大鼠急性肺栓塞后SMP-30表达下降及其对细胞凋亡的影响

目的：研究大鼠急性肺栓塞模型肺组织中衰老标记蛋白质30(SMP-30)的表达变化及其对Fas诱导的细胞凋亡的影响。方法:建立大鼠急性肺栓塞模型，分别在急性肺栓塞后1、8、24和48 h进行支气管肺泡灌洗，然后开胸取出肺组织。常规提取肺组织的总RNA和总蛋白，以正常组为对照，采取半定量RT-PCR的方法研究SMP-30在mRNA水平表达的变化；采用Western blotting方法进一步验证SMP-30在蛋白水平表达的变化；采用免疫组织化学方法检测大鼠肺组织中SMP-30以及肺泡巨噬细胞中IL-8在肺栓塞前后表达的变化及其组织分布情况；采用TUNEL法研究急性肺栓塞后组织细胞的凋亡情况；最后采用ELISA法检测急性肺栓塞后肺泡灌洗液中sFasL的浓度变化。结果:在大鼠急性肺栓塞后的不同时点，SMP-30的mRNA水平和蛋白水平均逐渐降低，在24和48 h下降最为明显。免疫组化研究表明SMP-30主要分布在支气管黏膜上皮细胞和肺泡上皮细胞，急性肺栓塞后SMP-30在上述细胞内的表达均明显降低。TUNEL染色发现随着SMP-30表达的降低，肺组织内出现明显的细胞凋亡现象，同时肺泡灌洗液中sFasL的浓度升高，肺泡巨噬细胞内IL-8的表达也明显升高。结论:大鼠急性肺栓塞后肺组织内SMP-30的表达明显降低，可能促进Fas－FasL细胞凋亡系统的活化。     

慢性重型乙肝病人血清sFas-sFasL复合体 ELISA检测方法的建立及应用

目的建立血清sFas-sFasL复合体ELISA检测方法,并探讨其在慢性重型乙肝病人发病机制研究中的意义.方法采用多抗体夹心ELISA法,检测血清sFas-sFasL复合体.检测了健康献血员37例、41例无症状已肝病毒携带者、40例慢性乙型重型肝炎及42例乙肝病毒相关性肝细胞癌患者血清sFas-sFasL复合体.结果慢性重型肝炎病人血清sFas-sFasL复合体阳性率显著高于健康对照组、无症状乙肝病毒携带者、乙肝相关性肝细胞肝癌组.结论本研究建立的ELISA方法可以较好地检测出血清中的sFas-sFasL复合体;慢性乙型重型肝炎患者血清中存在sFas-sFasL复合体.     

Acacetin inhibits the proliferation of Hep G2 by blocking cell cycle progression and inducing apoptosis

Flavonoids are a broadly distributed class of plant pigments, universally present in vascular plants and responsible for much of the coloring in nature. They are strong antioxidants that occur naturally in foods and can inhibit carcinogenesis in rodents. In this study, we examined acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, for its effect on proliferation in a human liver cancer cell line, Hep G2. The results showed that acacetin inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. Enzyme-linked immunosorbent assay showed that acacetin significantly increased the expression of p53 and p21/WAF1 protein, contributing to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand and soluble Fas ligand, as well as Bax protein, was responsible for the apoptotic effect induced by acacetin. Taken together, our study suggests that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of acacetin in Hep G2 cells.     

多器官功能障碍综合征患者血清sFas和sFasL浓度的变化

目的探讨多器官功能障碍综合征（multiple organ dysfunction syndrome，MODS）患者血清sFas和sFasL浓度在MODS诊断以及病情严重程度和预后判断中的临床意义以及与血清肿瘤坏死因子-α（TNF-α）水平的相关性。方法采用酶联免疫吸附法（ELISA）测定36例MODS患者血清sFas、sFasL和TNF-α浓度，以急性生理学与慢性健康状况评分系统Ⅲ（APACHEⅢ）评分和改良MODS评分评估病情严重程度，比较MODS组与对照组[包括非全身炎症反应综合征（SIRS）对照组和健康对照组]以及MODS组内死亡和存活患者之间血清sFas、sFasL浓度的差异，分析患者血清sFas、sFasL和TNF-α浓度与病情严重程度及血清sFas和sFasL与TNF-α浓度的相关性。结果MODS患者血清sFas、sFasL浓度均明显高于非SIRS对照组和健康对照组（P〈0．05或P〈0．01），且与病情严重度评分均呈正相关（P均〈0．01）。MODS患者中死亡组较存活组血清sFas、sFasL浓度明显升高（P均〈0．05）。MODS患者血清TNF-α浓度较非SIRS对照组和健康对照组明显增高，并与MODS组血清sFas、sFasL浓度呈高度正相关（P均〈0．01）。MODS患者血清sFas、sFasL浓度随器官衰竭个数增加而升高。结论血清sFas、sFasL可用于临床MODS辅助诊断和协助判断病情严重程度及预后。TNF-α可能促进了MODS中Fas／FasL系统表达上调。     

内毒素对严重烧伤后细胞因子、可溶性粘附分子及sFas/sFasL的影响

目的　探讨内毒素对严重烧伤后细胞因子、可溶性粘附分子及sFas sFasL的影响。方法　选择烧伤总面积 (TBSA) >30 %患者 2 8例 ,健康志愿者 4 0例作为正常对照组。分别于伤后不同时相点 (伤后 1、 3、 5、 7、 14、 2 1、 2 8和 35d)测定患者血中内毒素、细胞因子 (TNF α、IL 1β、IL 6、IL 8、IL 12和IL 18)、可溶性粘附分子 (ICAM 1和VCAM 1)以及sFas sFasL的水平。按内毒素水平将其分为三组 :A组 ,脂多糖 (LPS)为 0～ 0 2 0 0U ml;B组 ,LPS为 0 2 0 0～ 0 4 0 0EU ml;C组 ,LPS >0 4 0 0EU ml。结果　与A组和正常对照组比较 ,B组和C组的TNF α、IL 1β、IL 6、IL 8、IL 18和ICAM 1水平在不同时相点均呈显著性上升 (P <0 0 5～ 0 0 1) ;而B组和C组患者血中VCAM 1和sFas sFasL水平在部分时相点呈升高趋势 ,与正常对照组和A组比较 ,其差异具有显著性意义 (P <0 0 5～ 0 0 1) ;B组和C组患者血中IL 12水平在烧伤早期呈下降的趋势 ,与A组和正常对照组比较 ,其差异具有显著性意义 (P <0 0 5～ 0 0 1) ,但在烧伤中后期则呈上升趋势 ;血浆LPS主要与血清中TNF、IL 1β、IL 6、IL 8、IL 18水平呈显著正相关 (P <0 0 5～0 0 1)。结论　烧伤后内毒素直接或间接影响机体细胞因子、可溶性粘附分子及sFas     

Peripheral blood serum markers for apoptosis and liver fibrosis: Are they trustworthy indicators of liver realness?

BACKGROUND/AIMS: No reliable serum markers for liver inflammation, apoptosis and fibrosis have been established yet, although a large number have been evaluated. Moreover, it is not clear if a molecule detected and quantified in peripheral vein blood is a really trustworthy marker of the liver condition. To answer to this question, we had the opportunity to study paired serum samples drawn simultaneously during haemodynamic study from the right hepatic vein and from a peripheral vein from patients with hepatitis C virus related cirrhosis. METHODS: The serum levels of transforming growth factor beta-1, tumour necrosis factor-alpha, hyaluronic acid, soluble (s)human leukocyte class I antigens, soluble FAS ligand, and stumour necrosis factor related ligand were assessed in a consecutive series of 15 patients with hepatitis C virus related cirrhosis. RESULTS: No statistically significant differences were found between hepatic vein and peripheral vein levels for the cytokines, substance or soluble molecules evaluated, excepted for shuman leukocyte class I antigens. Instead a strong correlation between hepatic vein and peripheral vein levels was present for: hepatic vein, shuman leukocyte class I antigens, tumour necrosis factor-alpha, soluble FAS ligand and stumour necrosis factor related ligand, but not for transforming growth factor beta-1. CONCLUSIONS: Our results show that peripheral vein measurements seem to reflect the liver compartment in a large majority of cases, but not for all molecules and probably for any liver diseases. Further studies on this line are warranted in particular for new molecules.     

Association among Fas expression in leucocytes,serum Fas and Fas‐ligand concentrations and hepatic inflammation and fibrosis in chronic hepatitis C

Background: Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC) may impair immune functions and establish persistent infection. The aim of this study was to assess the influence of HCV on PBMC and their susceptibility to apoptosis in relation to liver inflammation and fibrosis. Methods: Eighty‐one patients with chronic hepatitis C (CHC) were enrolled in this study. Flow cytometry was used to determine the amount of T cells (CD4⁺, CD8⁺), B cells (CD19⁺), monocytes (CD14⁺) and natural killer cells (CD16⁺) in the peripheral blood and the expression of CD95⁺ (CD95/APO‐1) in each subset. Serum concentrations of sFas and sFasL were assessed by the enzyme‐linked immunosorbent assay method. Results: An increased expression of Fas was observed in CD4⁺ and CD8⁺ cells in CHC. There was a more prominent expression of Fas on CD4⁺ cells in HCV genotype 1b in contrast to 3a. Increased Fas expression on CD4⁺ cells was seen in advanced stages of liver disease. Fas expression on monocytes was lower in advanced stages of liver inflammation and fibrosis. Serum sFas concentration was higher in CHC compared with the control group. There was an association between sFasL concentration and inflammatory activity in the liver. Serum sFasL concentration correlated positively with the mean intensity of fluorescence of the Fas receptor in CD4⁺ and CD8⁺ cells, granulocytes and monocytes. Conclusion: These findings indicate that there is an increased susceptibility of PBMC to apoptosis, which can be attributed to the constant contact of leucocytes with the inflamed liver tissue, or from direct HCV influence.     

肾综合征出血热患者外周血淋巴细胞FasL的检测及与血清sFasL的相关性

目的：探讨ＦａｓＬ介导的细胞凋亡在肾综合征出血热（ＨＦＲＳ）致病机制中的作用。方法：采用ＳＡＢＣ免疫组化法检测ＨＦＲＳ患者外周血淋巴细胞ＦａｓＬ的表达；采用ＥＬＩＳＡ法测定ＨＦＲＳ患者血清ｓＦａｓＬ的浓度。结果：ＨＦＲＳ患者发热期、低血压休克期及少尿期淋巴细胞ＦａｓＬ的表达明显增强，与正常对照组比较差异有统计学意义（Ｐ＜０．００１），多尿期与正常对照组比较差异也有统计学意义（Ｐ＜０．０１）；ＨＦＲＳ患者血清ｓＦａｓＬ的浓度在发热期及低血压休克期明显增高，与其他各期及正常对照组比较差异有统计学意义（Ｐ＜０．０１）；ＨＦＲＳ患者淋巴细胞ＦａｓＬ的表达与血清ｓＦａｓＬ的浓度采用直线相关分析，二者呈高度正相关（Ｐ＜０．０１）。结论：ＨＦＲＳ的致病过程中淋巴细胞ＦａｓＬ的高表达及血清ｓＦａｓＬ的浓度增高是机体清除病毒的主要机制之一；血清ｓＦａｓＬ主要来源于外周血淋巴细胞。     

Levels of soluble FasL and FasL gene expression during the development of graft-versus-host disease in DLT-treated patients

Three patients with different clinical symptoms of graft-versus-host disease (GVHD) who had received donor lymphocyte transfusion (DLT) for the treatment of relapsed leukaemia after an allogeneic bone marrow transplantation (BMT) from HLA-matched sibling donors were analysed for the presence of soluble FasL (sFasL) in the sera and for the expression of the Fas ligand (FasL) gene in the peripheral blood mononuclear cells (PBMNC). Two patients who demonstrated liver damage with increased levels of serum bilirubin showed significantly increased levels of serum sFasL. The increase in the sFasL level was observed prior to the increase in the bilirubin during the clinical courses of both patients. The high dose of methyl predonisolone administered to one of these patients greatly reduced the levels of sFasL in the serum. The bilirubin levels were also reduced thereafter. The third patient (without liver damage) did not show any increase in the serum sFasL level. The expression of the FasL gene in the PBMNC of these three patients was examined. All three patients showed increased levels of the FasL gene expression during their clinical courses. However, only one patient showed a parallel alteration of FasL gene expression with sFasL in the serum. These cases provide evidence that the Fas/FasL system is closely associated with human GVHD, especially in the development of liver GVHD.     

黄芪对尿毒症患者血浆sFasL及外周血淋巴细胞亚群状态的作用

目的 :通过测定血浆中sFasL水平和外周血T淋巴细胞亚群状态的分析 ,探讨黄芪对改善尿毒症患者肾功能可能的机制。方法 :采用流式细胞仪和ELISA方法测定 2 5例尿毒症患者黄芪治疗前后及 2 0例正常人血浆中sFasL水平和外周血淋巴细胞亚群状态 ,以及患者治疗前后的肾功能变化。结果 :与正常人比较 ,尿毒症患者血浆中可溶性Fas配体 (sFasL)水平明显升高 ;CD3 CD4 细胞的百分率明显下降 ,CD8 细胞的百分率明显升高 ,CD4 /CD8 的比例小于 1(P <0 .0 1)。 2 5例患者黄芪治疗后血浆中sFasL水平降低 (P <0 .0 5 )。CD4 /CD8 的比例升高 (P <0 .0 1) ;肌酐 (Scr)和尿素氮 (BUN)水平亦有不同程度降低 (P <0 .0 5 )。且动态观察 12例尿毒症患者血肌酐和尿素氮的下降与血浆中sFasL水平的下调呈正相关 (r =0 .5 6 ,P <0 .0 1)。结论 :黄芪能改善尿毒症患者的肾功能 ,提高患者的细胞免疫功能及减慢患者的细胞凋亡是其可能的机制之一。