扁桃甙对大鼠肝贮脂细胞增殖和产生胶原的影响

目的探讨桃仁有效单体扁桃甙抗肝纤维化的作用机理．方法用链霉蛋白酶和胶原酶原位灌注大鼠肝脏，以１１％Ｍｅｔｒｉｚａｍｉｄｅ分离出贮脂细胞，传一代培养后分别加入１０－４－１０－９ｍｏｌ／Ｌ浓度的扁桃甙液温育７２ｈ，分别作细胞增殖和细胞产生胶原的测定．结果低浓度药物能减少［３Ｈ］胸腺嘧啶核苷细胞内掺入量及抑制［３Ｈ］脯氨酸掺入于细胞分泌的胶原酶敏感蛋白和细胞层胶原酶敏感蛋白中，其中尤以１０－８ｍｏｌ／Ｌ浓度最为明显，其对贮脂细胞增殖的抑制率达２５０％，培养液和细胞内胶原产生的抑制率分别为２４２％和２６８％．结论扁桃甙能抑制活化贮脂细胞的增殖和产生胶原     

贮脂细胞和肝细胞胶原合成能力的比较

目的为明确肝纤维化过程中细胞外基质（ｅｘｔｒａｃｅｌｕｌａｒｍａｔｒｉｘ，ＥＣＭ）的细胞来源以及贮脂细胞（ｆａｔｓｔｏｒｉｎｇｃｅｌ，ＦＳＣ）和肝细胞（ｈｅｐａｔｏｃｙｔｅ，ＨＣ）在肝纤维化胶原合成中的作用．方法用胶原酶和链霉蛋白酶Ｅ消化大鼠肝脏，结合密度梯度离心法分离培养大鼠ＨＣ和ＦＳＣ，在此基础上，用３Ｈ脯氨酸掺入结合胶原酶消化法和斑点杂交法，观察ＨＣ和ＦＳＣ胶原的合成及Ⅰ，Ⅲ，Ⅳ型前胶原基因的表达．结果发现体外培养的ＦＳＣ合成胶原的能力较ＨＣ强，约为ＨＣ的两倍（Ｐ＜００５）；ＦＳＣ胶原基因表达的水平同样较ＨＣ高（Ｐ＜００５）．结论ＦＳＣ在ＥＣＭ胶原合成中起主要作用，但ＨＣ的作用也不容忽视．     

纤维粘连蛋白和层粘连蛋白对原代培养血管平滑肌细胞胶原基因表达的影响

为了研究纤维粘连蛋白（Ｆｎ）和层粘连蛋白（Ｌｎ）对血管平滑肌细胞（ＶＳＭＣ）胶原基因表达的影响。我们应用Ｎｏｒｔｈｅｒｎ杂交的方式观察不同剂量的Ｆｎ和Ｌｎ处理的ＶＳＭＣｓⅠ、Ⅲ型前胶原［Ｐｒｏα１（Ⅰ）、Ｐｒｏα１（Ⅲ）］基因ｍＲＮＡ的改变。发现Ｆｎ能明显地促使Ｐｒｏα１（Ⅰ）基因的转录，Ｆｎ的这种作用与剂量呈正比，α１（Ⅲ）基因的５．８ｋｂｍＲＮＡ在Ｆｎ４．０μｇ组比对照组增加明显，４．８ｋｂｍＲＮＡ和５．４ｋｂｍＲＮＡ在Ｆｎ４００μｇ组较Ｆｎ４０μｇ组增加，三种剂量下５．８ｋｂｍＲＮＡ量变化不大。Ｌｎ也能使ＶＳＭＣｓ的α１（Ⅰ）基因ｍＲＮＡ量增加，但与剂量无关，对α１（Ⅲ）基因ｍＲＮＡ量的改变在Ｌｎ２．４μｇ组与２４μｇ组差异无明显性，但比对照组增加，在Ｌｎ２４０μｇ组则４．８ｋｂｍＲＮＡ量增加，５．８ｋｂｍＲＮＡ量变化不明显。结果提示：Ｆｎ和Ｌｎ都能影响ＶＳＭＣｓＰｒｏα１（Ⅰ）、α１（Ⅲ）基因的表达，它们的作用机制可能有联系，但不完全相同。     

纤维粘连蛋白和层粘连蛋白对原代培养血管平滑肌细胞胶原基…

为了研究纤维粘连蛋白（Ｆｎ）和层粘连蛋白（Ｌｎ）对血管平滑肌细胞（ＶＳＭＣ）胶原基因表达的影响。我们应用Ｎｏｒｔｈｅｒｎ杂交的方式观察不同剂量的Ｆｎ和Ｌｎ处理的ＶＳＭＣｓＩ、Ⅲ型前胶原［Ｐｒｏα１（Ｉ）、Ｐｒｏα１（Ⅲ）］基因ｍＲＮＡ的改变。发现Ｆｎ能明显地促使Ｐｒｏα１（Ｉ）基因的转录，Ｆｎ的这种作用与剂量呈正比，α１（Ⅲ）基因的５．８ｋｂ ｍＲＮＡ在Ｆｎ４．０μｇ组比对照组增加明显，４．８ｋｂ     

肝症口服液含药血清对TGFα诱导SMMC-7721细胞增殖和ERK蛋白的影响

目的观察肝症口服液含药血清对TGFα诱导人肝癌细胞增殖和信号传导因子ERK影响.方法采用血清药理学方法,以病理鼠血清作对照,应用MTT比色法观察中药鼠血清对TGFα诱导人肝癌细胞SMMC-7721增殖的抑制作用.用免疫组化方法检测中药血清对信号传导因子ERK蛋白表达影响.结果中药血清作用24 h对SMMC-7721细胞抑制率达25%,P＜0.05;作用48 h抑制率达30%,P＜0.001;中药血清作用24 h对1μg·L-1 TGFα诱导的SMMC-7721细胞增殖抑制率为12.3%,P＞0.05;作用48 h抑制率为16%,P＜0.05;中药血清作用24 h对5μg·L-1TGFα诱导的SMMC-7721细胞增殖抑制率为8.9%,P＞0.05,作用48 h抑制率为17.2%,P＜0.001.中药血清对TGFα诱导的肝癌细胞SMMC-7721增殖有明显的抑制作用,抑制效应呈时间依赖性.中药血清能抑制ERK蛋白在细胞核中的表达.结论肝症口服液含药血清能够抑制TGFα诱导的人肝癌细胞增殖,阻断TGFα在细胞内的信号传导.     

透明质酸、Ⅲ型前胶原、层粘连蛋白对肝纤维化诊断价值的研究

应用放射免疫法测定８８例各种肝病患者血清透明质酸（ＨＡ）、Ⅲ型前胶原（ＰＣⅢ）和层粘连蛋白（ＬＮ）水平。结果发现随着肝病慢性化的进展，三者均逐渐增高，肝硬化时达最高值。在３３例经病理学证实的病例中，慢性活动性肝炎及肝硬化时ＨＡ、ＰＣⅢ、ＬＮ与肝纤维组织增生程度密切相关（Ｐ＜０．０５），且与白蛋白／球蛋白比值呈显著负相关（Ｐ＜０．０１）。提示ＨＡ、ＰＣⅢ、ＬＮ对肝纤维化诊断有较高价值。     

血清透明质酸、层粘连蛋白、Ⅳ型胶原和Ⅲ型前胶原水平与肝纤维化的关系

目的:探讨4项血清肝纤维化指标的诊断价值。方法:采用放射免疫法对478例各型肝病患者血清透明质酸(HA)、层粘连蛋白(LN)、Ⅳ型胶原(Ⅳ-C)和Ⅲ型前胶原(PCⅢ)指标进行了联合检测。结果:HA、Ⅳ-C和PCⅢ3项指标在慢性肝炎轻度、急性肝炎、慢性肝炎中度、重度和重型肝炎之间依次升高,各组间均有显著性差异(P<0.01)。重型肝炎组HA、PCⅢ和Ⅳ-C显著高于原发性肝癌(HCC)组和肝炎肝硬变(HLC)组(P<0.05)。而LN在对照组、急性肝炎、慢性肝炎轻度、中度和重度间均无显著性差异(各组间P<0.05),在重型肝炎组、HLN组和HCC组LN水平则显著高于急性肝炎、慢性肝炎轻、中、重度组(P<0.01),说明LN可作为区分慢性肝炎和肝硬变的辅助指标之一。4项肝纤维化指标联合检测结果中,2项和3项以上阳性在慢性肝炎轻度、中度、重度和肝炎肝硬变、重型肝炎之间依次升高,各组间均有显著性差异(P<0.01或P<0.05)。结论;4项指标联合检测可极大增加肝纤维化临床诊断的准确性和可靠性。     

血清PCⅢ,HA,LN联合检测对肝纤维化诊断价值的初步探讨

血清ＰＣⅢ、ＨＡ、ＬＮ联合检测对肝纤维化诊断价值的初步探讨李兵顺，王继，刘金星，孙述强，魏梅新，刘晓梅，裴秀，王麟士为了寻找早期诊断肝纤维化敏感而可靠的血清学指标，我们对１４８例各种类型肝病联合检测了血清Ⅲ型前胶原（ｓｅｒｕｍｔｙｐｅⅢｐｒｏｃｏｌｌ...     

Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM To study the effects of hypoxia,hyperoxia on theregulation of expression and activity of matrixmetalloproteinase-2 (MMP-2) in hepatic stellate cells(HSC).METHODS The expressions of MMP-2,tissue inhibitor ofmatrix metalloproteinass-2 (TIMP-2) and membrane typematrix matalloproteinass-1 (MT1-MMP) in cultured rat HSCwere detected by immunocytochemistry (ICC) and in situhybridization (ISH).The contents of MMP-2 and TIMP-2 inculture supernatant were detected with ELISA and theactivity of MMP-2 in supernatant was revealed byzymography.RESULTS In the situation of hypoxia for 12h,theexpression of MMP-2 protein was enhanced (hypoxiagroup positive indexes:5.7±2.0,n=10;control:3.2±1.0,n=7;P<0.05),while TIMP-2 protein was decreasedin HSC (hypoxla group positive indexes:2.5±0.7,n=10;control:3.6±1.0,n=7;P<0.05),and the activity(total A) of MMP-2 in suparnatant declined obviously(hypoxla group:7.334±1.922,n=9;control:17.277±7.424,n=11;P<0.01).Compared the varied duration ofhypoxia,the changes of expressions Including mRNA andprotein level as well as activity of MMP-2 were mostnotable in 6 h group.The highest value (Ahypoxla~-Acontrol) ofthe protein and the most intense signal of mRNA were inthe period of hypoxia for 6 h,along with the lowestactivity of MMP-2.In the situation of hyparoxia for 12 h,the contents (A_(450)) of MMP-2 and TIMP-2 in supernatantwere both higher than those in the control,especially theTIMP-2 (hyperoxla group:0.0499±0.0144,n=16;control:0.0219±0.0098,n=14;P<0.01),and so wasthe activity of MMP-2 (hyperoxia group:5.252±0.771,n=14;control:4.304±1.083,n=12;P<0.05),and the expression of MT1-MMP was increased.CONCLUSION HSC is sensitive to the oxygen,hypoxiaenhances the expression of MMP-2 and the effect is moremarked at the early stage;hyperoxia mainly raises theactivity of MMP-2.     

Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats

ＩＮＴＲＯＤＵＣＴＩＯＮＬｉｖｅｒｆｉｂｒｏｓｉｓｉｓｔｈｅｃｏｍｍｏｎｐａｔｈｏｌｏｇｉｃａｌｆｅａｔｕｒｅｏｆｃｈｒｏｎｉｃｌｉｖｅｒｄｉｓｅａｓｅｓ,ａｎｄｉｓｃｌｏｓｅｌｙａｓｏｃｉａｔｅｄｗｉｔｈｃｈａｎｇｅｓｏｆｌｉｖｅｒｃｅｌｆｕｎｃｔ...     

双环醇对慢性乙型肝炎患者肝星状细胞活化和胶原合成的影响

目的:观察双环醇对慢性乙型肝炎(乙肝)患者肝组织炎症、纤维化程度、肝星状细胞活化和胶原合成的影响,探讨其抗纤维化作用机制。方法:20例慢性乙肝患者以双环醇150mg/d治疗24周,治疗前、后分别行肝穿刺活体组织病理学检查,评估治疗前、后肝组织炎症及纤维化程度;并应用免疫组织化学技术和彩色病理图文分析系统观察治疗前、后肝组织内α平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)表达状况和Ⅰ、Ⅲ型胶原含量的变化。结果:慢性乙肝患者经双环醇治疗后,其血清丙氨酸氨基转移酶(ALT)和γ谷氨酰转移酶(γ-GT)水平可显著降低,复常率分别达95%和70%,不仅肝组织炎症和纤维化程度明显改善,且肝组织内α-SMA和TGF-β1的表达强度明显减弱,同时Ⅰ、Ⅲ型胶原的含量也较治疗前明显降低。结论:双环醇治疗可减轻慢性乙肝患者肝脏炎症反应和纤维化程度,同时明显降低肝组织内TGF-β1、α-SMA的表达,阻止肝星状细胞活化和Ⅰ、Ⅲ型胶原的合成,从而发挥抗纤维化作用。     

肝星状细胞激活与ICAM-1的表达

目的探讨肝星状细胞（ＨＳＣ）的活化与细胞间粘附分子１（ＩＣＡＭ１）表达的关系．方法用链酶蛋白酶和胶原酶原位灌流，Ｎｙｃｏｄｅｎｚ密度梯度离心分离大鼠ＨＳＣ，并进行体外培养，应用免疫组织化学方法观察静息或活化状态下的ＨＳＣ中ＩＣＡＭ１表达．结果静息的ＨＳＣ不表达ＩＣＡＭ１，而活化的ＨＳＣ表达ＩＣＡＭ１，且随着培养时间的延长ＩＣＡＭ１表达量逐渐增加．结论ＩＣＡＭ１表达与ＨＳＣ活化及肝纤维化的发生有关．     

木犀草素抑制肝星状细胞增殖及其胶原合成

目的 研究木犀草素对体外培养的肝星状细胞(hepatic stellate cells,HSC)增殖及其胶原表达、合成的影响。方法 从Wistar大鼠肝脏分离培养HSC,并用~3H-TdR和~3H-pro同位素掺入实验,基因探针原位杂交等技术研究了木犀草素对HSC增殖、胶原基因表达合成的影响。结果 当木犀草素的浓度分别达到10 μmol/L和20 μmol/L 时抑制HSC增殖(t=2.542,P＜0.05)和胶原合成(t=3.650,P＜0.01),其作用具有剂量依赖关系;25 μmol/L木犀草素使Ⅰ、Ⅲ型前胶原mRNA的表达降低,其中Ⅰ型前胶原基因表达降低具有统计学差异(x~2=6.850,P＜0.01)。结论 木犀草素在体外抑制HSC增殖和胶原表达合成,在体内可能会具有预防或冶疗肝纤维化的作用。     

Cell survival,activation and apoptosis of hepatic stellate cells: modulation by extracellular matrix proteins

Aim: Cytokines and growth factors released by various hepatic cells exert both paracrine and autocrine effects on hepatic stellate cell (HSC) activation during liver injury. The aim of the present study was to examine whether the surrounding extracellular matrix (ECM) influences the activation, transdifferentiation and survival of HSCs. Methods: An in vitro model system of isolated HSCs maintained in culture on different matrix protein substrata was employed. Results: The rate of loss of HSC‐specific retinol uptake activity and gain of myofibroblast‐like activity such as ³⁵[S] proteoglycan synthesis varied in cells maintained on different matrix proteins and was in the order collagen I > collagen IV ≥ laminin. ³[H]‐thymidine incorporation by HSCs maintained on different matrix proteins varied and was in the order collagen I > collagen IV > laminin. MTT assay revealed that the growth inhibition in response to curcumin was significantly low in cells maintained on collagen I. Apoptotic marker activities such as DNA fragmentation, 4′,6′‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining, annexin staining and caspase‐3 activities showed that cells maintained on collagen I showed minimal apoptosis than those maintained on collagen IV, laminin and polylysine, showing the influence of ECM on HSC apoptosis. Experiments using blocking antibodies showed that the collagen I effect was mediated through α₂β₁ integrin. Conclusions: These results indicate that ECM influences activation, transdifferentiation and survival of HSCs, and suggest that apart from diffusible factors, the surrounding ECM also influences HSC behavior critical in both the progression of the fibrosis and the restitution of the liver during recovery after hepatic injury.     

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of ...