virological response and had not changed their regimen were enrolled retrospectively. Their corresponding PBMCs in 2009 (T1), 2010 (T2) and 2013 (T3) were included. Sequencing of the entire mtDNA using next-generation sequencing revealed the spectrum of mtDNA variants. Results: The trend showed that the number of mtDNA mutations during ART occurred as T1 < T2 < T3 (P=0.086). Interestingly, the numbers of whole mtDNA mutations at T3 (median 41, range 24-62) were significantly greater than at T1 (34, 25-46, P=0.029). A positive correlation was found between total mtDNA mutations and treatment time (r=0.352, P=0.002). During the observation period, mtDNA mutations more frequently occurred in the D-loop, cytochrome b (CYTB) and 12S rRNA regions. The heteroplasmic ratio of T3 was higher than that of T1 in CYTB and 12S rRNA (P=0.034 and P=0.042, respectively). High heteroplasmic population levels were found at nt 263 (A263G, D-loop) and nt 8860 (A8860G, ATPase6). A significant difference in heteroplasmy between T1, T2 and T3 occurred at nt 14783 (T14783C, CYTB, P=0.048, T3 > T2 > T1). Conclusions: Our findings reveal the spectrum of mtDNA variants in HIV-1-infected children who had an excellent virological response. mtDNA mutations accumulated during ART may play an important role in facilitating the occurrence of mitochondrial dysfunction.

We have analyzed mutations in whole mitochondrial (mt) genomes of blood samples from HIV-1-infected children treated with long-term antiretroviral therapy (ART), who had an excellent virological response. HIV-1-infected children who have undergone ART for 4 y with an excellent virological response (group A; 15 children) and ten healthy children (controls) without HIV-1 infection were enrolled retrospectively. Peripheral blood mononuclear cells (PBMCs) were obtained and mt DNA mutations were studied. The total number of mtDNA mutations in group A was 3 H more than in the controls (59 vs. 19, P<0.001) and the same trend was seen in all mtDNA regions. Among these mtDNA mutations, 140 and 28 mutations were detected in group A and the controls, respectively. The D-loop, CYTB and 12s rRNA were the three most common mutation regions in both groups, with significant differences between the groups observed at nucleotide positions C309CC, T489C CA514deletion, T16249C and G16474GG (D-loop); T14783C, G15043A, G15301A, and A15662G (CYTB); and G709A (12s rRNA). G15043A and A15662G had been associated with mitochondrial diseases. Our findings suggest that mtDNA mutations occur frequently in long-term ART-treated, HIV-1-infected children who have an excellent virological response, although they did not have obvious current symptoms. The CYTB region may play an important role in mtDNA mutation during ART, which might contribute to the development of subsequent mitochondrial diseases.

Objective To carry out expression profile microarray analysis after hepatoma carcinoma cell line-HepG2 cell are treated by all-trans retinoic acid ( ATRA ) in order to explore its effects on gene expression profile of hepatoma carcinoma cells. Methods The HepG2 cells were treated by 80μM ATRA ( trial group) and equal volume of ethanol ( control group) for 24h,then the total mRNA was extracted from the treated cells. The cDNA microarray technique was used to detect and compare the differentially expressed genes between the two groups. Results After HepG2 cells were treated by ATRA,a total 661 differentially expressed genes were screened out, moreover these genes were closely correlated to cell signal transduction, cell proliferation and cell differentiation. Conclusion The cDNA microarray technique is used to screen successfully the differentially expressed genes after HepG2 cells are treated by ATRA, which establishes the foundation for exploring further the action mechanism of ATRA on hepatocellular carcinoma, and provides also an important theoretical basis for the clinical application of ATRA to induce differentiation of liver cancer cells.

目的 探讨角蛋白18(keratin 18,K18)在33和52位丝氨酸(Ser)磷酸化水平变化的作用及其与肝纤维化的关系.方法 6周龄Balb/C小鼠30只,分为3组(对照组和2个实验组),每组10只.对照组腹腔内注射橄榄油;实验组腹腔内注射四氯化碳(CCl4)和橄榄油的混合液(1:9),10 ml/kg,每周2次,连续4周,分别在2周和4周处死小鼠.应用组织化学免疫荧光法检测正常对照小鼠和肝纤维化小鼠肝组织中K18及其磷酸化Ser33和Ser52的表达及其相对亚细胞定位;免疫印迹法(Western blotting)检测正常对照小鼠和肝纤维化小鼠肝组织的K18及其磷酸化Ser33和Ser52水平.结果 组织化学免疫荧光结果显示,K18在正常对照小鼠和肝纤维化小鼠肝组织中均有表达,但在小鼠肝纤维化不同阶段无明显差异;在正常对照小鼠肝组织中表达比较弱,而随着肝纤维化的进展表达增强.West-ern blotting结果,肝纤维化小鼠肝组织中的Ser33和Ser52磷酸化的K18表达水平显著增加,尤其是Ser33表达增加更为明显.

目的 探讨p53凋亡刺激蛋白2(apoptosis-stimulating protein of p53-2,ASPP2)对人肝星状细胞活化的调节作用及机制.方法 人永生的肝星状细胞系LX-2在孵箱中培养,转染ASPP2腺病毒和单荧光自噬指示体系(GFP-LC3)质粒,免疫印迹法检测ASPP2、α-平滑肌肌动蛋白(α-SMA)、自噬基因表达相关蛋白(Beclin-1)的表达水平.免疫荧光检测,LX-2细胞共转染ASPP2腺病毒和GFP-LC3质粒后,ASPP2过表达对LX-2细胞自噬的影响,M30染色检测ASPP2过表达对LX-2细胞凋亡的影响.结果 转染ASPP2腺病毒后LX-2细胞内ASPP2蛋白表达明显增加;与转染对照空载腺病毒的LX-2细胞相比,转染ASPP2腺病毒的LX-2细胞自噬和α-SMA的表达明显减少,肝星状细胞的活化受到抑制;转染ASPP2腺病毒后LX-2细胞的凋亡水平增加明显.结论 ASPP2过表达在体外具有抑制肝星状细胞活化的作用.

肝纤维化和肝硬化是肝脏损伤后的修复反应,其病理特征主要表现为细胞外基质的过度沉积,Ⅲ型前胶原蛋白[procollagen a1（Ⅲ）,cola1（Ⅲ）]是细胞外基质的主要成分[1]。通过检测血液或肝组织的表达量的变化可以观察肝纤维化及肝硬化的进展情况,指导临床诊疗和基础研究工作。因此,制备cola1（Ⅲ）单克隆抗体（mAb）对于肝纤维化和肝硬化的特异性检测具有重要意义。我们于2012年9月

ASPP2 is a pro-apoptotic member of the p53 binding protein family. ASPP2 has been shown to inhibit autophagy, which maintains energy balance in nutritional deprivation. We attempted to identify the role of ASPP2 in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In a NAFLD cell model, control treated and untreated HepG2 cells were pre-incubated with GFP-adenovirus (GFP-ad) for 12 hrs and then treated with oleic acid (OA) for 24 hrs. In the experimental groups, the HepG2 cells were pre-treated with ASPP2-adenovirus (ASPP2-ad) or ASPP2-siRNA for 12 hrs and then treated with OA for 24 hrs. BALB/c mice fed a methionine- and choline-deficient (MCD) diet were used to generate a mouse model of NAFLD. The mice with fatty livers in the control group were pre-treated with injections of GFP-ad for 10 days. In the experimental group, the mice that had been pre-treated with ASPP2-ad were fed an MCD diet for 10 days. ASPP2-ad or GFP-ad was administered once every 5 days. Liver tissue from fatty liver patients and healthy controls were used to analyse the role of ASPP2. Autophagy, apoptosis markers and lipid metabolism mediators, were assessed with confocal fluorescence microscopy, immunohistochemistry, western blot and biochemical assays. ASPP2 overexpression decreased the triglyceride content and inhibited autophagy and apoptosis in the HepG2 cells. ASPP2-ad administration suppressed the MCD diet-induced autophagy, steatosis and apoptosis and decreased the previously elevated alanine aminotransferase levels. In conclusion, ASPP2 may participate in the lipid metabolism of non-alcoholic steatohepatitis and attenuate liver failure.

血管生成是指在原有的血管结构基础上，内皮细胞以出芽方式，伴随内皮细胞的迁移、扩增、管腔化，形成新的血管的过程。血管生成可分为生理性和病理性两类，病理性血管生成见于以广泛、持续的炎症坏死和纤维化为特征的各种慢性肝脏疾病，包括：慢性乙型肝炎、慢性丙型肝炎、自身免疫性肝炎和原发性胆汁性肝硬化。新血管的形成与不同病因引起的慢性肝病纤维化发展模式紧密相关[1]，最终导致肝硬化组织异常血管结构的逐步形成。因此，在现代疾病进展的评价和治疗靶点的研究中，纤维化和血管生成的关系至关重要[2]。血管生成的程度可能会对疾病是否进展到肝硬化产生重要影响，并且是决定纤维化可否逆转的关键因素。

Background & Aims: Directed migration of hepatic myofibroblasts (hMFs) contributes to the development of liver fibrosis. However, the signals regulating the motility of these cells are incompletely understood. We have recently shown that sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) are involved in mouse liver fibrogenesis. Here, we investigated the role of S1P/S1PRs signals in human liver fibrosis involving motility of human hMFs.Methods: Si P level in the liver was examined by high-performance liquid chromatography. Expression of S1PRs was characterized, in biopsy specimens of human liver and cultured hMFs, by immunofluorescence and real-time RT-PCR or Western blot analysis. Cell migration was determined in Boyden chambers, by using the selective S1P receptor agonist or antagonist and silencing of S1PRs expression with small interfering RNA.Results: Si P level in the human fibrotic liver was increased through up-regulation of sphingosine kinase (SphK), irrespective of the etiology of fibrosis. S1P receptors type 1, 2, and 3 (S1P(1,2,3)) were expressed in human hMFs in vivo and in vitro. Interestingly, S1P(1,3) were strongly induced in human fibrotic samples, whereas expression of S1P(2) was massively decreased. S1P exerted a powerful migratory action on human hMFs. Furthermore, the effect of S1P was mimicked by SEW2871 (an S1P(1) agonist), and blocked by suramin (an S1P(3) antagonist) and by silencing S1P(1,3) expression. In contrast, JTE-013 (an S1P(2) antagonist) and silencing of S1P(2) expression enhanced S1P-induced migration.Conclusions: SphK/S1P/S1PRs signaling axis plays an important role in human liver fibrosis and is involved in the directed migration of human hMFs into the damaged areas. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.