复方良附颗粒含药血清诱导人胃癌细胞凋亡研究

【作者】 庄严；

【导师】 孙颖立；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2007， 博士

【摘要】 胃癌(gastric cancer)是最常见的恶性肿瘤之一，目前世界范围内胃癌的发病率有所下降，但我国仍为胃癌的高发地区。手术切除是治疗胃癌的首选方法，但由于临床缺乏特异性症状和体征，早期诊断率较低，手术对进展期胃癌的治疗效果并不理想，根治手术后有50％患者5年内出现局部复发和／或远处转移，常见的复发和转移部位是切除部位、肝脏和腹膜表面。因此，除手术和放疗外，化疗仍是胃癌治疗的主要手段，但严重不良反应使患者难以耐受，中医药的应用越来越受到重视。现阶段中医防治肿瘤工作已有很大进展，不仅表现在临床疗效的提高，诊治手段的多样化，还体现在理论实验研究的深入，从而挖掘了中医药所蕴藏的科学内涵，用现代理论阐明了中医药治疗机理，使中医药抗癌步入了一个新的阶段。1目的以人胃癌细胞BGC-823为靶细胞，以新加良附颗粒为受试药物，通过体外细胞孵育观察不同浓度含药血清对人胃癌细胞BGC-823的抑制作用，以形态学观察及流式细胞仪检测来评定凋亡，以Western blot测含药血清干预后BGC-823细胞的Bcl-2和Akt的蛋白表达，探讨新加良附颗粒抗肿瘤的机理。2方法以新加良附颗粒饲养大鼠7天后获得含药血清，用MTT法观察作用48 h，72 h，96h后不同浓度的含药血清对人胃癌细胞BGC-823细胞增殖的抑制作用。用倒置显微镜和电子显微镜观察细胞的形态及流式细胞仪检测对人胃癌细胞BGC-823细胞凋亡作用。应用免疫蛋白印记方法研究含药血清对Bcl-2和Akt表达的影响。3结果不同浓度含药血清可抑制BGC-823细胞生长和增殖，具有剂量依赖性。A值分别为0.52±0.08、0.44±0.06，与对照组相比P＜0.05。抑制率为40.35±6.45、30.29±4.89，与CTX组相比P＞0.05。出现凋亡典型的超微结构改变如核碎片。流式细胞检测结果表明新加良附颗粒有诱导BGC-823细胞凋亡作用，随剂量的增加，细胞凋亡增加，凋亡率35.92±0.87、29.51±1.02(％)，与对照组相比差异显著，P＜0.05。Bcl-2和Akt表达下降且与剂量相关。4结论新加良附颗粒抑制人胃癌细胞系BGC-823生长并诱导其凋亡及Bcl-2和Akt表达下调是新加良附颗粒抗肿瘤的机制之一。更多还原

【Abstract】 Gastric cancer is one of the common malignant tumors in digestive system. At present, theincidence of gastric cancer is declining in the worldwide, however, China is still a highincidence of gastric cancer area. Surgical resection of the gastric carcinoma is best choice. Dueto the lack of specificity of clinical symptoms and signs, the lower rate of early diagnosis,surgery for advanced gastric cancer treatment is not satisfactory. There are 50% of patientshad local recurrence and/or distant metastasis with in 5years after Radical surgery. Commonsite of recurrence and metastasis is resection site, liver and peritoneal surface. Chemotherapyis still the main treatment method for advanced gastric cancer in addition to surgery andradiation therapy. The application of Chinese medicine has drawn increasing attention asserious adverse reactions. It is greatly improved to prevent and cure cancer by Chinesemedicine now. It is exhibited to not only increase the clinic curative effect, diverse ways ofdiagnosis and treatment, but also go deep into the academic research. Chinese doctors havetaped the Chinese medicine latent science knowledge, and have elucidated the Chinesemedicine treating mechanism by modern theory, and have developed the Chinese medicinetreating cancer to a new stage.1 ObjectiveTo take the human gastric carcinoma cell line BGC-823 as the target cell, to take theXinjialiangfukeli as the experimental drug, to observe the inhibiting action of pharmaceuticsserum at different concentrations on the proliferation of human gastric cancer cells BGC-823 invitro, apoptosis was assessed with cell morphology and flow cytometry, detecting the proteinexpression of Bcl-2 and Akt of the Human gastric carcinoma cell line BGC-823 by WesternBlot, so to discuss its anticancer mechanisms.2 MethodsHuman gastric carcinoma cell line BGC-823 was treated with the pharmaceutics serumobtained of the Male Rats after feeding it with Xinjialiangfukeli for 7 days at differentconcentrations for 48h、72h and 96h respectively. The inhibiting rate of cells was observed byMTT. Observation cell morphology changes by morphological characteristics under phasecontrast microscope and transmission electron microscope(TEM). Apoptosis Detection ofHuman gastric carcinoma cell line BGC-823 by Flow cytometry. The expression of Bcl-2 andAkt protein were examined by Western blot respectively.3 ResultsObtain pharmaceutics serum at different concentrations can inhibit the growth andproliferation of BGC-823 cells with a dose-dependent manner. A value were 0.52±0.08,0.44±0.06, and be compared with control P＜0.05. The rate of inhibition were 40.35±6.45,30.29±4.89, and be compared with CTX P＞0.05. The typical changes of ultra structureincluded peripheral accumulation of nuclear chromatins, chromatins fragmentation ofnuclear.Obtain pharmaceutics serum may induce BGC-823 apoptosis with a dose-dependentmaimer, the rate of apoptosis was 35.92±0.87、29.51±1.02(%), and be compared with control P＜0.05. The Bcl-2 expressions of protein down-regulated with a dose-dependent manner byWestern blot. The Akt expression of protein was down-regulated with a dose-dependentmanner by Western blot.4 ConclusionsXinjialiangfukeli can inhibit the growth of BGC-823; induce BGC-823 apoptosis,down-regulating the expression of Akt and Bcl-2 probably is one of its molecularmechanisms.更多还原