Notch信号通路促进小鼠胸骨软骨细胞终末期成熟分化的实验研究

目的研究Notch信号通路对小鼠胸骨软骨细胞终末期成熟分化的促进作用及其成软骨分化的抑制作用。方法用鼠龄4 d昆明新生小鼠,分离和培养新生小鼠胸骨软骨细胞;NICD1慢病毒转染,提高细胞内Notch信号通路活性。实时聚合酶链反应(RT-PCR)方法检测细胞内终末期成熟分化因子和成软骨因子基因的表达;用碱性磷酸酶(ALP)染色和Alcian蓝染色方法检测软骨细胞内ALP和蛋白多糖的分泌。将细胞分为2组:对照组和NICD1组,比较两组细胞内各因子基因和蛋白的表达不同。结果新生小鼠的胸骨软骨细胞具有自发性终末期成熟分化的特点;经NICD1慢病毒转染后,软骨细胞内NICD1的蛋白表达显著提高。与对照组相比,NICD1组细胞内终末期成熟分化因子的基因表达和ALP分泌明显增加(P0.05);而成软骨分化因子的基因表达和蛋白多糖分泌明显降低(P0.05)。结论 Notch信号通路可促进小鼠胸骨软骨细胞终末期成熟分化,并抑制其向成软骨分化。     

人Hes1-shRNA,Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的 构建人Hes1-shRNA和Hes5-shRNA慢病毒表达载体,为Notch-Hes信号通路的相关研究奠定基础.方法 根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR /U6入门载体.通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列.将含有效干扰序列的入门载体与pLenti6/BLOCK-iT-DEST载体进行LR重组构建Hes1-shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒.用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达.结果 分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对Hes1,Hes5蛋白的表达有显著抑制作用.结论 成功构建了Hes1-shRNA和Hes5-shRNA慢病毒表达载体.     

mTOR shRNA慢病毒载体的构建及包装

目的:构建mTORshRNA慢病毒表达载体,为探讨RNA干扰技术抑制T细胞mTOR基因的相关研究奠定基础.方法:设计合成针对小鼠mTOR基因的shRNA片段,与酶切后的pLKD.UBC.GFP.U6载体片段进行连接并转化DH5α,提取阳性克隆质粒,测序,转染293T细胞.通过GFP表达水平测定病毒滴度.结果:DNA测序结果及PCR鉴定证实成功构建小鼠mTOR-shRNA重组慢病毒载体,对其进行包装后测定慢病毒滴度为1.38E+ 08 TU/ml.结论:成功构建并包装小鼠mTOR-shRNA重组慢病毒表达载体.     

小鼠CXCR4基因的克隆和慢病毒介导的表达

目的:克隆小鼠CXCR4基因并建立其慢病毒表达系统.方法:设计合成PCR引物, 从小鼠骨髓有核细胞来源的cDNA中扩增并克隆小鼠CXCR4基因的编码区.构建CXCR4-IRES-GFP表达单元后通过转染细胞, 观察GFP的表达证实其表达效能.然后建立慢病毒表达载体, 包装慢病毒后感染培养的细胞, 用流式细胞术分析其在被感染的细胞中的表达.结果:成功扩增了小鼠CXCR4基因的编码区.克隆入质粒载体后经DNA序列测定证实了其序列.通过转染细胞和流式细胞术以及荧光显微镜观察证实了CXCR4-IRES-GFP的表达效能.成功构建了CXCR4慢病毒表达载体, 感染细胞后经流式细胞术分析, 证实被感染的细胞可以表达小鼠CXCR4.结论:成功扩增、克隆了小鼠CXCR4基因, 并成功建立起其慢病毒表达系统, 为后续的基础和应用研究奠定了基础.     

小鼠Foxp3基因慢病毒载体构建及其在DC2.4细胞的表达

目的:构建小鼠Foxp3基因的慢病毒载体,体外感染树突状细胞DC2.4,制备Foxp3+DC细胞,为进一步研究其免疫调节作用奠定基础。方法:将小鼠Foxp3基因克隆到慢病毒pGC-FU载体,通过PCR和测序鉴定获得连接正确的克隆。将鉴定后的重组表达质粒pGC-FU-Foxp3与pHelper1.0质粒和pHelper2.0质粒共转染293T细胞,制备携带Foxp3基因的慢病毒Lentivirus-Foxp3。Lentivirus-Foxp3感染体外培养的DC细胞,流式细胞术(FCM)检测感染后的DC细胞Foxp3表达。结果:构建的慢病毒载体pGC-FU-Foxp3经PCR鉴定和测序正确,包装慢病毒Lentivirus-Foxp3滴度为2×108TU/mL。慢病毒Lentivirus-Foxp3感染DC2.4细胞后Foxp3表达明显增加。结论:成功构建了小鼠Foxp3基因的慢病毒表达载体,慢病毒Lentivirus-Foxp3能有效感染DC细胞。     

Notch信号通路与Slug在调控宫颈鳞状细胞癌上皮-间充质转变中的意义

目的探讨Notch信号通路与Slug在宫颈鳞状细胞癌上皮-间充质转变(EMT)中的作用机制。方法收集临床慢性宫颈炎及宫颈鳞状细胞癌标本,分别用免疫组化及Western blot检测Notch1、NICD1、Slug、E-cadherin、α-SMA蛋白表达情况,Real-Time PCR检测Notch1、SlugmRNA表达情况。结果与慢性宫颈炎比较,Notch1、NICD1、Slug和α-SMA在宫颈鳞状细胞癌中表达增加(P<0.01),E-cadherin蛋白在癌组织中的表达降(P<0.01)。结论 Notch信号通路和Slug可能通过调控EMT在宫颈鳞状细胞癌的侵袭转移过程中发挥作用     

下调Notch1信号抑制人神经母细胞瘤SH-SY5Y的增殖能力

 目的 探讨Notch1基因与人神经母细胞瘤SH-SY5Y细胞增殖能力的关系。方法 应用?-分泌酶抑制剂DAPT或表达Notch1-shRNA慢病毒载体阻断SH-SY5Y细胞Notch1信号的活化,RT-PCR和Western blot法行Notch1及Hes1表达水平的检测,MTT法和裸鼠体内种植检测瘤细胞体内外增殖能力的变化。结果 DAPT处理和表达Notch1-shRNA慢病毒载体转染均能减少细胞内NICD和Hes1表达,其细胞体外增殖能力受到明显抑制(P<0.01),Notch1RNAi细胞体内种植瘤重为0.20±0.13g,明显轻于对照组的0.89±0.58g（P<0.01)。结论 Notch1信号的活化与人神经母细胞瘤SH-SY5Y细胞的增殖能力密切相关,有望成为神经母细胞瘤基因治疗的潜在靶点。     

Notch1对神经胶质瘤U251细胞干性及化疗药物敏感性的调节

目的:探讨Notch1对人胶质瘤U251细胞干性和药物敏感性的影响。方法:用高表达Notch1胞内段(Notch1 intracellular domain,NICD1)和Notch1-shRNA慢病毒表达载体感染体外培养的人胶质瘤U251细胞,Western blot和免疫荧光染色法鉴定高表达NICD和Notch1沉默细胞。通过流式细胞术检测分析CD133+细胞的比例、免疫荧光染色法检测nestin和GFAP的表达情况、检测肿瘤细胞球的形成率和SCID小鼠体内种植致瘤情况,分析Notch1对细胞干性的调节。并采用MTT法检测各组细胞对化疗药物替尼泊苷(VM-26)和卡莫司汀(BCNU)的敏感性。结果:NICD表达增加的瘤细胞干性表型增强,如CD133+细胞的比例增加、nestin表达增强而GFAP表达减弱、肿瘤细胞球的形成率和SCID小鼠种植致瘤率增加,并伴有对VM-26和BCNU的敏感性降低。而Notch1基因表达下调的瘤细胞干性表型受到明显抑制,而对VM-26和BCNU的敏感性增高。结论:Notch1高表达可增加人胶质瘤U251细胞的干性,减弱U251细胞对化疗药物的敏感性。     

大鼠微小RNA miR-16的克隆及其慢病毒表达载体的构建

目的 克隆微小RNA rno-miR-16,构建其慢病毒表达载体pLV-miR-16并包装成慢病毒颗粒,为进一步研究miR-16的功能奠定了实验基础.方法 从大鼠细胞基因组中用PCR 的方法扩增miR-16 的前体,构建了miR-16的重组表达载体pLV-miR-16,脂质体法将重组慢病毒载体和包装质粒混合物(pPACK-GAG、pPACK-REV和pVSV)共转染包装细胞293TN细胞,包装产生慢病毒,以293TN细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达水平测定病毒滴度.结果 经PCR扩增检测阳性菌落和测序证实,成功构建携带大鼠miR-16基因重组慢病毒载体.倒置荧光显微镜下观察可见包装细胞293TN呈绿色荧光,并测得108＞ifu/ml.结论 成功构建大鼠慢病毒载体pLV-miR-16,为深入研究rno-miR-16的生物学功能奠定了基础.     

人C-Met胞外区慢病毒穿梭载体构建及在293T细胞中的表达

本研究构建含人肝细胞生长因子受体(C-Met)胞外区基因的慢病毒表达载体,并将其转染293T细胞,真核表达并纯化获得人C-Met胞外段蛋白。采用RT-PCR扩增人C-Met胞外区(ED)基因,构建慢病毒表达载体p RRL-CMV-ED,磷酸钙法转染293T细胞,RT-PCR及Western blot法检测ED基因在293T细胞中mRNA的转录和蛋白表达。PCR扩增及测序结果表明成功构建了p RRL-CMV-ED慢病毒表达载体,PCR扩增的目的基因大小为2 700bp左右;转染p RRL-CMV-ED的293T细胞上清经镍柱亲和纯化,SDS-PAGE分析纯化产物在105kD处有明显蛋白条带;Western及ELISA结果显示,纯化蛋白为C-Met胞外区蛋白且能与肝细胞生长因子(HGF)特异性结合。本研究结果可能为制备C-Met单克隆抗体以及筛选阻断HGF/C-Met信号通路的中和抗体奠定基础。     

稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体GluA1亚基单克隆细胞株的构建

目的 构建稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体GluA1亚基的细胞株。 方法 PCR扩增GluA1目的基因,并将其装载至慢病毒表达载体PLV.0-绿色荧光蛋白（GFP）,采用4质粒系统转染HEK293T细胞包装慢病毒。嘌呤霉素抗性筛选细胞,挑选单克隆细胞株用Real-time PCR、Western blotting、免疫荧光和全细胞膜片钳鉴定GluA1的表达与功能。 结果 菌落PCR鉴定扩增产物与GluA1分子量（2724 bp）一致,经测序插入慢病毒载体序列与GluA1序列一致;慢病毒感染并抗性筛选的HEK293T细胞经Real-time PCR、Western blotting和免疫荧光实验证明,GluA1在过表达GluA1组的HEK293T细胞正确表达,空载体组不表达。单克隆挑选的稳定细胞株免疫荧光染色显示,各细胞膜表面GluA1蛋白表达相对一致。单克隆细胞在电压钳模式下,细胞外液加10 mmol/L谷氨酸诱导,空载体组检测不到电信号,而过表达GluA1组可记录5~40 pA电信号。 结论 成功构建了稳定表达AMPA受体GluA1亚基的HEK293T细胞株。     

HIV-1包膜蛋白ENV慢病毒载体的构建及表达

目的:构建表达HIV-1包膜蛋白ENV的慢病毒载体,感染人胚肾细胞HEK293T,观察env基因在HEK293T中的表达。方法:通过点突变获得HIV-1 env完整基因,将env基因亚克隆至慢病毒穿梭载体pLVX-IRES-ZsGreen1的EcoRⅠ、XhoⅠ位点,构建重组质粒pLV-env,采用脂质体转染法转染HEK293T,经RT-PCR、Western blot检测目的基因表达,同时利用激光共聚焦技术对env基因的表达进行了定位。结果:成功获得了HIV-1 env基因,构建了重组慢病毒质粒pLV-env,RT-PCR、Western blot检测均表明外源基因能够表达,并具有抗原性,同时env基因表达后可以分泌到细胞膜表面膜上。结论:成功构建了含有HIV-1包膜蛋白env基因的重组慢病毒质粒,并验证了其表达,为下一步慢病毒的包装以及细胞模型和动物模型的构建奠定了基础。     

Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno‐associated virus (AAV8) (Y733F)‐chicken β‐actin (CBA)‐MIF or AAV8 (Y733F)‐CBA‐enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP‐injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta‐like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split‐1 (Hes‐1) increased, but the ERG a‐ and b‐wave amplitudes decreased in AAV8.CBA.MIF‐injected EAU mice. The Notch inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) reduced the expression of NICD, Hes‐1 and proinflammatory cytokines. Further, a MIF antagonist ISO‐1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over‐expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over‐expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF–Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.     

Noncyclic Notch activity in the presomitic mesoderm demonstrates uncoupling of somite compartmentalization and boundary formation

To test the significance of cyclic Notch activity for somite formation in mice, we analyzed embryos expressing activated Notch (NICD) throughout the presomitic mesoderm (PSM). Embryos expressing NICD formed up to 18 somites. Expression in the PSM of Hes7, Lfng, and Spry2 was no longer cyclic, whereas Axin2 was expressed dynamically. NICD expression led to caudalization of somites, and loss of Notch activity to their rostralization. Thus, segmentation and anterior-posterior somite patterning can be uncoupled, differential Notch signaling is not required to form segment borders, and Notch is unlikely to be the pacemaker of the segmentation clock.     

过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响

BACKGROUND: Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cells. OBJECTIVE: To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cells. METHODS: The Notch1 intracellular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned into BamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cells to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cells by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION: N1-ICD coding sequence was successfully generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cells, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Willebrand factor, an endothelial cell differentiation gene, and mildly increased the expression of smooth muscle 22α, a smooth muscle cell differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cells, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cell biology.      

利用同源重组构建大鼠甘油二酯激酶γ(DGKγ)慢病毒过表达载体

文题释义： 甘油二酯激酶γ(DGKγ)：属于第Ⅰ类DGK亚型,主要分布于神经系统,参与浦肯野细胞树突的发育、突触可塑性调节、神经上皮细胞的增殖和迁移、神经元分化等功能活动。 同源重组技术：一种利用同源重组的原理,进行无缝克隆的技术。该技术无需考虑插入片段的酶切位点,可将插入片段PCR产物定向克隆至任意载体的任意位点。同源重组技术还能实现多个DNA片段的一步组装,操作简单,重组效率高。 背景：慢病毒载体作为外源性转基因载体已被广泛应用,但是大鼠甘油二酯激酶γ(diacylglycerol kinase γ,DGKγ)基因慢病毒载体未见报道。 目的：用同源重组的方法构建大鼠DGKγ慢病毒过表达载体。 方法：提取成年SD大鼠脑组织总RNA,以反转录得到的cDNA作为模板,通过PCR反应分段扩增大鼠DGKγ基因CDS区5'端1 029 bp和3'端1 362 bp,用同源重组技术将这2个片段与线性化载体进行定向连接,构建CMV-rat DGKγ-GFP慢病毒载体并进行PCR扩增及测序鉴定。经293T细胞包装后产生慢病毒,收集慢病毒感染293T细胞,荧光显微镜下观察细胞中GFP的表达并应用实时荧光定量PCR和Western blotting法检测细胞中DGKγ mRNA和蛋白的表达。 结果与结论：CMV-rat DGKγ-GFP慢病毒载体经PCR扩增和测序鉴定构建成功;经CMV-rat DGKγ-GFP慢病毒感染后的293T细胞,荧光显微镜下呈GFP阳性,实时荧光定量PCR显示DGKγ mRNA的表达较空载体组显著升高(P < 0.01),Western blotting显示DGKγ蛋白表达较空载体组极显著升高(P < 0.001)。提示：成功构建了大鼠DGKγ慢病毒过表达载体,DGKγ在293T细胞有效高表达。 ORCID: 0000-0001-9352-3043(李蕾) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

目的构建含有小鼠Fas Ligand(FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础。方法从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达。结果通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Western blot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293。结论重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础。     

大鼠ferroportin 1过表达慢病毒载体构建及其转染PC12细胞

目的:构建含大鼠ferroportin 1(FP1,膜铁转运蛋白1)基因和EGFP(绿色荧光蛋白)基因的过表达慢病毒载体并转染PC12细胞。方法:化学合成含有目的基因FP1的质粒,将酶切获得的目的基因连接入线性化慢病毒载体,构建重组质粒PGC-FU-FP1并进行测序鉴定;鉴定正确的阳性克隆采用Lipofectamine2000转染293T细胞,通过荧光显微镜和Western Blot检测FP1表达情况;在脂质体介导下将PGC-FU-FP1、pHelper1.0和pHelper2.0三质粒系统共转染入293T细胞,包装产生慢病毒,并通过实时荧光定量PCR法检测病毒滴度。以重组慢病毒载体质粒PGC-FU-FP1转染PC12细胞,荧光显微镜下观察EGFP的表达,实时荧光定量PCR和Western Blot法分别检测FP1mRNA和蛋白表达情况。结果:FP1基因序列经测序后与GeneBank报道的序列完全一致;重组质粒PGC-FU-FP1中携带有正确的FP1基因并能在293T细胞中表达;病毒滴度为2×108TU/ml。荧光显微镜、实时荧光定量PCR和Western Blot法证实目的基因FP1能被重组慢病毒高效导入PC12细胞并得到过表达。结论:成功构建携带FP1基因的慢病毒载体,并获得FP1基因修饰的PC12基因工程细胞。为进一步研究FP1在帕金森病中的作用及基因治疗奠定基础。