慢病毒介导N2ICD过表达的人胃癌MKN45细胞稳定株的构建及鉴定

[目的]构建慢病毒介导Notch2受体胞内段((Notch2 intracellular domain,N2ICD)过表达的人胃癌MKN45细胞稳定株并进行鉴定。[方法]采用PCR法扩增N2ICD基因并克隆至慢病毒载体PTYE-EF1α-IRES-EGFP(简写为pLV)中,通过酶切及测序鉴定后,三质粒共转染HEK293T细胞包装病毒,收集且浓缩病毒毒液用于感染人胃癌MKN45细胞并筛选稳定细胞株,用Western Blot验证。[结果]成功构建慢病毒重组质粒N2ICD/pLV;包装病毒后,荧光显微镜下观察到大部分HEK293T细胞发出绿色荧光;收集病毒毒液感染MKN45细胞,显微镜下观察有部分细胞表达绿色荧光蛋白;流式分选技术筛选得到稳定细胞株,Western Blot鉴定结果显示:相比于空白对照组,稳定株的N2ICD过表达明显。[结论]成功构建慢病毒表达质粒N2ICD/pLV,并建立稳定过表达N2ICD的胃癌细胞株N2ICD/pLV-MKN45。     

PSF不同功能片段融合蛋白的构建及其在细胞内的分布

目的将人类PSF基因的不同功能片段定向连入pEGFP—C2质粒,使PSF蛋白的各功能片段与绿色荧光蛋白在HeLa细胞内融合表达,观察其在HeLa细胞中的表达及定位。方法以重组质粒pEGFP—C2-PSF为模板,PCR法扩增出目的基因,将扩增片段双酶切后连接到质粒pEGFP—C2上,构建重组质粒pEGFP—C2-PSF（I—V）。将构建成功的pEGFP—C2-PSF（I—V）质粒脂质体法转染HeLa细胞,Western印迹检测融合蛋白的表达,并在荧光显微镜下观察融合蛋白的定位与分布。结果成功构建质粒pEGFP—C2-PSF（I～V）,并在HeLa细胞中实现表达;Western印迹检测到融合蛋白GFP—PSF（I～V）;在激光共聚焦显微镜下观察到绿色的融合蛋白表达和定位。结论人类PSF基因的不同功能片段的重组质粒pEG—FP—C2-PSF（I～V）构建成功,可用于标记PSF蛋白的不同功能片段,为进一步研究PSF在信号转导中的作用机制以及其生物学功能奠定基础。     

人Notch2受体胞内区基因N2ICD的原核表达、纯化和鉴定

[目的]构建含Notch2胞内区基因N2ICD的原核重组质粒,并在E.coli中表达。[方法]根据Gen Bank上N2ICD基因序列设计引物,用PCR从真核重组质粒p CMV-Tag4/N2ICD中扩增目的基因,克隆至携带GST标签的pGEX-4T-1原核表达载体中并转化E.coli BL21。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,收集菌体经反复冻融、溶菌酶破菌、超声破碎后,通过SDS-PAGE电泳检测目的蛋白的表达形式,表达产物经谷胱甘肽琼脂糖树脂glutathione sepharose 4B纯化并经Western Blot鉴定。[结果]PCR获取了目的基因人N2ICD,并成功构建了重组表达质粒pGEX-4T-1/N2ICD,目的基因在E.coli中成功表达并以部分可溶性形式表达在上清中,纯化后经Western Blot鉴定表达产物分子量在110 k Da。[结论]重组N2ICD在E.coli中成功获得可溶性表达,为研究Notch2受体在细胞信号转导中的作用奠定了基础。     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

重组pEGFP—C2-p100-TSN点突变质粒构建及表达

目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段,将其连人pEGFP—C2质粒载体中,再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功;②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。     

Notch家族4个蛋白质对人胰腺癌细胞HPAC中YY1和EGFR表达的影响

Notch信号通路是肿瘤形成过程中一种重要的信号通路,其中心分子为Notch受体.Notch受体为细胞膜上的单次跨膜蛋白,介导细胞间信号转导,哺乳动物细胞内包括Notch1、Notch2、Notch3和Notch4的4个成员.Notch家族4个蛋白质在结构和功能上存在差异.前期研究显示,Notch1信号通路与转录因子YY1(YING-YANG 1)、表皮生长因子受体(EGFR)间存在调控作用.本研究在人胰腺癌细胞HPAC中,采用RNA干扰技术,分别降低细胞中Notch家族4个蛋白质的表达,检测YY1和EGFR在m RNA和蛋白质水平上的表达;并构建相应的激活形式的Notch受体——Notch胞内结构域(Notch intracellular domain,NICD)真核表达质粒,在HPAC细胞中分别过表达4种NICD,检测其对YY1和EGFR表达水平的影响.结果显示,降低细胞中Notch1或Notch3的表达,均使HPAC细胞中EGFR m RNA和蛋白质水平升高(P0.05),而降低Notch2和Notch4后,EGFR m RNA和蛋白质水平没有改变(P0.05).分别降低4个Notch的表达,对YY1的蛋白质和m RNA表达水平均没有改变(P0.05).在HPAC细胞中过表达N1ICD和N3ICD后,YY1和EGFR的蛋白质水平降低(P0.05),而过表达N2ICD和N4ICD后,YY1和EGFR蛋白质水平没有改变(P0.05).分别过表达4种NICD均没有改变YY1和EGFR的m RNA表达水平(P0.05).初步得出结论是,在HPAC细胞中,Notch信号通路经Notch1和Notch3影响EGFR的表达.Notch1和Notch3对EGFR的表达可能具有负调控作用.在Notch1和Notch3过度激活时,这种调控作用通过YY1介导.本文可为深入研究Notch信号通路对胰腺癌发生发展的作用机制提供有意义的参考.     

绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究     

荷包猪SLA-2-HB01真核表达载体的构建及在PK15细胞中的表达

为构建荷包猪SLA-2-HB01真核细胞表达载体,观察其在PK15细胞中的表达情况。首先参考SLA-2-HB01基因编码区序列设计了引物对,以SLA-2-HB01-pMD 18-T为模板PCR扩增获得SLA-2-HB01编码区基因片段,经TA克隆及菌落PCR筛选得到阳性克隆菌。测序正确后大量双酶切回收目的基因片段并与真核表达载体pEGFP N3相连接获得重组质粒SLA-2-HB01/pEGFP N3,重组质粒转化至大肠杆菌经大量克隆后,抽提阳性重组质粒并通过脂质体介导转染至PK15细胞,通过荧光显微镜观察转染后PK15细胞的荧光强度,进一步通过Western Blotting检测PK15细胞中SLA-2-HB01蛋白的表达情况。结果显示PCR成功扩增得到SLA-2-HB01,大小为1 104 bp,并成功构建重组质粒SLA-2-HB01/pEGFP N3,酶切鉴定证实插入片段大小为1 092 bp。SLA-2-HB01/pEGFP N3重组质粒转染PK15细胞后具有绿色荧光,Western Blotting显示SLA-2-HB01-EGFP融合蛋白分子量大小为70 kD,与理论值相符。成功构建了SLA-2-HB01/pEGFP N3重组质粒,并初步确认SLA-2-HB01-EGFP融合蛋白成功在PK15细胞中表达,为下一步进行SLA-2-HB01递呈多肽表位的研究奠定基础。     

Notch 家族四个蛋白质在人胰腺癌细胞 HPAC 中对 YY1 和 EGFR水平的影响

Notch信号通路是肿瘤形成过程中一种重要的信号通路,其中心分子为Notch受体. Notch受体为细胞膜上的单次跨膜蛋白,介导细胞间信号转导,哺乳动物细胞内包括Notch1、Notch2、Notch3和Notch4的4个成员. Notch家族4个蛋白质在结构和功能上存在差异.前期研究显示,Notch1信号通路与转录因子YY1（YING-YANG 1）、表皮生长因子受体（EGFR）间存在调控作用. 本研究在人胰腺癌细胞HPAC中,采用RNA干扰技术,分别降低细胞中Notch家族4个蛋白质的表达,检测YY1和EGFR在mRNA和蛋白质水平上的表达;并构建相应的激活形式的Notch受体--Notch胞内结构域（Notch intracellular domain,NICD）真核表达质粒,在HPAC细胞中分别过表达4种NICD,检测其对YY1和EGFR表达水平的影响. 结果显示,降低细胞中Notch1或Notch3的表达,均使HPAC细胞中EGFR mRNA和蛋白质水平升高（P<0.05）,而降低Notch2和Notch4后,EGFR mRNA和蛋白质水平没有改变（P>0.05）.分别降低4个Notch的表达,对YY1的蛋白质和mRNA表达水平均没有改变（P>0.05）. 在HPAC细胞中过表达N1ICD和N3ICD后,YY1和EGFR的蛋白质水平降低（P<0.05）,而过表达N2ICD和N4ICD后,YY1和EGFR蛋白质水平没有改变（P>0.05）.分别过表达4种NICD均没有改变YY1和EGFR的mRNA表达水平（P>0.05）.初步得出结论是,在HPAC细胞中,Notch信号通路经Notch1和Notch3影响EGFR的表达. Notch1和Notch3对EGFR的表达可能具有负调控作用. 在Notch1和Notch3过度激活时,这种调控作用通过YY1介导. 本文可为深入研究Notch信号通路对胰腺癌发生发展的作用机制提供有意义的参考.     

稳定表达HCV 1a NS5A和NS5A-△ISDR细胞株的构建

以pBRTM／HCV-3011模板,通过PCR法扩增ns5α、LISDR(左端序列)和RISDR(右端序列),分别经XhoⅠ／EcoRⅠ、XhoⅠ／HindⅢ和EcoRⅠ／HindⅢ双酶切,连入穿梭质粒pEGFP—N3中,构建重组质粒pEGFP—N3-ns5a和pEGFP—N3-ns5a—△ISDR。对重组质粒进行酶切分析和序列测定。将这2种重组质粒通过电穿孔法转染HeLa细胞,然后在G418选择压力下进行有限稀释法筛选,用RT-PCR和荧光显微镜鉴定。经酶切鉴定和基因测序证实,重组穿梭质粒已插入目的片段ns5a、LISDR和RISDR。RT-PCR和荧光显微镜检测到目的基因的表达。以上结果说明成功构建了真核表达载体pEGFP—N3-ns5a和pEGFP—N3-ns5α—△ISDR,目的基因在HeLa细胞中得到表达,为研究HCV NS5A中是否存在抗干扰素治疗的功能蛋白提供了实验材料。     

N3ICD with the transmembrane domain can effectively inhibit EMT by correcting the position of tight/adherens junctions

EMT allows a polarized epithelium to lose epithelial integrity and acquire mesenchymal characteristics. Previously, we found that overexpression of the intracellular domain of Notch3 (N3ICD) can inhibit EMT in breast cancer cells. In this study, we aimed to elucidate the influence of N3ICD or N3ICD combined with the transmembrane domain (TD+N3ICD) on the expression and distribution of TJs/AJs and polar molecules. We found that although N3ICD can upregulate the expression levels of the above-mentioned molecules, TD+N3ICD can inhibit EMT more effectively than N3ICD alone. TD+N3ICD overexpression upregulated the expression of endogenous full-length Notch3 and contributed to correcting the position of TJs/AJs molecules and better acinar structures formation. Co-immunoprecipitation results showed that the upregulated endogenous full-length Notch3 could physically interact with E-ca in MDA-MB-231/pCMV-(TD+N3ICD) cells. Collectively, our data indicate that overexpression of TD+N3ICD can effectively inhibit EMT, resulting in better positioning of TJs/AJs molecules and cell-cell adhesion in breast cancer cells.

Abbreviations: EMT: Epithelial-mesenchymal transition; TJs: Tight junctions; AJs: Adherens junctions; aPKC: Atypical protein kinase C; Crb: Crumbs; Lgl: Lethal (2) giant larvae; LLGL2: lethal giant larvae homolog 2; PAR: Partitioning defective; PATJ: Pals1-associated TJ protein     

斑马鱼notch3基因真核表达载体的构建及其表达分析

为了进一步研究notch3基因在斑马鱼中的功能,构建了斑马鱼notch3真核表达载体并在真核细胞中成功表达.其中斑马鱼notch3基因编码序列(coding sequence,CDS)从NCBI的在线数据库中获得,根据序列克隆其胞内段(notch intracellular domain,NICD),接着利用同源重组技...     

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.     

Calcium depletion dissociates and activates heterodimeric notch receptors

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.     

Lysosome-dependent degradation of Notch3

Notch signaling plays an essential role in diverse biological processes during development and in pathogenesis of diseases ranging from cancer to cerebrovascular disorders. Precise regulation of Notch signaling is essential for normal function and requires both timely activation and inactivation of the intracellular domain (ICD) of Notch receptors. In addition, inappropriate buildup of Notch3 ectodomain is a hallmark pathological feature of the stroke and dementia disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Thus, a clear understanding of mechanisms of Notch protein turnover is essential for understanding normal and pathological mechanisms of Notch function. Previous studies showed that the degradation of ICDs of Notch1 and Notch4 is controlled by the ubiquitin–proteasome system (UPS), though more recent work demonstrated that Notch1 ICD is also controlled by lysosomal degradation. The mechanism of degradation of Notch3 has not yet been identified. Here we report that the degradation of ICD of Notch3 (N3-ICD) is mediated by lysosomes. Lysosome inhibitors chloroquine and NH₄Cl led to the accumulation of transfected N3-ICD in 293 cells and endogenous N3-ICD in C2C12, H460, and HeLa cell lines; in addition, inhibition of lysosome function by chloroquine and NH₄Cl delayed the degradation of N3-ICD. In contrast, N3-ICD was not affected by proteasome inhibitors MG132 and lactacystin. Furthermore, we find that the Notch3 extracellular domain (N3-ECD) is also subjected to lysosome-dependent degradation. In sum, our experiments demonstrate a critical role for lysosomes in the degradation of Notch3, which distinguishes it from Notch1 and Notch4.     

Notch1 provides myocardial protection by improving mitochondrial quality control

Mitochondrial quality control is a new target for myocardial protection. Notch signaling plays an important role in heart development, maturation, and repair. However, the role of Notch in the myocardial mitochondrial quality control remains elusive. In this study, we isolated myocardial cells from rats and established myocardial ischemia reperfusion injury (IRI) model. We modulated Notch1 expression level in myocardial cells via infection with recombinant adenoviruses Ad-N1ICD and Ad-shN1ICD. We found that IR reduced myocardial cells viability, but Notch1 overexpression increased the viability of myocardial cells exposed to IRI. In addition, Notch1 overexpression improved ATP production, increased mitochondrial fusion and decreased mitochondrial fission, and inhibited mitophagy in myocardial cells exposed to IRI. However, N1ICD knockdown led to opposite effects. The myocardial protection role of Notch1 was related to the inhibition of Pink1 expression and Mfn2 and Parkin phosphorylation. In conclusion, Notch1 exerts myocardial protection and this is correlated with the maintenance of mitochondrial quality control and the inhibition of Pink1/Mfn2/Parkin signaling.     

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.     

Notch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines

In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 intracellular domain (ICD) to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels. In contrast, pharmacologically blocking Notch signaling reversed this situation and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression.