高脂膳食对人肺腺癌细胞SPCA1增殖活性影响及其机制的血清生理学研究
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摘要
目的:
用血清生理学(serophysiology)方法研究高脂膳食对人肺腺癌细胞系SPCA1增殖活性的影响,并且从整体水平和细胞水平上探讨其机制。
方法:
1 用灭活及非灭活的雌性大鼠血清各三个浓度(10%、15%、20%)培养人肺腺癌细胞SPCA1,通过MTT比色法实验及计算细胞倍增时间观察血清是否灭活以及不同血清浓度对细胞增殖的影响。
2 将20只SD雌性大鼠分为高脂组和对照组,对照组自由进食基础饲料,高脂组自由进食高脂饲料(猪油:基础饲料=1:9,w/w),喂养7周后分离大鼠血清并培养人肺腺癌细胞系SPCA1,用MTT比色法、~3H-TdR掺入法、细胞流式术检测方法从细胞增殖活力、细胞DNA合成以及细胞周期分布等方面观察高脂膳食喂养的雌性大鼠血清(RatSerum Treated with High-fat Diet,RSTHFD)对癌细胞增殖活性的影响。
3 采用酶法测定血脂、放免法测定雌激素、胰岛素水平,观察高脂膳食对大鼠体重、体脂分布、血脂及血清激素水平的影响,从整体水平上探讨高脂膳食促进癌细胞增殖的可能机制。
4 观察癌细胞雌激素受体表达情况及RSTHFD对细胞凋亡的影响,从细胞水平上分析探讨高脂膳食促进癌细胞增殖的可能机制。
结果:
Objective:The present study was designed to assess the effect of the diet high in fat on proliferative activities of lung adenocarcinoma cell line SPCA1 by means of serophysiological method, and to define the mechanisms separately from whole level and cellular level.Methods:1 Effects of activated or inactivated rat serum (10%, 15%, 20%) on cell proliferation were detected separately by MTT assay and doubling time to define the suitable conditions for cell culture.2 Female Sprague-Dawley rats were fed with common chow or high-fat diet (lard:common chow = 1:9, w/w) for 7 weeks, and serum was obtained by centrifugal method. MTT assay, [~3H] thymidine incorporation method and flow cytometric analysis were performed to identify cell proliferative activity, DNA duplication and cell cycle distribution, which were examined to discuss the effects of
Rat' s Serum Treated with High-fat Diet (RSTHFD) on proliferation of cancer cells.3 Lipids, insulin and estrogen levels in serum were examined and body weights were weighed to study the whole level mechanism on the effects of high-fat diet.4 Flow cytometric analysis was performed to measure the percentage of hypodiploid cells (apoptotic cells). Intracellular localization of estrogen receptor(ER) was determined by immunohistochemical staining.Results:1 The rat serum concentration supplemented in culture medium was 15%, and serum was inactivated.2 RSTHFD can accelerate DNA duplication, cell proliferation and cell mitosis.3 Subcutaneous and visceral fat accumulated in rats which were fed with high-fat diet. There were no significant differences in body weight, serum lipids and insulin levels between two groups. Rats fed with high-fat diet significantly had an elevated circulating level of estrogen, which was involved in the mechanism of cell proliferation.4 ER was localized in the cytoplasm and partly in nuclei, and might be a key contributor to cell proliferation. In the meantime, RSTHFD was found to decrease the number of apoptotic cells, then subsequently displayed activity of promoting cell proliferation.
Conclusions:1 Fifteen percent of inactivated rat serum is suitable condition for culturing SPCA1 cells.2 High-fat diet can stimulate the proliferative activity of SPCA1 cells in the present study.3 The mechanisms may be as follows:■ High-fat diet elevated estrogen concentrations which highly contributed to cell proliferation.■ ER exsists in SPCA1 cells, which indicated cell proliferation might be induced by ER-dependent pathway.■ The findings presented by flow cytometry showed that RSTHFD inhibited cell apoptosis and ultimately promoted cell proliferation.
用血清生理学(serophysiology)方法研究高脂膳食对人肺腺癌细胞系SPCA1增殖活性的影响,并且从整体水平和细胞水平上探讨其机制。
方法:
1 用灭活及非灭活的雌性大鼠血清各三个浓度(10%、15%、20%)培养人肺腺癌细胞SPCA1,通过MTT比色法实验及计算细胞倍增时间观察血清是否灭活以及不同血清浓度对细胞增殖的影响。
2 将20只SD雌性大鼠分为高脂组和对照组,对照组自由进食基础饲料,高脂组自由进食高脂饲料(猪油:基础饲料=1:9,w/w),喂养7周后分离大鼠血清并培养人肺腺癌细胞系SPCA1,用MTT比色法、~3H-TdR掺入法、细胞流式术检测方法从细胞增殖活力、细胞DNA合成以及细胞周期分布等方面观察高脂膳食喂养的雌性大鼠血清(RatSerum Treated with High-fat Diet,RSTHFD)对癌细胞增殖活性的影响。
3 采用酶法测定血脂、放免法测定雌激素、胰岛素水平,观察高脂膳食对大鼠体重、体脂分布、血脂及血清激素水平的影响,从整体水平上探讨高脂膳食促进癌细胞增殖的可能机制。
4 观察癌细胞雌激素受体表达情况及RSTHFD对细胞凋亡的影响,从细胞水平上分析探讨高脂膳食促进癌细胞增殖的可能机制。
结果:
Objective:The present study was designed to assess the effect of the diet high in fat on proliferative activities of lung adenocarcinoma cell line SPCA1 by means of serophysiological method, and to define the mechanisms separately from whole level and cellular level.Methods:1 Effects of activated or inactivated rat serum (10%, 15%, 20%) on cell proliferation were detected separately by MTT assay and doubling time to define the suitable conditions for cell culture.2 Female Sprague-Dawley rats were fed with common chow or high-fat diet (lard:common chow = 1:9, w/w) for 7 weeks, and serum was obtained by centrifugal method. MTT assay, [~3H] thymidine incorporation method and flow cytometric analysis were performed to identify cell proliferative activity, DNA duplication and cell cycle distribution, which were examined to discuss the effects of
Rat' s Serum Treated with High-fat Diet (RSTHFD) on proliferation of cancer cells.3 Lipids, insulin and estrogen levels in serum were examined and body weights were weighed to study the whole level mechanism on the effects of high-fat diet.4 Flow cytometric analysis was performed to measure the percentage of hypodiploid cells (apoptotic cells). Intracellular localization of estrogen receptor(ER) was determined by immunohistochemical staining.Results:1 The rat serum concentration supplemented in culture medium was 15%, and serum was inactivated.2 RSTHFD can accelerate DNA duplication, cell proliferation and cell mitosis.3 Subcutaneous and visceral fat accumulated in rats which were fed with high-fat diet. There were no significant differences in body weight, serum lipids and insulin levels between two groups. Rats fed with high-fat diet significantly had an elevated circulating level of estrogen, which was involved in the mechanism of cell proliferation.4 ER was localized in the cytoplasm and partly in nuclei, and might be a key contributor to cell proliferation. In the meantime, RSTHFD was found to decrease the number of apoptotic cells, then subsequently displayed activity of promoting cell proliferation.
Conclusions:1 Fifteen percent of inactivated rat serum is suitable condition for culturing SPCA1 cells.2 High-fat diet can stimulate the proliferative activity of SPCA1 cells in the present study.3 The mechanisms may be as follows:■ High-fat diet elevated estrogen concentrations which highly contributed to cell proliferation.■ ER exsists in SPCA1 cells, which indicated cell proliferation might be induced by ER-dependent pathway.■ The findings presented by flow cytometry showed that RSTHFD inhibited cell apoptosis and ultimately promoted cell proliferation.
引文
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4 刘巍,吴树岭,孙喜文,等.哈尔滨市南岗区居民肺癌发病死亡10年动态分析.中国肿瘤 2003:12(7):384-386.
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7 Mulder I, Jansen MC, Smit HA, et al. Role of smoking and diet in the cross-cultural variation in lung-cancer mortality: the Seven Countries Study. Seven Countries Study Research Group. Int J Cancer 2000 Nov 15;88(4):665-671.
8 Kesteloot H, hesaffre E, Joossens JV. Dairy fat, saturated animal fat, and cancer risk. Prey Med 1991Mar:20(2):226-36.
9 Alavanja M, Brown C, Swanson C, et al. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. J Natl Cancer Inst1993;85:1906-1916.
10 World Cancer Research Fund/Amercan Institute of Cancer Research. Food, nutrition and the prevention of cancer:a global perspective. Washington, DC: WCRF, 1997.
11 Smith-Warner SA, Ritz J, Hunter DJ, et al. Dietary fat and risk of lung cancer in a pooled analysis of prospective studies. Cancer Epidemiol Biomarkers Prey 2002 Oct;11(10 Pt 1):987-92.
12 Zock PL. Dietary fats and cancer. Curr ODin Lipidol 2001;12(1):5-10.
13 Wynder EL, Cohen LA, Winters BL. The challenges of assessing fat intake in cancer research investigations: J Am Diet Assoc 1997 Jul;97(7 Suppl):S5-8.
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2 孙燕.肺癌流行病学与肺癌预防.中国肺癌杂志,2000,3(6):404-405.
3 马尔健,王莉丽,姚辉.合肥市居民1987～2001年恶性肿瘤死亡趋势分析.疾病控制杂志,2003,7(4):318-320.
4 刘巍,吴树岭,孙喜文,等.哈尔滨市南岗区居民肺癌发病死亡10年动态分析.中国肿瘤 2003:12(7):384-386.
5 井立滨,宇广华,郭长胜.辽宁省部分城市历年恶性肿瘤发病和死亡趋势分析.中国肿瘤,2003,12(7):393-394.
6 王启俊,祝伟星,刑秀梅.北京城区居民1982-1997年癌症发病趋势.中国肿瘤,2001,10(9):507-509.
7 Mulder I, Jansen MC, Smit HA, et al. Role of smoking and diet in the cross-cultural variation in lung-cancer mortality: the Seven Countries Study. Seven Countries Study Research Group. Int J Cancer 2000 Nov 15;88(4):665-671.
8 Kesteloot H, hesaffre E, Joossens JV. Dairy fat, saturated animal fat, and cancer risk. Prey Med 1991Mar:20(2):226-36.
9 Alavanja M, Brown C, Swanson C, et al. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. J Natl Cancer Inst1993;85:1906-1916.
10 World Cancer Research Fund/Amercan Institute of Cancer Research. Food, nutrition and the prevention of cancer:a global perspective. Washington, DC: WCRF, 1997.
11 Smith-Warner SA, Ritz J, Hunter DJ, et al. Dietary fat and risk of lung cancer in a pooled analysis of prospective studies. Cancer Epidemiol Biomarkers Prey 2002 Oct;11(10 Pt 1):987-92.
12 Zock PL. Dietary fats and cancer. Curr ODin Lipidol 2001;12(1):5-10.
13 Wynder EL, Cohen LA, Winters BL. The challenges of assessing fat intake in cancer research investigations: J Am Diet Assoc 1997 Jul;97(7 Suppl):S5-8.
14 贺玉琢.日本汉方药“血清药理学”、“血清药化学”的研究概况.国外医学中医中药分册,1998,20(5):3-7.
15 吴善芳.人体肺腺癌细胞系SPCA1的建立及其生物学特性.中国科学(B辑),1982,10:913.
16 王宁生.关于血清药理学的若干思考.中国中西医结合杂志,1999,19(5):264.
17 陈长勋.用血清药理学实验方法观察附子的强心作用.中国中医药科技,1996,3(3):12.
18 周明眉,杨奎,姜远平.中药血清药理学方法学研究—含药血清低温保存和血清灭活的影响.中药药理与临床,1999,15(2):44—46.
19 魏于全,赵霞.流式细胞术在细胞生物学和分子生物学研究上的应用.华西医学,1996,11(4):436～438.
20 Visskorpi T. High cell proliferation activity determined by DNA flow cytometry and prognsis in epidermoid lung carcinoma, Acta Oncologica 1995; 34:605-609.
21 粱之彦主编.生理化学(下).上海:上海科学技术出版,1990.367.
22 Nagata C, Takatsuka N, Kawakami N, et al. Relationships between types of fat consumed and serum estrogen and androgen concentrations in Japanese men[J]. Nutr Cancer, 2000, 38(2): 163-167.
23 Kaaks R. Nutrition, hormones, and breast cancer:is insulin the missing link? Cancer Causes Control 1996;7:605-625,
24 Simoncikova P, Wein S, Gasperikova D, et al.Comparison of the extrapancreatic action of gamma-linolenic acid and n-3 PUFAs in the high fat diet-induced insulin resistance[J]. Endocr Regul, 2002, 36(4): 143-149.
25 Adeline Seow, Wee-Teng Poh, Ming Teh, et al. Diet, reproductive factors and lung cancer risk among Chinese women in Singapore: evidence for a protective effect of soy in nonsmokers. Int J Cancer 2002;97:365-371.
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