ampk对小鼠乳腺上皮细胞增殖和乳腺发育影响word格式论文.docxVIP

ABSTRACTObjective:AMP-activated protein kinase (AMPK) is considered as an fuel sensor in eukaryotes. Thus, the present study aims to construct AMPK dominant negative transgenic mice, explore the effect of AMPK in the normal mouse MECs and mammary gland development, observe the changes of mammary gland development treated by metformin and track whether the malignancies occur.Methods:Generation and identification of transgenic mice. The AMPK dominant negative transgenic mice(short for DN below) contain a mutant AMPK sequence with ectogenic α-flag fragment. We identified the transgenic mice with the expressions of target genes or proteins in transgenic mice by PCR and immunohistochemistry(IHC).The effect of AMPK DN in mammary gland development. Morphological changes were observed between various phases of WT and DN mice with whole mount, such as 4~30w virgin mice, pregnancy 5d/10d/17d and lactation 2d. To explore the effect of AMPK DN on the construction and proliferation of MECs, HE staining and PCNA staining were made for further researches.Moreover, intraperitoneal injection of metformin with 5 mg/ml/d into WT mice a week was made to observe the morphologic changes of the ducts during the puberty and pregnancy.Results:RT-PCR identify the genotype of WT and DN primarily, target bands are 500bp. Then Q-PCR showed no expression of α-flag mRNA in mammary gland of WT mice. Also, IHC indicated the successful construction of AMPK DN mice with α-flag protein expression and decrease in p-AMPK expression in DN compared with WT.In puberty, AMPK DN has no significant changes in the invasion and branching of ducts in mammary glands. However, in 5w DN mice, the number of TEBs increase compared with WT, p 0.05. The average areas of TEBs in 5w/6wDN mice increase compared with WT, p 0.05. Moreover, AMPK DN promotes the cell proliferation and incrassation of mature ductal epithelium by HE staining and PCNA staining. In pregnancy, AMPK DN has no significant changes in the developme