日本血吸虫可溶性虫卵抗原体外诱导巨噬细胞分泌TNF-α活化肝星状细胞及黄芪总苷对其影响

发布时间：2018-01-08 14:28

本文关键词：日本血吸虫可溶性虫卵抗原体外诱导巨噬细胞分泌TNF-α活化肝星状细胞及黄芪总苷对其影响　出处：《安徽医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病是一种在热带或亚热带国家和地区广泛传播并严重危害人类健康的寄生虫病,肝纤维化是其严重的病变后果。纤维化的形成是通过巨噬细胞、淋巴细胞、纤维细胞和其它细胞产生的细胞因子动态调节的过程[1.2]。TNF-α作为重要的炎症介质可直接参与肝纤维化中的关键细胞肝星状细胞(hepatic stellate cell, HSC)的活化,促进其增殖。黄芪总苷(astrogalosides, AST)是黄芪抗肝纤维化的主要成份,研究已证实AST具有显著的抗炎和抗肝纤维化等作用。本文观察日本血吸虫可溶性虫卵抗原(soluble egg antigen, SEA)体外刺激巨噬细胞后,TNF-α表达和分泌的改变以及SEA活化的腹腔巨噬细胞条件培养基(soluble egg antigen induced macrophage conditioned medium, SEA-MCM)对HSC增殖刺激胶原合成的影响,并在此基础上,观察AST对其的作用。 1 SEA诱导大鼠腹腔巨噬细胞活化和TNF-α表达及AST对其影响制备SEA(0.17g·L-1),采用HE染色、免疫细胞化学染色、逆转录聚合酶链反应(RT-PCR)技术及放射免疫法,观察在SEA(10 mg·L-1)的刺激下,AST与大鼠腹腔巨噬细胞(peritoneal macrophage, PMФ)在体外共育3h、6h和9h后,大鼠PMФs形态改变及对TNF-α表达的影响。结果显示:(1)SEA(10 mg·L-1)作用3h、6h和9h可明显刺激PMΦ活化和增加TNF-α的表达(;2)AST(10,20 mg·L-1)能明显降低SEA(10 mg·L-1)刺激的PMΦTNF-α的表达。提示SEA体外可以活化PMΦ,使其增加TNF-α的表达,AST可抑制这种TNF-α的表达。 2.SEA-MCM诱导HSC活化及AST对其抑制作用大鼠腹腔注射SEA(0.17mg·ml-1,0.2ml/只),制备SEA-MCM。利用肝星状细胞株HSC-T6细胞,采用MTT和3H-脯氨酸掺入法,观察SEA-MCM诱导HSC-T6细胞增殖和胶原合成的作用及AST对其影响。结果显示:(1)不同稀释度的SEA-MCM(终稀释比1:2、1:4、1:8、1:16)均可明显促进HSC-T6细胞增殖和胶原合成;(2)AST(10和20 mg·L-1)呈浓度依赖性抑制SEA-MCM(1:4)刺激的HSC-T6细胞增殖和胶原合成。表明SEA可以通过活化的巨噬细胞促进HSC增殖和胶原合成,这种作用可被AST抑制。 3.抗TNF-α抗体对SEA-MCM诱导的HSC-T6细胞增殖和胶原合成影响在SEA-MCM(1:4)中加入不同稀释度TNF-α抗体,采用MTT、免疫印记(Western blotting)和放射免疫法检测SEA-MCM对HSC-T6细胞增殖和胶原合成的作用。结果显示:TNF-α抗体(1:100和1:200)均可明显抑制SEA-MCM对HSC-T6细胞的增殖和胶原合成作用,且呈浓度依赖性趋势。表明SEA通过巨噬细胞分泌TNF-α促进HSC的增殖和产生胶原可能是日本血吸虫性肝纤维化的重要机制之一。
[Abstract]:Schistosomiasis is a parasitic disease widely spread in tropical or subtropical countries and regions and seriously endangers human health. Hepatic fibrosis is the result of its serious pathological changes. The formation of fibrosis is through macrophages. The process of dynamically regulating cytokines produced by lymphocytes, fibroblasts, and other cells. [1. TNF- 伪, as an important inflammatory medium, can be directly involved in hepatic stellate cell (HSC) hepatic stellate cell in hepatic fibrosis. Astragaloside astrogalosides (AST) is the main anti-hepatic fibrosis component of Astragalus membranaceus. It has been proved that AST has significant anti-inflammatory and anti-hepatic fibrosis effects. The soluble egg antigen of Schistosoma japonicum soluble egg antigen was observed. After stimulation of macrophages in vitro by sea. Changes in expression and secretion of TNF- 伪 and conditioned medium for SEA activated peritoneal macrophages. Soluble egg antigen induced macrophage conditioned medium. On the basis of the effects of SEA-MCM on collagen synthesis stimulated by HSC proliferation, the effects of AST on collagen synthesis were observed. 1 SEA induced activation and expression of TNF- 伪 in rat peritoneal macrophages and effects of AST on it SEA(0.17g 路L -1 was prepared by HE staining, immunocytochemical staining, reverse transcriptase polymerase chain reaction (RT PCR) and radioimmunoassay. The co-culture of SEA(10 with peritoneal macrophage (PM) in vitro for 3 h was observed. After 6 and 9 hours, the morphologic changes of PM and its effect on the expression of TNF- 伪 were observed in rats. The results showed that 10 mg 路L -1 of SEAA (10 mg 路L ~ (-1)) was treated with 10 mg 路L ~ (-1) of TNF- 伪 for 3 h. The activation of PM 桅 and the expression of TNF- 伪 were significantly stimulated at 6h and 9h. 2the expression of PM 桅 TNF- 伪 stimulated by SEA(10 mg 路L ~ (-1) was significantly decreased by SEA (20 mg 路L ~ (-1)), suggesting that SEA could activate PM 桅 in vitro. By increasing the expression of TNF- 伪, AST could inhibit the expression of TNF- 伪. 2. SEA-MCM induces HSC activation and AST inhibits it. SEA-MCM was prepared by intraperitoneal injection of SEA(0.17mg 路ml-1 (0.2 ml / rat). Hepatic stellate cell line HSC-T6 cells were used. MTT and 3H-proline incorporation were used. The effects of SEA-MCM on the proliferation and collagen synthesis of HSC-T6 cells and the effects of AST on them were observed. The results showed that the final dilution ratio of SEA-MCMs with different dilution was 1: 2. 1: 4 + 1: 1: 1: 16) could significantly promote the proliferation and collagen synthesis of HSC-T6 cells. AST 10 and 20 mg 路L -1) inhibited SEA-MCM 1: 4 in a concentration-dependent manner. It is suggested that SEA can promote HSC proliferation and collagen synthesis through activated macrophages. This effect can be inhibited by AST. 3. Effects of anti-TNF- 伪 antibody on proliferation and collagen synthesis of HSC-T6 cells induced by SEA-MCM TNF- 伪 antibody with different dilution was added to SEA-MCM-1: 4) and MTT was used. Western blotting and radioimmunoassay were used to detect the effects of SEA-MCM on the proliferation and collagen synthesis of HSC-T6 cells. 1: 100 and 1: 200) could significantly inhibit the proliferation and collagen synthesis of HSC-T6 cells induced by SEA-MCM. The results indicated that SEA could promote the proliferation of HSC and produce collagen through macrophage secreting TNF- 伪, which might be one of the important mechanisms of schistosomiasis hepatic fibrosis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R285.5;R392

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