胃泌素在结肠癌患者中的表达及其受体拮抗剂对人结肠癌细胞株的抑制作用及其对P38信号转导通路的影响

Expression of gastrin in colon cancer and its effect on human colon cancer cell proliferation and P38 signal transduction pathway

背景结肠癌(colon cancer,CC)是我国常见消化系统恶性肿瘤,早期缺乏特异性症状诊断率较低,导致患者丧失根治性机会,病死率较高,极大危害患者生命健康.胃泌素主要是由胃肠道G细胞分泌一种激素,与胃泌素受体结合后可刺激胃酸分泌,促进胃肠道黏膜生长.丝裂原活化蛋白激酶是一组能被胃泌素等激素激活的丝氨酸-苏氨酸蛋白激酶,负责细胞表面与细胞核内部间信号传递.目的分析胃泌素在CC患者中的表达,并探讨其受体拮抗剂对人CC细胞株的抑制作用及对P38信号转导通路的影响.方法将2016-01/2018-10我院病理科30例CC组织标本根据世界卫生组织恶性肿瘤分化程度标准分为低分化标本、中分化标本、高分化标本,采用免疫组化技术检测观察胃泌素在CC组织中表达情况,体外培养人CC细胞株SW480,将细胞分为对照组(不进行任何药物处理)、胃泌素组(分别加入6.25-200.00 mg/L胃泌素进行处理)、丙谷胺组(分别加入8.00-256.00 mg/L丙谷胺进行处理)、胃泌素联合丙谷胺组(加入12.5 mg/L胃泌素与8.00-256.00 mg/L丙谷胺进行处理),统计SW480中胃泌素受体/胆囊收缩素-B受体表达情况,比较各组CC细胞株SW480活力、细胞增殖指数、P38信号转导通路表达情况P38蛋白、磷酸化-P38蛋白、B淋巴细胞瘤-2(B lymphocyte tumor-2,SBcl-2)、细胞凋亡促进基因(BAX).结果CC组织分化程度越高,胃泌素表达阳性率越高;胃泌素组6.25-200.00 mg/L范围内SW480 OD值均高于对照组(P<0.05);胃泌素组12.50 mg/L时SW480 OD值最高(P<0.05);胃泌素组25.00-200.00 mg/L范围内SW480 OD值比较差异无统计学意义(P>0.05);丙谷胺组8.00-256.00 mg/L范围内SW480 OD值比较差异无统计学意义(P>0.05);胃泌素组联合丙谷胺组在12.50 mg/L胃泌素联合16.00 mg/L丙谷胺时,SW480OD值最低,低于对照组(P<0.05),之后随着丙谷胺浓度增加,SW480 OD值比较差异无统计学意义(P>0.05);胃泌素组(12.50 mg/L)细胞增殖指数高于丙谷胺组(16.00 mg/L)、胃泌素组联合丙谷胺组(12.5mg/L+16.00 mg/L)(P<0.05);胃泌素组(12.50 mg/L)P38蛋白、磷酸化-P38蛋白、BAX蛋白低于对照组、丙谷胺组(16.00 mg/L)、胃泌素组联合丙谷胺组(12.5mg/L+16.00 mg/L),Bcl-2蛋白表达高于对照组、丙谷胺组(16.00 mg/L)、胃泌素组联合丙谷胺组(12.5 mg/L+16.00 mg/L)(P<0.05).结论胃泌素可抑制人CC细胞株SW480的凋亡,且在CC组织中的表达与肿瘤分化程度有关,分化程度越高,其表达量越高,胃泌素受体拮抗剂在一定浓度范围内可拮抗胃泌素促增殖效应,其机制与上调P38、磷酸化-P38、BAX表达及下调Bcl-2表达有关.

Background Colon cancer(CC)is a common malignant tumor of the digestive system in China.The early diagnosis is low due to nonspecific symptoms,which leads to the loss of chance of radical surgery and a high mortality rate,greatly harming patients’life and health.Gastrin is a hormone that is mainly secreted from G cells in the gastrointestinal tract.Upon binding to gastrin receptors,it stimulates gastric acid secretion and promotes gastrointestinal mucosal growth.Mitogen-activated protein kinases(MAPKs)are a group of serine-threonine protein kinases that are activated by hormones such as gastrin and are responsible for signal transduction between the cell surface and the nucleus.AIM To analyze the expression of gastrin in CC patients,and to investigate the inhibitory effect of gastrin receptor antagonist on human CC cell line and the P38 signal transduction pathway.Methods From January 2016 to October 2018,30 CC specimens collected from the Department of Pathology of our hospital were divided into poorly,moderately,and highly differentiated specimens according to the criteria of the World Health Organization’s malignant tumor differentiation.The immunohistochemical technique was used to detect the expression of gastrin in these specimens.The human CC cell line SW480 was cultured in vitro,and the cells were divided into a control group(no drug treatment),a gastrin group(6.25-200.00 mg/L of gastrin was added),a proglumide group(8.00-256.00 mg/L of proglumide for treatment),and a gastrin plus proglumide group(12.5 mg/L gastrin and 8.00-256.00 mg/L proglumide for treatment).The expression of gastrin receptor/cholecystokinin-B receptor in SW480 cells was detected,and SW480 cell viability,proliferation index,and expression of P38 signal transduction pathway molecules(P38 protein,phosphorylated P38 protein,Bcl-2,and BAX)in different groups were compared.Results The higher the degree of differentiation of CC tissues,the higher the positive rate of gastrin expression.The OD values of SW480 cells treated with gastrin at concentrations ranging from 6.25 to 200.00 mg/L were significantly higher than those in control cells(P<0.05).Gastrin at a concentration of 12.50 mg resulted in the highest OD value in SW480 cells(P<0.05).There was no significant difference in OD values of SW480 cells treated with gastrin at concentrations between 25.00 and 200.00 mg/L(P>0.05).There was no significant difference in OD values of SW480 cells treated with glutamine at concentrations of 8.00-256.00 mg/L(P>0.05).Gastrin at 12.50 mg/L combined with 16.00 mg/L of proglumide resulted in the lowest OD value in SW480 cells,which was significantly lower than that in the control group(P<0.05),but this significant difference disappeared with the increase of proglumide concentration(P>0.05).The cell proliferation index of the gastrin group(12.50 mg/L)was significantly higher than those of the proglumide group(16.00 mg/L)and the gastrin plus proglumide group(12.5 mg/L+16.00 mg/L)(P<0.05).The levels of P38 protein expression and phosphorylation and BAX protein expression in the gastrin group(12.50 mg/L)were significantly lower than those of the control group,proglumide group(16.00 mg/L),and gastrin plus proglumide group(12.5 mg/L+16.00 mg/L),while Bcl-2 protein expression was significantly higher that in the control group,proglumide group(16.00 mg/L),and gastrin plus proglumide group(12.5 mg/L+16.00 mg/L)(P<0.05).Conclusion Gastrin can inhibit the apoptosis of human CC cell line SW480,and its expression in CC is related to the degree of tumor differentiation.The higher the degree of differentiation,the higher the expression level.Gastrin receptor antagonist can antagonize the proliferative effect of gastrin via mechanisms possibly related to upregulation of P38 expression,phosphorylation of P38,and BAX expression and down-regulation of Bcl-2 expression.