本文選題：重組人粒細(xì)胞集落刺激因子　切入點(diǎn)：MCF-10A　出處：《河南科技大學(xué)》2015年碩士論文

【摘要】：目的:1.研究重組人粒細(xì)胞集落刺激因子(Rh G-CSF)對(duì)人類正常乳腺細(xì)胞株MCF-10A和乳腺癌細(xì)胞株SK-BR-3增殖作用的影響。2.探討Rh G-CSF對(duì)乳腺癌模型MMTV-erb B2轉(zhuǎn)基因鼠乳腺發(fā)育的影響。方法:1.體外培養(yǎng)人正常乳腺上皮細(xì)胞株MCF-10A和乳腺癌細(xì)胞株SKBR-3細(xì)胞株,將含不同濃度的Rh G-CSF的培養(yǎng)液與MCF-10A和SK-BR-3共同培養(yǎng)不同時(shí)間,然后利用MTT法,研究Rh G-CSF對(duì)人乳腺正常細(xì)胞MCF-10A和乳腺癌細(xì)胞株SK-BR-3增殖率的影響。2.選擇鼠齡12周的健康雌性MMTV-er Bb2轉(zhuǎn)基因鼠60只,隨機(jī)分為三組,分別為低劑量組:Rh G-CSF藥物小鼠皮下注射(劑量為0.125μg,每日一次);高劑量組:Rh G-CSF藥物小鼠皮下注射(劑量為0.25μg,每日一次);對(duì)照組:生理鹽水小鼠皮下注射(劑量為0.25μg,每日一次)。Rh G-CSF藥物皮下注射8周后,每組取6只小鼠解剖,取小鼠腹部左下側(cè)完整乳腺組織行Whole mount染色,觀察小鼠乳腺大體形態(tài)結(jié)構(gòu)的變化,然后應(yīng)用HE染色法觀察小鼠乳腺導(dǎo)管結(jié)構(gòu)發(fā)育情況,用免疫組織化學(xué)染色SP法檢測(cè)各組小鼠乳腺組織內(nèi)PCNA蛋白表達(dá)情況,用實(shí)時(shí)定量PCR(Real Time-PCR)法,檢測(cè)cyclin D1、c-myc、VEGF、G-CSF3R在各組小鼠乳腺組織中的表達(dá)。結(jié)果:1.經(jīng)不同濃度的Rh G-CSF藥物處理細(xì)胞后,高低劑量的Rh G-CSF藥物對(duì)人類正常乳腺細(xì)胞MCF-10A的增殖作用均不明顯,而與對(duì)照組相比,乳腺癌細(xì)胞株SK-BR-3在低劑量時(shí)細(xì)胞增殖作用明顯。2.Whole mount結(jié)果顯示,用藥8周后,小鼠乳腺形態(tài)以對(duì)照組為參考,低劑量組乳腺小葉發(fā)育旺盛,小鼠乳腺導(dǎo)管數(shù)目增多(P=0.000),而高劑量組小鼠乳腺導(dǎo)管增殖民作用不明顯(P=0.027)。3.各組小鼠乳腺組織內(nèi)增殖細(xì)胞核抗原(Proliferating Cell Nuclear Antigen PCNA)與對(duì)照組相比,低劑量組PCNA高表達(dá)(P=0.000),高劑量組PCNA陽(yáng)性不明顯(P=0.004),Real Time-PCR結(jié)果顯示細(xì)胞增殖基因和相關(guān)通路因子如cyclin D1、c-myc、VEGF、G-CSF3R的m RNA表達(dá)水平在乳腺組織中與對(duì)照組相比,低劑量時(shí)高表達(dá),高劑量時(shí)作用不明顯(P0.05)。結(jié)論:1.Rh G-CSF對(duì)人類正常乳腺細(xì)胞MCF-10A的增殖作用不明顯,對(duì)乳腺癌細(xì)胞株SK-BR-3低劑量時(shí)具有增殖作用。2.Rh G-CSF對(duì)MMTV-erb B2轉(zhuǎn)基因鼠乳腺發(fā)育具有影響,在低劑量明顯促進(jìn)小鼠乳腺發(fā)育,而高劑量時(shí)乳腺發(fā)育增殖作用不明顯。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【共引文獻(xiàn)】

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