华支睾吸虫3-磷酸甘油醛脱氢酶与肝纤维化的关系  

The relationship between Clonorchis sinensis 3⁃phosphoglycerate dehydrogenase and liver fibrosis

作　　者：胡月 黄艳 李文芳 HU Yue;HUANG Yan;LI Wenfang(Division of Management Infection,Hefei Second People's Hospital,Hefei,Anhui 230011,China)

机构地区：[1]合肥市第二人民医院院感处,安徽合肥230011 [2]中山大学中山医学院寄生虫学教研室,广东广州510080 [3]中山大学附属第五医院检验科,广东珠海519000

出　　处：《热带医学杂志》2025年第1期6-11,F0002,共7页Journal of Tropical Medicine

基　　金：广东省自然科学基金(2019A1515110300,2023A1515140139)。

摘　　要：目的探讨华支睾吸虫3-磷酸甘油醛脱氢酶(Cs.GAPDH)与肝纤维化之间的关系,为华支睾吸虫病的防治提供理论依据和实验基础。方法采用腹腔注射法建立Cs.GAPDH诱导大鼠肝纤维化模型,以注射磷酸盐缓冲液(PBS)作为阴性对照组,以注射猪血清作为阳性肝纤维化模型对照组,通过HE染色、Masson染色和免疫组织化学法观察大鼠肝脏炎症及坏死反应的严重程度;利用免疫组织化学实验观察肝脏组织石蜡切片的Ⅲ型胶原表达情况;通过免疫细胞化学实验观察Cs.GAPDH与人肝星状细胞LX‐2的结合情况,以及这种结合是否依赖其纤溶酶原结合特性;通过CCK‐8细胞增殖和流式细胞法评估不同浓度Cs.GAPDH(0、2、5、10、20、40μg/mL)对人肝星状细胞LX‐2分别在24、48、72 h的影响;通过RT‐PCR方法和ELISA检测Cs.GAPDH刺激RAW264.7细胞分泌细胞因子白细胞介素-33(IL‐33)的转录和表达水平变化。结果通过对实验组大鼠肝脏HE染色和Masson染色发现,与对照组相比,注射Cs.GAPDH大鼠肝脏组织中出现了纤维胶原的沉积与炎症细胞的浸润;免疫组织化学染色显示,Cs.GAPDH组肝组织可见Ⅲ型胶原成条索状小片状分布在肝窦间隙中;Cs.GAPDH与LX‐2细胞结合并不依赖于其纤溶酶原结合特性;低浓度(2μg/mL)的Cs.GAPDH可以在短期内诱导LX‐2细胞的增殖(F=11.69,P<0.05),而持续高浓度(20μg/mL)则未能促进细胞增殖反而抑制了细胞的增殖,并诱导LX‐2细胞的凋亡(F=15.88,P<0.05);在体外刺激RAW264.7细胞24 h后,与PBS组相比,IL‐33在蛋白转录表达水平上显著升高且差异有统计学意义(t=5.69,P<0.05)。结论Cs.GAPDH通过诱导RAW264.7细胞分泌IL‐33,进一步介导肝星状细胞的增殖和凋亡,参与华支睾吸虫长期慢性感染引起的肝纤维化发生与发展,为深入了解肝纤维化机制及华支睾吸虫病的防治提供了潜在的研究方向。Objective To investigate the relationship between Clonorchis sinensis glyceraldehyde-3-phosphate dehydrogenase(Cs.GAPDH)and liver fibrosis,providing theoretical and experimental foundations for the prevention and treatment of clonorchiasis of clonorchiasis.Methods A Cs.GAPDH-induced rat liver fibrosis model was established via intraperitoneal injection,with phosphate buffered saline(PBS)serving as the negative control group and porcine serum as the positive control group.HE staining,Masson staining,and immunohistochemistry were used to observe the severity of liver inflammation and necrosis in rats.Immunohistochemical analysis was conducted to examine typeⅢcollagen expression in liver tissue paraffin sections.Immunocytochemistry was used to study the binding of Cs.GAPDH to human hepatic stellate cells(LX-2)and to determine whether this binding depends on its plasminogen-binding properties.The effects of different concentrations of Cs.GAPDH(0,2,5,10,20,and 40μg/mL)on LX-2 cells over 24,48,and 72 hours were evaluated using the CCK-8 cell proliferation assay and flow cytometry.RT-PCR and ELISA were used to detect changes in the transcription and expression levels of cytokine interleukin 33(IL-33)secreted by RAW264.7 cells stimulated with Cs.GAPDH.Results HE and Masson staining of the liver tissues in the experimental group revealed significant collagen deposition and inflammatory cell infiltration in rats injected with Cs.GAPDH,compared to the control groups.Immunohistochemical staining showed that typeⅢcollagen in the Cs.GAPDH group was distributed in streaks and small patches within the liver sinusoidal spaces.The binding of Cs.GAPDH to LX-2 cells was not dependent on its plasminogen-binding properties.Low concentrations(2μg/mL)of Cs.GAPDH appeared to transiently promote the proliferation of LX-2 cells(F=11.69,P<0.05),whereas sustained high concentrations(20μg/mL)inhibited cell proliferation and induced apoptosis in LX-2 cells(F=15.88,P<0.05).Stimulation of RAW264.7 cells in vitro with Cs.GAPDH resulted in

关 键 词：华支睾吸虫 3-磷酸甘油醛脱氢酶 肝纤维化 白细胞介素-33 

分 类 号：R383.22[医药卫生—医学寄生虫学]