肝细胞生长因子在肿瘤坏死因子相关凋亡诱导配体作用下对原代肝星状细胞增殖及凋亡的影响**☆

背景：活化的肝星状细胞是肝纤维化的关键因素,研究表明肝细胞生长因子能促进星状细胞凋亡,其具体机制可能与增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导星状细胞凋亡有关。 目的：观察肿瘤坏死因子相关凋亡诱导配体作用下,肝细胞生长因子对原代肝星状细胞增殖、凋亡的影响并初步探讨其可能机制。 方法：将SD大鼠原代肝星状细胞复苏、传代,细胞增殖明显时用于实验。实验分为4组：空白对照组为单纯肝星状细胞培养;肝细胞生长因子组：将100 μg/L肝细胞生长因子作用于肝星状细胞;TRAIL组：将2 mg/L的TRAIL作用于肝星状细胞;肝细胞生长因子+TRAIL组：将肝细胞生长因子预先刺激肝星状细胞24 h,再加入2 mg/L TRAIL。 结果与结论：MTT检测显示肝细胞生长因子及TRAIL分别在50~200 μg/L、0.5~1.5 mg/L各浓度下对肝星状细胞增殖抑制率无影响,TRAIL在2 mg/L作用下对肝星状细胞有抑制作用。流式细胞仪检测肝细胞生长因子+TRAIL组的中晚期凋亡率明显高于空白对照组及肝细胞生长因子组(P < 0.05);肝细胞生长因子+TRAIL组DR5荧光强度明显高于其他3组(P < 0.01)。提示在TRAIL作用下,肝细胞生长因子能促进肝星状细胞的凋亡、抑制其增殖。可能与肝细胞生长因子上调活化肝星状细胞表面DR5表达有关。      

霉酚酸及其衍生物对人T淋巴细胞及混合淋巴细胞免疫应答的影响

目的探讨霉酚酸及霉酚酸衍生物对人T淋巴细胞和混合淋巴细胞免疫应答的影响。方法从正常人外周血中分离单个核淋巴细胞(PBMC),在抗CD3刺激下和混合淋巴细胞反应体系中,加入或不加霉酚酸及其衍生物共培养,应用ELISA和流式细胞术检测CD4+和CD8+T细胞细胞因子的产生、细胞活化和增殖情况。结果经抗CD3刺激后,PBMC产生的细胞因子明显增加,加入霉酚酸及其衍生物后,IFN-γ及TNF-α明显降低,IL-2产生略有增加。在混和淋巴细胞反应体系中,3种细胞因子产生皆被抑制。抗CD3刺激和混和淋巴细胞反应后,T细胞表面活化分子CD25及CD69表达增加,加入霉酚酸及其衍生物共培养24、48 h后,T细胞CD25及CD69的表达并未被抑制。霉酚酸及其衍生物可抑制抗CD3刺激下和混和淋巴细胞反应后T细胞的增殖。结论霉酚酸及其衍生物对T淋巴细胞和混合淋巴细胞体系中因子的产生和细胞增殖都有抑制作用,为其在临床自身免疫疾病及移植排斥中的广泛应用提供了理论依据。     

下调肝星状细胞中SOX12的表达可抑制肝硬化

目的研究SOX12在肝星状细胞与正常肝细胞中的表达,并探讨SOX12通过介导TGF-β1(转化生长因子β1)的表达在调节肝星状细胞的活性中对肝硬化的影响。方法培养细胞LX-2(肝星状细胞)与Changliver cell(正常肝细胞),使用PCR及Western blot检测细胞中SOX12与免疫组化检查肝组织中肝硬化各项指标的表达,同时探讨SOX12与TGF-β1之间的关系;用细胞转染的方法研究抑制SOX12基因的表达后对TGF-β1的影响。结果1) SOX12蛋白在人类肝细胞和星状细胞中均有高表达; 2)抑制SOX12基因的表达可以使肝星状细胞中TGF-β1以及collagenⅠ(胶原蛋白Ⅱ)表达量减少; 3) SOX12可调控肝星状细胞的活性和TGF-β1的表达,导致细胞外基质的积累。结论下调肝星状细胞中SOX12的表达可抑制肝硬化。     

TCRVβ8.4 基因转染人PBMC对肝癌细胞杀伤活性的研究

目的：探讨转染hTCRVβ8.4基因后健康人PBMC的免疫学特性及其对起源于乙肝病毒的肝癌细胞株BEL-7402杀伤活性的改变。方法：将hTCRVβ8.4基因克隆至真核表达载体pcDNA3．1(-)上，并转染健康人PBMC，流式细胞术检测转染后PBMC中TCRVβ8.4蛋白表达，乳酸脱氢酶释放活性法检测重组质粒转染后PBMC对癌细胞BEL-7402的杀伤活性。结果：TCRVβ8.4在基因转染后淋巴细胞中表达显著增高；与BEL-7402共培养后，基因转染组CD3^ TCRVβ8.4T细胞增殖明显高于对照组；BEL-7402刺激后免疫细胞活化，表达CD122的细胞数量增多，表达CD19(B细胞活化的标志)的B细胞增加；重组质粒转染后，PBMC对肝癌细胞BEL-7402杀伤活性增强；透射电镜观察发现，重组质粒转染的PBMC使BEL-7402凋亡。结论：hTCRVβ8.4基因修饰可显著增强淋巴细胞在超抗原BEL-7402刺激下的增殖及免疫细胞活化，基因修饰后T淋巴细胞杀伤活性明显增强。     

云芝丹参的体外促增殖试验及其临床应用研究

目的 通过云芝丹参的体外促增殖试验及对乳腺癌患者的临床应用研究,观察其对机体免疫调节活性的影响.方法 采用MTT比色法、流式细胞分析法和ELISA法,分别检测云芝丹参对PHA刺激后的健康成人外周血单个核细胞增殖作用及服用云芝丹参胶囊后的乳腺癌患者外周血淋巴细胞亚群和血浆sIL-2R的浓度.结果 体外实验,中低浓度的云芝丹参药液(6.25～200 μg/mL)对健康成人PBMC有促增殖作用,高浓度(400 μg/mL)时则产生抑制作用;而临床试验中,乳腺癌患者血浆sIL-2R浓度、CD8 细胞百分率均显著降低,而B淋巴细胞数量及百分率、CD4 /CD8 比值和Th细胞的百分率都明显升高.结论 云芝丹参胶囊对正常人及乳腺癌患者都具有免疫增强作用.     

TCRVβ7.1基因修饰T细胞对乳腺癌细胞杀伤作用的研究

目的：观察TCPVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性的影响。方法：脂质体包裹PdWA3.1vβ7.1后转染健康人PBMC，流式细胞仪检测PdWA3.1Vβ7.1基因表达，改良MIT法检测TCRVβ7．1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性。结果：TCRVβ7．1基因转染可显著增加正常人PBMC该基因表达，转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性有显著性差异。结论：用TCR基因修饰可明显提高正常人PBMC对乳腺癌细胞杀伤作用。     

IL-23单独或联合IL-2对人PBMC增殖及抗白血病活性影响的研究

目的比较单独应用IL-23或联合应用IL-23和IL-2对人外周血单个核细胞(PBMC)增殖及抗白血病活性的影响。方法密度梯度离心法分离人PBMC,以不同的细胞因子培养液培养,MTT比色法测定PBMC的增殖情况及PBMC对白血病K562细胞株的杀伤活性,流式细胞术分析诱导前后PBMC的细胞表型变化。结果细胞因子各组培养PBMC 1 d、3 d、5 d后,PBMC的增殖以及对K562细胞的杀伤活性均增强(P<0.05),IL-23与IL-2联合后PBMC的增殖以及杀伤活性进一步增强(P<0.05);IL-23(50 ng/ml)与IL-23+IL-2(50 ng/ml+100 IU/ml)组作用于PBMC 5 d后,检测CD3+细胞为(80.7±4.37)%和(83.2±4.04)%,CD16+CD56+细胞为(8.4±0.28)%和(12.7±0.9)%,CD4+细胞为(45.2±1.4)%和(47.0±1.72)%,CD8+细胞为(34.5±2.53)%和(35.4±2.11)%;与对照组相比,两组对PBMC细胞表型的增加有显著性差异(P<0.05);两组间比较CD16+CD56+细胞的表达上有显著性差异(P<0.05),对CD3+、CD4+、CD8+细胞的表达以及CD4+/CD8+比值的变化无显著性差异(P>0.05)。结论 IL-23对PBMC的增殖,以及PBMC对K562细胞的杀伤均有促进作用,与IL-2联合有协同作用。IL-23单独或联合IL-2诱导后PBMC中CD3、CD16/56、CD4、CD8抗原的表达均增加,二者联合后可以进一步增加CD16/56抗原的表达。     

促肝细胞生长素及其S4组份对人PBMC表型的影响

本文采用生物素 链霉亲和素 (BSA )免疫细胞化学法及流式细胞仪技术检测CD3+、CD4+、CD8+、HLA DR+及CD2 5 +细胞百分率 ,藉以研究促肝细胞生长素 (pHGF )及其组分S4对免疫活性细胞的作用。结果表明 ,pHGF可使脐血中CD4+、CD8+及HLA DR+细胞数有明显增长 ;对正常人及肿瘤患者PBMC的HLA DR+细胞数亦有促进作用 ,与PHA的刺激作用相近似。用FPLC及HPLC等方法分离的单一成分S4对成人PBMC表面CD2 5抗原表达有明显促进作用。     

骨髓间充质干细胞调控尿激酶型纤溶酶原激活物表达及其对大鼠肝星状细胞凋亡的影响**

背景：骨髓间充质干细胞主要通过旁分泌及激活肝细胞生长因子促进肝星状细胞凋亡。 目的：观察骨髓间充质干细胞对尿激酶型纤溶酶原激活物合成的调控及肝细胞生长因子活性的影响,探讨其诱导肝星状细胞凋亡的机制。 方法：实验分为5组：①共培养组：大鼠骨髓间充质干细胞与肝星状细胞建立上下双层细胞共培养体系。②肝星状细胞单独培养组。③纤维原细胞对照组。④UK122干预组：在骨髓间充质干细胞与肝星状细胞共培养6 h前加入尿激酶型纤溶酶原激活物特异性抑制剂UK122。⑤骨髓间充质干细胞空白对照组。 结果与结论：共培养组尿激酶型纤溶酶原激活物mRNA表达较肝星状细胞单独培养明显增加(P < 0.01)。与肝星状细胞单独培养相比,共培养组的肝星状细胞在与骨髓间充质干细胞共培养24 h后表现明显增殖抑制(P < 0.01),且呈现时间依赖性;骨髓间充质干细胞与肝星状细胞共培养后,活性肝细胞生长因子的蛋白表达及肝星状细胞凋亡增加(P < 0.01)。UK122干预后活性肝细胞生长因子表达及肝星状细胞凋亡减少(P < 0.01)。提示骨髓间充质干细胞通过促进分泌尿激酶型纤溶酶原激活物,增加活性肝细胞生长因子表达,促进肝星状细胞凋亡。     

转化生长因子-β在肝纤维化形成中的作用及意义

转化生长因子 β(TGF β)是一类多肽生长因子 ,通过与其受体结合发挥生物学作用。Mφ、内皮细胞、胆管上皮细胞、淋巴细胞、血小板、肝细胞及活化的肝星状细胞可能是肝脏TGF β的来源细胞。TGF β在肝纤维化形成过程中的主要作用是促进细胞外基质的合成 ,其机制可能与其通过不同途径增加胶原基因表达和增加肝星状细胞增殖有关。TGF β可能成为反映肝纤维化的血清学指标 ,TGF β反义基因治疗可能成为肝纤维化的血清学指标 ,TGF β反义基因治疗可能成为肝纤维防治的一种有效措施。     

Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts

Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.     

The epigenetic regulation of stem cell factors in hepatic stellate cells

The epigenetic regulation by DNA methylation is an important mechanism to control the expression of stem cell factors as demonstrated in tumor cells. It was recently shown that hepatic stellate cells (HSC) express stem/progenitor cell factors and have a differentiation potential. The aim of this work was to investigate if the expression of stem cell markers is regulated by DNA methylation during activation of rat HSC. It was found that CD133, Notch1, and Notch3 are regulated via DNA methylation in HSC, whereas Nestin shows no DNA methylation in HSC and other undifferentiated cells such as embryonic stem cells and umbilical cord blood stem cells from rats. In contrast to this, DNA methylation controls Nestin expression in differentiated cells like hepatocytes and the hepatoma cell line H4IIE. Demethylation by 5-Aza-2-deoxycytidine was sufficient to induce Nestin in H4IIE cells. In quiescent stellate cells and embryonic stem cells, the Nestin expression was suppressed by histone H3 methylation at lysine 9, which is another epigenetic mechanism. Apart from the known induction of Nestin in cultured HSC, this intermediate filament protein was also induced after partial hepatectomy, indicating activation of HSC during liver regeneration. Taken together, this study demonstrates for the first time that the expression of stem cell-associated factors such as CD133, Notch1, and Notch3 is controlled by DNA methylation in HSC. The regulation of Nestin by DNA methylation seems to be restricted to differentiated cells, whereas undifferentiated cells use different epigenetic mechanisms such as histone H3 methylation to control Nestin expression.     

Effects of retinol and hepatocyte-conditioned medium on cultured rat hepatic stellate cells

Hepatic stellate cells (HSC) become activated in liver injury, proliferating and secreting components of connective tissue. Activated HSC lose their native retinol and fat storing capacity. Signals from hepatocytes and/or Kupffer cells injured (eg, by iron overload) may contribute to the so-called activated HSC phenotype. Primary rat HSC cultures were treated with retinol to determine if this could produce a quiescent cell for controlled in vitro studies of activation. Retinol resulted in suppressed DNA synthesis in proliferating HSC, a reorganization of actin filaments, and a return of fat storage. However, it did not suppress the expression of fibrogenic genes such as those for collagens type I and IV, and TGF-beta1. Furthermore, retinol-treated cells may increase expression of these genes in response to conditioned medium from hepatocyte cultures. The effect is especially apparent for collagen type I mRNA, and with conditioned medium from iron-loaded hepatocytes. Thus, retinol may be a two-edged sword in iron overload, potentially suppressing HSC proliferation on the one hand, and sensitizing a fibrogenic pattern of gene expression on the other. Factors influencing this balance merit further study.     

Activation of cultured rat hepatic stellate cells by tumoral hepatocytes

Hepatocellular carcinoma (HCC) is the main type of primary liver cancer, and it develops from hepatocytes. The stroma of HCC is infiltrated by myofibroblasts. In other settings, such as liver fibrosis, myofibroblasts are derived mainly from the activation of hepatic stellate cells (HSC). In this study, we investigated whether tumoral hepatocytes were able to activate HSC. HSC were isolated from normal rats and were plated in dishes coated with Matrigel, to prevent their spontaneous activation. HSC were exposed to conditioned medium (CM) from the rat HCC lines Fao and H5. Tumor cell CM elicited major morphologic changes, such as spreading and generation of cytoplasmic processes. Fao and H5 CM increased HSC proliferation to 1.60 and 1.76 times control values, respectively. The expression of alpha-smooth muscle actin was low or undetectable in control cells and was markedly increased by both tumor cell CM but not by normal rat hepatocyte CM. Desmin expression was also enhanced. Gelatinase A secretion was significantly increased 1.20-fold by Fao CM and 1.55-fold by H5 CM. Expression of beta-type platelet-derived growth factor receptor mRNA was increased 5.8-fold by H5 CM but was decreased to 13% of control levels by Fao CM. HSC activation by tumor cell CM was not prevented by urokinase or matrix metalloproteinase inhibitors, suggesting that Matrigel degradation was not central to the activation process. Finally, a blocking antibody to transforming growth factor-beta1 did not impede Fao CM-induced activation but significantly blocked the increase in matrix metalloproteinase-2 expression induced by H5 CM. Our results show that tumoral rat hepatocyte CM is able to induce the activation of rat HSC in culture. The lack of induction of beta-type platelet-derived growth factor receptor mRNA by Fao CM indicates that, in some cases, tumor-induced activation differs from classic fibrosis-type activation. Our data thus suggest that HSC recruitment and activation in HCC could be under the control of tumor cells.     

Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.     

Patterns of alpha-1-adrenergic receptor expression in regenerating and neoplastic hepatic tissue.

As norepinephrine is a potent hepatocyte comitogen through binding to the alpha 1-adrenergic receptor, we have examined mRNA levels of the alpha 1a- and alpha 1b-adrenergic receptor subtypes in normal and regenerating rat hepatocytes as well as in several different rat hepatoma cell lines. All rat hepatomas examined lacked both alpha 1a- and alpha 1b-receptor message and receptor binding in radioligand binding experiments, suggesting that the growth of dedifferentiated neoplastic rat hepatocytes is not regulated by the alpha 1-adrenergic receptor. Interestingly, unlike the rat hepatomas analyzed, the human hepatocellular carcinoma cell line, HepG2, was positive for both alpha 1a and alpha 1b message at 4.5 kb, yet this cell line lacked receptor binding in radioligand binding assays. While normal and regenerating liver is negative for alpha 1a-receptor expression, it is positive for alpha 1b expression and is characterized by the presence of two bands at approximately 4.0 and 3.2 kb which peaked between 20 and 48 h after partial hepatectomy. A dramatic decrease in message level of the lower band and the continued presence of the upper band between 6 and 12 h after partial hepatectomy, and before the peak in DNA synthesis in regenerating rat liver, may correspond with observed differences in alpha 1-receptor function during liver regeneration.     

CD40 expression in HCV-associated chronic liver diseases

CD40 is expressed primarily on B cells and plays an important role in antigen presentation, B cell proliferation, and T cell activation. It has been reported that the CD40 signal modulates apoptosis and has an anti-viral effect in certain cells. Therefore, we investigated the expression and the function of CD40 in HCV-associated chronic liver disease. The expression of CD40 on liver tissues was determined through immunohistochemistry on 50 liver specimens obtained from HCV-positive patients. The effect of CD40 signaling on apoptosis of HepG2 cells was assessed using the MTT assay. The effect of CD40 stimulation on NF-kappaB activation was determined in NF-kappaB reporter gene-transfected HepG2 cells with the Luciferase assay. CD40 positive hepatocytes were observed in both periportal and lobular areas, accompanied by inflammation. In both areas, CD40 staining intensity became significantly stronger, correlating with the histological grading. Similarly, it became stronger with the progression of the histological staging in F1, F2 and F3 cases; however, the expression level decreased in F4 cases. CD40 ligation induced apoptosis in HepG2 cells in the presence of 500 ng/ml of actinomycin D, while CD40 ligation alone could not. Anti-CD40 monoclonal antibody caused NF-kappaB activation in HepG2 cells in a dose-dependent manner. These results suggest that hepatocyte over-expression of CD40 might play an important role in regulating hepatocyte survival and death in HCV-associated chronic liver diseases.     

Morphologic features in the regenerating liver—a comparative intravital,lightmicroscopical and ultrastructural analysis with focus on hepatic stellate cells

Different cell types play a role in the liver regeneration. The present study reveals morphological key steps of liver regeneration by correlating intravital, light, and electron microscopic with immunohistochemistry results focusing on hepatic stellate cells (HSCs). In Lewis rats, liver regeneration was induced by a 2/3-hepatectomy. Animals (n = 7 each) were killed after 0, 1, 2, 3, 4, 7, and 14 days. Morphological features were investigated by light microscopy, immunohistochemistry [α-smooth muscle actin (α-SMA), Desmin, vascular endothelial growth factor (VEGF)/VEGF receptor, Ki-67, ssDNA], intravital microscopy (sinusoid density, number of hepatocytes, and HSC), and electron microscopy focussed on cell-to-cell interactions. During liver regeneration, HSC were activated at day 3 showing a loss of autofluorescence and simultaneously an increased α-SMA expression and direct cell contact to hepatocytes. HSC activation was followed by increasing VEGF expression and sinusoid density. After 14 days, liver architecture and ultrastructure was restored and HSCs were deactivated showing decreased α-SMA expression as well as increased apoptosis and no more direct cell contact to hepatocytes. HSCs play a central role in the regenerating liver by governing angiogenesis and extracellular matrix remodeling. A direct cell contact to hepatocytes seems to be essential for HSC activation, whereas deactivation is accompanied by loosening of hepatocyte contact and increased apoptosis.     

Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers

Liver injury activates quiescent hepatic stellate cells (Q-HSC) to proliferative myofibroblasts. Accumulation of myofibroblastic hepatic stellate cells (MF-HSC) sometimes causes cirrhosis and liver failure. However, MF-HSC also promote liver regeneration by producing growth factors for oval cells, bipotent progenitors of hepatocytes and cholangiocytes. Genes that are expressed by primary hepatic stellate cell (HSC) isolates overlap those expressed by oval cells, and hepatocytic and ductular cells emerge when HSC are cultured under certain conditions. We evaluated the hypothesis that HSC are a type of oval cell and, thus, capable of generating hepatocytes to regenerate injured livers. Because Q-HSC express glial fibrillary acidic protein (GFAP), we crossed mice in which GFAP promoter elements regulated Cre-recombinase with ROSA-loxP-stop-loxP-green fluorescent protein (GFP) mice to generate GFAP-Cre/GFP double-transgenic mice. These mice were fed methionine choline-deficient, ethionine-supplemented diets to activate and expand HSC and oval cell populations. GFP(+) progeny of GFAP-expressing precursors were characterized by immunohistochemistry. Basal expression of mesenchymal markers was negligible in GFAP(+)Q-HSC. When activated by liver injury or culture, HSC downregulated expression of GFAP but remained GFP(+); they became highly proliferative and began to coexpress markers of mesenchyme and oval cells. These transitional cells disappeared as GFP-expressing hepatocytes emerged, began to express albumin, and eventually repopulated large areas of the hepatic parenchyma. Ductular cells also expressed GFAP and GFP, but their proliferative activity did not increase in this model. These findings suggest that HSC are a type of oval cell that transitions through a mesenchymal phase before differentiating into hepatocytes during liver regeneration. Disclosure of potential conflicts of interest is found at the end of this article.