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目的观察外周血单个核细胞(PBMC)中p38丝裂原活化蛋白激酶(p38 MAPK)及其活化形式磷酸化p38MAPK(P-p38 MAPK)在急、慢性胰腺炎炎症反应中的水平变化。方法选择2010年11月至2014年12月收治的胰腺炎患者82例,依据临床表现将患者分为慢性胰腺炎组29例、轻型急性胰腺炎组25例、重症急性胰腺炎组28例。选取同期在本院健康体检正常的37例为对照组。取静脉血,分离PBMC,采用实时聚合酶链反应(Real-time PCR)法检测各组p38 MAPK mRNA在PBMC的表达;采用Western Blot检测各组p38 MAPK、P-p38 MAPK蛋白在PBMC的表达。结果 (1)对照组、慢性组、轻型组、重症组p38 MAPK mRNA相对表达量(2-△△Ct)分别为0.33±0.08、0.35±0.04、0.34±0.07、0.42±0.05,重症组明显高于其余3组,差异有统计学意义(F=12.793,P<0.05),其中重症组均值较对照组增加了27.2%。对照组、慢性组、轻型组3组之间比较差异无统计学意义(P>0.05)。(2)p38 MAPK蛋白表达水平(相对表达量,以β-actin为内参):重症组明显高于对照组,差异有统计学意义(P<0.05),其均值较对照组增加了23.4%,对照组、慢性组、轻型组3组之间比较,差异无统计学意义(P>0.05)。P-p38 MAPK蛋白水平表达(相对表达量,以β-actin为内参):重症组P-p38 MAPK蛋白水平明显高于其他3组,差异有统计学意义(P<0.05,P<0.01),其中重症组均值较对照组、慢性组、轻型组分别增加了455.6%、51.5%、47.1%。慢性组和轻型组P-p38 MAPK蛋白水平高于对照组,差异有统计学意义(P<0.05),其均值较对照组分别增加了266.7%、277.8%。慢性组和轻型组比较,差异无统计学意义(P>0.05)。结论胰腺炎患者PBMC中p38MAPK及其磷酸化后的P-p38 MAPK,与病情的严重程度、病程的长短密切相关。对p38 MAPK和P-p38 MAPK在mRNA、蛋白水平表达的检测,有望成为重症胰腺炎临床指标和药物治疗的新靶点。
Objective To observe the changes of p38 mitogen activated protein kinase (p38 MAPK) and its activated form phosphorylated p38 MAPK (P-p38 MAPK) in the acute and chronic pancreatitis inflammatory reaction in peripheral blood mononuclear cells (PBMCs). Methods Eighty-two patients with pancreatitis were selected from November 2010 to December 2014. The patients were divided into chronic pancreatitis group (n = 29), light acute pancreatitis group (n = 25) and severe acute pancreatitis group (n = 28) according to the clinical manifestations. Select the same period in our hospital normal physical examination 37 cases as control group. The venous blood was collected and PBMCs were isolated. Real-time PCR was used to detect the expression of p38 MAPK mRNA in PBMCs. Western blot was used to detect the expression of p38 MAPK and P-p38 MAPK protein in PBMC. Results (1) The relative expression of p38 MAPK mRNA in the control group, chronic group, light group and severe group were 0.33 ± 0.08,0.35 ± 0.04,0.34 ± 0.07,0.42 ± 0.05 respectively, significantly higher in severe group In the other three groups, the difference was statistically significant (F = 12.793, P <0.05), in which the average value of severe group increased 27.2% compared with the control group. There was no significant difference between the control group, the chronic group and the light group (P> 0.05). (2) The expression level of p38 MAPK protein (relative expression, β-actin as internal reference): severe group was significantly higher than the control group, the difference was statistically significant (P <0.05), the average increase of 23.4% There was no significant difference between the three groups in the control group, the chronic group and the light group (P> 0.05). P-p38 MAPK protein expression (relative expression, β-actin as an internal control): Severe group P-p38 MAPK protein levels were significantly higher than the other three groups, the difference was statistically significant (P <0.05, P <0.01) Among them, the average value of severe group increased 455.6%, 51.5% and 47.1% respectively compared with control group, chronic group and light group. The protein levels of P-p38 MAPK in chronic group and mild group were significantly higher than those in control group (P <0.05), and their mean values were 266.7% and 277.8% higher than those in control group respectively. Chronic group and light group, the difference was not statistically significant (P> 0.05). Conclusion The expression of p38 MAPK and phosphorylated P-p38 MAPK in PBMC of patients with pancreatitis is closely related to the severity of the disease and the duration of the disease. The detection of p38 MAPK and P-p38 MAPK at the mRNA and protein levels is expected to become a new target of clinical treatment of severe pancreatitis and drug therapy.