三七提取物抑制人胃粘膜上皮细胞恶性转化的作用研究

【作者】 王志斌；

【导师】 李军祥；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2004， 硕士

【摘要】 胃癌是人类发生率和死亡率最高的恶性肿瘤之一，也是我国最常见的的恶性肿瘤。由于胃癌的病因一级的预防还比较困难。因此，加强对胃癌癌前病变的研究和预防就显得尤为重要。 目前西医对于胃癌前病变的逆转治疗尚无特别有效的药物。中医药在治疗胃癌前病变的临床和机理研究很多，但多为复方研究，有关单味中药治疗胃癌前病变的报道极少。针对以上问题，我们通过细胞培养方法，从研究灌服三七提取物后的犬血清对胃癌前细胞的抑制作用入手，找出三七入血后的最佳作用时间点(段)，并进一步研究其促凋亡机理，为开发治疗胃癌的新药提供一些理论依据。 1．灌服三七提取物的犬血清对GES-1及MC细胞增殖抑制作用 实验中采用永生化的人胃粘膜上皮细胞系GES-1细胞以及MNNT进一步转化GES-1细胞后获得的MC细胞作为胃癌前病变细胞的体外研究模型。采用三七水提物给彼格犬三七水提物一次性灌胃的方法，取给药前的血清、给药后5分钟、10分钟、30分钟、60分钟、120分钟、180分钟、240分钟、300分钟、360分钟、480分钟、600分钟、720分钟、840分钟、960分钟、1200分钟、1440分钟各时点的血清，灭活补体后作为实验药物血清。 以MTT法分别检测每个采血时间点上不同的药物血清的浓度不同(8％、4％、2％)对GES-1及MC细胞作用72小时后的抑制情况，并根据结果找出最佳抑制的药物血清时点。结果显示其中在2小时和6小时血清的含药血清刺激下的抑制率均为最高，两个时间点含8％药物血清的培养基对两种细胞的抑制作用均十分显著，发现这两个时间点含药物血清的培养基对两种细胞的抑制作用均十分显著，其中2小时8％-GES-1组抑制率为70.8％(p<0.01)；2小时8％-MC组抑制率为45.3％(p<0.01)；6小时8％-GES-1组抑制率为88.5％(p<0.01)；6小时8％-MC组抑制率为42.4％(p<0.01)。为了进一步探讨灌服三七提取物的犬血清对胃癌前细胞抑制增殖作用的机理，我们做了下面实验。 2．灌服三七提取物的犬血清对MC细胞的促凋亡作用 根据上个实验的分析所获的两个最佳药效时点，应用这两个时段不同浓度的含药物血清对MC细胞进行促凋亡实验。采用PI单染法对刺激后的细胞染色，以流式细胞分析仪分析药物血清对MC细胞的促凋亡作用。结果发现两个时间点的含药血清能明显促进MC细胞的凋亡，和同样条件下用无药血清培养的MC细胞相比其凋亡比例显著性增加(p<0.05)。在药物血清的作用下两个时点组的MC细胞在G0／G1期细胞比例明显下降(p<0.05)；而G2／M期细胞显著增加(p<0.05)；但s期并没有一致性的变化。 综上所述，对胃癌前病变的体外细胞模型研究结果表明，采用灌服三七提取物后2、6小时取的药物犬血清对GES-1及MC细胞的抑制作用最强；两个时点的药物血清对MC细胞都有显著的促凋亡作用，且能显著降低MC细胞在G0／G1期的中文摘要比例及显著增加GZ/M期的比例，这可能是其抑制MC细胞增殖及促凋亡的原因之更多还原

【Abstract】 Gastric cancer, the most-commonly seen in China, is one of the malignant cancers with the highest incidence and mortality. Because the first-grade prevention targeted at pathogeny is relatively difficult, it’s important to strenghen the research and prevention of precancerous lesions.At present, Western medicine shows little effect in reversing precancerous lesions .There are many clinical studies about the mechanism of Traditional Chinese Medicine(TCM) in treating gastric precancerous lesions. Most of these studies are about compound prescriptions and there is few reports about the study of simple prescriptions. In order to solve this problem, we studied the inhibiting effects on precancerous gastric cells by the serum of the dog being fed with distilled substances of SanQi(penex notogingseng) through the method of cells culture and tried to find the points or periods of time when the SanQi’s function was the strongest.The mechanism of promoting apoptosis was further investigated, providing some theoretic foundation for developing new medicine.l.The inhibiting effects of the dog serum on the proliferation of GES-1 and MC cellsThe experiments adopted eternalized human gastric mucosa epithelium GES-1 cells and MC cells gained from GES-1 cells transformed by MNNT as the model of precancerous lesions for study in vitro. Feeding the dog with SanQi distilled substances one time, we took the serum befor feeding, and 5, 10, 30, 60, 120, 180, 240, 300, 360, 480, 600, 720, 840, 960, 1200, 1440 minites afer feeding as the medicated serum after destroying alexins.We examined the inhibiting effects 72 hours after function on GES-1 and MC cells of different concentration of medicated serum(8 % * 4 % , 2% )gained from different points of time by means of MTT, so as to find the best functional points of time.The results show that GES-1 and MC cells inhibition rate is the highest at 2 hour and 6 hour- medicated serum.At these two points, the culture medium containing 8% medicated serum has prominent inhibiting effects and quantity-effect relation are observed at each point. In order to further discuss the mechanism of inhibiting effect of medicated serum on proliferation of precancerous gastric cells, the following experiments are implemented. 2. The apoptosis-promoting effects of the dog serum on MC cellsAt these two points of time, different concentration of medicated serums were used to promote the apoptosis. Results showed that the serums has significant effects of promoting apoptosis at these two points of time.Compared with non-medicated serums under same conditions, the proportion of apoptosis had a significant increase(p<0.05. MC cells’ proportion was decreased in GO/Gl period (p<0.05 ) and increased in the G2/M period ( p<0.05 ) ,while no consistant changes are observed in S period.To summarize, the results of the study on the cell models in vitro of gastric precancerous lesions showed that the points of time with the strongest inhibiting effects is 2 hours and 6 hours after being fed with the SanQi substance.At these two points, the serums had significant effects of promoting apoptosis, Dramatically decreasing MC cells’ proportion in GO/Gl period and increasing it in the G2/M period were probably responsible for the effects of inhibiting the proliferation of MC cells and promoting apoptosis.更多还原