整合素α5β1与大鼠肝纤维化发生的实验研究

[目的]肝星状细胞（HSC）活化是肝纤维化的病理基础，本研究旨在探讨整合素受体在HSC活化及肝纤维化过程中的作用。[方法]制作大鼠肝硬化模型，同时进行体外细胞株HSC—T6实验。用免疫组化法观察仅平滑肌肌动蛋白（α-SMA）、整合素α5β1及纤维连接蛋白（FN）的表达。HSC细胞株在FN包被的皿上培养，测定细胞活化、α-SMA、整合素α5β1蛋白表达。[结果]（1）模型组肝组织FN、α-SMA和α5β1蛋白表达增多。（2）FN包被明星促进HSC—T6的活化、α—SMA及α5β1的表达。[结论]FN促进整合素α5β1表达是活化HSC、促进肝纤维化的重要途径。     

酶谱法测定肝星状细胞合成的明胶酶活性

目的建立检测肝星状细胞（HSC）合成的明胶酶（MMP-2,MMP-9）活性的方法。方法用含SDS-明胶的聚丙烯酰胺凝胶电泳为基础的酶谱法,检测体外培养的HSC所合成的明胶酶活性。结果酶谱法能灵敏地检测到HSC分泌到培养基中的MMP-2和MMP-9,培养基中MMP-2活性高于MMP-9,并观察到药物作用以后酶活性的改变。结论酶谱法是研究HSC调节肝纤维化过程中细胞外基质（ECM）代谢的有效手段。     

姜黄素对血小板衍生生长因子诱导活化的肝星状细胞细胞外基质的影响

目的 研究姜黄素对血小板衍生生长因子（PDGF）诱导活化的肝星状细胞细胞外基质沉积的干预机制。方法 以Western blotting和RT-PCR方法分别检测姜黄素对PDGF诱导活化的肝星状细胞中α-平滑肌肌动蛋白（α-SMA）、细胞外基质主要成分I型前胶原（α1I胶原）和纤黏蛋白的表达,以及对调控细胞外基质降解平衡的基质金属蛋白酶（MMP）-2、MMP-9与其组织抑制剂（TIMP-1）蛋白与mRNA表达的影响。结果 姜黄素可显著下调α-SMA、α1I胶原和纤黏蛋白的蛋白与mRNA表达,减少细胞外基质的沉积;还可调节细胞外基质的降解平衡,表现为TIMP-1表达下调和MMP-2的表达上调。结论 姜黄素能够抑制PDGF诱导的肝星状细胞合成与分泌细胞外基质增加,促进细胞外基质降解,减少细胞外基质在肝脏中的沉积,进而发挥治疗肝纤维化的作用。     

肝星状细胞凋亡信号途径及药物 reversine 的研究进展

肝星状细胞（ hepatic stellate cells ,HSC）在肝纤维化的发生发展中起着重要作用。正常情况下HSC处于静止状态,当致肝病因子造成肝细胞损伤时,激活的Kupffer细胞、窦内皮细胞及损伤的肝细胞等分泌多种细胞因子和化学介质共同作用于HSC,使HSC激活并转化为肌成纤维细胞,激活的HSC通过自分泌和旁分泌,促进HSC增殖,合成大量细胞外基质（ECM）并在肝内沉积,导致肝纤维化［1－2］。因此,HSC的活化是肝纤维化形成的关键,是肝纤维化发生发展的中心环节［3－7］,那么阻断HSC活化并促进活化HSC的凋亡对逆转肝纤维化具有实用价值。研究发现,逆转素（ reversine ）能够让高度分化的成熟细胞回复到脱分化的状态,已证实re-versine在体内外均具有抑制HSC增殖的作用［8］,但reversine抑制HSC增殖及促进HSC凋亡的具体机制仍不清楚。本文就HSC凋亡的信号途径中,有关于Bax、Bcl－2、Caspase－3、TNF－α、NF－κB、TIMP－1与HSC凋亡的关系以及reversine的研究进展作相关综述,以期为研究reversine治疗肝纤维化可能的机制奠定理论基础。     

肝纤维化相关因子与治疗展望

中国是乙肝大国，肝纤维化是多种慢性肝病发展至肝硬化的必经阶段，肝细胞、内皮细胞、枯否细胞等能够通过产生转化生长因子．血小板衍生生长因子、肝细胞生长因子、肿瘤坏死因子等细胞因子直接或间接地促进肝星状细胞（HSC）活化，继而原癌基因和核因子等转录因子被激活，导致HSC增殖，细胞外基质（ECM）降解减少并沉积。因此，针对特定的细胞因子，在体细胞基因水平进行调控，可以阻止肝纤维化病理进程。研究肝纤维化及细胞因子的关系，可以为通过相关因子治疗肝纤维化提供理论基础。     

Smad蛋白与肝纤维化关系的研究进展

肝星形细胞(HSC)是肝纤维化发生发展的关键细胞,转化生长因子β1(TGF-β1)是促进HSC活化、细胞外基质合成的主要因子。在肝纤维化形成的过程中,TGF-β1/Smad信号转导对HSC的作用非常重要。深入研究TGF-β1/Smad信号转导通路可进一步阐述肝纤维化的发病机制,为肝纤维化的防治提供新的有效途径。     

针对肝星状细胞抗肝纤维化治疗的研究进展

肝星状细胞（HSC）是肝纤维化中细胞外基质的主要细胞来源。HSC从静止向激活状态的转化是肝纤维化发生的一个中心环节,因此,抑制HSC激活是治疗肝纤维化的关键步骤。本综述扼要介绍目前常用的针对HSC抗肝纤维化治疗的研究进展情况。     

苦参与茯苓对肝纤维化的作用及机制的研究

目的研究苦参、茯苓两种中药抗肝纤维化的作用及其机制。方法以生理盐水作为对照,将苦参和茯苓两种中药用于大鼠肝纤维化模型和大鼠肝星状细胞(HSC)。观察肝组织形态变化;检测血透明质酸酶、Ⅳ型胶原含量;MTT法检测HSC细胞增殖抑制率;免疫组化法检测基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶组织抑制因子-1(TIMP-1)、转化生长因子(TGF)β1、血小板衍生生长因子(PDGF)的表达。结果与对照组比较,中药处理组的HSC细胞增殖抑制率升高(P0.05);血清透明质酸、Ⅳ型胶原含量减少(P0.05);肝组织TIMP-1、TGFβ1及PDGF的表达均减少(P0.05)。结论两种中药均能减缓大鼠肝纤维化的发生,其机制可能与这些中药能下调TGFβ1及PDGF表达、抑制HSC增殖活化、促进细胞外基质降解、减少肝纤维结缔组织沉积有关。     

整合素在肝纤维化中的作用

整合素为一类位于细胞表面的糖蛋白受体家族分子,介导细胞-细胞及细胞-细胞外基质成分之间的相互作用。整合素在正常肝脏实质和间质细胞中均有表达,在肝纤维化发生发展过程中,整合素表达种类及其水平均有不同程度变化。肝损伤早期细胞外基质成分的变化通过整合素调节肝脏细胞的表型功能,尤其是促进肝星状细胞活化以促进肝纤维化发生;而且肝脏细胞又可通过整合素调节其迁移粘附的病理生理功能。选择性干预异常ECM成分与整合素的结合及其细胞内信号转导,可抑制肝纤维化的发生,促进肝脏内环境稳定与肝脏细胞功能恢复,可望成为肝纤维化防治的新途径。     

缺氧诱导因子-1α及其下游分子在大鼠肝纤维化组织中的表达

目的观察缺氧诱导因子-1α（HIF-1α）及其下游分子在大鼠肝纤维化组织中的表达情况，分析HIF-1α及其下游分子与肝纤维化程度的关系。方法 用CCl4诱发大鼠肝纤维化模型，建模结束后（实验8周末）根据纤维化程度将模型组分为S2、S3、S4亚组，然后分别应用免疫组织化学和逆转录酶PCR方法检测HIF-1α、基质金属蛋白酶-2（MMP-2）、血管内皮生长因子（VEGF）蛋白和HIF-1α、MMP-2 mRNA在大鼠肝纤维化组织中的表达变化。结果 与对照组（S0组）比较。模型组（S3、S3、S4亚组）HIF-1α、MMP-2、VEGF蛋白和HIF-1α、MMP-2 mRNA表达均明显增强（P〈0．01）；HIF-1α、MMP-2、VEGF的表达与肝纤维化程度呈正相关（r值分别为0．923、0．848、0．878，均P〈0．001）；MMP-2、VEGF的表达与HIF-1α也呈正相关（r值分别为0．862、0．993、0．892，均P〈0．001）。结论 HIF-1α可能通过调节MMP-2及VEGF的表达促进了肝纤维化的发展。     

Expression of transforming growth factor-beta1 in renal fibrosis of human mesengial proliferative glomerulonephritis]

OBJECTIVE: To explore the possible effect of transforming growth factor-beta(1) (TGF -beta(1)) on the development of renal fibrosis in human mesengial proliferative glomerulonephritis (MsPGN). METHODS: Immunohistochemistry method, sirius red staining polarization microscopy and the computer imaging analysis system were used to detect the expression of TGF-beta(1), the distribution of collagen I, collagen III and collagen IV. RESULT: In MsPGN with renal fibrosis, collagen IV was increased markedly,and collagen I and collagen III appeared in the expanded mesengial matrix abnormally. Collagen III and collagen IV were increased markedly in tubulointerstitium. TGF-beta(1) expression was positively correlated with the expression of collagen I, collagen III and collagen IV in tubulointerstitium (r=0.82 0.92,P<0.01), and negatively correlated with I/III, I/IV and III/IV (r=-0.83,-0.92, P<0.001). CONCLUSION: Abnormal increase of TGF-beta(1) may be one of the important factors associated with glomerular sclerosis and tubulointerstitial fibrosis through the increment and abnormal distribution of collagen I, collagen III and collagen IV.     

All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal

Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.     

丹参素对PDGF-BB刺激下肝星状细胞的抑制作用

目的观察丹参素对PDGF-BB刺激下大鼠肝星状细胞活化增殖,Ⅰ、Ⅲ型胶原合成与分泌,ERK1/2和PI3K通道蛋白表达的影响,探讨丹参素抑制肝纤维化的分子机制。方法用不同浓度丹参素(0.250、0.125、0.062 mmol/L)、ERK通道特异性抑制剂UO126(20 nmol/L)和PI3K通道特异性抑制剂LY294002(20 nmol/L)分别作用于经血小板衍生生长因子PDGF-BB(10 ng/ml)处理的大鼠肝星状细胞(HSC)48 h,CCK8法检测HSC增殖活性,免疫化学染色法测定Ⅰ、Ⅲ型胶原合成,酶联免疫吸附法观察Ⅰ、Ⅲ型胶原分泌,Western blot测定ERK1/2、p-ERK1/2、AKT和p-AKT蛋白表达。结果 PDGF-BB 10 ng/ml处理后,HSC增殖明显增加(0.139±0.009),丹参素能抑制PDGF-BB刺激大鼠HSC增殖的作用,并随丹参素浓度增加(0.062、0.125、0.250 mmol/L)细胞增殖受到的抑制作用越强(0.102±0.008、0.083±0.007、0.066±0.014,P<0.05),Ⅰ、Ⅲ型胶原合成及分泌均显著减少(P<0.05),p-ERK1/2和p-AKT蛋白表达均显著降低(P<0.05)。结论丹参素可通过抑制PDGF-BB诱导的HSC增殖和活化,同时抑制与肝纤维化密切相关的MAPK、PI3K途径中ERK、AKT蛋白的磷酸化,负性调控肝纤维化过程,其机制可能与抑制ERK1/2和PI3K信号转导通路有关。     

Effect of angiotensin Ⅱ and angiotensin Ⅱ type 1 receptor antagonist on the proliferation,contraction and collagen synthesis in rat hepatic stellate cells

Background Angiotensin Ⅱ （Ang Ⅱ） is a very important vasoactive peptide that acts upon hepatic stellate cells （HSCs）, which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang Ⅱ and angiotensin Ⅱ type 1 receptor antagonist （AT1RA） on the proliferation, contraction and collagen synthesis in HSCs.
Methods HSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by ^3H-thymidine incorporation. The effects of angiotensin Ⅱ and AT1RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen Ⅰ （Col Ⅰ）, collagen Ⅲ （Col Ⅲ） and transforming growth factor β1 （TGF-β1） by enzyme linked immunosorbent assay. HSC was harvested to measure collagen Ⅰ, collagen Ⅲ and tissue inhibitor of metalloproteinase-1 （TIMP-1） mRNA expression.
Results Ang Ⅱ （（1×10^-10-1×10^-4）mol/L）stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT1RA inhibited angiotensin Ⅱ induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin Ⅱ （1×10^-9-1×10^-5 mol/L） and with time over 48 hours. AT1RA blocks angiotensin Ⅱ induced contraction of collagen lattice. Col Ⅰ, Col Ⅲ and TGF-β1 levels of the Ang Ⅱ group were higher than those of control group and this increase was downregulated by AT1RA. The mRNA expressions of Col Ⅰ, Col Ⅲ and TIMP-1 were higher in HSCs from the Ang Ⅱ group than the control group and downregulated by AT1RA.
Conclusions Angiotensin Ⅱ increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col Ⅲ and TGF-β1 lev     

TGF-β1和Ⅳ型胶原在慢性移植肾病患者肾组织中的表达及意义

OBJECTIVE: To investigate the expressions of transforming growth factor (TGF)-beta1 and collagen IV in the renal tissues of patients with chronic allograft nephropathy (CAN). METHODS: Immunohistochemical method and computer-assisted image analysis system were used to detect the expressions of TGF-beta1 and collagen IV in the renal tissues of patients with CAN, and the association between TGF-beta1 and collagen IV expressions as well as that between their expressions and the pathological grading of CAN were analyzed. RESULTS: The expressions of TGF-beta1 and collagen IV were significantly higher in the renal tissues of the patients than in normal renal tissues (P<0.001), and the expressions tended to increase with the pathological grades of CAN; TGF-beta1 and collagen IV expressions in both the renal glomeruli and the tubulointerstitium were in patients with CAN positively correlated with normal renal tissues (r=0.943, P<0.001; r=0.910, P<0.001). CONCLUSIONS: Abnormal collagen IV deposition is one of the major factors associated with renal fibrosis in CAN, and TGF-beta1 might play an important role in renal fibrosis in CAN through up-regulation of collagen IV in the renal tissues.     

Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells

Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.     

冬虫夏草菌丝对二甲基亚硝胺诱导的大鼠肝纤维化的作用

目的:探讨冬虫夏草菌丝对二甲基亚硝胺诱导的大鼠肝纤维化的作用。方法:将SD大鼠分为正常对照组、模型组、冬虫夏草菌丝治疗组。采用二甲基亚硝胺诱导的大鼠肝纤维化模型。每组大鼠给予相应的药物。4周后,大鼠肝脏用胶原染色观察胶原纤维沉积的变化,盐酸水解法检测肝组织羟脯氨酸含量,Envi-sion两步法测定肝组织Ⅳ型胶原含量和金属蛋白酶组织抑制因子2(tissueinhibitorofmetalloproteinase-2,TIMP-2)含量;酶图法检测肝组织基质金属蛋白酶2(matrixmetalloproteinases-2,MMP-2)活性,Western印迹法检测肝组织Ⅰ型胶原。结果:模型组大鼠肝组织羟脯氨酸、Ⅳ型胶原和TIMP-2含量,I型胶原蛋白表达,较正常对照组均增加,而MMP-2含量降低;冬虫夏草菌丝治疗组大鼠肝组织羟脯氨酸、Ⅳ型胶原和TIMP-2含量,I型胶原蛋白表达较模型组均有不同程度的下降,而MMP-2含量升高。结论:冬虫夏草菌丝有显著的抗肝纤维化作用,其作用机制与促进胶原降解有关。     

高糖对人肝星状细胞的激活作用及机制

目的 探讨高糖是否可以激活人肝星状细胞（hepatic stellate cells,HSCs）及其机制.方法 实验分为正常对照组（NG组）、高糖组（HG组）和渗透压对照组（OC组）;体外培养的人HSCs高糖处理后,应用western-blot方法检测TGF-β1、α-SMA、I型胶原和MMP-2的蛋白表达量;酶联免疫吸附试验（ELISA）测定TGF-β1的分泌水平.结果 应用30 μmol/L D-葡萄糖处理人HSCs细胞48 h,可以激活HSCs,表现为：α-SMA、I型胶原和MMP-2的蛋白表达增加,TGF-β1的分泌水平升高.结论 高糖可以激活人HSCs,进而促进肝纤维化的发生,此激活过程可能是通过TGF-β1通路.