Mammary Gland Morphological and Gene Expression Changes Underlying Pregnancy Protection of Breast Cancer Tumorigenesis. Rattus norvegicus

Pregnancy has been shown to decrease the risk of mammary carcinogenesis in human rretrospective epidemiological studies. In rodents, pregnancy prior to carcinogen administration or after carcinogen challenge has also been shown to reduce the incidence of palpable carcinomas. In this study our objective to determine the underlying genomic signature of the pregnancy and reproductive hormones on the mammary gland that contribute to the protection against mammary gland carcinogenesis. We used the rat microarray technology to observe total transcriptome changes after the pregnancy and exogenous reproductive hormone stimulation of the mammary gland. Overall design: Fifteen 3 month old post-pubertal virgin Lewis rats were randomly assigned to three groups (5 rats per group): control (C), pregnancy (P) and hormone treatment (H). The P group animals had a full-term pregnancy (21-23 days) and rats in the group H were implanted subcutaneously on the dorsal midline with two silastic capsules [(0.078 inch inner diameter, 0.125 inch outer diameter) x 2 cm long; Dow Corning, Midland, MI) filled separately with 100 μg ethynyl estradiol (Sigma, St. Louis, MO) packed in a cellulose matrix (Sigma) and 30 mg of megesterol acetate (Sigma) for 21 days. The control animals had neither the hormone treatment nor being pregnant. The animals in C and P groups were also implanted with sham capsules filled with cellulose matrix only. The capsules were surgically implanted at the beginning of the experiment and removed from all animals after 21 days except that the capsules were removed from the P group following parturition (21-23 days). The delivered pups in the P group were euthanized within 4-6 hours of delivery to avoid suckling. After the removal of capsules all groups were rested a total of ~49 days before euthanasia. All animals were euthanized during metestrus stage, determined by vaginal cytology and total RNA was extracted from the mammary gland tissues using Trizol reagent.

现有回顾性流行病学研究表明，妊娠可降低人类乳腺癌发生风险。在啮齿类动物中，致癌剂给药前或给药后妊娠同样可降低可触及性乳腺癌的发病率。本研究旨在探究妊娠与生殖激素对乳腺的潜在基因组特征，该特征可赋予乳腺抗致癌保护作用。我们采用大鼠基因芯片（microarray）技术，观察妊娠及外源性生殖激素刺激后乳腺的全转录组变化。

总体实验设计：将15只3月龄性成熟未产Lewis大鼠随机分为3组（每组5只）：对照组（C）、妊娠组（P）与激素处理组（H）。P组大鼠经足月妊娠（21~23天）；H组大鼠于背部中线皮下植入2根硅橡胶囊（内径0.078英寸，外径0.125英寸，长2cm；Dow Corning，密歇根州米德兰市），分别填充100μg乙炔基雌二醇（Sigma，密苏里州圣路易斯市，以纤维素基质[Sigma]包裹）与30mg醋酸甲地孕酮（Sigma），植入时长为21天。对照组大鼠既未接受激素处理，也未妊娠。C组与P组大鼠同样植入仅填充纤维素基质的假手术囊。实验开始时通过外科手术植入所有囊体，21天后从所有动物体内取出囊体，但P组的囊体在分娩后（21~23天）取出。P组产下的幼崽在分娩后4~6小时内实施安乐死，以避免幼崽吮吸母乳。囊体取出后，所有组大鼠均休息约49天，随后实施安乐死。所有动物均在动情间期（metestrus stage）实施安乐死，通过阴道细胞学检查确定该阶段；采用Trizol试剂从乳腺组织中提取总RNA。