人miR-146b慢病毒载体构建及人脂肪细胞中表达验证

目的 构建人miR-146b慢病毒表达载体,包被病毒并检测其在人脂肪细胞中过表达效果。方法 以人基因组DNA为模板,PCR高保真扩增包含miR-146b区域上下游各100bp左右的基因组DNA,亚克隆入慢病毒表达载体,与包装质粒共转染HEK-293T细胞,包装病毒,收取病毒上清,纯化后感染人脂肪前体细胞,36 h开始观察荧光标记,72 h收取细胞,抽提RNA,Realtime PCR检测慢病毒感染下miR-146b的相应表达量。结果 成功构建人miR-146b慢病毒表达载体,包装获得的病毒感染人脂肪细胞的效率可达到85%以上,miR-146b过表达水平可接近4倍。结论 本研究成功构建了人miR-146b慢病毒表达载体,包被的慢病毒可以在人脂肪细胞中实现过表达效果,为后续功能研究奠定了基础。     

经尿道灌注慢病毒转染豚鼠膀胱的研究

目的 观察慢病毒介导绿色荧光蛋白（GFP）转染膀胱的效果, 并确定取得较好转染效果的病毒量。方法 豚鼠经尿道灌注及静脉注射不同量的携带 GFP 基因的慢病毒, 饲养 7 d 后取膀胱、 肝、 肺、 肾等组织冰冻切片,激光共聚焦显微镜下观察各组织 GFP 分布。结果 滴度为 4×108的慢病毒, 经尿道膀胱灌注 30 μL 时可见组织黏膜下有绿色荧光, 40 μL 时组织肌层有广泛绿色荧光分布, 50 μL 时组织肌层有更强绿色荧光分布, 静脉注射 25 μL 时组织肌层可见有广泛绿色荧光分布; 经尿道灌注各病毒量下未在膀胱外组织见有明显绿色荧光分布, 静脉注射下在肝、 肺等组织见有较膀胱更强绿色荧光。结论 慢病毒可以成功介导 GFP 转染豚鼠膀胱, 并且经尿道灌注可以避免静脉注射带来的病毒在膀胱外组织广泛分布, 能够为膀胱疾病的临床治疗带来新的研究方向和手段。     

Molecular Studies of HTLV-1 Replication: An Update

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. Studies on HTLV-1 have been instrumental for our understanding of the molecular pathology of virus-induced cancers. HTLV-1 is the etiological agent of an adult T-cell leukemia (ATL) and can lead to a variety of neurological pathologies, including HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP). The ability to treat the aggressive ATL subtypes remains inadequate. HTLV-1 replicates by (1) an infectious cycle involving virus budding and infection of new permissive target cells and (2) mitotic division of cells harboring an integrated provirus. Virus replication initiates host antiviral immunity and the checkpoint control of cell proliferation, but HTLV-1 has evolved elegant strategies to counteract these host defense mechanisms to allow for virus persistence. The study of the molecular biology of HTLV-1 replication has provided crucial information for understanding HTLV-1 replication as well as aspects of viral replication that are shared between HTLV-1 and human immunodeficiency virus type 1 (HIV-1). Here in this review, we discuss the various stages of the virus replication cycle—both foundational knowledge as well as current updates of ongoing research that is important for understanding HTLV-1 molecular pathogenesis as well as in developing novel therapeutic strategies.     

麦胚凝集素慢病毒载体的构建及其对脂肪干细胞的感染

目的探讨麦胚凝集素（WGA）慢病毒载体的构建与表达的方法,以及WGA在神经系统中跨膜示踪的能力。方法 采用基因工程方法,将WGA的基因插入到慢病毒表达载体Plvx-IRES-ZsGreen1 中。再采用慢病毒包装,将构建好的质粒 和其他3 种包装质粒（RPEV、PRRE、VSVG）在293T细胞中进行共转染,获取病毒液后感染人源的脂肪干细胞（hADSCs）, 将感染后的hADSCs 注入小鼠的脑卒中模型,修复神经损伤。结果显微镜下检测到绿色荧光报告蛋白表示感染成功。 对感染后的hADSCs 进行免疫组化显色,结果显示WGA已经成功的在hADSCs 中实现了表达,在MCAO小鼠模型脑损伤 处追踪到感染了WGA的hADSCs。结论慢病毒包装WGA蛋白的方法可靠高效,可以作为一种高效的示踪技术广泛应 用于各类细胞。     

稳定表达HBx的人肝星形细胞系的建立及其生物学特性初探

目的建立稳定表达HBVX蛋白（HBx）的LX-2细胞系,并初步研究HBx对于肝星形细胞（HSC）的增殖及其I型胶原蛋白表达的影响。方法构建包含HBx基因的重组慢病毒载体LV5-X,并转染LX-2细胞,同时用对照质粒转染LX-2细胞,经嘌呤霉素筛选后获得HBx稳定表达的LX-2-X和对照LX-2-C细胞系。荧光显微镜下观察感染效率,Western印迹检测细胞株中HBx的表达。在LX-2细胞系的生物学特性探索方面,应用CCK_8法检测细胞的增殖;Real—timePCR检测细胞中I型胶原蛋白mRNA的表达;ELISA法愉测细胞培养上清中I型胶原蛋白的表达。结果成功构建重组慢病载体LV5-X,感染LX-2细胞后建立细胞系LX-2-X和LX-2-C,嘌呤霉素筛选3d后,荧光显微镜显示近100％细胞发出绿色荧光,Western印迹证实LX-2-X细胞稳定表达HBx。培养72h和96h后LX-2-C细胞增殖明显高于LX-2和LX-2-C细胞组（P〈0．05）;PCR证实LX-2X细胞I型胶原蛋白mRNA合成高于LX-2和LX-2-C细胞组（P〈0．05）;ELISA结果显示LX一2一X细胞I型胶原蛋白表达高于LX-2和LX-2-C细胞组（P〈0．05）。结论成功构建了稳定表达HBx的LX-2细胞系;HBx能够促进HSC的增殖,并增加I型胶原蛋白的表达。     

慢病毒介导的调节因子XI过表达及其对胶质瘤细胞增殖的抑制作用

目的：构建调节因子X1(regulatory factor X1,RFX1)过表达慢病毒载体,并观察其对F98细胞增殖的影响。方法：运用PCR技术扩增RFX1,并将其插入慢病毒空载体质粒pITA,构建pITA-RFX1慢病毒质粒,经琼脂糖凝胶电泳和测序鉴定正确。将包装慢病毒共转染293T细胞,然后感染F98细胞。利用Western印迹和激光共聚焦验证RFX1过表达情况,用流式细胞仪和CCK-8试剂盒观察转染前后细胞增殖情况。 结果：电泳和测序显示慢病毒载体pITA-RFX1构建成功,将其感染F98细胞后过表达RFX1蛋白,同时可以明显抑制细胞的增殖。结论：过表达慢病毒载体构建成功,上调RFX1可以降低恶性胶质瘤细胞的增殖。     

靶向ANGPTL4基因shRNA慢病毒载体抑制SiHa细胞迁移与侵袭

目的探讨慢病毒介导短发夹RNA(shRNA)沉默血管生成素样蛋白4(ANGPTL4)基因对人宫颈癌细胞系Si Ha细胞迁移与侵袭的影响。方法实验分为3组,即空白对照组、阴性对照组及LV3/ANGPTL4组。用ANGPTL4shRNA慢病毒干扰载体感染Si Ha细胞,Western blot检测Integrinβ1、VEGF及MMP9蛋白表达,划痕实验检测Si Ha细胞迁移能力,Transwell实验检测Si Ha细胞迁移与侵袭能力。结果重组慢病毒载体成功感染Si Ha细胞,与空白对照组及阴性对照组比较,LV3/ANGPTL4组Si Ha细胞Integrinβ1、VEGF及MMP9蛋白表达水平降低(P0.01),细胞迁移与侵袭能力减弱(P0.05)。结论靶向ANGPTL4基因shRNA慢病毒载体可降低Si Ha细胞Integrinβ1、VEGF及MMP9蛋白表达水平,抑制Si Ha细胞迁移与侵袭能力。     

重组慢病毒LV5-NE的构建及NE促进NB4细胞增殖

目的构建重组慢病毒LV5-NE筛选纯NB4/LV5-NE细胞株,探讨中性粒细胞弹性蛋白酶(NE)对人急性早幼粒细胞白血病细胞株NB4增殖的影响,为研究NE致白血病的可能机制奠定基础。方法以质粒p CMV-myc-NE为模板得到PCR产物NE,将NE基因连接到穿梭载体p GLV10/U6/GFP/Puro后酶切验证,将序列正确的质粒与包装质粒p Gag/Pol、pRev、p VSV-G共转染293T细胞。测定病毒滴度。实验分为空白组、LV5-NC组和LV5-NE组,病毒感染NB4细胞72 h后用嘌呤霉素筛选稳定表达的细胞。用Western blot检测NE在NB4细胞中的蛋白表达以及其切割作用;CCK-8法观察NB4细胞增殖能力;流式细胞术测定NB4细胞周期和凋亡;Western blot检测p Akt和Akt以及Bax和Bcl-2蛋白表达。结果成功构建表达NE的慢病毒LV5-NE,筛选得到纯NB4/LV5-NE细胞株,NE对NB4细胞的增殖有明显促进作用(P0.05)结论 NE在NB4细胞株中稳定表达后中可促进NB4细胞的增殖,其增殖作用可能通过激活PI3K/Akt通路调节。     

Vascular endothelial growth inhibitor affects the invasion, apoptosis and vascularisation in breast cancer cell line MDA-MB-231

Background Breast cancer is one of the most common malignant female diseases worldwide.It is a significant threat to every woman＇s health.Vascular endothelial growth inhibitor （VEGI） is known to be abundant in endothelial cells.According to previous literature,overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models,but there has been limited research on the significance of VEGI in the breast cancer.Methods In our study,cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed.The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo.The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting.The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth,invasion,adhesion,and migration assay with subcutaneous tumor-bearing nude mice models.Then the growth curves of the subcutaneous tumors were studied.Expressions of VEGI,CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.Results Infection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI.As can be seen in the invasion,adhesion and migration assay,the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration,adhesion and invasion.The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups.Immunohistochemistry analysis of the tumor biopsies cleady showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly.In vitro and in vivo,the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.Conclusions Taken together,recombinant lentivirus that were successfully constructed,demonstrated up-regulated VEGI gene expression in breast cancer cells.Lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration,adhesion and invasion.Over-expression of VEGI diminished the tumorigenic capacity of breast cancer cells in vivo.Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis.