CAJ | 학술논문

目的：通过研究噎膈血瘀证、脾气虚证患者及健康人血清对食管癌 EC9706细胞增殖、磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/核因子kappaB（NF-κB）信号通路相关分子mRNA及蛋白表达的调控，探讨噎膈血瘀证、脾气虚证的分子机制。方法本研究于2014年6—12月选择来源于唐山市人民医院、迁安燕山医院肿瘤内科的病理确诊的食管鳞癌患者。按照《中华人民共和国中医药行业标准———中医病症诊断疗效标准》确定噎膈血瘀证或脾气虚证标准，选取血瘀证、脾气虚证患者各10人，另选取健康志愿者10人（来自两院体检人员）。将EC9706细胞于37℃，5％饱和湿度的二氧化碳（ CO2）培养箱孵育24 h，饥饿24 h，分别加入倍比梯度的噎膈血瘀证、脾气虚证患者和健康人血清，噻唑蓝（MTT）染色法检测细胞增殖变化；实时荧光定量聚合酶链反应（PCR）检测PI3K、Akt、NF-kB表达；免疫印迹（Western blot）法检测PI3K/Akt/NF-κB信号通路相关分子蛋白表达。结果血瘀证患者血清刺激细胞的50％增殖率为71.1μl/ml，脾气虚证为118.0μl/ml；血瘀证患者血清上调细胞 PI3K、Akt 和NF-κB mRNA表达水平，与各对照组比较差异有统计学意义（ P﹤0.05），脾气虚证患者血清无上调mRNAs作用；血瘀证患者血清促进细胞表皮生长因子受体（EGFR）、PI3K、Akt、磷酸化的蛋白激酶B（p-Akt）和 NF-κB等蛋白表达水平增高，与各对照组比较差异有统计学意义（ P﹤0.05），脾气虚证患者血清无促进此五蛋白表达作用。结论噎膈血瘀证患者血清能够促进细胞增殖，该证候的分子机制可能与PI3K/Akt/NF-κB信号通路过度激活有关；脾气虚证患者血清无刺激细胞增殖作用，其证候分子机制有待探讨。
목적：통과연구일격혈어증、비기허증환자급건강인혈청대식관암 EC9706세포증식、린지선기순3-격매（PI3K）/단백격매B（Akt）/핵인자kappaB（NF-κB）신호통로상관분자mRNA급단백표체적조공，탐토일격혈어증、비기허증적분자궤제。방법본연구우2014년6—12월선택래원우당산시인민의원、천안연산의원종류내과적병리학진적식관린암환자。안조《중화인민공화국중의약행업표준———중의병증진단료효표준》학정일격혈어증혹비기허증표준，선취혈어증、비기허증환자각10인，령선취건강지원자10인（래자량원체검인원）。장EC9706세포우37℃，5％포화습도적이양화탄（ CO2）배양상부육24 h，기아24 h，분별가입배비제도적일격혈어증、비기허증환자화건강인혈청，새서람（MTT）염색법검측세포증식변화；실시형광정량취합매련반응（PCR）검측PI3K、Akt、NF-kB표체；면역인적（Western blot）법검측PI3K/Akt/NF-κB신호통로상관분자단백표체。결과혈어증환자혈청자격세포적50％증식솔위71.1μl/ml，비기허증위118.0μl/ml；혈어증환자혈청상조세포 PI3K、Akt 화NF-κB mRNA표체수평，여각대조조비교차이유통계학의의（ P﹤0.05），비기허증환자혈청무상조mRNAs작용；혈어증환자혈청촉진세포표피생장인자수체（EGFR）、PI3K、Akt、린산화적단백격매B（p-Akt）화 NF-κB등단백표체수평증고，여각대조조비교차이유통계학의의（ P﹤0.05），비기허증환자혈청무촉진차오단백표체작용。결론일격혈어증환자혈청능구촉진세포증식，해증후적분자궤제가능여PI3K/Akt/NF-κB신호통로과도격활유관；비기허증환자혈청무자격세포증식작용，기증후분자궤제유대탐토。
Objective To investigate the molecular mechanisms of Blood-Stagnation Syndrome( BSS)and spleen-Qi-dificiency syndrome( SQDS)patients with Esophageal cancer( EC)through observation on cells proliferation,mRNAs and proteins expression in PI3K/Akt/NF-κB signaling pathway regulated by sera from patients. Methods From June to December, 2014,we enrolled patients who were definitely diagnosed with esophageal squamous cell carcinoma in oncology department of Tangshan People's Hospital and Yanshan Hospital. According to TCM Professional Standard of People's Republic of China/ TCM diagnosis and efficacy standard,we determined the standard of blood stasis syndrome and spleen-yang deficiency syndrome in patients with dysphagia. We enrolled 10 patients of blood stasis syndrome,10 patients with spleen-yang deficiency syndrome, and another 10 healthy controls(people who received physical examination from the two hospitals). EC9706 cells were put into CO2 incubator with a temperature of 37℃ and a saturation humidity of 5% and were cultured for 24 hours and kept hungury for 24 hours. Then serum of patients of blood stasis syndrome and spleen-yang deficiency syndrome and healthy controls was put into the incubator by proportional gradient,and MTT staining method was used to detect the changes in cell proliferation;the expression levels of PI3K,Akt and NF-κB mRNA were measured by Real-time PCR,and the protein expression of relevant molecules of PI3K/Akt/NF-κB signalling pathway was detected by Western blot method. Results Half maximal ( 50%) promotion concentration(PC50)of BSS was 71. 1 μl/ml,PC50 of SQDS was 118. 0 μl/ml. Sera from BSS patients promoted PI3K,Akt and NF-κB mRNAs expression,being significantly different from those in control group(P ﹤0. 05),however,sera from SQDS patients didn't work. Western blot assay showed that BSS patients'sera stimulated over-expression of EGFR,PI3K,Akt, p-Akt and NF-κB,showing significant difference from control group(P﹤0. 05),however,sera from SQDS patients didn't work. Conclusion Sera from BSS patients stimulates EC9706 cells proliferation. Molecular hypostasis of BSS relates to over -activation of PI3K/Akt/NF-κB signaling pathway. Sera from SQDS patients can't stimulate EC9706 cells proliferation,and molecular hypostasis of it needs further study.