慢病毒载体介导的脂肪细胞SOCS-3RNA干扰及对瘦素反应的研究

体内存在瘦素抵抗是大多数肥胖者的共同特征,但如何缓解瘦素抵抗目前还无有效方法.通过应用慢病毒载体介导的RNA干扰技术对大鼠脂肪细胞中瘦素信号转导通路的负反馈抑制因子—细胞因子信号转导抑制因子-3(suppressor of cytokine signaling 3,SOCS-3)基因进行敲减,观察其对大鼠重组瘦素作用的反应.研究发现,阴性对照慢病毒和SOCS-3shRNA慢病毒对成熟脂肪细胞的感染率约为80%.利用real-time PCR和Westernblot法检测发现SOCS-3 shRNA慢病毒通过在成熟脂肪细胞内表达shRNA而造成SOCS-3基因在mRNA和蛋白水平的敲减,有效率分别为72%和57%;经50nmol/L瘦素处理6h后,感染阴性对照病毒的脂肪细胞SOCS-3mRNA表达有明显升高(P0.05),而感染SOCS-3 shRNA慢病毒的脂肪细胞SOCS-3mRNA表达则无明显变化(P0.05).结果表明,利用SOCS-3shRNA慢病毒去敲减脂肪细胞中SOCS-3的表达可能对于解除肥胖者外周瘦素抵抗具有一定的作用.     

慢病毒载体介导RNAi的研究进展

RNAi通过双链RNA的介导,特异性阻抑相关序列的表达,从而导致转录后水平的基因沉默.广泛存在于真菌、植物和动物等真核生物中.慢病毒载体是理想的真核细胞基因转移工具,被广泛应用于相关的RNAi研究领域,例如抗病毒研究、癌症及其治疗、遗传性疾病的治疗、基因治疗.现已发现,慢病毒载体能够介导组织特异、时间特异的RNAi,在疾病的基因靶向性治疗上必有广阔的前景.     

慢病毒载体介导的RNA干扰

RNA干扰(RNAinterference)是指由双链RNA分子抑制同源基因的表达。慢病毒载体(lentivirusvector)则是高效的基因转导工具,能将外源序列稳定导入分裂相和非分裂相细胞。将慢病毒载体和RNA干扰结合,能在哺乳动物各类细胞中,特异性抑制同源基因的表达;也是基因功能研究和基因治疗的有力手段。     

慢病毒介导的小鼠PINK1基因RNAi载体的构建及在NIH3T3细胞中的筛选

目的:构建筛选靶向特异PINK1-siRNA慢病毒表达载体,感染小鼠胚胎细胞(NIH3T3)验证该病毒载体的敲减效率,为研究帕金森病的发病机制奠定基础。方法:构建2对靶向小鼠PINK1-siRNA序列(KD1和KD2),将这2对序列连接在GV118上,将重组载体和病毒包装的辅助质粒共转染293 T细胞,获得慢病毒颗粒,再将该病毒颗粒转染入NIH3T3细胞,用qRT-PCR验证细胞内PINK1 mRNA表达水平以验证敲减效果。结果:筛选出可以用于后续实验的高效靶向KD2序列,并成功转染到小鼠NIH3T3细胞中,在感染复数(MOI)为50时,KD2的沉默效果最好,敲减率达到66.4%。结论:成功构建了高效靶向小鼠PINK1-siRNA慢病毒载体,其可稳定转染小鼠NIH3T3细胞,可高效抑制PINK1 mRNA的表达。为下一步利用其感染神经细胞或注入动物脑内,进一步研究PINK1基因在帕金森病发病过程中的作用环节和机制提供了分子生物学的技术基础。     

慢病毒载体介导RNAi稳定抑制XIAP表达及对胰腺癌细胞增殖、凋亡影响的实验研究

目的:探讨运用慢病毒载体介导的RNA干扰技术对X-连锁凋亡抑制蛋白(XIAP)的抑制效率及对胰腺癌细胞增殖、凋亡的影响,建立XIAP表达稳定抑制的胰腺癌细胞株.方法:应用pGJCSIL-PUR慢病毒载体构建针对XIAP的ShRNA载体,转染包装细胞293T,收集病毒上清转染胰腺癌细胞系SW1990,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量PCR和western-blot免疫印迹检测癌细胞内XIAP的表达:四甲基偶氮唑盐(MTT)比色法检测细胞增殖;caspase3/7活性测定和DAPI染色检测细胞凋亡.结果:成功构建3个XIAP-ShRNA慢病毒栽体(X1、X2、X3)及XIAP表达稳定抑制的胰腺癌细胞株,对XIAP的抑制效率均达70%以上;MTT检测显示X1、X3稳定抑制XIAP后胰腺癌细胞增殖明显减慢,但caspase3/7活性及细胞凋亡并没有明显增加.结论:慢病毒栽体介导的靶向XIAP的RNAi可有效抑制XIAP表达,降低胰腺癌细胞的增殖能力;成功建立的XIAP表达稳定抑制的胰腺癌细胞株为进一步研究打下基础.     

慢病毒介导RNA干扰SOCS3基因在猪前体脂肪细胞和成肌细胞中的表达

构建细胞信号抑制因子3(suppressor of cytokine signaling 3,SOCS3)慢病毒干扰载体,获得有感染性的病毒颗粒,感染猪前体脂肪细胞和成肌细胞,并检测其对前体脂肪细胞的干扰效率.首先设计并合成3对针对目的基因SOCS3的siRNA序列,退火后连接于LentiH1上,测序验证后,与包装质粒△8.9和vsv-g共转染到293T细胞中进行包装和浓缩,纯化后测定病毒滴度,然后感染猪前体脂肪细胞和成肌细胞.重组慢病毒载体LentiH1-siRNA经酶切和测序鉴定正确,病毒滴度为3×107tu/mL,感染猪成肌细胞和前体脂肪细胞后,可见报告基因GFP的表达;RT-PCR和Western印迹分析表明,前体脂肪细胞中SOCS3的表达被显著下调,其中LentiH1-siRNA3介导对SOCS3基因mRNA和蛋白的干扰效率分别达53%和71%.本研究成功构建了猪SOCS3慢病毒干扰载体,感染猪前体脂肪细胞能稳定沉默SOCS3基因的表达,为深入研究SOCS3的功能奠定了基础.     

慢病毒介导的大鼠肝BRL-3A 细胞Tmub1 基因沉默及稳定感染细胞 系的建立

目的:构建Tmub1基因慢病毒干扰载体,建立稳定转染细胞系,检测大鼠肝BRL-3A细胞中Tmub1基因表达的干扰效果。方法:设计并构建4对针对大鼠Tmub1基因的特异性shRNA干扰质粒,酶切鉴定、DNA测序所得质粒。将由pRSV-Rev、pMDLg-pRRE、pMD2G和pll3.7干扰质粒组成的包装系统共转染293T细胞,产生慢病毒。所得慢病毒感染大鼠正常肝细胞BRL-3A,Western Blot检测不同靶点RNAi后Tmub1蛋白表达情况,确定有效靶点。针对有效靶点大量包装慢病毒。测定病毒滴度并以最适感染复数(multiplicity of infection,MOI)感染BRL-3A细胞后,G418抗生素筛选稳定感染细胞系BRL-3A/256。RT-PCR和Western Blot检测各组细胞Tmub1 mRNA和蛋白质的表达差异。结果:结果显示Tmub1 RNAi慢病毒载体构建成功,C0020Sh2-Hops-256干扰靶点RNAi效果最强。成功包装Tmub1基因RNAi慢病毒,测定病毒滴度为2.3×108TU/ml,对293T细胞的最适感染复数为60。成功建立Tmub1 RNAi慢病毒载体稳定感染细胞系BRL-3A/256,且在该细胞系中Tmub1 mRNA和蛋白质表达明显降低。结论:成功构建Tmub1 RNAi慢病毒载体,有效干扰BRL-3A细胞中Tmub1 mRNA和蛋白表达;成功筛选出Tmub1RNAi慢病毒稳定感染细胞系BRL-3A/256。     

慢病毒介导的靶向NDV P基因shRNA对NDV复制的抑制作用

小干扰RNA(siRNA)诱导的RNA降解可以特异性地抑制病毒感染,它作为一种有效的抗病毒治疗方法正被广泛研究。为了探讨慢病毒介导的shRNA对NDV复制的抑制效果,从而为新城疫病毒的抗病毒研究奠定基础,本研究以新城疫病毒(Newcastle disease virus,NDV)P基因为靶基因,构建了靶向NDV P基因的shRNA重组慢病毒表达载体RNAi-341和RNAi-671。将其与辅助细胞共转染293T细胞,获得包装好的重组慢病毒;在鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)和SPF鸡胚上进行了干扰实验,并通过荧光定量PCR和病毒滴度测定检测shRNA对NDV的抑制效果。结果发现,RNAi-341和RNAi-671均能抑制FLAG-P蛋白在293T细胞中的瞬时表达。在CEF细胞感染后16h后,NDV的病毒滴度分别降低了66.6倍和30.6倍;在鸡胚感染48h后,RNAi-341和RNAi-671组NDV病毒的增殖量分别减少99%和98%。RNAi-341与RNAi-671不仅能抑制P基因的转录,还能显著降低NP、M、F、HN和L基因的转录水平。与RNAi-671相比,RNAi-341的抑制效果更好。研究结果表明,慢病毒介导的靶向P基因的shRNA具有抗病毒作用,能够抑制NDV在CEF和鸡胚中的复制,从而为临床防治NDV提供一个新方法。     

靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响

目的 构建携带促血管生成素2-小干扰RNA（Ang2-siRNA）慢病毒载体,观察其对恶性黑色素瘤细胞中Ang2基因表达的干扰作用.方法 将经XbaⅠ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒（pNL-EGFP）载体与pSilencer 1.0-U6启动子-促血管生成素2-小干扰RNA（pSilencer 1.0-U6-Ang2-siRNA）重组质粒连接,产生加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅰ（pNL-EGFP-U6-Ang2-Ⅰ）、加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅱ（pNL-EGFP-U6-Ang2-Ⅱ）慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒、水疱性口炎病毒G蛋白（pVSVG）包膜质粒和pHelper包装质粒共转染293T细胞,产生pNL-EGFP-U6-Ang2-Ⅰ、pNL-EGFP-U6-Ang2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Ang2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Ang2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.0×103/ml.实时荧光定量RT-PCR结果显示：Ang2-siRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Ang2基因的表达（P＜0.05）.结论 成功构建了Ang2-SiRNA慢病毒载体,体外研究显示Ang2-SiRNA慢病毒载体能抑制恶性黑色素瘤细胞中Ang2 mRNA的表达,为下一步进行裸鼠恶性黑色素瘤移植瘤生长的干预实验奠定基础,为肿瘤的基因治疗提供实验依据.     

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.     

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector‐mediated RNAi

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin‐signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector‐based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic‐cell nuclear transfer (SCNT) studies. Sh‐RNA positive cells were screened by puromycin selection. Using real‐time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down‐regulation in sh2 shRNA‐treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin‐targeting siRNA produced endogenously could efficiently down‐regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus‐mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:452–459, 2015     

Germ line transmission and expression of an RNAi cassette in mice generated by a lentiviral vector system

We have used a lentiviral delivery system (LentiLox3.7) to generate transgenic mice harbouring RNA interference (RNAi) against the hepatocyte nuclear factor 4 gamma (HNF4γ). HNF4γ is a nuclear receptor with unknown function. Our analyses performed on founder (F₀) and first generation (F₁) mice revealed mosaicism in F₀ founders and a low efficiency of transgenesis (6%) in F₁ mice. These data, together with the observation of multiple silenced transgenes, do not favour the use of LentiLox3.7 lentivirus for transgenesis. Despite the low efficiency of transgenesis, we achieved a tissue-dependent knockdown of HNF4γ expression in some mice. Milen Kirilov and Minqiang Chai contributed equally to this work.     

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in color-ectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data col-lectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.     

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.     

Knockdown of human p53 gene expression in 293-T cells by retroviral vector-mediated short hairpin RNA

RNA interference （RNAi） is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA （shRNA） transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vectormediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, HI-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.     

SOCS家族在中枢神经系统的研究进展

细胞因子信号抑制因子（SOCS）家族是一类对细胞因子信号通路具有负反馈调节作用的蛋白分子,参与多种细胞因子、生长因子和激素的信号调节。细胞因子对中枢神经系统中的各种生物效应具有广泛多样的调节作用,SOCS家族的许多成员在发育时期和成年的脑内均有表达,SOCS家族不仅与细胞因子信号调节及中枢神经系统多种功能的调节密切相关,而且可能是神经发育和分化的重要调控因子,并参与神经免疫内分泌调节。本文综述了SOCS家族的发现、结构特点、脑内分布以及在中枢神经系统中的功能等方面的研究进展。     

SOCS1在氯胺酮减轻小胶质细胞活化中的机制研究

目的:探讨麻醉药氯胺酮(Ketamine, KET)对暴露于脂多糖(Lipopolysaccharide, LPS)中的小胶质细胞活化水平的影响,并观察细胞因子信号转导抑制因子1(Suppressor of Cytokine Signaling 1, SOCS1)在其中的作用。方法:本研究选用N9小胶质细胞系,将其暴露于浓度为10 ng/mL的LPS中,模拟炎症反应,同时给与浓度为1 m M的KET,采用Western blot、酶联免疫吸附检测(Enzyme-Linked Immunosorbent Assay, ELISA)小RNA干扰和免疫细胞染色等方法,观察KET对暴露于LPS中的小胶质细胞活化水平的影响,及SOCS1分子在其中的作用。结果:将细胞分为3组,分别为正常培养的Control组、LPS组和KET+LPS组,研究发现,将N9小胶质细胞暴露于含10 ng/mL的细胞培养基中24 h后,细胞诱导型一氧化氮合酶(Inducible Nitric Oxide Synthase, i NOS)表达和培养基肿瘤坏死因子α(Tumor Necrosis Factorα, TNF-α)含量显著增加(P0.05),而KET可显著降i NOS蛋白表达和培养基TNF-α含量(P0.05)。随后,将细胞分为5组,分别为Control组、LPS组、KET+LPS组、SOCS1-siRNA+KET+LPS组和乱序siRNA(SC-siRNA)+KET+LPS组,我们发现,LPS可显著增加小胶质细胞TNF-α和白细胞介素1β(Interleukin-1β, IL-1β)的释放、增加SOCS1和核因子κB(Neuclear FactorκB, NF-κB)表达(P0.05),而KET可显著逆转LPS对炎症因子释放和NF-κB表达的影响,并进一步增加SOCS1表达(P0.05),而SOCS1-siRNA显著逆转了KET的上述作用(P0.05),SC-siRNA未对KET产生的上述作用造成显著影响(P0.05)。结论:KET可降低LPS对小胶质细胞的活化作用,上述作用可能通过SOCS1分子介导。