胆红素对肝星状细胞-T6增殖及细胞周期的影响

[目的]观察胆红素对肝星状细胞(HSC)-T6增殖及细胞周期影响.[方法]将培养细胞分成正常组和胆红素不同浓度(10 μmol/L、30 umol/L、50 /μmol/L、70 μmol/L、100 μmol/L)干预组,采用MTT法观察胆红素对HSC-T6增殖的影响,流式细胞仪观察各组细胞周期的变化.[结果]①不同浓度胆红素对HSC-T6均有促进增殖作用,且呈一定的量效关系,与正常组比较差异有统计学意义(P＜0.05);②10 μmol/L、50 μmol/L、100 μmol/L浓度胆红素作用HSC-T6后,G0/G1期减少,S期增加,G2/M期增加,与正常组比较均P＜0.05.[结论]胆红素对HSC-T6均有促进增殖作用.     

调节性T细胞对肝星状细胞增殖及透明质酸分泌的影响

目的研究CD4+CD25+调节性T细胞(Treg)对肝星状细胞(HSC)增殖及透明质酸(HA)分泌的影响。方法 MASC法分离慢性乙型肝炎患者血中CD4+CD25+Treg细胞;传代培养人HSC LX-2(空白组),不同比例CD4+CD25+Treg细胞与HSC直接共培养(共培养组),不同剂量的重组人转化生长因子(TGF)β1和HSC共培养(对照组);使用CCK-8法检测HSC增殖、放射免疫法检测HA分泌情况。组间比较采用单因素方差分析。结果共培养组与空白对照组(0.6623±0.02702)相比,在比例为2.5∶1时HSC增殖最为明显(0.6836±0.00560),比例为1∶1、5∶1及7.5∶1时HSC增殖均明显减少,且随着比例升高其增殖水平逐渐降低,各组与空白对照组相比有统计学差异(F=11.421,5.272,84.514,490.639;均P<0.05)。不同浓度TGFβ1对照组的HSC均比空白对照组(0.3883±0.00189)细胞增殖明显(0.6720±0.00753,0.6773±0.00585,0.5373±0.00387),并于8 ng/μl时增殖达到高峰,各亚组与空白组相比有统计学差异(F=196.175,799.624,236.461;P<0.01)。共培养组与对照组中细胞上清液中HA浓度与HSC增殖呈正相关(r=0.661、0.871,均P<0.01)。结论 CD4+CD25+Treg细胞促HSC增殖并促进其分泌HA,并在体外证实CD4+CD25+Treg细胞在慢性乙型肝炎进展过程中发挥重要作用。     

大鼠肝星状细胞大电导钙激活钾通道特性与细胞增殖的关系

目的探讨肝星状细胞（HSC）钙激活钾通道特性,以及钾通道对HSC增殖的影响。方法以胶原酶循环灌流法分离大鼠HSC,用结蛋白抗体及α-抗平滑肌抗体进行分离细胞鉴定,采用单通道细胞膜片钳技术检测传代HSC钾通道的特性,通过MTT法、流式细胞术分别检测钾离子通道阻滞剂四乙胺（TEA）对HSC的增殖、细胞周期的影响。结果所分离细胞为HSC。传代活化的HSC有单通道存在,其电导值为（210.86±5.49）pS（n=7）,属于大电导通道;其通道表现出明显的电压依赖性以及对钙敏感性,TEA可明显减少外向钾离子通道开放概率。TAE对HSC的增殖抑制效应具有时间依赖与剂量依赖的特点,可使细胞周期阻滞于G0/G1期。结论 HSC上大电导钙激活钾通道可能参与了HSC活化增殖。     

PAX6通过MEK/ERK信号通路抑制肝星状细胞活化和增殖实验研究

目的 探讨PAX6通过丝裂原活化蛋白激酶(MEK)/细胞外调节蛋白激酶(ERK)信号通路抑制肝星状细胞活化和增殖的效果。方法 取LX2肝星状细胞,分为对照组、PAX6 inhibitor组和PAX6 mimics组。采用CCK-8试剂盒测定细胞增殖,采用油红O染色测定细胞分化水平,使用流式细胞仪分析细胞凋亡,采用RT-PCR法和蛋白印迹法测定细胞PAX6、MEK和ERK mRNA和蛋白表达水平。结果 PAX6 inhibitor组细胞PAX6 mRNA水平、凋亡率和G1期分别为(1.49±0.23)、(2.70±0.85)%和(59.02±1.25)%,显著低于LX2肝星状细胞组【分别为(1.85±0.19)、(3.40±0.47)%和(64.66±1.41)%,P<0.05】,而细胞增殖、存活率、分化率、MEK和ERK mRNA和蛋白水平分别为(0.79±0.03)、(73.35±9.74)%、(49.37±4.24)%、(2.55±0.43)、(3.90±0.49)、(0.89±0.15)和(1.17±0.17),显著高于LX2肝星状细胞组【分别为(0.58±0.05)、(6...     

Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity

Purpose. Alterations in the kinetics of hepatic stellate cells (HSCs) after the cells are activated once have not been well documented. We investigated the characteristic profiles of cell proliferation of once-activated HSCs in contrast to the in fibrogenesis activity. Methods. HSCs from male Wistar rats were submitted to primary culture for 14 days and to secondary culture for 7 days. The potential for cell proliferation was evaluated by the number of the cells in G2/M phase, based on flow cytometric analysis of the cell cycle. The fibrogenesis activity was assessed by Northern blot analysis of the expression of type I and type III procollagen mRNA. Results. The number of HSCs in G2/M phase was maintained at a low level in primary culture after 6 days, while a significantly (P < 0.05) elevated number of HSCs in G2/M phase was observed on days 3 to 4. In secondary culture, the number of HSCs in G2/M phase was also consecutively maintained at a decreased level. By contrast, HSCs showed progressively increased type I and type III procollagen mRNA expression during the experimental periods of primary culture. Conclusions. These results clearly demonstrated consecutively decreased proliferative activity, evaluated by the potential for cell mitogenesis, in once-activated HSCs, in contrast to their progressively increased fibrogenesis activity. Received: April 12, 2000 / Accepted: December 22, 2000     

Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection

Summary.  Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection ( n  = 44) and HCV-negative controls with normal liver histology ( n  = 9). We used immunohistochemical techniques to identify activated (α-smooth muscle actin⁺), proliferative (Ki-67⁺) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling⁺) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, ×200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0–3.1 vs 1.1, 0.2–3.5 cells/HPF, P  =   0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis ( P  = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01–0.28 cells/HPF) per increase in fibrosis stage ( P  = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.     

人骨髓间充质干细胞上清液对体外肝星状细胞增殖周期及MMP-1表达的影响

目的 研究骨髓间充质干细胞(BMSC)对肝星状细胞(HSC)增殖周期及基质金属蛋白酶-1(MMP-1)活性的影响,以探讨其防治肝纤维化的作用机制.方法 采用密度梯度离心法分离鉴定人BMSC,收集原代培养7d的BMSC培养上清,加入HSC中培养24 h及48 h.通过流式细胞仪观察HSC增殖周期,ELISA方法检测MMP-1浓度.结果 与HSC单独培养组相比,共培养48 h组G1期细胞显著增加(P＜0.01),S期细胞显著减少(P＜0.01),并且共培养组MMP-1表达明显增加,与对照组相比差异均具有统计学意义(P＜0.05).结论 BMSC可抑制HSC的增殖,并且可能通过增加MMP-1的产生,从而起到抗纤维化的作用.     

Inhibition of hepatic stellate cells proliferation by mesenchymal stem cells and the possible mechanisms

Aim: During fibrosis, hepatic stellate cells (HSCs) undergo a complex activation process characterized by increased proliferation and extracellular matrix deposition. Previous studies have suggested that mesenchymal stem cells (MSCs) may ameliorate fibrogenesis and represent a promising strategy for cell therapy. However, the underlying mechanisms are not fully understood. Methods: Hepatic stellate cells were treated with or without MSCs. Then cell proliferation and cell cycle were analyzed. Production of soluble factors by MSCs and its relation with cell proliferation suppression was evaluated by transwell co‐culture and RNA interference. Effects of MSCs on the gene expression of collagen were also evaluated. Results: MSCs induced G₀/G₁ arrest of HSCs growth partly through secreting soluble factors TGF‐β3 and HGF, which resulted in up‐regulation of p21^Cip1 and p27^Kip1 expression and down‐regulation of cyclinD1. MSCs inhibited the phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2 and reduced gene expression of collagen type I and III. MSCs did not reverse the proliferation and collagen type I gene expression of HSCs provoked by PDGF. Conclusions: The growth inhibition of HSCs induced by MSCs through an arrest in the G₀/G₁ phase of the cell cycle is partially mediated by secretion of TGF‐β3 and HGF. MSCs inhibit HSCs activation through decreasing phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. These results further support MSCs may be used as a novel therapy for treating fibrotic diseases in human.     

牛磺酸通过调控细胞周期蛋白抑制肝星状细胞增殖

目的进一步研究牛磺酸对肝星状细胞(HSC)增殖抑制作用的机制。方法用四甲基偶氮唑盐法检测细胞增殖;流式细胞仪测定细胞周期;免疫细胞化学和实时荧光定量PCR测定细胞周期调控蛋白Cyclin D1和P21waf1表达。结果牛磺酸对HSC增殖具有抑制作用,在浓度为5、10、20,30、40、50 mmol/L 作用48h时的抑制率分别为6.7%、14.4%、23.3%、32.2%、36.7%和45.6%,t值为2.939～6.369,P<0.05～0.01。流式细胞仪检测发现牛磺酸可阻滞HSC由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少。G0/G1期、S期细胞,牛磺酸浓度为40 mmol/L时,分别为(68.2±1.4)%和(26.2±1.3)%,与对照组分别为(56.2±1.7)%和(38.5±0.8)%,差异有统计学意义,t≥5.422,P<0.01。牛磺酸可抑制Cyclin D1表达、促进P21waf1表达,用免疫细胞化学染色结合数码图像分析系统软件分析发现牛磺酸浓度在40 mmol/L时HSC的Cyclin D1表达的平均吸光度为0.13±0.02,P21waf1为0.19±0.02,对照组分别为0.18±0.02和0.14±0.01,差异有统计学意义,t=6.689和t=6.528,P<0.01。实时荧光定量PCR检测也发现经40 mmol/L牛磺酸处理的HSC的Cyclin D1 mRNA表达量(拷贝数与106磷酸甘油醛脱氢酶比值)降低为5776.7±3345.0,对照组为18 400.6±1374.8,而P21waf1 mRNA表达量(拷贝/106磷酸甘油醛脱氢酶)增多为44 866.7±3910.7,对照组为16 933.3±960.9。结论牛磺酸通过抑制Cyclin D1表达、促进P21waf1表达,使HSC阻滞于G0/G1期,而抑制HSC增殖。     

清肝活血方对酒精性肝纤维化大鼠肝星状细胞增殖与凋亡的影响

目的:观察酒精性肝纤维化大鼠肝星状细胞(HSC)的增殖与凋亡以及清肝活血方的调节作用。方法:雄性Wistar大鼠60只,随机分为空白对照组、模型对照组以及清肝活血方高、中、低剂量组,采用复合因素制备酒精性肝纤维化动物模型。观察清肝活血方对酒精性肝纤维化大鼠肝功能、病理、α-平滑肌动蛋白(α-SMA)以及HSC增殖与凋亡的影响。结果:清肝活血方各剂量组能显著降低酒精性肝纤维化大鼠血清ALT、AST水平(P<0.05);清肝活血方高剂量组能显著降低血清γ-GT水平(P<0.05);各药物组均能在不同程度上减轻肝脏炎症和纤维化,减少肝组织α-SMA的表达(P<0.05),抑制HSC增殖并提高HSC的凋亡率。结论:清肝活血方能有效改善酒精性肝纤维化大鼠肝功能,减轻纤维化程度。其作用机制可能是通过抑制HSC的增殖并促进活化HSC凋亡实现的。     

斑蝥酸钠维生素B6对人肝星状细胞增殖的影响及其机制

目的 探讨斑蝥酸钠维生素B6注射液对人肝星状细胞LX-2增殖活化的影响及其机制.方法 培养人肝星状细胞LX-2,将其分为3个观察组（斑蝥酸钠维生素B6的浓度分别为1、5、10 μg/mL）和1个空白对照组;培养24、48、72 h后,采用MTT法检测细胞增殖活性,采用荧光定量PCR方法检测Ⅰ型胶原、Ⅲ型胶原、转化生长因子β1基因表达变化.结果 与空白对照组比较,观察组人肝星状细胞LX-2增殖活化被显著抑制（P均＜0.05）,Ⅰ型胶原、Ⅲ型胶原、转化生长因子β1mRNA表达下降（P均＜0.05）.结论 斑蝥酸钠维生素B6可以抑制人肝星状细胞LX-2增殖活化,其机制可能与其抑制、转化生长因子β1信号通路有关.     

苦参碱对人涎腺腺样囊性癌细胞增殖抑制及PCNA、c-myc、p53蛋白表达的影响

目的探讨苦参碱对肿瘤细胞的增殖抑制作用及机制。方法体外培养人涎腺腺样囊性癌（SACC）细胞系ACC-M细胞,用不同质量浓度的苦参碱进行干预,采用MTT及流式细胞仪检测细胞增殖情况,采用免疫细胞化学染色、间接免疫荧光定量检测等方法检测增殖细胞核抗原（PCNA）、c—myc与p53蛋白表达。结果经苦参碱处理后的ACC-M细胞生长速度减慢,细胞周期明显阻滞;其PCNA与c-myc蛋白表达明显下降,而p53蛋白表达则明显上升。结论苦参碱对ACC-M细胞具有明显的增殖抑制作用,可能机制为调控PCNA、c-myc及p53蛋白表达;此为临床治疗SACC提供了理论依据。     

Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

AIMTo investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis.METHODSThe expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed.RESULTSThe expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs.CONCLUSIONELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.