Mechanism study of Pygo2 in mediation of Wnt signaling and expansion of breast cancer stem cells

Wnt经典信号通路是胚胎和成体发育过程中的一个关键性的信号传导通路，也是干细胞/前体细胞中重要的信号通路。Wnt信号通路异常将引起多种肿瘤发生。经典Wnt信号通路中的关键蛋白是Armadillo/β-catenin，Wnt信号促进β-catenin进入细胞核，与Wnt靶基因上的TCF/LEF结合，募集染色质修饰、重塑因子激活Wnt靶基因转录。 Armadillo/β-catenin的转录活性依赖于Pygo（Pygopus）。Pygo是Wnt信号通路中新发现的成员，通过BCL9/Lgs与β-catenin结合。在果蝇中Pygo和Legless/BCL9是Wnt信号通路的重要成员。然而哺乳动物细...The Wnt signaling pathway controls numerous cell fates both in embryonic and adult, and is also an important pathway in stem/ progenitor cell and cancer. Its inappropriate activity can lead to cancer in many human tissues. A key effector of the canonical Wnt pathway is Armadillo/β-catenin. Wnt signaling inhibits its phosphorylation and degradation; this allows it to associate with TCF/LEF factors ...学位：理学博士院系专业：生命科学学院生物医学科学系_细胞生物学学号：2162007015382

Construction of polybutycyanocrylate nanoparticles(PBCA-NPs) loaded P16a gene and expression in Hep-2 cell

目的制备携带P16A基因的聚氰基丙烯酸正丁酯纳米粒并在喉癌细胞中进行表达。方法选用乳化聚合法制备PbCA-nPS,以激光粒度分析仪及透射电子显微镜分析纳米粒的形态和粒径。用阳离子表面活性剂十六烷基三乙基溴化铵(CTAb)对纳米粒进行表面修饰。构建真核表达载体PIrES2-EgfP-P16A经过酶切鉴定及测序后与PbCA-nPS连接,转染喉癌细胞HEP-2,荧光显微镜检测转染效率,蛋白印迹法检验P16A基因的表达,流式细胞术检测细胞凋亡率。结果所制PbCA-nPS粒径均匀、zETA电位较高,较为理想;重组质粒PIrES2-EgfP-P16A经过酶切鉴定及测序后,表明真核表达载体构建正确;PbCA-nPS可以介导PIrES2-EgfP-P16A高效转染喉癌细胞,蛋白印迹检测表明转染后喉癌细胞能够表达外源P16A基因,在其介导下P16A能有效抑制喉癌细胞的增殖并能诱导细胞凋亡。结论聚氰基丙烯酸正丁酯纳米粒可以作为一种良好的基因载体,为喉癌的基因治疗提供了新思路。[Objective] To prepare polybutycyanocrylate nanoparticles(PBCA-NPs)loaded P16a gene as the gene delivery system,and to express the P16a protein in Hep-2 cell line.[Methods] PBCA-NPs were prepared by the emulsion polymerization method.Surface of PBCA-NPs was surveyed by transmission electron micrographs(TEM),the grain distribution and zeta potentials of PBCA-NPs were determined with the laser grain analyzer.The PBCA-NPs surface was modified by the cationic surfactant cetyltrimethylammonium bromide(CTAB).Construct the eukaryotic expression vector pIRES2-EGFP-P16a and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing,the constructed eukaryotic expression plasmid was transfected to Hep-2 line.Transfect ion efficiency was observed by fluorescence microscope and the expression of P16a gene was detected by Western blot.Apoptosis was detected by flow cytomery.[Results] Nps with even size and smooth surface were successfully obtained,holding the higher zeta electric potential.The new constructed vector was confirmed by restricted enzyme and sequencing.pIRES2-EGFP-P16 can be mediated by PBCA-NPs with high transfection efficiency.Exogenous P16a gene can be expressed in transfected Hep-2 cell line detected by Western blot.P16a mediated by Nps can effectively inhibit the proliferation of Hep-2 cell,and induce Hep-2 cell apoptosis in vitro.[Conclusions] PBCA-NPs could be a good vector and provided a new way for gene therapy of laryngocarcinoma.厦门市卫生局科研立项专项资金(序号wsk09)资

Construction of eukaryotic vector pcDNA3.1-flag-pygo2 and expression in C6 cell

目的构建pcDNA3.1-flag-pygo2真核表达载体并在C6细胞中进行表达。方法从小鼠脑胶质细胞中提取总RNA,RT-PCR法反转录合成c DNA,设计引物,调取目的片段,与pcDNA3.1-flag载体连接后转化大肠杆菌DH5α,LB平板筛选菌落,提取质粒。重组质粒pcDNA 3.1-flag-pygo2经过酶切鉴定及测序后,阳离子脂质体法转染C6细胞并经免疫细胞化学染色及蛋白印迹检测重组体的表达。结果重构质粒pcDNA 3.1-flag-pygo2经限制性核酸内切酶EcoRⅠ,HindⅢ酶切分析及测序检查,表明真核表达载体构建正确;瞬时转染C6细胞后,免疫细胞荧光染色及蛋白印迹检测表明转染细胞能够表达外源Pygo2基因。结论成功构建了pcDNA3.1-flag-pygo2真核表达载体并能够在真核细胞中进行表达,这为今后研究pygo2基因在胶质瘤中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector pcDNA3.1-flag-pygo2 and express the combined protein in C6 cell line.Methods To extract total RNA from primary glial cell of mouse and to synthesize c DNA by RT-PCR,then design primer and clone whole segment of pygo2 gene.After the targeted gene was inserted into vector pcDNA3.1-flag,the recombined plamid was transformed into E coli DH5α for LB agar plate screening and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing.,the constructed eukaryotic expression plamid was transfected to C6 cell line by lipofectamin method,finally the protein expression was detected by immunocytochemical staining as well as western blot.Results The new constructed vector was confirmed by restricted enzyme Eco R I,HindIII digestion assay and correct Pygo2 was verified by sequenceing.finally,pcDNA3.1-flag-hpygo2 can express exogenous pygo2 gene in glioma C6 cell line after transient transfection by the determination of immunocytofluorescent staining and western blot.Conclusion The new plamid pcDNA3.1-flag-pygo2 was constructed successfully,and can express fused protein in eukaryotic cell,which establish the foundation for future research on pygo2 gene function in human glioma

Establishment and Phenotype Analysis of a Pygo2 Transgenic Mouse Model

主要通过建立PygO2转基因小鼠的模型对其表型进行初步分析.首先构建k14-2xflAg-PygO的转基因构件,经酶切、纯化后构建PygO2转基因小鼠.出生后的仔鼠用PCr和WESTErn方法检测基因型,并通过进一步的免疫组化验证PygO2基因的表达.PCr检测获得7只转基因阳性鼠,6只WESTErn检测为阳性.对转基因小鼠子代的胚胎和成体进行免疫组化证明,PygO2基因在皮肤和乳腺组织中有过量表达.转基因小鼠的皮肤、乳腺以及鼠尾椎骨等组织出现了异常的表型.乳腺中有肿瘤组织的形成,且PygO2在肿瘤中有大量表达.该模型的成功建立为进一步研究PygO2的功能奠定了基础.Pygopus(Pygo) is a new component of Wnt signaling with conserved PHD domain,associates with Armadillo/β-catenin through the Legless/BCL9 adaptor.The objective of this study was to construct the specific expression of vector of Pygo2 and to establish a Pygo2 transgenic mouse model and check its phenotype.K14 promoter-2×flag-Pygo2 construct was constructed,linearized,purified and then injected to produce transgenic mouse.The genotypes of transgenic founders were identified by PCR and Western.71 offspring were born and 7 were proved to be positive founder.Protein expression of Pygo2 in tissues of transgenic mice were detected by immunohistochemistry.Pygo2 transgenic mice have been established successfully,6 in 7 viable offsprings were positive transgenic mice,the rate of positive mouse was 8.45%(6/71).In the skin and mammary gland of transgenic mice Pygo2 was over expressed.And there is abnormal phenotype in the skin,mammary gland and tail tissues in transgenic mice.Skin shows festering and abnormal phenotype of hair follicle,mammary gland shows early maturing and some transgenic mice occurs mammary gland tumor,including adenocarcinoma and adenoacanthoma.Pygo 2 protein is highly expressed in these tumor tissues.Taken together,these data indicate that the models will be contributed to the research of Pygo2 gene function.国家自然科学基金(30671173);福建省自然科学基金重点项目(2008J0007