慢病毒载体介导hTERT对肝细胞生物学特性的影响

目的：研究慢病毒介导人端粒酶逆转录酶（h-TERT）基因对人张氏肝细胞（chang liver）生物学特性的影响。方法：将表达h-TERT基因的重组慢病毒感染人张氏肝细胞,通过杀稻瘟筛选获得阳性克隆。分别采用实时荧光RT-PCR、TRAP-PCR及ELISA方法检测转染前后chang liver细胞的h-TERT-mRNA水平和端粒酶活性的变化。通过MTT法、流式细胞术（FCM）观察其形态变化及生长增殖情况。结果：转染后的chang liver细胞存在h-TERT基因过表达和端粒酶活性上调,体外培养条件下维持肝细胞原有形态,存活期显著延长,增殖能力较强。结论：外源性h-TERT基因的异位表达不仅上调chang liver细胞的端粒酶活性,而且促进其增殖。     

慢病毒介导的Survivin基因稳定干扰PC-3细胞系的建立

目的：构建生存素（Survivin）基因稳定干扰的PC-3细胞系。方法：设计针对Survivin基因的shR—NA序列，构建Survivin shRNA慢病毒载体并感染PC-3细胞，采用RT-PCR和流式细胞技术分别检测Survivin mRNA和蛋白表达水平，并与阴性对照组和空白对照PC-3细胞进行比较。结果：Survivin shRNA慢病毒载体感染PC-3细胞后，Survivin mRNA和蛋白水平分别为（5．89±0．12）％和（11．82±0．32）％，明显低于阴性对照组（55．17±0．87）％、（95．70±1．56）％和空白对照PC-3细胞（58．21±0．68）％、（95．90±1．78）％。结论：采用shR—NA慢病毒载体成功构建了Survivin基因稳定干扰的PC-3细胞系。     

α-Synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: Lessons from viral transduction of knockout mice

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinson's disease (PD). We have been exploring the normal function of the PD-related protein α-synuclein (α-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that α-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523–3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090–3099]. We recently began exploring the impact of α-Syn on TH in vivo, by transducing dopaminergic neurons in α-Syn knockout mouse (ASKO) olfactory bulb using wild type human α-Syn lentivirus. At 3.5–21 days after viral delivery, α-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of α-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated α-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with α-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated α-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal α-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.     

Immune selection of equine infectious anemia virus env variants during the long-term inapparent stage of disease

The principal neutralizing domain (PND) of equine infectious anemia virus (EIAV) is located in the V3 region of SU. Genetic variation in the PND is considered to play an important role in immune escape and EIAV persistence; however, few studies have characterized genetic variation in SU during the inapparent stage of disease. To better understand the mechanisms of virus persistence, we undertook a longitudinal study of SU variation in a pony experimentally inoculated with the virulent EIAV(Wyo). Viral RNA isolated from the inoculum and from sequential sera samples was amplified by RT-PCR, cloned, and individual clones were sequenced. Of the 147 SU clones obtained, we identified 71 distinct V3 variants that partitioned into five major non-overlapping groups, designated PND-1 to PND-5, which segregated with specific stages of clinical disease. Genotypes representative of each group were inserted into an infectious molecular clone, and chimeric viruses were tested for susceptibility to neutralization by autologous sera from successive times post-infection. Overall, there was a trend for increasing resistance to neutralizing antibody during disease progression. The PND genotype associated with recrudescence late in infection was resistant to both type-specific and broadly neutralizing antibody, and displayed a reduced replication phenotype in vitro. These findings indicate that neutralizing antibody exerts selective pressure throughout infection and suggest that viral strategies of immune evasion and persistence change in the face of an evolving and maturing host immune response.     

BimS慢病毒RNA干扰载体的构建及其感染效率和干扰效果的实验研究

目的:应用RNA干扰技术构建BimS慢病毒RNA干扰载体,探讨其感染效率和干扰效果.方法:针对人BimS设计3个干扰靶点,将单链引物退火成双链oligo序列,连接入Age Ⅰ和EcoR Ⅰ双酶切线性化载体中.将重组质粒进行病毒包装、并且通过感染ACC-2细胞观察其感染效率,定量PCR检测其对BimS mRNA干扰效果.结果:PCR产物经扩增电泳后阳性克隆得到337bp条带,插入序列片段与DNA测序结果完全相符.重组慢病毒载体在293T细胞中包装获得滴度为2×10s TU/ml的病毒颗粒,MOI=20,转染效率为85％.转染pFU-GV-BmS-1组、pFU-GV-BmS-2组及对照组BmS mRNA相对表达水平分别为:0.743±0.025、0.466±0.023、1.266±0.042(组间两两比较,P＜0.05).结论:BimS慢病毒RNA干扰载体构建成功,并能高效感染ACC-2细胞及下调BmS mRNA表达.     

慢病毒介导核受体相关因子1基因修饰骨髓间充质干细胞移植治疗帕金森病模型大鼠

目的探讨慢病毒介导核受体相关因子1(Nurrl)基因修饰骨髓间充质干细胞(BMSCs)移植至帕金森病(PD)模型大鼠纹状体后对PD大鼠的治疗作用。方法采用神经毒素6-羟多巴胺(6-OHDA)纹状体注射成功制备PD大鼠模型18只,将携带绿色荧光的慢病毒GV287-Nurr1感染大鼠BMSCs,然后随机移植入6只PD模型大鼠的纹状体内(实验组),设生理盐水组(假移植组)6只及感染空慢病毒GV287的BMSCs组(对照组)6只;术后第1、2、4周观察大鼠的行为改善情况;免疫组织化学染色法检测纹状体及黑质中Nurrl和酪氨酸羟化酶(TH)蛋白的表达变化;RT-PCR法检测黑质Nurrl mRNA和TH mRNA的表达变化。结果慢病毒感染后的BMSCs及上清中均检测到Nurr1蛋白;对照组与实验组大鼠在移植后第1、2、4周的旋转行为较假移植组均有所改善,且实验组比对照组改善更明显;实验组纹状体Nurr1阳性细胞有效存活并沿胼胝体向皮质及对侧脑组织迁移;实验组纹状体及黑质损毁侧Nurrl和TH蛋白及mRNA的表达较假移植组和对照组明显增高,差异均有统计学意义(P0.05)。结论慢病毒介导Nurr1基因修饰大鼠BMSCs移植治疗PD大鼠,能有效地改善PD模型大鼠的行为学症状,增加大鼠脑内纹状体和黑质区Nurr1和TH的表达。     

CCL21转染PANC-1细胞株及对其生物学行为的影响

目的观察CCL21重组慢病毒载体转染PANC-1细胞株及对其生物学行为的影响。方法构建的CCL21重组慢病毒载体转染胰腺癌细胞株PANC-1,通过PCR、Western blot进行验证。结果构建的重组CCL21-慢病毒成功转染胰腺癌细胞株并稳定表达,但转染后的胰腺癌细胞株PANC-1其生物学行为未发生明显变化。结论通过PCR、Western blot检测,构建的重组CCL21-慢病毒成功转染胰腺癌细胞株并稳定表达,但转染后的胰腺癌细胞株PANC-1其生物学行为未发生明显变化。     

胰腺癌CFPAC1细胞增殖诱导配体基因shRNA慢病毒表达载体的构建

目的 构建针对人胰腺癌细胞株CFPAC1增殖诱导配体(a proliferation-inducing ligand,APRIL)基因的shRNA慢病毒表达载体.方法 应用基因工程技术,筛选出针对APRIL基因的RNAi靶序列,与pGCL-GFP载体连接,构建慢病毒表达载体LV-shAPRIL;将连接产物转化到DHSα感受态细胞,经PCR筛选阳性克隆、测序鉴定.再用LV-shAPRIL、pHelper 1.0、pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒.重组慢病毒感染CFPAC1细胞,实时定量PCR和Western blotting检测CFPAC1细胞APRIL mRNA和蛋白的表达.结果 PCR和测序结果与构建的慢病毒载体的预期结果一致,经包装产生的病毒滴度为5×107TU/ml.构建的慢病毒载体感染CFPAC1细胞后第4天、第4周和第8周,APRIL mRNA表达量较空载体慢病毒感染组分别下降了73%、70%和71%;APRIL蛋白表达量分别下降了66%、63%和62%(P＜0.05).而各时间段未感染慢病毒的细胞组与空载体组相比无明显差异(P＞0.05).结论 成功构建了APRIL基因的shRNA慢病毒表达载体LV-shAPRIL.     

靶向B7-H3基因RNA干扰慢病毒的构建及人胰腺癌PaTu8988稳定感染细胞株的建立

目的 构建靶向人B7同源性3(B7-H3)基因的小发夹RNA(shRNA)的慢病毒载体,并建立稳定感染的胰腺癌PaTu8988细胞株.方法 根据GenBank提供的B7-H3 cDNA序列,设计4条靶向B7-H3的siRNA序列,构建重组干扰质粒pGCSIL-GFP-B7-H3-shRNA.将重组干扰质粒和过表达B7-H3质粒共同转染293T细胞,经蛋白质印迹法筛选出干扰效果最佳的重组干扰质粒.该质粒经慢病毒包装,并感染胰腺癌PaTu8988细胞株,建立稳定低表达B7-H3细胞株.应用实时定量PCR及蛋白质印迹法检测细胞B7-H3基因的表达抑制率.结果 携带干扰效果最好的shRNA的慢病毒感染PaTu8988细胞,建立了稳定低表达B7-H3基因的PaTu8988细胞株,其B7-H3 mRNA表达的抑制率达96.8％,B7-H3蛋白表达的抑制率达88.1％.结论 成功构建了人B7-H3-RNAi的慢病毒载体,并建立了稳定感染的低表达B7-H3基因的PaTu8988细胞株.     

RNA干扰Cdx2基因沉默对人胃癌裸鼠耐药模型Survivin和C-myc表达的影响

目的观察携带人尾型同源盒转录因子2（Cdx2）基因沉默的重组慢病毒颗粒（Cdx2-RNAi-LV）对人胃癌耐顺铂细胞SGC-7901／DDP裸鼠皮下移植瘤中Survivin和C—myc表达的影响。方法建立人胃癌耐顺铂细胞SGC-7901／DDP裸鼠皮下移植瘤模型,36只裸鼠随机分成3组（n=12）：PBS组、LV-RNAi组、Cdx2．RNAi-LV组;各组瘤体内分别注射磷酸盐缓冲液、慢病毒颗粒（0．5×10^8TU／m1）、含Cdx2基因重组慢病毒颗粒（0．5×10^8TU／m1）相应药物100μl,1次／3d,共6次;同时所有裸鼠腹腔注射药物顺铂（25mg／kg）,隔天1次,共10次;注射Cdx2-RNAi-LV病毒颗粒第20天后脱颈椎法处死裸鼠,完整切取肿瘤组织,并应用PT-PCR和Westernblot技术检测移植瘤中Survivin和C．myc基因的表达。结果Cdx2．RNAi．LV组、LV．RNAi组和PBS组的细胞均有Survivin、C—myc的mRNA和蛋白的表达,但各组细胞对应的Survivin、C—myc的mRNA和蛋白的表达水平有差别;Cdx2-RNAi—LV组Survivin、C．myc的mRNA和蛋白的表达量较PBS组和LV．RNAi组明显下调（P〈0．05）,而PBS组和LV—RNAi组比较差异元统计学意义（P〉0．05）。结论Cdx2基因沉默重组慢病毒颗粒瘤内注射下调人胃癌耐顺铂细胞裸鼠移植瘤Survivin和C-myc表达,可能是Cdx2基因沉默逆转肿瘤的耐药性机制之一。