细胞凋亡与骨髓增生异常综合征疾病演变的关系

目的探讨骨髓增生异常综合征（ＭＤＳ）骨髓细胞凋亡特征及其病理学意义。方法用原位末端脱氧核糖核苷酸转移酶介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ），ＤＮＡ梯子和体外培养等方法对３８例ＭＤＳ患者骨髓单个核细胞（ＢＭＭＣ）凋亡进行研究。结果ＭＤＳ患者ＢＭＭＣ的ＴＵＮＥＬ凋亡细胞阳性指数（ＰＩ）为２０．１９％±１１．０７％，显著高于正常人、ＭＤＳ转化的白血病、急性髓细胞白血病（ＡＭＬ）及缺铁性贫血（ＩＤＡ）组，（Ｐ＜０．００１和Ｐ＜０．０１）。ＴＵＮＥＬ和ＣＤ４１碱性磷酸酶抗碱性磷酸酶免疫酶标法（ＡＰＡＡＰ）双标记证明ＭＤＳ骨髓病态小巨核细胞发生了凋亡。动态观察１０例ＭＤＳ患者，转为白血病后凋亡细胞ＰＩ值显著下降，尚未转为白血病患者随病情恶化凋亡呈下降趋势。７例ＭＤＳ患者ＢＭＭＣ体外培养后，晚期ＰＩ（ＬＰＩ）显著增高，３例出现ＤＮＡ梯子，ＡＭＬ和ＩＤＡ组ＬＰＩ未见明显升高，未出现ＤＮＡ梯子。结论ＭＤＳ骨髓细胞凋亡过度与演变和转为白血病有关。原位检测细胞凋亡可作为监测ＭＤＳ预后指标     

化疗药物对肺癌细胞凋亡诱导作用的研究

目的探讨化疗药物鬼臼乙叉甙（ｅｔｏｐｏｓｉｄｅ，Ｖｐ１６）及中成药得力生对肺癌细胞凋亡的诱导作用。方法采用电镜、ＤＮＡ电泳、ＴＵＮＥＬ原位末端标记和流式细胞术（ＦＣＭ）检测凋亡细胞。结果Ｖｐ１６及得力生在一定的作用时间与剂量下均能诱导小细胞肺癌和腺癌细胞的凋亡，凋亡细胞有典型的形态学改变及ＤＮＡ梯形带形成，ＦＣＭ检测凋亡细胞处于低ＤＮＡ含量位置（ＰｒｅＧ１峰），ＴＵＮＥＬ荧光染色能将凋亡细胞的形态与定量计数结合起来。结论Ｖｐ１６及得力生均能成功诱导肺癌细胞凋亡，此种诱导作用呈药物作用浓度及时间依赖性     

细胞凋亡与细胞增殖在人肝细胞癌中的表达和意义

目的了解人肝细胞癌中细胞凋亡的形态与分布规律，探索细胞凋亡标记率和增殖标记率与肝细胞癌组织学分级的关系。方法 对２１例人肝细胞癌和１７例慢性肝病分别作ＴＵＮＥＬ标记和ＰＣＮＡ免疫组化检测，采用图像细胞仪作阳性细胞计数。结果 （１）细胞核染色质浓缩，边聚，胞浆嗜酸性变及核周空晕与ＴＵＮＥＬ标记有极好的相关性；（２）凋亡细胞胞多呈弥散分布，以癌坏死区周围多见，包膜浸润处少见；（３）增殖细胞率和凋亡细胞     

甘氨脱氧胆酸诱导大鼠肝细胞凋亡

目的 探讨甘氨脱氧胆酸（Ｇｌｙｃｏｄｅｏｘｙｃｈｏｌａｔｅ，ＧＤＣ）与大鼠肝细胞凋亡的关系。方法 以ＧＤＣ为处理因素，体外培养大鼠原代肝细胞，分别用光镜、电镜、ＴＵＮＥＬ原位杂交，流式细胞术和ＤＮＡ电泳技术观察和分析ＧＤＣ诱大鼠肝细胞凋亡的作用。结果 大鼠肝细胞在加入终浓度为１００μｍｏｌ／Ｌ的ＧＤＣ培养２ｈ后，光镜、电镜、ＴＵＮＥＬ原位杂交，可见凋亡细胞，终浓度为５０、１００、１５０μｍｏｌ／Ｌ     

低密度脂蛋白对人肾小球系膜细胞增殖的影响

用酶法测定了正常人、肾病综合征（ＮＳ）未服用糖皮质激素和服用糖皮质激素治疗患者的血清脂蛋白组分，用体外肾小球细胞培养的方法观察了正常人及ＮＳ患者的低密度脂蛋白（ＬＤＬ）对正常成年人肾小球系膜细胞（ＨＭＣ）增殖的影响。结果表明：（１）ＮＳ患者的ＬＤＬ构成成分发生改变，其对ＨＭＣ的增殖影响与正常人不同，小剂量时促进ＨＭＣ增殖作用较正常人增强，较大剂量时使ＨＭＣ增殖高峰的下降提前出现。（２）糖皮质激素如无治疗ＮＳ的疗效时，不影响ＮＳ患者ＬＤＬ的构成成分和对ＨＭＣ的作用。系膜细胞病变是肾小球疾病的重要病理部位，我们认为脂质代谢异常可能加速肾脏疾病的进展。     

rIFN－a对胃癌细胞表皮生长因子受体表达的调节

用中性红吸收分析法和放射配体结合法分析基因重组干扰素－α（ｒＩＦＮ－α）对胃癌细胞（ＳＧＣ７９０１、ＫＡＴＯⅢ）增殖及其表皮生长因子受体（ＥＧＦ－Ｒ）表达的影响。结果显示ｒＩＦＮ－α可明显抑制ＳＧＣ７９０１细胞增殖，与作用时间、剂量相关，而对ＫＡＴＯⅢ增殖无明显抑制作用。采用氯胺Ｔ法自行标记表皮生长因子（ＥＧＦ），配体结合实验Ｓｃａｔｃｈａｒｄ分析结果ＳＧＣ７９０１、ＫＡＴＯⅢ均高表达单一亲和性的ＥＧＦ－Ｒ。ｒＩＦＮ－α降低ＳＧＣ７９０１细胞ＥＧＦ－Ｒ数量，与作用时间、剂量相关，ＥＧＦ－Ｒ亲和性无变化，而对ＫＡＴＯⅢ细胞ＥＧＦ－Ｒ的数量及亲和性均无明显影响。结果提示ｒＩＦＮ－α抑制胃癌细胞生长可能和其下调细胞ＥＧＦ－Ｒ相关。     

硝苯地平对肝星状细胞分泌内皮素和一氧化氮的影响

一氧化氮 (ＮＯ)和内皮素 (ＥＴ)是调节肝星状细胞 (ＨＳＣ)收缩与舒张的重要因素。我们研究硝苯地平对ＨＳＣ分泌ＮＯ和ＥＴ的影响 ,探讨硝苯地平防治肝硬化门脉高压症的作用及其可能机制。一、材料与方法鼠肝星状细胞系ＨＳＣ Ｔ6 (美国旧金山总医院肝病研究中心Ｆｒｉｅｄｍａｎ教授惠赠 ) ,为ＳＶ 40转染ＳＤ大鼠ＨＳＣ。细胞表 1 　硝苯地平对ＨＳＣ分泌内皮素及一氧化氮的影响 ( ｘ±ｓ)分组样本数ＮＯ(ｍｍｏｌ/Ｌ)ＮＯＳ活性 (ｋｕ/Ｌ) ＥＴ1 ( μｇ/Ｌ)1:ＨＳＣ +对照 889.5± 5.5 2 .35± 0 .13 10 .2± 1.6 2 :ＨＳＣ +硝苯地…     

HCV核心蛋白诱导HepG2细胞凋亡作用的研究

多家实验室通过酵母双杂交等技术证实 ,Ｃ蛋白可与ＴＮＦＲ 1、ＬＴ βＲ、ＮＦ κＢ及ＡＰ 1等信号分子相互作用 ,影响有关细胞凋亡的信号转导 ,但这种影响的最终结果 ,是诱导凋亡还是抑制凋亡 ,尚有争议。在建立ＨＣＶＣ区转基因细胞模型的实验过程中 ,观察到Ｃ蛋白的表达对人肝源性肿瘤细胞具毒性作用 ,进而研究证实这种细胞毒作用是由于Ｃ蛋白诱导细胞凋亡所致。将原核表达的ＨＣＶＣ蛋白通过单细胞显微注射技术导入ＨｅｐＧ2细胞胞浆内 ,观察细胞生长与增殖变化 ,并用ＡＯ/ＥＢ荧光染色和ＴＵＮＥＬ试剂检测细胞凋亡情况。结果表明 ,与…     

褪黑素在急性肾缺血再灌注损伤中的防治作用及其对细胞凋亡的影响

目的：探讨急性肾缺血再灌注损伤（ ＡＲⅠ） 的机制及其与细胞凋亡的关系以及褪黑素（ ＭＥＬ） 对ＡＲⅠ的保护作用。　方法：用动脉夹夹闭大鼠双侧肾蒂４５ ｍｉｎ 再灌注２４ｈ 的手术方法制成ＡＲ Ⅰ动物模型,将动物分为假手术对照组、手术组、维生素Ｃ 预防组、ＭＥＬ 不同剂量预防组,动态检测血尿素氮（ＢＵＮ） 、肌酐（Ｃｒ） 、超氧化物歧化酶（ＳＯＤ） 、谷胱甘肽过氧化物酶（ＧＳＨＰｘ） 、丙二醛（ ＭＤＡ） 、肾组织光镜、电镜形态学观察及ＤＮＡ 电泳分析。结果：手术组ＢＵＮ、Ｃｒ 升高、ＳＯＤ、ＧＳＨＰｘ 下降,ＭＤＡ 上升,形态学显示肾小管细胞发生凋亡。ＭＥＬ 预防用药能不同程度地逆转上述改变,防止凋亡的发生,效应呈剂量依赖性,且较已知的抗氧化剂作用更明显。　结论：氧自由基（ＯＦＲ） 是ＡＲⅠ的主要机制之一,ＡＲⅠ确实存在着细胞凋亡的病理改变。ＭＥＬ 预防用药能减轻ＡＲⅠ,减少细胞凋亡的发生,效应呈剂量依赖性。     

小鼠病毒性心肌炎与细胞凋亡及氧自由基的关系

采用电镜技术及原位末端标记法（ＴＵＮＥＬ）对柯萨奇病毒Ｂ３（ＣＶＢ３）心肌炎小鼠不同时期心肌组织中细胞凋亡进行检测,并采用硫代巴比妥酸法检测外周血丙二醛（ＭＤＡ）。ＣＶＢ３感染１００只雄性Ｂａｌｂ／ｃ小鼠,ＶＭＣ发病率为８６％。各期病鼠心肌中均见到凋亡细胞,包括心肌细胞、血管内皮细胞及间质细胞。ＴＵＮＥＬ法检出率为７８．９４％,中、重度ＶＭＣ小鼠中阳性检出率明显高于轻度ＶＭＣ小鼠（Ｐ〈０．０５）。     

肝细胞对星状细胞凋亡的影响和复方861的干预作用

目的：研究肝细胞对星状细胞凋亡和影响中药复方861的干预作用。方法：体外采用分离培养的正常大鼠原代肝细胞和星状细胞系,将培养的肝细胞和复方861作用于不同的培养基再作用于星状细胞,用电镜观察和流式细胞仪检测细胞凋亡。临床研究对象是用复方861治疗6个月行治疗前后肝穿的慢性乙型肝炎病人。结果：原代培养的正常肝血细胞促进了星状细胞的凋亡（P＜0．05）。复方861可以增加这种促凋亡作用（P＜0．05）。临床研究显示慢性乙型肝炎患者肝星状细胞大量活化增生,用复方861治疗6个月后有肝细胞再生,同时活化的星状细胞数量显著减少,可观察到其凋亡。结论：正常肝细胞可以促进星状细胞凋亡,复方861对星状细胞有直接和间接的促凋亡作用。     

中药复方861抑制肝星状细胞NF-κB活性的体外研究

目的 研究中药方861对肝星状细胞NF－κB活性的影响。方法 将5mg/ml的复方861加入体外培养的大鼠肝星状细胞系作用48h,NF－κB活性的检测采用凝胶电泳移动抑制法,细胞培养液中细胞因子的检测采用ELISA法,细胞凋亡采用流式细胞仪和TUNEL法检测。复方861作用命名体外培养的肝星状细胞NF－κB结合活性比未加药的空白对照组显著减弱,细胞培养液中的IL－6和sICAM水平减低（P＜0.05）,星状细胞凋亡增加（P＜0.01）。结论 复方861显著抑制体外培养的肝星状细胞NF－κB活性可能为治疗肝纤维化中的重要机理之一。     

Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells.

Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.     

Role of the receptor for advanced glycation end products in hepatic fibrosis

AIM: To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. METHODS: In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGEBSA) and Nε-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-α (TNF-α). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. RESULTS: In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-α. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen α1(Ⅰ) mRNA by AGE-BSA. CONCLUSION: Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.     

Antifibrotic effects of green tea on in vitro and in vivo models of liver fibrosis

AIM: To examine the protective effect of green tea extract (GT) on hepatic fibrosis in vitro and in vivo in dimethylnitrosamine (DMN)-induced rats. METHODS: HSC-T6, a rat hepatic stellate cell line, was used as an in vitro assay system. Cell proliferation,collagen content, and type 1 collagen expression were examined in activated HSC-T6 cells. Collagen was determined by estimating the hydroxyproline content.In rats with DMN-induced hepatic fibrosis, serum aspartate aminotransferase and alanine aminotransferase concentrations, liver hydroxyproline and lipid peroxides were determined. Pathologic changes were examined by hematoxylin & eosin staining.RESULTS: GT administration prevented the development of hepatic fibrosis in the rat model of DMN-induced liver fibrosis. These results were confirmed both by liver histology and by quantitative measurement of hepatic hydroxyproline content, a marker of liver collagen deposition. Accordingly, inhibition of proliferation, reduced collagen deposition, and type 1 collagen expression were observed in activated HSC-T6 cells following GT treatment. These results imply that GT reduced the proliferation of activated HSC and down regulated the collagen content and expression of collagen type 1, thereby ameliorating hepatic fibrosis. tea administration can effectively improve liver fibrosis caused by DMN, and may be used as a therapeutic option and preventive measure against hepatic fibrosis.     

肝星状细胞凋亡机制研究进展

肝纤维化是肝脏对慢性损伤的一种修复反应,其特征性变化为细胞外基质过度沉积。肝星状细胞（HSC）活化是肝纤维化形成的中心环节,诱导活化的HSC凋亡有望逆转肝纤维化。本文从死亡受体途径、线粒体途径、内质网途径、神经生长因子途径、过氧化物酶体增生物激活受体-γ途径、核因子-κB途径对HSC的凋亡机制作一综述。     

Hepatic stellate cell behavior during resolution of liver injury

Acute self-limiting and chronic liver injury are both associated with activation and proliferation of hepatic stellate cells (HSCs). In chronic injury, activated stellate cells are the major source of the collagens that comprise fibrosis and cirrhosis, as well as of the tissue inhibitors of metalloproteinases (TIMPs) which inhibit collagen degradation. Recovery from acute and chronic injury is characterized by apoptosis of activated HSCs, which removes extracellular matrix-producing cells that are also expressing TIMPs, thereby relieving the inhibition of matrix degradation. HSC apoptosis is regulated in progressive injury and counterbalances cell proliferation. Apoptosis probably also represents a default pathway for the HSCs. The survival of activated HSCs in liver injury is dependent on soluble growth factors and cytokines, and on components of the fibrotic matrix. Additionally, stimulation of death receptors expressed on HSCs can precipitate their apoptosis. Our increasing understanding of the process of stellate cell behavior in recovery from injury is likely to be important to the design of antifibrotic therapies.     

肝损伤修复过程中p75神经营养因子受体的作用

p75神经营养因子受体(p75NTR)是肝星状细胞(HSC)表面表达的与神经生长因子(NGF)特异结合的受体,在肝脏损伤修复过程中发挥重要作用。在肝脏损伤修复过程中,p75NTR能提高肝组织肝细胞生长因子的表达,促进受损肝细胞的再生和肝组织结构的恢复;对于静息状态的HSC,p75NTR能促进HSC活化;在肝损伤晚期,随着再生肝细胞和活化HSC的增多,NGF分泌明显增加,NGF与p75NTR结合能促进活化的HSC凋亡和肝纤维化逆转。     

Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.     

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.