【作者】 杨小珊；

【导师】 糜漫天；

【作者基本信息】 第三军医大学 ， 营养与食品卫生学， 2009， 博士

【摘要】 癌症是危害人类健康的重大疾病,在女性因肿瘤死亡的疾病谱中乳腺癌处在第二位,威胁着妇女的健康和生命。近年来,乳腺癌发病呈增加趋势,其病因与遗传、生殖、性激素、营养、电离辐射等多种因素有关。流行病学和实验研究表明大约3/4的乳腺癌与生活方式有关,如饮食因素和环境因素等。随着营养科学的发展,膳食营养物质与肿瘤的关系日益受到研究人员的重视。研究人员发现多种膳食营养因素与人类肿瘤的发生、发展和预后关系密切。许多流行病学研究和实验室资料研究显示,膳食营养物质n-3多不饱和脂肪酸(n-3 PUFA)和三羟异黄酮(GEN)均表现出抑制乳腺癌、前列腺癌、大肠癌等多种与体内激素相关肿瘤的发生、发展。目前,有关研究证实膳食中富含n-3 PUFA与乳腺癌发病呈高度负相关,可使乳腺癌发病危险性降低。Rose等指出,含n-3 PUFA饲料喂养裸鼠,可以使移植的癌细胞生长受抑,转移发生率和严重程度降低(n-6 PUFA组与之相反)。Lu等也发现n-6 PUFA喂养可促进MNU诱导的大鼠乳腺癌发生。鉴于乳腺癌的发生与雌激素直接有关,多项研究还探讨了这类脂肪酸对体内雌激素水平的调节作用。Hilakivi-Clarke等报道,孕鼠摄入高水平n-3 PUFA能显著增加17-β-estradiol水平(与n-6 PUFA组比较有显著性差异),且发现高n-3 PUFA组喂养的孕鼠所产的子代乳腺细胞分化更好,同时子代暴露于化学致癌剂后诱导的肿瘤发生率明显低于n-6 PUFA喂养组。Nakagawa还发现具有雌激素活性的三羟异黄酮与EPA在抗MCF-7和MDA-MB-231乳腺癌细胞增殖方面有协同效应,而目前已有人将DHA与抗癌药物结合(如与Paclitaxel共价相连)用于肿瘤治疗。肿瘤流行病学研究发现,亚洲豆类食物摄入多的人群中乳腺癌的发病率显著低于美国和欧洲豆类食物摄入低的人群,这提示豆类食物消费量和癌症的发生风险存在负相关。大量研究证实大豆及大豆制品中的黄酮类物质,尤其是三羟异黄酮对化学诱癌剂诱导的肿瘤发生具有延缓作用。有研究报道,Hilakivi-Clarke等却发现青春期暴露于三羟异黄酮可降低由DMBA诱发的小鼠乳腺癌细胞恶变,这可能与其促进乳腺癌细胞分化有关。Fielden等报道,在宫内和哺乳期暴露于人体摄入水平的三羟异黄酮对子代乳腺的发育无不良影响,反而对子代暴露于化学诱癌剂时有防护效应。研究发现,n-3 PUFA和三羟异黄酮均具有拮抗肿瘤的作用,拮抗肿瘤的机制共同之处可能与二者均参与体内性激素调节作用有关。植物雌激素与雌激素有着密切关系,可同时作用于靶器官,竞争结合雌激素受体,对机体雌激素起双向调节作用。而n-3 PUFA是体内合成激素的前体物质,二者对机体性激素的影响可能影响机体对化学诱癌剂的反应。基于以上分析及本实验室的前期研究,结合国内外研究进展,本研究结合人们日常膳食特点,研究探讨了n-3 PUFA和三羟异黄酮对乳腺癌发生、发展、转移的影响。在SD大鼠孕期给予n-3 PUFA和三羟异黄酮进行营养干预,获得经孕期营养干预的雌性子代大鼠。利用化学诱癌剂在体内诱导动物乳腺癌模型和体外细胞实验,采用组织病理学、激光共聚焦、流式细胞仪、RT-PCR、Western blotting以及免疫组织化学等技术方法从体内、外不同侧面系统探讨n-3 PUFA和三羟异黄酮孕期干预SD大鼠对子代在化学诱癌剂作用下乳腺癌发生、发展的影响及可能分子机制。本研究主要实验结果如下:1、发现n-3 PUFA和三羟异黄酮孕期营养干预对妊娠期母鼠的体重增长、妊娠时间、产子数量以及产子的性别比均无异常影响,n-3 PUFA增加断乳时母鼠血清雌二醇和孕酮水平,而三羟异黄酮却降低断乳时母鼠血清雌二醇和孕酮水平,混合干预组表现为降低血清雌二醇和孕酮水平。n-3PUFA和三羟异黄酮孕期营养干预后对对雄性仔鼠有促进生长作用;孕期营养干预后雄性仔鼠的AGD值在三羟异黄酮干预组和混合干预组间有差异,表明孕期营养干预对雄鼠的作用较明显,其原因与雄鼠对体内雌激素水平的变化更为敏感,孕期营养干预改变了体内血清雌激素水平有关。与对照组比较,n-3 PUFA和三羟异黄酮孕期营养干预可以促进雌性仔鼠乳腺的发育,可以促使乳腺腺管分化成熟时间提前,促进乳腺细胞发育。上述结果提示孕期营养干预可通过改变母体相关激素水平影响胎儿的生长发育及器官分化。2、采用MNU一次性腹腔注射给药,在n-3 PUFA和三羟异黄酮孕期营养干预后的子代体内成功复制大鼠乳腺癌模型,经病理检查确认为乳腺导管癌。研究发现孕期给予n-3 PUFA和三羟异黄酮进行营养干预可以延长MNU诱导的F1代雌性大鼠乳腺癌的潜伏期,降低乳腺癌的发病率,减少乳腺癌的多发率,表明n-3 PUFA和三羟异黄酮孕期营养干预可以抑制化学诱癌剂MNU诱导的子代乳腺癌的发生。但在试验末期,结合母鼠断乳时血清雌二醇和孕酮水平,雌性子代血清雌二醇和孕酮水平变化表现与母鼠不一致,雌二醇和孕酮代谢紊乱可能是MNU诱导发生乳腺癌所致。3、体外研究显示,150μmol/L DHA和40、80μmol/L的三羟异黄酮对MCF-7、MDA-MB-231人乳腺癌细胞都有抑制作用,这种抑制作用在24小时内表现一致,在以后的时间段,对MDA-MB-231细胞有一定时效关系,但是对MCF-7乳腺癌细胞则未表现出时效关系。DHA和三羟异黄酮对两种乳腺癌细胞细胞周期的影响各不相同,对MCF-7细胞表现出阻滞细胞于G0/G1期,即影响DNA合成;对MDA-MB-231细胞则表现为增加G2/M期细胞比例,即通过向前推进细胞周期,使细胞周期被阻断于G2/M期,从而使大部分细胞失去进入有丝分裂的能力。其原因与这两种肿瘤细胞雌激素受体表达不同有关。4、发现n-3 PUFA和三羟异黄酮孕期营养干预后,其子代乳腺癌组织蛋白表达与模型对照组比较,各营养干预组能降低子代乳腺癌组织ERα的mRNA和蛋白,同时能降低PR、C-erbB-2蛋白的表达。但是与阴性对照组比较,则乳腺癌组织中雌激素相关受体蛋白表达增高,这提示发生乳腺癌后会引起雌激素相关蛋白表达增加。n-3 PUFA和/或三羟异黄酮孕期营养干预后,其子代乳腺癌组织的增殖能力(PCNA、Ki-67蛋白)较模型对照组降低表明孕期营养干预可以抑制乳腺癌组织的增殖能力。但在孕期营养干预后的子代乳腺癌组织中抑癌基因BRCA1的表达增加,提示n-3 PUFA和三羟异黄酮孕期干预后抑制MNU诱导乳腺癌的发生发展可以通过增强抑癌基因BRCA1的表达来实现。该发现与体外实验结果具有一致性,提示n-3 PUFA和GEN可以通过雌激素受体途径影响乳腺癌的发生发展。综上所述,n-3 PUFA和三羟异黄酮孕期营养干预可以通过影响母鼠血清雌激素水平,促进雌性子代乳腺的发育分化,降低MNU诱导子代乳腺癌的发生,同时延长子代乳腺癌的潜伏期,降低肿瘤多发率。体内外实验也证实,n-3 PUFA和三羟异黄酮能通过影响雌激素相关受体的表达以及提高抑癌基因BRCA1水平影响乳腺癌的发生发展。该项研究从生命早期膳食营养干预的途径为预防乳腺癌的发生提供了重要理论和实验依据,有助于人们合理利用n-3 PUFA和三羟异黄酮。更多还原

【Abstract】 Human breast cancer is the second leading cause of cancer death in women, but the etiology of human breast cancer is largely unknown. Genetic susceptibility, hormonal effects and environmental factors appear to be major determinants. Many dietary components have been evaluated for their influence on breast cancer in epidemiological and experimental studies. In the last several decades, increasing attention has been paid to the intake of n-3 polyunsaturated fatty acid (n-3 PUFA) and the risk of cancers, which has indicated that dietary fat intake can influence incidence of the hormone-related cancers(e.g., breast cancer, prostate cancer and colon cancer). The dietary isoflavones may be playing very important role in the decreasing cancer occurrence, people whose diets are rich in soy products have low incidence of breast cancer, prostate cancer and colon cancer.It also showed in epidemiological and experimental studies that high fish consumption and genistein in people’s diet resulted in low incidence of breast cancer, prostate cancer and colon cancer. Studies suggest that dietary fat and dietary phytochemicals affect the etiology of breast cancer. Somebody reports that dietary of high dose n-3 PUFA could increase the concentration of 17-β-estradiol in pregnancy rats ( higher than the n-6 PUFA group), and the mammary gland cell divided more well in high n-3 PUFA pregnancy mouse offspring, whose offspring reveals in occurrence rate guided by the carcinogens raise a group obviously lower than n-6 PUFA.Epidemiological studies indicate that incidence and mortality of breast cancer is higher in Western countries than in some Asian countries. It is generally thought that Asian people whose diets are rich in soy products have low incidence of clinically manifested carcinoma. Genistein can postpone cancer occurrence in animal experiment. It also found that genistein maybe the most anticancer component, and it can suppress many cancer cell lines. Such findings suggest that estrogens play an important role in the etiology of breast cancer. Estrogen exposure early in life, when endogenous estrogen levels are low, may have profound effects, including effects on breast carcinogenesis. Relationships between dietary factors and breast cancer may result from effects that occur before maturity when the breasts are still developing. For example, it was increased the incidences of DMBA-induced breast cancer when genistein was injected to pregnancy rats. However, it can decrease the incidences of DMBA-induced breast cancer when genistein exposed on puberty mice. Genistein maybe effected the mammary cell differentiation. Fielden et al reported that genistein at the dose of dietary intake at gestation and lactation have no harmful effects, furthermore, it can protect from chemical carcinogens.Both n-3 PUFA and genistein can anticancer, and the same method was that both of them are closely link with body hormone. Genistein can be direct compete with body inner estrogen acceptor, lighten estrogen’s thereby urging the tumour proliferation of cells effect, reduce the cancer morbidity danger being related to estrogen. However, n-3 PUFA is the sex hormone effect possibility reaction. Both of them can effect hormone and effect the reaction to chemical carcinogens.Based on the above analysis, combining the progress of research both at home and abroad, in vivo study, fetus were exposed to n-3 PUFA and genistein in uterus. Then, using MNU-induced breast cancer models on female offspring, RT-PCR, immunohistochemistry, western blotting methods were used to study the effects of n-3 PUFA and genistein onMNU-induced mammary tumors. In vitro study, using MCF-7 and MDA-MB-231 breast cancer cells study the effects of n-3 PUFA and genistein on the proliferation and apoptosis. Further, we focused on the influence of MNU-induced breast cancer on female offspring which exposed to n-3 PUFA and genstein in uterus and its mechanism.The results and conclusions are as followed:1. N-3 PUFA and genistein developed no harmful effects on the pregnancy rats and their offspring. N-3 PUFA and genistein have no abnormally influence on the gestation period, weight increasing, and sex ratio of offspring. N-3 PUFA increased the E2 and P4 concentrations in the serum on ablation rats, but genistein decreased the E2 and P4 concentrations in the serum on ablation rats, both of them decreased the concentration. It can help the male offspring’s growth at the lactation and prepubescence period. The male offspring’s AGD was affected between the genistein group and the n-3 PUFA and genistein group. The results suggested that the influence of exponential to male offspring was more obvious than to the female offspring. N-3 PUFA and genstein can affect the fetus, but the effects display later. The mammary gland tissues of the female offspring were better differentiation than the control group. It can urge the mammary gland to be in charge of mature time of differentiation, boosting the mammary gland cell developing ahead of time.2. Fifty-day-old female Sprangue-Dawley rats were received intraperitoneal injection of methyl-nitrosourea (MNU) at 50 mg/kg body weight one time to establish the female F1 rat model of mammary carcinogenesis. The breast tumors induced by MNU were confirmed to be mammary ductal carcinomas by histopathological evaluation of hematoxylin and eosin (HE) staining. Meanwhile, the study showed that exposing to n-3 PUFA and genistein treatment in utero could diversely influence incidence of MNU-induced breast tumors in F1 female rats. There was a longer latency period, less tumor incidence and tumor multiplicity on exposing to n-3 PUFA and genstein. Because of MNU-induced breast cancer, the E2 and P4 concentrations in F1 female serum were different with it in their mother serum.3. Both DHA and geinistein inhibited the proliferation of MCF-7, MDA-MB-231 breast cancer cell. The inhibitions were the same in 24 hours, but time-effect relationship was just on MDA-MB-231 cell line. DHA and genistein can postpone cell cycle on G0/G1 of MCF-7 cell line, but they block the MDA-MB-231 cell line at G2/M. This can inhibit the cell cycle and suppress the proliferation of MCF-7 and MDA-MB-231.4. Compared with the MNU-induced breast cancer model group, n-3 PUFA and genistein inhibited the expression of C-erbB2, ERαmRNA and protein, reduced the level of PR, PCNA, Ki-67 and increased the expression of BRCA1 mRNA and protein in mammary tumor tissues. The results suggested that n-3 PUFA and genistein reduced ERαexpression by directly inhibiting the expressions of ERαmRNA in karyoplast. On the other hand, n-3 PUFA and genistein can also reduced mutation and tumor formation by increasing the expressions of BRCA mRNA and protein.In summary, when female offspring were exposed to n-3 PUFA and genistein treatment in utero, latency period of MNU-induced breast cancer was prolonged and the incidence, invasion of MNU-induced breast tumors were decreased. It suggested that the mammary gland tissues of the female offspring were better differentiation than the control group if the pregnancy rats exposed to n-3 PUFA and genistein. N-3 PUFA and genistein decreases the formation of breast cancer by inhibiting the ERαgene and protein. N-3 PUFA and genistein can inhibit the proliferation of breast cancer cell, change the expressions of PCNA, C-erbB-2, BRCA1, and ERαare correlated to ERαpathway.更多还原