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Expression of Perforin in the Peripheral Blood of Patients with Primary Biliary Cirrhosis and Its Clinical Significance
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摘要:
目的 研究原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)患者外周血穿孔素(perforin, 又称pore-forming protein, PFP)的表达, 探讨PFP在PBC发病机制中的作用及其临床意义。 方法 应用荧光定量PCR检测86例PBC、56例慢性乙型肝炎(chronic hepatitis type B, CHB)和69名健康对照者的外周血单个核细胞(peripheral bloodmono nuclear cells, PBMCs)中PFP mRNA基因表达的差异; 采用流式细胞术分别检测CD3-CD56+自然杀伤细胞(natu-ral killer cells, NK)、CD3+CD8+细胞毒性T细胞(cytotoxic Tlymphocytes, CTL)和CD3+CD56+自然杀伤性T细胞(natural killer Tlymphocytes, NKT)的PFP蛋白表达量。 结果 PBC组PBMCs中PFP mRNA基因表达较健康对照者升高(P < 0.05);PBC、CHB患者和健康对照者NK、CTL和NKT占PBMCs的比例无明显差别(P>0.05), PBC患者NK、CTL和NKT中表达PFP蛋白的细胞较健康对照者明显升高(P < 0.01)。PFPmRNA基因表达量及NK、CTL和NKT表达PFP蛋白的细胞比例与PBC患者Mayo危险评分呈显著负相关(P < 0.01), PBC患者总胆红素水平与NK、CTL表达PFP蛋白的细胞比例呈负相关(P < 0.05)。 结论 PBC患者PFP的异常表达, 提示PFP可能参与PBC的发病。PBC患者NK、CTL和NKT表达PFP蛋白的细胞比例可做为PBC患者生存预后的有效指标。 Abstract:Objective To study the expression of perforin (also known as pore-forming protein, PFP) in the peripheral blood of patients with primary biliary cirrhosis (PBC) and to analyze the clinical significance of PFP in the pathogenesis of PBC. Methods Peripheral blood mononuclear cells PFP mRNA were detected by real-time RT-PCR assay in 86 PBC patients, 56 chronic hepatitis B patients, and 69 health controls. The expressions of PFP protein in CD3-CD56+natural killer cells (NK), CD3+CD8+cytotoxic T lymphocytes (CTL), and CD3+CD56+natural killer T lymphocytes (NKT) were examined by tricolour flow cytometry. Results The mRNA expression of PFP was significantly higher in PBC patients (P < 0.05). The percentage of lymphocyte subsets was not significantly different among the three groups (P>0.05), while the percentage of PFP in the three lymphocyte subsets in PBC patients was significantly higher than those in health controls (P < 0.01). The Mayo risk score was significantly correlated with the gene expression of PFP and the percentage of PFP in three types of cells in PBC patients (P < 0.05). The percentage of PFP in NK and CTL cells were also positively correlated with the levels of total bilirubin in PBC patients (P < 0.05). Conclusions PFP shows abnormal expression in PBC patients, suggesting that PFP may be involved in the pathogenesis of PBC. The percentages of PFP-positive NK, CTL, and NKT cells can be used as effective indicators in predicting the survival of PBC patients. -
图 1 PFP基因和GAPDH基因扩增产物凝胶电泳图
1~3.PFP基因扩增产物；4~6. GAPDH基因扩增产物；PFP：穿孔素；GAPDH：甘油醛-3-磷酸脱氢酶；M：标准参照物
图 4 流式细胞术检测PBMCs中PFP蛋白表达
A.以PBMCs中CD3-细胞圈门，CD3-CD56+NK细胞PFP的表达；B.以PBMCs中CD3+细胞圈门，CD3+CD56+NKT细胞PFP的表达；C以PBMCs中CD3+CD8+CTL细胞PFP的表达PBMCs：外周血单个核细胞；PFP同图 1
图 5 PBC患者PFP mRNA基因表达的量、PFP蛋白表达比例与肝脏功能相关指标的相关性
A. PFP mRNA基因表达量与MRS相关性；B. NK细胞表达PFP蛋白比例与MRS相关性；C NK细胞表达PFP蛋白比例与TBil相关性；D. CTL细胞表达PFP蛋白比例与MRS相关性；E. CTL细胞表达PFP表达PFP蛋白比例与TBil相关性；F. NKT细胞表达PFP蛋白比例与MRS相关性；采用Spearson秩相关分析
表 1 原发性胆汁性肝硬化患者及对照者基本资料
表 2 外周血PF P蛋白表达(%, $ \overline x \pm s $)
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