RhG-CSF对MMTV-erbB2转基因鼠乳腺发育的影响

发布时间：2018-03-30 21:34

本文选题：重组人粒细胞集落刺激因子　切入点：MCF-10A　出处：《河南科技大学》2015年硕士论文

【摘要】：目的:1.研究重组人粒细胞集落刺激因子(Rh G-CSF)对人类正常乳腺细胞株MCF-10A和乳腺癌细胞株SK-BR-3增殖作用的影响。2.探讨Rh G-CSF对乳腺癌模型MMTV-erb B2转基因鼠乳腺发育的影响。方法:1.体外培养人正常乳腺上皮细胞株MCF-10A和乳腺癌细胞株SKBR-3细胞株,将含不同浓度的Rh G-CSF的培养液与MCF-10A和SK-BR-3共同培养不同时间,然后利用MTT法,研究Rh G-CSF对人乳腺正常细胞MCF-10A和乳腺癌细胞株SK-BR-3增殖率的影响。2.选择鼠龄12周的健康雌性MMTV-er Bb2转基因鼠60只,随机分为三组,分别为低剂量组:Rh G-CSF药物小鼠皮下注射(剂量为0.125μg,每日一次);高剂量组:Rh G-CSF药物小鼠皮下注射(剂量为0.25μg,每日一次);对照组:生理盐水小鼠皮下注射(剂量为0.25μg,每日一次)。Rh G-CSF药物皮下注射8周后,每组取6只小鼠解剖,取小鼠腹部左下侧完整乳腺组织行Whole mount染色,观察小鼠乳腺大体形态结构的变化,然后应用HE染色法观察小鼠乳腺导管结构发育情况,用免疫组织化学染色SP法检测各组小鼠乳腺组织内PCNA蛋白表达情况,用实时定量PCR(Real Time-PCR)法,检测cyclin D1、c-myc、VEGF、G-CSF3R在各组小鼠乳腺组织中的表达。结果:1.经不同浓度的Rh G-CSF药物处理细胞后,高低剂量的Rh G-CSF药物对人类正常乳腺细胞MCF-10A的增殖作用均不明显,而与对照组相比,乳腺癌细胞株SK-BR-3在低剂量时细胞增殖作用明显。2.Whole mount结果显示,用药8周后,小鼠乳腺形态以对照组为参考,低剂量组乳腺小叶发育旺盛,小鼠乳腺导管数目增多(P=0.000),而高剂量组小鼠乳腺导管增殖民作用不明显(P=0.027)。3.各组小鼠乳腺组织内增殖细胞核抗原(Proliferating Cell Nuclear Antigen PCNA)与对照组相比,低剂量组PCNA高表达(P=0.000),高剂量组PCNA阳性不明显(P=0.004),Real Time-PCR结果显示细胞增殖基因和相关通路因子如cyclin D1、c-myc、VEGF、G-CSF3R的m RNA表达水平在乳腺组织中与对照组相比,低剂量时高表达,高剂量时作用不明显(P0.05)。结论:1.Rh G-CSF对人类正常乳腺细胞MCF-10A的增殖作用不明显,对乳腺癌细胞株SK-BR-3低剂量时具有增殖作用。2.Rh G-CSF对MMTV-erb B2转基因鼠乳腺发育具有影响,在低剂量明显促进小鼠乳腺发育,而高剂量时乳腺发育增殖作用不明显。
[Abstract]:Objective 1. To study the effects of recombinant human granulocyte colony stimulating factor (Rh G-CSF) on the proliferation of human breast cancer cell line MCF-10A and breast cancer cell line SK-BR-3. 2. To investigate the effect of Rh G-CSF on breast development in breast cancer model MMTV-erb B2 transgenic mice. Methods 1. Human breast epithelial cell line MCF-10A and breast cancer cell line SKBR-3 cell line were cultured in vitro. The culture medium containing different concentrations of Rh G-CSF was co-cultured with MCF-10A and SK-BR-3 for different time, and then MTT method was used. To study the effect of Rh G-CSF on the proliferation rate of human breast normal cell MCF-10A and breast cancer cell line SK-BR-3. 2. Sixty healthy female MMTV-er Bb2 transgenic mice of 12 weeks of age were selected and randomly divided into three groups. Low dose group (0.125 渭 g, once a day); high dose group (0.25 渭 g, once a day); control group: normal saline mice subcutaneously (0.25 渭 g). Rh G-CSF was injected subcutaneously once a day for 8 weeks. Six mice in each group were dissected, and the intact breast tissues of the left and lower abdomen of the mice were stained with Whole mount to observe the changes of the gross morphology of the mammary glands of the mice, and then to observe the development of the ductal structure of the mammary glands of the mice by HE staining. Immunohistochemical staining SP method was used to detect the expression of PCNA protein in the mammary gland of mice in each group, and real-time quantitative PCR(Real Time-PCR method was used to detect the expression of cyclin D1c-mycfon G-CSF3R in the mammary gland of mice in each group. Results: 1. After the cells were treated with different concentrations of Rh G-CSF, the expression of GCSF3R in mammary gland of each group was detected. The effects of Rh G-CSF at high and low doses on the proliferation of MCF-10A in human normal breast cells were not obvious. However, compared with the control group, the proliferation of breast cancer cell line SK-BR-3 was obvious at low dose. 2. Whole mount showed that, after 8 weeks of treatment, the proliferation of breast cancer cell line SK-BR-3 was significantly higher than that of control group. The mammary gland morphology of mice was referred to the control group, and the development of mammary lobule in low dose group was strong. The number of mammary ducts increased in mice, but the proliferative effect of mammary ducts in high dose group was not obvious. The proliferating Cell Nuclear Antigen PCNA (proliferating Cell Nuclear Antigen PCNA) in mammary gland of mice in each group was higher than that in control group. In the low dose group, the expression of PCNA was higher than that in the control group, and the expression of m RNA in the high dose group was higher than that in the control group. The results showed that the expression of proliferative gene and related pathway factors such as cyclin D1 c-myct G-CSF3R in the breast tissue was higher than that in the control group, and the expression of m RNA in the high dose group was higher than that in the control group. Conclusion: 1. Rh G-CSF has no obvious effect on the proliferation of MCF-10A in human normal breast cells, but it has a proliferative effect on breast cancer cell line SK-BR-3 at low dose. 2. Rh G-CSF has an effect on mammary gland development of MMTV-erb B2 transgenic mice. The development of mammary gland in mice was obviously promoted at low dose, but the proliferation of mammary gland was not obvious at high dose.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

【共引文献】