摘要
胃癌是发病率较高的恶性疾病。当代胃癌治疗以手术及化学药物治疗为主,但在化学药物治疗上并没有合成一种针对胃癌有特殊疗效的药物,而当前应用的化学药物副反应较大,造成了机体的“二次打击”。我国传统医学有着几千年的历史,中医药的疗效也为大家所承认,尤其中药在调节机体免疫力进而抗肿瘤方面作用突出,因此,在中药中提取有效成份治疗恶性肿瘤成为热点。本文从细胞水平及分子水平对核桃楸皮的抗肿瘤尤其治疗胃癌的机制方面进行系统的研究,以期得到治疗胃癌的有效药物。
本文通过MTT法对比研究不同浓度核桃楸皮水提取物及乙酸乙酯提取物对胃癌细胞的增殖抑制作用及其对正常胃粘膜上皮细胞的作用、流式细胞仪检测细胞周期及细胞凋亡,探讨核桃楸皮水提取物及乙酸乙酯提取物对胃癌细胞作用机理。同时成功诱导小鼠原位种植胃癌模型,进一步研究核桃楸皮水提取物及乙酸乙酯提取物对小鼠特异性免疫及非特异性免疫功能的调节机理,通过RT-PCR及Westernblot方法明确核桃楸皮水提取物及乙酸乙酯提取物抗胃癌作用的分子机制,为临床开发高效低毒的抗胃癌药物提供充分的理论依据。
通过实验研究发现桃楸皮水提取物及乙酸乙酯提取物具有明确的抑制胃癌细胞增殖的作用,同时在24小时内能够促进正常胃粘膜上皮细胞的增殖。其抑制机理为通过调节P53、Bcl-2及BaxmRNA及相应蛋白表达水平促进胃癌细胞的凋亡、同时增加机体自身抗肿瘤的免疫反应而治疗胃癌。
Gastric Carcinoma is one of the most common malignant tumor throughout the world. China is a country which has a high incidence of Gastric Carcinoma with the average death rate of 16 / 100000, and even 60 / 100000 in some districts with the highest incidence of Gastric Carcinoma. Gastric Carcinoma is also the most common alimentary canal cancer in China and causes more deaths than any other malignant tumor. Reports of assessment of the worldwide case rate of common cancers conducted by WHO in 2001 show that people suffering from Gastric Carcinoma take up more than 30% of those suffering from all kinds of alimentary canal cancers in the world, and the death rate caused by Gastric carcinoma in China is among the highest throughout the world. Gastric carcinoma leads to quite low survival rate, so it threatens people's health and living standard badly. Therefore, study on Gastric carcinoma epidemic and the causes and pathogeny of its molecular biology has become one of the most important topics in the field of medicine. Till now, no specific drug aiming at curing Gastric carcinoma with high performance and low toxicity has been developed yet. Nowadays, developing new medicine from traditional Chinese medicine is becoming a hot spot in the field of medicine.
Juglans mandshurica Maxim is a kind of vegetation of Juglans family. Its fruits, walnuts, are rich in unsaturated fatty acid, protein, mineral element, vitamin and so on, and are of high nutrition and quite good for health. Thus, they are liked by people greatly. Juglans mandshurica Maxim , as a kind of traditional Chinese medicine, has the effect of heat-clearing and detoxicating, antibiosis and dephlogisticate, improving acuity of vision, etc. Juglans mandshurica Maxim is an important source of medical materials in China, and the immature skin of its fruits, epidermis of its roots, skin of twigs, barks and leaves can all be used as medical materials. A famous doctor called Meng Xian in Tang Dynasty said that walnuts "can clear out meridian and vessels, moist blood vessels and the frequent use of them can help keep the skin in silky condition.; Li Shizhen, the sage in medicine in Ming Dynasty also spoke highly of the medical effect of walnuts as "reinforcing QI and blood, moistening the dryness and dissipating phlegm, moistening the lung and intestineand so on". For many years, among the common people in China and Korea, the epidermis of the roots of Juglans mandshurica Maxim is used in curing cancers effectively and also in India and some African countries, extract from many parts of the bark of Juglans mandshurica Maxim has been used as folk medicine for a long period of time. Juglans mandshurica Maxim, as a kind of traditional Chinese medicine, with effect of heat-clearing and detoxicating, antibiosis and dephlogisticate, improving acuity of vision, is clinically used in curing diseases like enteritis, bacillary dysentery, bone tuberculosis, hordeolum, fluor albus and conjunctival congestion. The bark, epidermis of roots, leaves, fruits and skin of fruits (also called Qing Longyi), can all be used as medical materials. Pharmacology study proves that aqueous extract and acetic ether extract from the bark of Juglans mandshurica Maxim, leaves and immature skin of the fruits of Juglans mandshurica Maxim indeed has the activity of restraining the proliferation of cancer cells.
The study will mainly discuss the effect and the mechanism of effect of aqueous extract and acetic ether extract from the bark of Juglans mandshurica Maxim on Gastric carcinoma cells, study the restraint effect and mechanism of extract from the bark of Juglans mandshurica Maxim on Gastric carcinoma cells of the mice and provide sufficient theoretical grounds for clinical development of anti-Gastric carcinoma medicine with high performance and low toxicology.
Firstly, we extract the effective ingredients from the bark of Juglans mandshurica Maxim. It is reported in some documents that aqueous extract and acetic ether extract from the bark of Juglans mandshurica Maxim has the effect of anticancer, thus we extract effective ingredients from it and prepare physic liquor and examine its effect at different time on the mice's pre-Gastric carcinoma cells and the normal human being's gastric epithelium cells,and then see whether it can restrain the cancer cells and also whether it has toxicity influence on normal cells. Meanwhile, we examine the effect of extract from the bark of Juglans mandshurica Maxim on the cell cycle and decay of the cells, and clarify the mechanism of effect on the cells level by means of DNA ladder.
After we successfully grow the model of mice with Gastric carcinoma, we check the immunity of mice by examining whether the extract can restrain the cancer cells according to different levels of concentration of the two kind of extract. Also, we observe the influence of the extract on T cell subset of the mice with Gastric carcinoma cells and cell factor level by means of flow cytometry and clarify the effect and mechanism of effect of the extract from the bark of Juglans mandshurica Maxim on immunity. Besides, we make it clear on molecular level whether the exact can exert the effect of anticancer through improving the decay of cancer cells.
Results of the experiment show that: 1. Study on the influence of extract from the bark of Juglans mandshurica Maxim on proliferation of mice's Gastric carcinoma cells by means of MTT demonstrates that the effect of restraint rises with the concentration of extract and the extension of time, but acetic ether extract causes comparatively strong toxic effect to the normal gastric epithelium cells. The toxic effect is weaker than 5-FU (P<0.05) .The concentration with the best effect of restraint is: aqueous extract 100μg/ml (24h), 200μg/ml (48h); acetic ether extract 200μg/ml (24h). 2. By using flow cytometry, we clarify for the first time that extract form the bark of Juglans mandshurica Maxim restrains the proliferation of mice's pre-Gastric carcinoma cells by restraining cell cycle that causes block in G1 phase and S phase. 3.It is clarified by means of flow cytometry that extract from the bark of Juglans mandshurica Maxim restrains the proliferation of cells through causing the decay of cancer cells. The effect of restraint increases with the concentration and extension of time, so it appears to be the relationship of Time-Quantity-Effect. 4. It is made clear by means of DNA electrophoresis that extract from the bark of Juglans mandshurica Maxim has the effect of accelerating the decay of cancer cells. 5. This study successfully grows Gastric carcinoma in Kun Ming mice's gastric wall by injecting cancer cells, and it is an economic and easy method with high survival rate. 6. Through experiment on animals, it is discovered that extract from the bark of Juglans mandshurica Maxim has evident effect of restraint on solid tumor of Gastric carcinoma; the effect increases with the concentration without causing toxicology to normal mice. And this is safer than 5-FU under the circumstance that the effect of cure is not harmed. 7. Extract from the bark of Juglans mandshurica Maxim has marked effect on adjusting immunity by increasing the activity of NK cell and the level of IL-12 and TNF-αand reducing the level of IL-4. It adjusts specific immunity and nonspecific immunity, accelerates the anticancer immunity and restrains the proliferation of cancer cells. 8. Extract from the bark of Juglans mandshurica Maxim raises the level of CD3~+ and CD4~+, and the ratio of CD4~+ to CD8~+, and improves the immunity of cells by adjusting the split up of T cell subset. 9.By means of RT-PC, we clarify that extract from the bark of Juglans mandshurica Maxim accelerates the decay of cancer cells and restrains the proliferation of them through restraining the express of mutant gene of P53 and gene of Bcl-2, and raising that of Bax.
In general, By using flow cytometry, this study clarifies for the first time that extract form the bark of Juglans mandshurica Maxim restrains the proliferation of mice's pre-Gastric carcinoma cells by restraining cell cycle that causes block in G1 phase and S phase. It is clarified by means of flow cytometry that extract from the bark of Juglans mandshurica Maxim restrains the proliferation of cells through causing the decay of cancer cells. The effect of restraint increases with the concentration and extension of time, so it appears to be the relationship of Time-Quantity-Effect. This study successfully cultivates Gastric carcinoma in Kunming mice's gastric wall by injecting cancer cells, and it is an economic and easy method with high survival rate. Through using flow cytometer, we clarify that extract from the bark of Juglans mandshurica Maxim raises the level of CD3~+ and CD4~+, and the ratio of CD4~+ to CD8~+, and improves the immunity of cells by adjusting the split up of T cell subset. It raises cell factor level and furthermore, adjusts specific immunity and nonspecific immunity. It is made clear by means of DNA electrophoresis that extract from the bark of Juglans mandshurica Maxim has the effect of accelerating the decay of cancer cells. By means of RT-PCR, We clarify that extract from the bark of Juglans mandshurica Maxim accelerates the decay of cancer cells and restrains the proliferation of them through restraining the express of mutant gene of P53 and gene of BCL-2, and raising that of Bax. This study provides theoretical grounds for clinical development of anti-Gastric carcinoma medicine with high performance and low toxicology by researching the effect and the mechanism of effect of aqueous extract and acetic ether extract from the bark of Juglans mandshurica Maximon Gastric carcinoma cells.
引文
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