人Semaphorin 4D慢病毒载体的构建及鉴定

目的:构建并制备能够有效表达Semaphorin 4D的重组慢病毒。方法:从人急性T细胞白血病Jurkat细胞DNA 扩增人Semaphorin 4D基因,克隆至pWPI GW慢病毒载体上,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装出重组慢病毒,将纯化后的重组病毒直接感染293T和HUVEC细胞,通过免疫印迹、免疫荧光染色和血管内皮细胞迁移分析等方法检测Semaphorin 4D的表达和诱导血管内皮细胞迁移的作用。结果: 重组慢病毒介导Semaphorin 4D在293T和HUVEC内获得表达,能介导血管内皮细胞迁移。结论:成功构建了表达Semaphorin 4D的重组慢病毒载体。     

KLF4siRNA慢病毒载体构建和鉴定

目的:构建上皮锌指蛋白4(Krüppel-like factor 4,KLF4)siRNA慢病毒载体并进行初步鉴定,为研究KLF4在宫颈细胞癌中的分子机制奠定基础。方法:利用公用网站中提供的RNA干扰序列设计原则,设计4个RNA干扰靶点序列,合成含干扰序列的单链DNA oligo,然后退火配对产生双链,再通过其两端所含酶切位点直接连入酶切后的RNAi慢病毒载体上;将连接产物转入制备好的细菌感受态细胞,PCR鉴定阳性重组子后,送测序验证,测序结果经比对确认正确的克隆,制备编码慢病毒颗粒的重组病毒质粒及其两种辅助包装原件载体质粒,共转染293T细胞,收集富含慢病毒颗粒上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。收集上清液感染宫颈癌He La细胞,通过q RT-PCR及Western Blot鉴定KLF4 siRNA慢病毒干扰效果。结果:成功构建KLF4 siRNA慢病毒载体。KLF4 siRNA慢病毒感染He La细胞后,q RT-PCR及Western Blot测定结果显示,KLF4表达明显降低。结论:KLF4 siRNA慢病毒载体构建及包装成功,可有效抑制KLF4表达,为研究KLF4生物学功能奠定基础。     

人肾脏相关基因Nox4过表达慢病毒载体的构建与定位检测

目的:克隆Nox4基因入pLenti6.3慢病毒表达载体,为探索Nox4基因在ROS产生中的作用提供实验基础。方法:根据NCBI人Nox4 mRNA序列设计引物,再利用酶切连接反应将Nox4插入到入门载体pENTR3C中,成功构建pENTR3C-Nox4后,通过LR反应,将Nox4和EGFP tag插入到慢病毒表达载体pLenti6.3中,经酶切和测序验证正确后,将重组表达质粒转染入人Hela细胞,通过Western-Blot验证Nox4的表达情况,免疫荧光验证Nox4在细胞内的定位情况。结果:入门载体及表达质粒测序比对完全正确,转染Hela细胞后可见明显的表达条带,并且主要定位于细胞器内质网中。结论:成功构建了带有EGFP tag的Nox4基因慢病毒重组表达载体,转染Hela细胞后,其能正确表达并定位于内质网中,为研究Nox4在调节ROS产生中的作用奠定了基础。     

hNoc4L基因重组慢病毒表达载体的构建与鉴定

核糖体前体的形成和核运输需要多种核仁复合物的参与,hNoc4L是酿酒酵母S.cerevisiae的核仁复合物相关蛋白4的同源蛋白,并含有保守的Noc结构域,但其功能未知。为了构建hNoc4L基因过表达的慢病毒载体,本实验通过将EF1α启动子替换原shRNA慢病毒载体pll3.7的U6启动子,成功构建了慢病毒表达载体pll3.7-EF1,并进一步得到了hNoc4L基因过表达的慢病毒载体。利用慢病毒包装系统对不同物种的细胞进行感染,以此检测该重组慢病毒载体的包装效率,并通过构建的hNoc4L过表达的RAW264.7稳定细胞系检测了该载体的免疫原性和稳定转染能力。结果表明成功构建了高效、长期稳定表达和免疫原性低的hNoc4L特异性表达的慢病毒载体,为进一步研究hNoc4L蛋白在哺乳动物核糖体生物发生中的调控作用奠定了基础。     

miR-455慢病毒表达载体的构建与鉴定

目的：构建人源microRNA-455（miR-455）慢病毒载体,并鉴定成熟has-miR-455在细胞内的表达水平。方法：提取siHa细胞中的人基因组DNA,设计并合成人miR-455的上下游引物,PCR扩增目的基因,将其中表达miR-455的结构经酶切后插入慢病毒转移质粒pWPT-GFP,构建成pWPT-GFP-pri-miR-455,在293T细胞中与pMD2G、pSPAX2包装产生慢病毒,并用含慢病毒的上清感染SiHa细胞。结果：测序结果证明插入质粒载体中的miR-455前体序列完全正确,慢病毒载体构建成功并获得相应的慢病毒;重组慢病毒质粒pWPT-GFP-pri-miR-455感染SiHa细胞后上调miR-455的表达近40倍。结论：构建了miR-455的慢病毒载体,并能在293T细胞中表达,产生的慢病毒能成功感染SiHa细胞。为进一步研究miR-455的功能,以及利用慢病毒进行基因治疗奠定了基础。     

Netrin-1慢病毒表达载体的构建及鉴定

目的:构建携带有Netrin-1基因的逆转录病毒载体,为研究Netrin-1在神经发育中的作用奠定基础。方法:PCR扩增Netrin-1基因片段后,将其克隆入慢病毒表达载体pLXSN;通过PCR、酶切、测序鉴定重组质粒。重组质粒转染PA317包装细胞后获得包装的病毒颗粒。病毒颗粒感染人脑胶质瘤细胞SW038-C2,经Western blot证明重组病毒在真核细胞内表达Netrin-1的情况。结果:经PCR扩增、酶切和测序验证,重组质粒构建正确,命名为pLX-NT。Western blot证明在感染细胞泳道有一特异性条带。结论:成功构建了能表达Netrin-1的慢病毒载体。     

PIG7慢病毒表达载体的构建

目的:构建一个含有PIG7基因ORF区的表达载体pPRRL-PIG7-IRES,为研究PIG7基因的功能打下基础。方法:采用RT-PCR方法从经苯丁酸钠处理过的Kasumi-1细胞获得PIG7基因SIMPLE转录本的ORF片段,再用酶切-连接的方法将目的片段亚克隆入慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结果:PIG7基因ORF区成功亚克隆入了慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结论:成功完成了PIG7慢病毒表达载体的构建,为研究PIG7基因的功能打下了基础。     

小鼠RNA干扰基因RelA慢病毒载体的构建与鉴定

目的：构建小鼠RelA 基因的RNA 干扰慢病毒载体,转染小鼠成骨样细胞并鉴定。方法：针对小鼠RelA 基因序列,设计特异 性的shRNA 序列,应用基因重组技术插入慢病毒载体GV-248。得到的重组质粒转化感受态大肠杆菌DH5-alpha,筛选得到阳性克隆 并扩大培养。所得质粒进行测序分析确定载体构建成功。重组质粒载体及包装辅助质粒转染293T 细胞,得到目的病毒并测定相 应病毒滴度。慢病毒转染MC3T3-E1 细胞后,Real-time PCR 及Western blot 检测MC3T3-E1 细胞RelA 基因及成骨相关基因 ALP、OCN、RANKL的表达。结果：成功构建小鼠RelA 基因的RNA干扰慢病毒载体,感染MC3T3-E1 细胞后,RelA 基因的表达 明显受到抑制,同时RANKL基因表达水平明显下降,ALP、OCN基因表达水平明显上升。结论：成功构建了小鼠RelA 基因的 RNA 干扰慢病毒载体。当小鼠成骨细胞RelA基因表达被干扰,NF-资B 通路被抑制后,小鼠成骨细胞成骨相关基因ALP、OCN的 表达明显上升,成骨功能增强;同时RANKL 的表达明显下降,其介导的破骨细胞骨吸收功能减弱。     

小鼠Smad6基因RNAi慢病毒载体的构建与鉴定

构建小鼠Smad6基因RNA干扰（RNAi）慢病毒载体,有效沉默骨髓树突状细胞（BMDC）的Smad6基因表达,为构建骨髓致耐受DC用于哮喘等自身免疫疾病的研究。设计小鼠Smad6 shRNA序列,合成、退火,得到双链DNA,与经酶切后的Psih1-H1-copGFP shRNA Vector载体连接产生LV-shSmad6慢病毒载体,并测序鉴定。转染293TN细胞,包装产生慢病毒,测定滴度。感染小鼠骨髓树突细胞,检测Smad6基因的表达状况成功构建Smad6 shRNA的慢病毒载体LV-shSmad6。包装慢病毒,并显著抑制Smad6 mRNA水平及蛋白水平的表达。成功构建出小鼠Smad6基因shR-NA慢病毒载体,为后期研究Smad6基因在哮喘发病机制及新治疗方法提供了稳定的转染细胞载体。     

hsa-miR-20a低表达慢病毒载体的构建及其表达鉴定

目的:构建hsa-mi R-20a低表达慢病毒载体,检测其在HL-60中表达。方法:采用In-fusion重组交换克隆法设计并合成hsa-mi R-20a前体序列的扩增引物,扩增获得目的片段插入慢病毒GV159中,得到重组的LV-hsa-mi R-20a表达载体,通过与包装质粒共转染293T细胞,获得携带hsa-mi R-20a的重组慢病毒并测定病毒滴度。取对数生长期HL-60细胞根据病毒滴度及细胞MOI值感染慢病毒,感染后24 h、48 h、72 h、96 h镜下观察荧光表达情况,判断感染效率,q RT-PCR检测HL-60细胞hsa-mi R-20a的表达变化。结果:成功构建LV-hsa-mi R-20a低表达慢病毒载体,其病毒滴度为(8E+8)TU/m L。该病毒感染HL-60细胞的效率可高达到80%,并可有效降低HL-60细胞hsa-mi R-20a表达水平。结论:成功构建了hsa-mi R-20a低表达慢病毒载体,包被的慢病毒可以在HL-60细胞中实现低表达效果,为后续功能研究奠定了基础。     

pWPT-GFP慢病毒载体的改构及鉴定

目的:为实现慢病毒感染后可用抗生素筛选外源基因稳定整合的细胞,我们对慢病毒载体质粒(pWPT-GFP)进行了改构。方法:将三种抗生素抗性基因(puro、hygro、neo)连同其启动子分别克隆入慢病毒载体(pWPT-GFP)。酶切鉴定载体构建正确后,将这三种改构载体分别与辅助质粒psPAX2、pMD2.G共转染293T包装细胞,收获病毒上清并感染靶细胞,使用相应抗生素筛选细胞验证抗性基因的插入。结果:成功构建了重组慢病毒载体pWPT-GFP-puro、pWPT-GFP-hygro、pWPT-GFP-neo,通过调整病毒上清的用量可以达到靶细胞100%的感染效率;用相应抗生素筛选细胞证明了抗性基因的表达。结论:我们成功改构了慢病毒载体(pWPT-GFP),从而使病毒感染后用抗生素筛选稳定整合的细胞成为可能。     

Sema4C在神经干细胞中的表达

目的：探讨Sema4C及其相关蛋白在神经干细胞中的表达。方法：利用RT-PCR、WestemI,blot、免疫荧光等方法对神经干细胞中内源性Sema4C及其相关蛋白的表达进行检测。结果：Sema4C及其相关蛋白在神经干细胞中表达,并且Sema4C在神经干细胞终末分化细胞神经元中表达,但在星形胶质中不表达。结论：Sema4C及其相关蛋白在神经干细胞中表达,提示Sema4C及其相关蛋白可能在神经干细胞中起作用。     

梅花鹿S100A4 基因RNAi 重组慢病毒载体的构建

本研究针对东北梅花鹿S100A4 基因筛选出若干条RNAi 靶位点,并利用NCBI 中的BLAST 工具去除脱靶情况,最终得到2 条高分靶位点。然后在T4 连接酶的作用下,将其与载体质粒pLVTHM 的酶切产物进行连接,并通过PCR 鉴定及测序筛选出阳性质粒。pLVTHM 阳性重组质粒与pMD2. G 及pCMV-dr8.91 共转染到293 T 细胞中。在倒置荧光显微镜下观察转染效果并进行收获病毒质粒。结果显示在转染24 h 后,在293 T 细胞中观察到了绿色荧光。本研究成功构建出针对梅花鹿S100A4 基因的慢病毒载体,为以后进一步研究S100A4 基因在鹿茸再生中的具体功能与机制打下了基础。     

A new versatile and compact lentiviral vector

During the past decade, lentiviral vectors based on the HIV-1 genome have been developed to become highly useful tools for efficient and stable delivery of transgenes to dividing and nondividing cells in a variety of experimental protocols. The vector system has been progressively and substantially improved, mainly to meet growing concerns over safety issues. However, the actual design and size of the lentiviral transfer vector often makes transgene cloning and DNA preparation a troublesome task. In this study, the pHR transfer vector used for lentivirus production in many laboratories was modified to contain a more versatile polylinker than the one present in the original pHR vector. In addition, the vector was significantly reduced in size from 12 to 7 kb, by replacing the original vector backbone with sequence from the multipurpose pUC18 vector. These modifications allowed for easier cloning and higher DNA yields without compromising the fundamental ability of this vector system to transduce cells in vitro and in vivo. Finally, the trimmed vector sequence was fully characterized by sequencing the vector in its entirety. In both cultured cells and directly into the rat striatum, transduction with this lentivirus, based on the modified pHsCXW vector, was as efficient and durable as with the pHR vector-based virus. In conclusion, the modified lentiviral transfer vector pHsCXW holds promise as a new valuable tool for the research community in the field of gene transfer.     

A systematic expression analysis implicates Plexin-B2 and its ligand Sema4C in the regulation of the vascular and endocrine system

Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand-receptor pair.     

Sema4D/Plexin-B1 promotes the progression of osteosarcoma cells by activating Pyk2-PI3K-AKT pathway

Objectives:Osteosarcoma (OS) is one of the two most common malignant bone tumors among children and teens but it is still a rare disorder. Semaphorin 4D (Sema4D) has been reported to play a specific role in human cancers. The aim of this study was to explore the function of Sema4D in the tumorigenesis and development of OS.Methods:10 pairs of OS tissues and paracancerous normal tissues from human OS samples and OS cell lines were used. Western blot assay was performed to detect the protein expression of Sema4D, Plexin-B1, and associated proteins of Pyk2-PI3K/AKT pathway. To explore the effect of Sema4D in the progression of OS, we reduced the expression of Sema4D. The effect of Sema4D knockdown on cell proliferation was explored by CCK-8 assay and clone formation assay. The effect of Sema4D knockdown on cell migration and invasion was assessed by Transwell assay.Results:Sema4D was overexpressed in OS tissues and cell lines. Sema4D knockdown notably suppressed cell proliferation in OS cells. Cell migration and invasion were reduced by Sema4D knockdown. Sema4D/Plexin-B1 facilitated OS, progression by promoting Pyk2-PI3K/AKT pathway.Conclusion:Sema4D/Plexin-B1 promoted the development of OS so Sema4D might be a potential target of treatment for patients with OS.     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.