大鼠移植骨髓细胞向肝细胞转化的实验研究

目的 探讨体内骨髓细胞向肝细胞转化的可行性。方法 将雌性 SD大鼠随机分为3组,每组15只。①R+BMT(全身照射+骨髓移植);②2-AAF+R+BMT:③2-AAF+PH(部分肝切)+BMT。进行交叉性别骨髓细胞移植,雄性骨髓植入雌性受体,分别于第5、10、20天处死雌鼠。以雄性性别决定基因sry作为细胞标记,用原位杂交和FISH作为检测方法对骨髓细胞的肝细胞转化进行分析。结果 PCR移植效果初步分析可见,R+ BMT组11例中有10例PCR阳性;2AAF+PH+BMT组11例中有7例阳性,2AAM+R+BMT组10例中有6例阳性。sry原位杂交染色发现,第5天各组雌性受体肝索中均未见sry阳性的肝细胞。第10天R+BMT组可见1例sry阳性的细胞位于肝细胞索,FISH染色可见这一细胞白蛋白mRNA阳性。第20天各组PCR阳性各例均可在肝索中检测到sry阳性的细胞。FICH染色可见白蛋白mRNA阳性。经统计学分析第20天各组sry阳性细胞数无明显差异。结论 在R+BMT、2-AAF+PH+BMT和2-AAF+R+BMT模型中移植的骨髓细胞均可以植入肝脏,并存在于肝细胞索。植入肝索的骨髓细胞最早可见于移植后第10天,并发生转分化,表达白蛋白mRNA。不经过全身照射的2-AAF+PH+BMT组,移植的骨髓细胞也可以进入肝脏发生转分化,因此全身照射并不一定是移植骨髓细胞活化、植入和转化的必须条件。

肝细胞转化中细胞骨架的改变

用组织化学染色法显示新生大鼠肝细胞和诱发转化细胞的细胞骨架。正常肝上皮细胞呈现直的纤维细丝状、成束平行,整齐排列,由核向四周放射,布满于整个细胞质中。细胞经用 MNNG 处理,不仅显示去接触抑制、微绒毛形成等一系列生物学性状的变化,而且出现了 AFP 水平复升等表型改变。此时,处理细胞的纤维丝状细胞骨架逐渐减少,且排列不规则,也较模糊,呈点状或粗网状。对照细胞未见有变化。

灵芪合剂影响肝细胞生长因子/上皮间质转化因子通路抑制结直肠癌

目的:探索灵芪合剂对结直肠癌发展的作用机制,并且明确灵芪合剂是否可通过肝细胞生长因子(HGF)/上皮间质转化因子(c-Met)信号通路抑制结直肠癌发展。方法:将直肠癌大鼠分为灵芪合剂高中低剂量组,同时进行中西药对比的治疗效果,通过蛋白质印迹法(Western Blotting)检测、免疫荧光检测和凋亡的检测等技术手段的检测与分析,观察肿瘤的生长、细胞凋亡情况和HGF/c-Met通路蛋白的表达情况。结果:Western Blotting检测肿瘤组织中HGF蛋白表达增强,c-Met蛋白表达减少;在灵芪合剂治疗后,HGF的表达减少,c-Met的表达增强,这与免疫荧光表现结果一致,差异有统计学意义(P<0.05)。结论:灵芪合剂通过抑制HGF的表达,上调c-Met的表达,抑制HGF/c-Met信号通路从而抑制结直肠癌细胞的侵袭和转移,实现对结直肠癌的预防和治疗。

乙型肝炎病毒驱动甲胎蛋白表达诱导肝细胞恶性转化的分子机制

肝癌细胞的恶性转化与感染乙型肝炎病毒(hepatitis B virus,HBV)和丙型肝炎病毒密切相关.但是HBV没有直接诱导肝癌发生的生物学功能,HBV可通过其x蛋白(HBx)激活生长信号,促进癌基因的表达从而诱导肝细胞恶性转化.在肝细胞恶性转化过程的早期,甲胎蛋白(alpha fetoprotein,AFP)基因被激活,而AFP能激发PI3K/AKT信号传递,由于PI3K/AKT信号途径具有促进细胞恶性转化的作用,所以AFP的表达在HBV诱导肝细胞恶性转化过程发挥关键性作用.本文就HBV通过优先驱动AFP表达促进肝癌细胞增殖和自然重编程从而诱发肝癌的分子机制进行阐述,对认识AFP在HBV相关性肝癌发生过程中的作用以及预警肝癌发生有重要的科学意义.

甲胎蛋白具有诱导肝细胞恶性转化的生物学功能及其作为肝癌治疗的新靶点

甲胎蛋白(alpha fetoprotein,AFP)在肝癌发生过程中所起的生物学作用一直是个谜.由于其是肝癌细胞高特异性表达的蛋白质,临床上作为诊断肝癌的金标准,但是AFP在肝癌发生过程如何发挥作用并不清楚.最近作者的研究发现,乙型肝炎病毒(hepatitis B virus,HBV)诱导肝细胞恶性转化的过程,优先激活AFP基因表达;而且AFP通过抑制人第10号染色体缺失的磷酸酶及张力蛋白同源的蛋白(phosphatase and tensin homolog deleted on chromosome ten)活性促进磷脂酰肌醇-3-激酶(phosphoinositide-3-kinase)/蛋白激酶B(protein kinase B)等生长信号途径;笔者研究也发现A F P具有信息调控分子样作用,A F P不仅能与维甲酸受体-(retinoic acid receptor-beta,RAR-)结合,阻遏RAR-进入细胞核内调节靶基因的表达,同时也能抑制Caspase3的活性,阻断凋亡信号的传递.靶向抑制AFP表达能增加肝癌细胞对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis inducing ligand)和全反式维甲酸(all trans retinoid acid)的敏感性.提示AFP不仅有驱动肝细胞恶性转化的生物学作用,而且其还具有抗凋亡诱导的功能.新发现AFP的这些功能,预示其是肝细胞恶性转化的先锋因子以及肝癌细胞耐药的新靶点,为肝癌的生物治疗提供新的思路和策略.

2型糖尿病患者肝细胞生长因子与转化生长因子β1的测定及其意义

目的探讨血清肝细胞生长因子（HGF）、转化生长因子β1（TGF-β1）、基质金属蛋白酶-2（MMP-2）、MMP-9及金属蛋白酶抑制物-1（TIMP-1）在糖尿病不同肾损害期中的差异及其临床意义。方法将113例2型糖尿病患者根据尿微量白蛋白含量分为正常白蛋白尿组（NA组）38例、微量白蛋白尿组（MA组）40例和大量自蛋白尿组（CP组）35例，同时随机分为糖耐量受损（IGT）组40例和正常对照（NC）组30例，并采用ELISA法检测血清HGF、TGF-β1、MMP-2、MMP-9、TIMP-1和Ⅳ型胶原（IVC）含量。结果IGT组和NA组HGF含量显著高于NC组（P〈0.01），而MA组与CP组含量显著低于NC组（P〈0．01），以CP组含量最低；NA、MA和CP组TGF—β1含量显著高于NC组与IGT组（均P〈0．01），以CP组含量最高；2型糖尿病3组间HGF和TGF-9、含量的差异均有统计学意义（P〈0.01或0.05）；CP组和MA组MMP-2、MMP-9含量显著低于NC、IGT及NA组（P〈0．01），而TIMP-1与IVC含量显著高于NC、IGT和NA组（均P〈0.01）。结论糖尿病不同肾损害期患者存在HGF与TGF—β1、及MMP／TIMP系统的失衡，与糖尿病肾病的进展有关。

NAFLD与肝细胞恶性转化相关研究新进展

随着人们生活方式和饮食结构的改变,非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)[1]或代谢相关性脂肪性肝病(metabolic dysfunction-associated fatty liver disease,MAFLD)[2]发病率逐年增长,并呈低龄化趋势。真实世界NAFLD发病率可能较高,应引起重视。脂质及其代谢产物的分析鉴定、脂质功能与代谢调控(关键基因/蛋白/酶)、脂质代谢途径及网络调控等方面的研究非常活跃,成为代谢组学重要的分支之一。NAFLD与HCC发生的问题也已引起了广泛的关注。NAFLD是非酒精性脂质代谢异常导致肝细胞脂肪堆积、影响肝细胞的正常功能发挥、诱发炎症而引起的最常见的慢性肝病[3]。长期肝细胞脂质堆积,脂质毒性的影响将启动肝细胞恶性转化相关信号通路,引起致癌基因激活和免疫功能失调等一系列的改变,但其中确切的发生机制仍在探究之中。新发现线粒体功能[4]、非编码RNA(non-coding RNA,ncRNA)、肠道菌群和免疫系统,尤其是T淋巴细胞(T细胞)及其亚群等参与了NAFLD相关的肝细胞恶性转化[5]。

脂肪积聚肝恶性转化模型制备及其生物信息学分析

目的:制备非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)和诱导肝细胞恶性转化模型,观察基因表达谱动态变化。方法:雄性Sprague-Dawley(SD)大鼠分别以高脂饲料喂饲制备非酒精性脂肪积聚,以含0.05%2-乙基氨基芴(2-fluorenylacetamide,2-FAA)高脂饲料喂饲诱发鼠肝细胞恶性转化,根据肝病理学苏木精-伊红(Hematoxylin&Eosin,HE)染色检查结果分为对照(normal control,NC)、NAFLD、肝细胞变性(hepatocyte degeneration,H-deg)、癌前(precanceraion,Pre-c)和癌变(liver cancer,LC)组,脂质以油红O染色行定量分析,从肝组织抽提RNA并合成cRNA、标记、杂交和扫描,利用基因芯片分析各组差异基因表达谱动态变化,对差异表达基因行基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析。结果:SD鼠摄入脂肪后,肝脂肪积聚与NC组比较,NAFLD组升高9倍(t=14.78,P<0.001)、H-deg组2.4倍(t=5.96,P<0.001)、Pre-c组2.3倍(t=8.37,P<0.001)和LC组8倍(t=11.97,P<0.001)。与NC组比较全基因表达谱变化,差异表达基因NAFLD组163个,H-deg组934个,Pre-c组1452个,LC组1738个。生物学过程主要集中在细胞对刺激反应的代谢调节。肝细胞恶化转化中信号通路主要有固醇合成、P53、细胞周期信号通路,其中固醇合成通路Cyp51,Tm7sf2显著下调,Ccnb1和周期蛋白依赖性激酶1(cyclin-dependent kinases 1,CDK1)与P53通路和细胞周期相关。癌变相关标志甲胎蛋白(alpha-fetoprotein,AFP)、磷脂酰肌醇蛋白多糖-3(glypican-3,GPC-3)、CD44和Wnt3a等表达,在Pre-c组和LC组中明显上调。结论:脂肪积聚肝细胞恶性转化过程中,众多基因和信号通路参与癌变发生过程。

柴胡皂苷d对人肝细胞损伤的保护作用研究及抗肝纤维化机制探讨

目的观察柴胡皂苷d(SSd)对人肝细胞损伤的保护作用及抗肝纤维化机制,探讨L02细胞氧化应激损伤与细胞焦亡、上皮-间质转化(EMT)的关系,为SSd在治疗肝纤维化疾病的应用提供临床依据。方法体外培养人正常肝细胞L02,采用H_(2)O_(2)诱导L02细胞损伤模型,干预组加入中药单体SSd(低、中、高剂量组)孵育24 h后再经过H_(2)O_(2)处理细胞,CCK8法观察SSd干预的损伤L02细胞后的活力、丙二醛(MDA)水平、上清液谷丙转氨酶(ALT)、谷草转氨酶(AST)检测,Western Blot法、RT-PCR法检测细胞焦亡相关指标半胱氨酸蛋白酶1(Caspase-1)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)及上皮间质转化(EMT)相关指标转化生长因子(TGF-β1)、钙黏附蛋白(Ecadherin)、波形蛋白(Vimentin)、肌劲蛋白(α-SMA)的表达。结果SSd能抑制L02细胞损伤引起的细胞活力下降,在有效剂量内无明显肝毒性(ALT、AST未见明显升高);与对照组比较,氧化应激损伤的L02细胞焦亡相关指标Caspase-1、IL-1β、IL-18表达明显升高(P<0.05,P<0.01),EMT相关指标TGF-β1、Vimentin、α-SMA的表达升高,Ecadherin表达下调(P<0.05,P<0.01);SSd干预后,Caspase-1、IL-1β、IL-18、TGF-β1、Vimentin、α-SMA的表达明显下调,Ecadherin表达上调(P<0.05,P<0.01)。结论氧化应激损伤的L02细胞活力下降可能与细胞焦亡相关,SSd有抑制损伤的L02细胞焦亡、控制EMT过程,发挥抗肝纤维化的作用。

让肝细胞“返祖”动物实验成功

日本国立癌症研究中心最近成功将实验鼠的成熟肝细胞转化成肝脏祖细胞，并通过移植肝脏祖细胞明显改善了患慢性肝炎实验鼠的肝功能。这一技术今后有望用于肝脏再生医疗，或许能部分替代肝脏移植。

癌胚型信号分子在监测肝细胞恶性转化中的临床价值

肝细胞癌(hepatocellular carcinoma,HCC)的预防、早期监测或诊断,仍是亟需解决的医学界难题,因为现有HCC诊断标志物早期监测价值有限。肝细胞恶性转化与多种信号通路激活密切相关。近年除非编码RNA(non-coding RNAs,ncRNAs)外,在慢性肝病恶性转化时,高迁移率族蛋白3(high mobility group box 3,HMGB3)、Wnt通路中Wnt3a、分泌型簇蛋白(secretory cluster protein,sCLU)、血管生成素-2(angiopoietin-2,Ang-2)、高尔基蛋白73(Golgi protein 73,GP73)和磷脂酰肌醇蛋白聚糖-3(glypican-3,GPC-3)等信号分子异常升高,与肝细胞恶性转化进展正相关。本文述评了部分具有监测肝细胞恶性转化相关信号分子及其临床价值。

HCV NS3 N端多肽诱导人肝细胞系转化及成瘤实验

目的 研究丙型肝炎病毒(HCV)非结构区3N端多肽(HCV NS3-5′)对人肝细胞株QSG7701的转化作用及致癌性。方法 通过脂质体介导将含有HCV NS3 N端cDNA的真核表达质粒(pRcHCNS3-5′)导入人源性肝细胞株QSG7701,G418筛选目的基因表达的细胞;聚合酶链反应(PCR)及免疫组织化学SP法检测细胞中HCV NS3基因及蛋白的表达;细胞计数,锚着非依赖性生长实验,成瘤性检测等鉴定其生物学行为变化,免疫组织化学S-P法检测所致肿瘤中HCV NS3及c-myc蛋白表达。结果 HCV NS3-5′转染的QSG7701细胞中NS3蛋白过度表达于胞质,质粒pRcHCNS3-5′转染细胞的倍增时间较pRcCMV转染细胞和未转染QSG7701细胞明显缩短(分别为12h,26h,28h)。pRcHCNS3-5′和pRcCMV转染细胞及未转染QSG7701在软琼脂中的克隆形成率分别为33.0％、1.5％、1.1％。pRcHCNS3-5′转染细胞的克隆率高于其他两种转染细胞(P<0.01)。三种细胞接种探鼠后,pRcHCNS3-5′转染细胞注射组出现肿瘤,为肝细胞癌,肿瘤组织有HCV NS3蛋白和c-myc蛋白的表达。阳性对照组亦出现肿瘤,而pRcCMV转染细胞及未转染QSG7701细胞注射组在注射40d后仍未见肿瘤发生。结论 HCV NS3 N端蛋白具有转化细胞和促进肿瘤发生的作用。

高迁移率族蛋白B1在高糖微环境诱导人肝细胞恶性转化过程中作用和机制的研究

目的探讨高糖微环境对人肝细胞释放高迁移率族蛋白B1(HMGB1)的影响及HMGB1诱导人肝细胞恶性转化和糖尿病增加肝癌发病风险的可能机制。方法将人肝细胞HL-7702分为对照组(Con)、高糖处理组(HG)、高糖+HMGB1中和抗体组(HG+HMGB1)、高糖+HMGB1中和抗体对照组(HG+IgY)。ELISA检测HMGB1含量,MTS检测细胞增殖活力,流式细胞术检测周期和凋亡率变化,Transwell检测细胞侵袭能力。结果与Con组比较,HG组上清HMGB1含量升高[(64. 45±11. 58)vs(433. 17±15. 18)pg/ml,P<0. 05],DNA合成期(S期)比例升高[(15. 27±2. 01)%vs(28. 68±0. 82)%,P<0. 05],早期凋亡率降低[(2. 31±0. 14)%vs(0. 88±0. 14)%,P<0. 05],侵袭能力增强。与HG组比较,HG+HMGB1组上清HMGB1含量降低[(433. 17±15. 18)vs(213. 80±24. 32)pg/ml,P<0. 05],S期比例降低[(28. 68±0. 82)%vs(17. 70±0. 64)%,P<0. 05],早期凋亡率升高[(0. 88±0. 14)%vs(2. 14±0. 13)%,P<0. 05],侵袭能力减弱。结论高糖诱导人肝细胞HL-7702胞内HMGB1释放至胞外,促进细胞增殖并干扰细胞周期和凋亡,使其发生恶性转化。

常用的抗衰老中药

何首乌 宋代《开宝本草》称之“久服长筋骨,益精髓,延年不老”。现代研究发现,何首乌能够促进神经细胞的生长,对神经衰弱及其他神经系统疾病有辅助治疗作用,并可调节血清胆固醇,降低血糖,提高肝细胞转化和代谢胆固醇的能力,还具有良好的抗氧化作用。

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-pi and a-smooth muscle actin in rats with hepatic fibrosis

AIM:To investigate the effect of interferon-a (IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCI4. METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-a in saline daily at the doses of 1× 105 U for 6 wk, group D (IFN-a treatment, n = 24) treated with intra-muscular injection of IFN-a in saline daily at the doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-pi (TGF- μ41) and α-smooth muscle actin (α-SMA). RESULTS: The expressions of TGF-pl, the number of activated hepatic stellate cells and a-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-a can inhibit the production of TGF-pl, decrease HSC activation and stimulate its apoptosis.

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.

Effects of thalidomide on angiogenesis and tumor growth and metastasis of human hepatocellular carcinoma in nude mice

AIM: To investigate the effects of thalidomide on angiogenesis, tumor growth and metastasis of hepatocellular carcinoma in nude mice.METHODS: Twenty-four nude mice were randomly divided into therapy group and control group, 12 mice in each group. Thalidomide dissolved in 0.5% sodium carboxyl methyl cellulose (CMC) suspension was administered intraperitoneally once a day at the dose of 200 mg/kg in therapy group, and an equivalent volume of 0.5% CMC in control group. Mice were sacrificed on the 30th d, tumor size and weight and metastases in liver and lungs were measured. CD34 and VEGF mRNA in tumor tissue were detected by immunohistochemistry and semi-quantitative RT-PCR respectively and microvessel density (MVD) was counted. Serum concentrations of TNF-α and ALT and AFP were also tested.RESULTS: MVD and VEGF mRNA in therapy group were less than those in control group (31.08±16.23 vessels/HP vs 80.00±26.27 vessels/HP, 0.0538±0.0165 vs 0.7373±0.1297,respectively, P＜0.05). No statistical difference was observed in tumor size and weight and metastases in liver and lungs.TNF-α was significantly lower in therapy group than in control group (28.64±4.64 ng/L vs42.69±6.99 ng/L, P＜0.05). No statistical difference in ALT and AFP was observed between groups.CONCLUSION: Thalidomide can significantly inhibitangiogenesis and metastasis of hepatocellular carcinoma.Italso has inhibitory effects on circulating TNF-α.

Relative predictive factors for hepatocellular carcinoma after HBeAg seroconversion in HBV infection

AIM： To determine the predictive factors for hepatocellular carcinoma （HCC） development in patients after spontaneous or therapeutic HBeAg seroconversion. METHODS： In 48 patients who seroconverted to anti- HBe positive during follow-up, the background factors for HCC development were analyzed. RESULTS： HCC was developed in six patients during follow-up （average follow-up after HBeAg seroconversion： 10.9±5.4 years）. The incidence of HCC evaluated by Kaplan-Meier analysis was significantly higher in patients with abnormal aspartate aminotransferase （AST〉 40 IU/L） level, lower platelet counts （PLT〈10×10^4/IJL）, lower albumin level （Alb〈30 g/L）, positive HBV-DNA or older age at seroconversion （〉40 years）. However, lower platelet count was the only predictive factor for HCC development shown by multivariate proportional-hazard analysis. CONCLUSION： Active hepatitis or advanced hepatitis at HBeAg seroconversion or progressive hepatitis even after HBeAg seroconversion would be the risk factors for HCC development. These predictive factors should be taken into account in determining the frequency of biochemical study or imaging studies for HCC surveillance.