血管紧张素Ⅱ对肝星状细胞迁移和增殖的影响

目的:探讨血管紧张素Ⅱ(ANGⅡ)对肝星状细胞(HSC)增殖、迁移活性的影响.方法:体外培养HSC,在不同浓度的ANGⅡ作用下,观察HSCs生长情况并绘制生长曲线,采用MTT法测定细胞增殖、Transwell小室检测细胞迁移.结果:1,2,4,8,10 umol/L ANGⅡ可显著促进HSC增殖,与空白对照组相比有显著差异(F= 2.305,P<0.05;F=4.003,6.833,8.855,21.066,P<0.01).随作用浓度的增加,细胞增殖活性显著提高,相关分析显示呈正相关(r=0.917,P<0.01).10,8,4μmol/L的ANGⅡ可显著诱导HSC迁移,与空白对照组相比差异显著(F= 22.084,15.155,10.392,P<0.01).随ANGⅡ浓度的增加,细胞迁移率显著升高,相关分析显示呈正相关(r=0.952,P<0.01).结论:ANGⅡ可显著诱导HSC增殖与迁移,并随剂量的增加作用加强.     

血管紧张素Ⅱ对肝星状细胞迁移和增殖的影响

目的探讨血管紧张素Ⅱ(ANGⅡ)对肝星状细胞(HSC)增殖、迁移活性的影响.方法体外培养HSC,在不同浓度的ANGⅡ作用下,观察HSCs生长情况并绘制生长曲线,采用MTT法测定细胞增殖、Transwell小室检测细胞迁移.结果1,2,4,8,10 μmol/LANGⅡ可显著促进HSC增殖,与空白对照组相比有显著差异(F=2.305,P＜0.05;F=4.003,6.833,8.855,21.066,P＜0.01).随作用浓度的增加,细胞增殖活性显著提高,相关分析显示呈正相关(r=0.917,P＜0.01).10,8,4 μmol/L的ANGⅡ可显著诱导HSC迁移,与空白对照组相比差异显著(F=22.084,15.155,10.392,P＜0.01).随ANGⅡ浓度的增加,细胞迁移率显著升高,相关分析显示呈正相关(r=0.952,P＜0.01).结论ANGⅡ可显著诱导HSC增殖与迁移,并随剂量的增加作用加强.     

骨髓间充质干细胞共培养对肝星状细胞增殖、凋亡和RohA表达的调控

目的:观察体外大鼠骨髓间充质干细胞(BMSCs)共培养对肝星状细胞(HSCs)增殖、凋亡和RohA表达的影响,探讨BMSCs旁分泌HGF在其中的作用机制.方法:贴壁筛选法培养、纯化SD大鼠BMSCs,传代至第4代使用;大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用.应用6孔塑料细胞培养盒,每孔使用半透膜(transwell insert)建立上下双层细胞共培养体系,常规培养.实验分4组:空白对照组、阴性对照组、BMSCs实验组、预处理实验组(c-met多克隆抗体预处理).用四甲基偶氮唑蓝(MTT)法检测HSCs细胞增殖能力;流式细胞仪检测细胞凋亡;RT-PGR、Western blot检测BMSCs与HSCs共培养后HSCs内RohA mRNA和蛋白的表达.酶联免疫吸附法(ELISA)检测BMSCs与HSCs共培养上清液中肝细胞生长因子(HGF)浓度.结果:BMSCs对HSCs增殖具有抑制作用,BMSCs与HSCs共培养后24 h、48 h的增殖抑制率分别为12.21%,35.43%,与空白对照组、实验对照组和C-met抗体预处理组比较有显著性差异(P<0.01).Annexin-V-FITC/PI双染法检测BMSCs与HSCs共培养48 h后HSCs的凋亡率为25.80%,与空白对照组、实验对照组与c-met抗体预处理组比较有显著性差异(P<0.01).BMSCs与HSCs共培养48 h,BMSGs组RohA mRNA的表达抑制明显,且显著低于空白对照组、实验对照组与C-met抗体预处理组,有显著性差异(P<0.01).BMSCs与HSCs共培养48 h RohA蛋白的表达明显抑制,且显著低于空白对照组、实验对照组与C-met抗体预处理组,有显著性差异(P<0.01).ELISA检测BMSCs与HSCs共培养24 h、48 h上清液中HGF浓度分别为250 ng/L与570 ng/L,明显高于单独BMSCs培养和单独HSCs培养,有显著性差异(P<0.01).结论:BMSCs与HSCs共培养能抑制HSCs的增殖,促进凋亡,抑制RohA表达,其机制可能是通过BMSCs旁分泌HGF发挥抑制大鼠HSCs增殖,促进凋亡的作用.     

Decorin对肝星状细胞和肝细胞周期的影响

目的探讨decorin对肝星状细胞(HSC-T6)和肝细胞(L-02)生长抑制的作用机制.方法细胞培养,经dccorin处理后用流式细胞仪分析细胞周期的变化.结果Decorin使细胞周期发生明显改变.G1期细胞百分率明显增加,S期细胞百分率降低G2期细胞百分率减少甚至缺失(P＜0.05,P＜0.01).结论Decorin对肝星状细胞(HSC-T6)和肝细胞(L-02)有细胞周期阻滞作用,通过细胞周期阻抑可以抑制细胞生长.     

复方861对肝星状细胞的增殖和凋亡的干预作用

目的 研究ＨＳＣ在体外和体内的增殖和凋亡及中药的干预作用。方法 体外研究对象为ＨＳＣ系。结果增殖采用ＭＴＴ比色法,细胞凋亡采用电镜观察,流式细胞仪和ＴＵＮＥＬ法检测。临床研究对象是量慢性乙型肝炎患者。结果 复方８６１显著抑制体外培养的ＨＳＣ增殖,随着药物剂量的增加和时间的延长,ＨＳＣ的凋亡明显增多,凋亡率增加,呈剂量和时间依赖,ＴＵＮＥＬ法检测,用５ｍｇ／ｍｌ复方８６１作用４８ｈ二,ＨＳＣ凋亡率为     

大鼠纤维化肝组织中PTEN的动态表达及其与肝星状细胞增殖和活化的关系

目的 探讨第10号染色体缺失的磷酸酶张力蛋白同源物基因(PTEN)在大鼠纤维化肝组织中的动态表达及其对在体肝星状细胞(HSC)活化、增殖的影响. 方法 采用胆总管结扎法建立大鼠肝纤维化模型;应用免疫组织化学染色、Western blot和实时荧光定量PCR技术测定大鼠肝组织中PTEN的表达;应用免疫荧光双标记共聚焦激光扫描显微术测定大鼠肝组织中活化HSC的PTEN表达;采用免疫组织化学染色检测大鼠肝组织中α-平滑肌肌动蛋白的表达.结果 免疫组织化学染色显示正常大鼠肝组织中PTEN有广泛表达,主要表达于细胞质,随着肝纤维化的发展,PTEN表达逐渐减少(P<0.01),而α-平滑肌肌动蛋白阳性细胞明显增多(P<0.01);造模1、2、3周及4周不同时间大鼠纤维化肝组织中PTEN的mRNA(分别为假手术组的0.66、0.53、0.44和0.37)及蛋白质表达(吸光度比值分别为1.20±0.13,1.07±0.16,0.88±0.08,0.73±0.07)均低于假手术组(P<0.01),并随着肝纤维化的进展逐渐降低(P<0.01);免疫荧光双标记共聚焦激光扫描显微术显示PTEN在活化HSC广泛表达,主要表达于细胞质,随着肝纤维化的进展,表达PTEN的活化HSC占总的活化HSC的比例逐渐减少(P<0.01). 结论 大鼠纤维化肝组织中PTEN的mRNA及蛋白质表达均下调;在体HSC的PTEN表达亦降低;肝组织中PTEN的动态表达与HSC的活化、增殖呈显著负相关.     

大鼠肝脏前体细胞通过直接共培养抑制肝星状细胞的活化

目的：观察大鼠肝脏前体细胞系（WB-F344）对肝星状细胞系（HSC-T6）活化及细胞外基质的影响。方法将慢病毒-GFP 空白载体转染的 HSC-T6与 WB-F344按1∶1与1∶2比例直接共培养3 d,使用流式细胞仪对其进行分选,慢病毒-GFP 空白载体转染的 HSC-T6单独培养作为对照,观察 HSC-T6活化指标及细胞外基质相关指标表达的变化。结果经流式细胞仪检验慢病毒-GFP 空白载体转染 HSC-T6的效率可达近90％;与 WB-F344直接共培养3 d 后,HSC-T6的细胞形态与对照组相比无差别;同时利用 GFP 对各组 HSC-T6细胞分选后,经流式细胞仪检验纯度可达94％;对分选后的细胞进行分析发现,直接共培养组 HSC-T6细胞活化标记物α-平滑肌肌动蛋白（α-SMA）在基因水平（1∶1与1∶2组分别是对照组的0．66与0．61倍,P＜0．05）和蛋白水平（1∶1与1∶2组分别是对照组的0．61倍与0．25倍,P＜0．05）的表达均下降;HSC-T6细胞的细胞外基质标记物 I 型胶原（Col-I）、基质金属蛋白酶-13（MMP-13）、金属蛋白酶组织抑制剂-1（TIMP-1）的表达与对照组比较差异无统计学意义。结论WB-F344细胞可以抑制 HSC-T6细胞的活化,但在一定时间内对其细胞外基质的表达没有影响。     

过表达HBx的肝细胞对肝星状细胞增殖和活化的影响及其机制

目的探讨HBV感染对肝星状细胞(HSC)活化的影响及作用机制。方法收集2020年11月—2021年1月慢性乙型肝炎患者血浆30份、乙型肝炎肝硬化患者血浆42份、肝细胞癌患者血浆30份及健康体检者(健康对照组)的血浆18份,ELISA法检测血浆和条件培养液中HBx、TGFβ1、多巴胺β羟化酶(DBH)和羟脯氨酸(Hyp)的含量。采用LO2细胞构建过表达HBx稳转株细胞,LO2细胞分为LO2-HBx组(稳定表达HBx)、阴性对照组(LO2-con)、空白组,分别制备LO2-HBx、LO2-con和LO2细胞(Mock)的条件培养基,孵育人HSC株LX-2,分为LX-2/LO2-HBx、LX-2/LO2-con、LX-2/Mock 3组,采用CCK-8法检测各组细胞增殖变化。采用rhTGFβ1刺激LX-2细胞,另采用TGFβ1受体抑制剂处理LX-2/LO2-HBx组细胞。荧光定量PCR或Western Blot法检测LO2细胞中的HBx及上述LX-2细胞中α-SMA、Col1A1、DBH和TGFβ1的表达。多组间比较采用单因素方差分析,进一步比较方法采用Bonferroni法; 2组间比较采用t检验;相关性分析采用Pearson法。结果 LO2-HBx可稳定表达HBx蛋白,其培养上清中TGFβ1含量升高(F=324.701,P 0.01);共培养LX-2/LO2-HBx组细胞发生明显细胞形态变化,出现细胞收缩,胞突明显伸长,胞内脂滴减少,与LX-2/LO2-con组比较其增殖活力明显增强(P 0.05),且α-SMA和Col1A1的mRNA(F值分别为144.712和76.680,P值均0.01)及蛋白(F值分别为234.142和528.708,P值均0.001)表达水平升高; LX-2/LO2-HBx组细胞中TGFβ1 mRNA(F=29.382,P 0.01)及DBH mRNA水平升高(F=42.662,P 0.01)。随着rhTGFβ1刺激浓度的增加,LX-2细胞中α-SMA(F=1 794.031,P 0.01)、Col1A1(F=91.340,P 0.01)及DBH(F=2 501.011,P 0.01)表达增加,在rhTGFβ1 10 ng/ml时达到峰值。在LO2-HBx组条件培养液中加入TGFβ1受体抑制剂后LX-2细胞中DBH和Col1A1的表达较对照组下调(t值分别为3.603、5.798,P值均0.05)。慢性乙型肝炎、乙型肝炎肝硬化、肝细胞癌患者的血浆TGFβ1(F=51.188,P 0.001)、HBx (F=39.227,P 0.001)、DBH(F=34.431,P 0.001)及Hyp(F=16.211,P 0.001)较健康对照组升高,血浆中HBx与TGFβ1、TGFβ1与DBH、Hyp与DBH的表达量呈正相关,r分别为0.931、0.863、0.765(P值均0.001)。结论 HBx蛋白可促进LO2细胞分泌TGFβ1,诱导LX-2的增殖和活化,促进肝纤维化的发生,并上调LX-2细胞中TGFβ1及DBH的表达;rhTGFβ1刺激可诱导LX-2活化和DBH表达上调。     

肝苏对人肝细胞和肝星状细胞增殖、氧应激以及细胞外基质表达的影响

背景：有研究显示肝苏对肝细胞具有保护、抗炎、抗氧化和抗肝纤维化的作用,但其作用机制未明。目的：探讨肝苏对人肝细胞和肝星状细胞（HSC）增殖、氧应激以及细胞外基质表达的影响。方法：分别用0．01～1．0mg／ml肝苏培养肝细胞和HSC,以M1丫r法检测肝苏对肝细胞和HSC增殖的影响：用次氮基三乙酸铁（Fe-NTa）和0．05—1．0mg／ml肝苏共同培养肝细胞和HSC,检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;用1．5mg／ml和2．5mg／ml肝苏培养HSC,以酶联免疫吸附测定（ELISA）检测细胞外基质透明质酸（HA）、层黏连蛋白（LN）、I型胶原、Ⅲ型胶原和细胞因子转化生长因子（TGF）-β1含量。结果：在0．05。1．0mg／ml浓度范围内,肝苏可促进肝细胞增殖,但各浓度肝苏对HSC增殖无明显影响。肝苏可增高肝细胞SOD活性,降低肝细胞和HSCMDA含量,但对HSCSOD活性无明显影响。同时肝苏可抑制HSC细胞外基质HA、LN和细胞因子TGF-β1的表达,而I型胶原、Ⅲ型胶原无明显差异。结论：肝苏可促进肝细胞增殖,保护肝细胞和HSC免受氧应激损伤,抑制HSC分泌HA、LN和TGF-β1,提示其具有肝细胞保护、抗氧化和抗肝纤维化作用。     

乙型肝炎病毒X基因对肝细胞系细胞凋亡及端粒酶活性的影响

目的探讨乙型肝炎病毒x基因对肝细胞恶性变的作用机制.方法将带C基因、S基因的载体电转染导入HepG2细胞,筛选表达细胞克隆,复苏带X基因的HepG2细胞.PCR-ELISA检测各株细胞的端粒酶活性.用反义寡核苷酸诱导细胞凋亡,流式细胞仪观测转染了x基因、C基因、S基因细胞的凋亡情况.结果表达细胞克隆经同步化处理,39.50%转染X基因的细胞进入细胞S周期,其端粒酶活性指数395±0.07明显高于其它各组细胞.反义寡核苷酸诱导后,转染X基因细胞凋亡峰明显减小,凋亡率仅1.75%;其细胞活性与反义寡核苷酸浓度成反比.结论乙型肝炎病毒X基因上调肝源细胞端粒酶活性,抑制细胞凋亡,这可能是诱导肝细胞恶性变的又一机制.     

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis delta virus(HDV) seems to strongly suppress hepatitis B virus(HBV)replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein(HBx) is essential for HBV replication, determination of HBV X gene(HBX)quasispecies complexity in HBV/HDV infection compared to HBV monoinfection may provide information on the interactions between these two viruses.AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected patients.METHODS Twenty-four untreated patients were included: 7/24(29.2%) with HBeAgnegative chronic HBV infection(CI, previously termed inactive carriers), 8/24(33.3%) with HBeAg-negative chronic hepatitis B(CHB) and 9/24(37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels.The HBX 5' region [nucleotides(nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing(MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidencebased indices(number of haplotypes and number of mutations), abundancebased indices(Hill numbers of order 1 and 2), and functional indices(mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL,interquartile range(IQR) 3.5-7.9] than CHD(3.4 logIU/mL, IQR 3-7.6)(P = n.s.)or CI(3.2 logIU/mL, IQR 2.3-3.5)(P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences(CHB2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3 G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.     

致纤维化生长因子对肝星状细胞移行的影响

目的观察肝纤维化过程中Disse间隙生长因子微环境的改变对肝星状细胞（HSC）移行的影响，从细胞移行角度探讨肝纤维化病变的新机制。方法运用改良的Boyden腔系统，在体外条件下模拟体内正常Disse间隙的微环境及肝纤维化时的相关改变，以人HSC为研究对象，通过细胞迁移实验、细胞增殖实验等方法，观察肝纤维化时致纤维化生长因子对HSC移行的影响。结果肝纤维化时增高的血小板衍化生长因子（PDGF）-BB，转化生长因子β1（TGF-β1）及上皮细胞生长因子（EGF）均可导致活化的HSC移行能力增强，而肝纤维化时同样也增高的内皮细胞生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）则无此效果。PDGF—BB诱导的HSC移行能力的增强与其导致的HSC增殖有关，而由TGF-β1和EGF诱导的这种能力的增强与细胞增殖无关。结论肝纤维化时Disse间隙微环境的改变促进了HSC的移行，TGF-β1、PDGF—BB和EGF具有促进HSC移行的作用，而bFGF和VEGF则无。     

氧化苦参碱对大鼠肝星状细胞增殖的影响

目的探讨氧化苦参碱对大鼠肝星状细胞增殖的影响及其抗肝纤维化的机理.方法用链酶蛋白酶和胶原酶原位灌流消化正常大鼠肝脏,Nycodenz密度梯度离心分离肝星状细胞,以MTT比色法观察氧化苦参碱对肝星状细胞毒性作用,3H-TdR法观察氧化苦参碱对肝星状细胞增殖的效应.结果氧化苦参碱浓度＞10-5mol/1时对肝星状细胞增殖有抑制作用(P＜0.05).结论氧化苦参碱可抑制肝星状细胞的增殖,有抗肝纤维化的作用.     

丹酚酸B对MDA刺激的肝星状细胞增殖的抑制作用

目的:研究丹酚酸B的抗氧化性对大鼠肝星状细胞(HSC)的影响．方法:采用原位灌注法消化大鼠肝脏,108 g／L Nycodenz密度梯度离心,分离肝星状细胞,分别以不同浓度MDA／SAB处理细胞,MTT法观察细胞的增殖能力,2’,7’-二氯二氢荧光素 (DCFH)掺入反映细胞内氧化水平,Western blot检测增殖细胞核抗原(PCNA)蛋白含量, 免疫组化法检测血小板衍生的生长因子受体 (PDGFR)含量．结果:MDA刺激后,细胞增殖明显增强, MTT结果显示,吸光度与对照组相比显著升高(0．253±0．016 vs 0．213±0．004,P<0．05), 而1 μmol／L SAB(0．182±0．006,P<0．01)和 10 μmol／L SAB(0．179±0．006,P<0．01)均可以显著抑制MDA刺激的HSC增殖．Western blot显示,PCNA蛋白在MDA刺激后明显增加(1．72±0．026 vs 1．223±0．025,P<0．01),而 1和10 μmol／L SAB可显著抑制PCNA蛋白表达的升高(分别是1．080±0．040和1．066± 0．025,P<0．01)．MDA可明显刺激细胞PDGF 受体的表达(5．5±0．653 vs 对照组3．3±0．616, P<0．01),提高HSC细胞内氧化水平(荧光强度: 4．721±0．385 vs 对照组2．413±0．662,P<0．01), 10 μmol／L SAB则可抑制PDGF受体的表达(2．723±0．326)和降低细胞内的氧化水平 (3．324±0．264)(P<0．01)．结论:丹酚酸B可通过影响PDGF信号通路而抑制体外培养HSC的增殖,且这种抑制作用与丹酚酸B的抗氧化作用有关．     

表达HBVX基因的小鼠模型的建立及对肝细胞凋亡因子的影响

目的:探讨乙型肝炎病毒X(HBVX)基因及其产物对肝细胞凋亡相关基因表达的影响.方法:将实验用SPF级成年♂KM小鼠分为实验组、空质粒对照组、生理盐水对照组.实验组以构建好的PCDNA3.1-HBVX质粒,空质粒对照组以PCDNA3.1质粒,生理盐水对照组以生理盐水,通过尾静脉高压注入动物体内,在第48小时处死小鼠后,取肝组织,以RT-PCR,凝胶回收测序及Westernblot检测HBVX表达,以RT-PCR半定量检测小鼠肝组织内bax、c-myc及bcl-2的表达.结果:实验组RT-PCR显示465bp处有清楚条带,肝组织内有HBVXmRNA的存在,两对照组无HBVXmRNA存在,Western blot检测实验组有HBVX蛋白的表达;两对照组则无HBVX蛋白表达.通过对小鼠肝组织凋亡相关因子的半定量RT-PCR,相对于空质粒对照组和生理盐水对照组,转染HBVX基因的小鼠肝组织bax、c-myc及bcl-2的mRNA相对表达量明显增高,差异有显著性意义(bax:1.3127±0.0900vs1.0023±0.1670,0.9094±0.1081;c-myc:1.6294±0.0672vs1.2869±0....     

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on

AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells.METHODS: The recombinant adenoviruses AdXC,Ad-X and Ad-C expressing HBV X-HCV C fusion gene,HBVX gene and HCV C gene were constructed,respectively.Hepatoma cells were infected with different recombinant adenoviruses.MTT,colonyforming experiment,FCM,TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells.RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells.Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells.FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of GIS phase in the HepG2 cell cycle.The apoptosis index of the Ad-XC,Ad-X,Ad-C group cells was significantly lower than that of the AdO and control group cells.Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in AdXC infected cells.Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells,but not in control mice.CONCLUSION: Ad-XC,Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro.The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C.Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.     

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the expression and clinical significance of B7-H4 and hepatitis B virus X(HBx) protein in hepatitis B virus-related hepatocellular carcinoma(HBV-HCC).METHODS: The expression of B7-H4 in the human HCC cell lines Hep G2 and Hep G2.2.15 were detected by western blot, flow cytometry, and immunofluorescence. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Paraffin sections were generated from 83 HBV-HCC patients(22 females and 61 males) enrolled in this study. The age of these patients ranged from 35 to 77 years, with an average of 52.5 ± 11.3 years. All experiments were approved by the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine.RESULTS: B7-H4 was significantly upregulated in Hep G2.2.15 cells compared to Hep G2 cells. Specifically, the protein expression of B7-H4 in the lysates of Hep G2 cells was more than that in Hep G2.2.15 cells. In addition, HBx was expressed only in Hep G2.2.15 cells. Similar data were obtained by flow cytometry. The positive rates of B7-H4 and HBx in the tissues of 83 HBV-HCC patients were 68.67%(57/83) and 59.04%(49/83), respectively. The expression of HBx was correlated with tumor node metastases(TNM) stage, and the expression of B7-H4 was positively correlated with HBx(rs = 0.388; p 0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues. The expression level of B7H4 was negatively related to tumor TNM stage.CONCLUSION: Higher expression of HBx and B7-H4 was correlated with tumor progression of HBV-HCC, suggesting that B7-H4 may be involved in facilitating HBV-related hepatocarcinogenesis.     

miR-21对肝星状细胞的增殖和成纤维化的影响

目的研究miR-21对肝星状细胞(hepatic stellate cells,HSC)的增殖和成纤维化的影响。方法将大鼠HSC-T6传代分为四组,分别为正常组、酒精组、miR-21模拟物组、miR-21抑制剂组。其中正常组不做处理;miR-21模拟物组和miR-21抑制剂组分别将miR-21模拟物和miR-21抑制剂瞬时转染入细胞;然后,再用含有酒精的培养基诱导酒精组、miR-21模拟物组、miR-21抑制剂组的HSC的活化;48 h后,分别做两部分实验,一部分是细胞增殖测试实验(cell counting kit-8,CCK8);另一部分是提取各组细胞蛋白,Western blotting检测因子增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、转化生长因子β1(transforming growth factorβ1,TGF-β1)、Smad3、平滑肌肌动蛋白(α-smooth muscles actin,α-SMA)、结缔组织生长因子(connective tissue growth factor,CTGF)的表达变化。结果CCK8结果显示,miR-21模拟物能够促进HSC的增殖与活化,从而促进纤维化的进展;而miR-21抑制剂能够抑制HSC的活化,降低HSC的活力值,缓解纤维化的进展。Western blotting检测结果显示,PCNA、TGF-β1、Smad3、α-SMA、CTGF在酒精组的表达量明显比正常组高(P<0.05),而在miR-21模拟物组则比酒精组高(P<0.05),miR-21抑制剂组比酒精组明显降低(P<0.05)。结论miR-21能够促进HSC的增殖,促进HSC成纤维化的进展。     

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM： To investigate the biological impact of hepatitis B virus X- hepatitis C virus core （HBV X-HCV C） fusion gene on hepatoma cells.
METHODS： The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBVX gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colony- forming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion aene on liver cells.
RESULTS： MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the AdO and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice.
CONCLUSION： Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.     

乙型肝炎病毒X基因的原核表达及血清抗HBx抗体的检测

目的 应用表达蛋白检测乙型肝炎患者血清抗-HBx抗体水平并探讨其临床意义。方法 通过PCR扩增获得HBVX基因，与原核表达载体Pet32a＋连接构建PET32a-HBX原核表达载体，转化E．coli BL21表达获得重组融合蛋白。经切胶透析纯化后，应用重组蛋白HBx建立检测血清中抗-HBx抗体的间接ELISA方法，分别检测正常人组、急性肝炎组、慢性肝炎组、肝硬化组和肝细胞癌组患者血清中的抗-HBx抗体。结果 获得具有免疫原性的HBx融合蛋白；ELISA检测表明，慢性肝炎组、肝硬化组和肝细胞癌组的抗-HBx抗体的水平均高于急性肝炎组，差异具有显著性；在三组之问，慢性肝炎组高于肝硬化组和肝细胞癌组，差异具有显著性，肝硬化组和肝细胞癌组的抗-HBx抗体水平无显著性差异。结论 HBV患者血清中抗-HBX抗体是乙型肝炎病毒感染的一种特异性抗体，是HBV感染的血清学指标之一，可以反映乙型肝炎肝炎患者病情的变化。