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目的检测磷酸酶和张力蛋白同源物(phosphatase and tensin homolog deleted on chromosome ten,PTEN)基因及蛋白在急性梗阻性化脓性胆管炎(acute obstructive suppurative cholangitis,AOSC)患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的表达情况,探讨其在脓毒血症发生过程中的意义。方法分离AOSC患者(n=25)治疗前及治愈后1周时PBMC和血清,以及同期健康自愿者(正常对照组,n=15)外周血PBMC和血清,分别采用蛋白印迹法(Western blot法)和实时荧光定量聚合酶链反应法(real-time quantitative polymerase chain reaction,q RT-PCR)检测PBMC中PTEN、核因子κB p65(nuclear fatorκB p65,NF-κB p65)和核转录因子κB抑制因子(inhibitor of NF-κB,IκB)蛋白和基因的表达以及磷酸化水平;并用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测血清中脂多糖(lipopolysaccharide,LPS)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白介素10(interleukin 10,IL-10)的含量。结果 AOSC组患者治疗前血清中LPS、TNF-α和IL-10的含量均明显高于正常对照组(P<0.05),治愈后1周时,上述指标则明显下降并接近正常水平。AOSC组患者治疗前PBMC中PTEN和IκB的蛋白和基因表达水平明显低于正常对照组(P<0.05);治愈后1周时,两者的蛋白和基因的表达水平恢复到正常水平,但是两者治疗前的磷酸化水平均明显高于正常对照组(P<0.05),治愈后1周时恢复正常;而NF-κB p65基因及蛋白在AOSC组患者治疗前其表达增高,治愈后1周时下降至正常水平。结论脓毒血症的发生可能与肠道LPS的移位有关,并引起机体促炎因子和抗炎因子的失衡;PTEN在脓毒血症发生过程中可能通过磷酸化降解IκB或直接激活NF-κB,在脓毒血症发生初期可作为治疗靶点之一。
Objective To detect the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene and protein in peripheral blood mononuclear cells (PBMCs) in patients with acute obstructive suppurative cholangitis (AOSC) mononuclear cell (PBMC), and to explore its significance in the process of sepsis. Methods Peripheral blood PBMCs and sera from patients with AOSC (n = 25) and PBMCs and serums at 1 week after treatment and peripheral blood mononuclear cells (PBMCs) and serum from healthy volunteers (n = 15) were detected by Western blot ) And real-time quantitative polymerase chain reaction (q RT-PCR) were used to detect the expression of PTEN, nuclear fatorκB p65 (NF-κB p65) and nuclear factor κB inhibitor (inhibitor of NF-κB, IκB) protein and gene expression and phosphorylation; ELISA and enzyme linked immunosorbent assay (ELISA) were used to detect serum lipopolysaccharide (LPS), tumor necrosis factor-α tumor necrosis factor-α, TNF-α and interleukin 10 (IL-10). Results The levels of LPS, TNF-α and IL-10 in serum of patients with AOSC before treatment were significantly higher than those of normal control group (P <0.05). At 1 week after treatment, the above indexes decreased significantly and approached the normal level. The protein and gene expression levels of PTEN and IκB in PBMC of AOSC patients before treatment were significantly lower than those of normal control group (P <0.05). At 1 week after treatment, the expression of PTEN and IκB protein and gene returned to normal level, The level of phosphorylation before treatment was significantly higher than that of the normal control group (P <0.05), and returned to normal one week after the treatment. The expression of NF-κB p65 gene and protein in AOSC group before treatment increased, When it dropped to normal levels. Conclusions The occurrence of sepsis may be related to the intestinal LPS translocation and cause imbalance of proinflammatory cytokines and anti-inflammatory cytokines. PTEN may phosphorylate IκB or directly activate NF-κB in sepsis, κB, in the early stages of sepsis can be used as a therapeutic target.