p27kip及关联蛋白在乳腺肿瘤原发与转移病灶屮功能研究

p27 as a cell cycle negative regulatory factor， it is closely related to cells into the S phase，and also it is an important medium to cause G1 arrest. P27 can be able to inhibit various CDKS, including the CDK4, CDK2 and of CDC2. They can block the phosphorylation of Rb protein, then make the tumor cells arrest in the G phase.There is closed relationship between p27 expression and tumor occurrence, differentiation, invasion and metastasis，and therefore also become a hot spot with the p27 gene and protein research. In this study, to confirm the biological function of p27 in canine mammary tumor cells, two levels will be surveyed with inhibit the expression of the p27 gene and p27 gene overexpression ， to determine p27 in primary tumor cells and metastasis of canine mammary tissue iexpress condition。 （1）Using molecular biology techniques, to make silence and over-expression of p27 gene, observe its biological function in breast tumor cells; simultaneously detect the expression of p27 protein in the organization of the clinical cases and the distribution in the cells, determine with tumor as well as play the position of the biological effects;to explore the PI3K/Akt signaling pathway on the regulation of p27 protein, that is to observe p27 degradation of the mRNA levels of key molecules reveal the adjustment mechanism in this pathway, （2）To analysis and comparison related genes polymorphisms of the cells derived from primary lesions and metastatic lesions with different parts of the tumor cells isolated the impact of from investigation p27 and joint p21, the p53 single nucleotide polymorphisms (single nucleotide polymorphism. SNP) . p27-depth research it is for individuals to provide a reliable molecular mechanisms and cancer susceptibility. Its significance not only for the of tumor immunology therapy and anticancer drug development provide the new role of the target, but also for individuals, analysis of tumor susceptibility to make early prevention of tumor treatment, prevention and control of more targeted .

p27为一种细胞周期负性调控因子，由于它的表达与肿瘤的发生、分化、浸润及转移密切相关，因此与p27基因和蛋白相关的研究也成为热点。本课题从抑制p27基因的表达和使p27基因的过表达两个层面，完整系统的确定p27在犬乳腺肿瘤细胞中的生物学功能，探究p27在犬乳腺原发肿瘤细胞和转移组织中的表达状况，来证实它是否参与了肿瘤的形成过程，并从两个方面阐述p27蛋白在肿瘤细胞中功能改变的机制：一是从p27自身基因多态性为切入点，观察其在原发肿瘤细胞组织和转移肿瘤细胞组织中的结构变化，联合研究较为广泛的p21、p53基因多态性，将三者进行对比研究，确定三者与乳腺肿瘤发生、发展、转移及预后的关系，为乳腺癌易发群体提供一种可靠的预测指标，第二就是从p27调节机制着手，研究p27基因转录情况，选取PI3K/Akt信号通路，确定p27蛋白功能降低甚至失活的分子机制，为肿瘤的预防和治疗提供理论依据。

肿瘤的发生发展是一个多因素、多步骤、多阶段的复杂过程，细胞周期异常调控可引起正常组织细胞异常增殖。p27kip、cyclinD1（cyclin 家族）作为细胞周期分别负向和正向调控作用的关键成员。PI3K/Akt信号通路从多个方面发挥对细胞的生长、增殖、细胞周期相关蛋白和凋亡的作用，是肿瘤发生、发展的关键通路之一。本研究以犬乳腺肿瘤为研究对象，采用RT-PCR方法扩增犬p27kip、cyclinD1基因的目的片段，并成功构建重组质粒经转化诱导顺利表达目的蛋白；通过ELISA、western blot以及免疫组化等方法表明p27kip和cyclinD1蛋白具有良好的特异性和反应原性；成功制备犬p27kip单克隆抗体和cyclinD1多克隆抗体；为进一步研究p27kip和cyclinD1的生物学特性提供研究材料。通过免疫组化方法对犬正常乳腺、犬乳腺肿瘤细胞系（CHMm、CHMp）、临床收集的84例犬乳腺肿瘤组织（良性肿瘤、导管内癌、浸润性导管癌、浸润性小叶癌、黏液癌、髓样癌、伴随淋巴细胞浸润、混合样癌）中p27kip、cyclin D1的检测；证明cyclin D1在良性肿瘤中的表达低于恶性，正常乳腺中基本无表达，p27kip在正常组织的表达高于良性肿瘤高于恶性。cyclinD1可用于区分导管内癌与浸润性小叶癌；p27kip可以区分混合样癌与导管内癌，此外，cyclinD1与p27kip共同应用于浸润性导管癌的诊断方面有价值，提示其在肿瘤发生的早期具有辅助性诊断的意义，为今后研究细胞周期调节失调与肿瘤发生关系提供实验材料。成功构建pcDNA3.1-PTEN质粒，并转染到犬乳腺肿瘤细胞系中，成功表达了PTEN蛋白，证明PTEN过表达能够抑制犬乳腺肿瘤细胞系的增殖、促进其凋亡；此外，PTEN过表达能下调PI3K/Akt信号通路，进而调控该通路下游相关细胞凋亡相关分子Bcl-2、Bax、caspase-3、caspase-9和周期相关分子CyclinD1、P27kip的表达，抑制犬乳腺肿瘤细胞的生长。。以上研究不仅成功制备了细胞周期蛋白p27kip、cyclinD1抗体，检测在犬乳腺肿瘤组织中的表达情况，及其细胞周期蛋白在PI3K/Akt信号通路中的作用，为今后犬乳腺肿瘤的早期诊断及其靶治疗提供新思路。