慢病毒介导的外源基因体外投递系统的建立

目的针对不同哺乳类细胞建立相应的慢病毒体外感染体系，以建立慢病毒介导的外源基因体外投递系统。方法按Invitrogen公司推荐的标准程序进行慢病毒（携带EGFP基因）包装（脂质体介导的瞬时转染）、超速离心浓缩和保存等，随后用病毒上清或浓缩后的病毒感染293FT细胞，24—48h后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功生产；将携带EGFP基因的病毒上清或浓缩后的病毒分别加入内含293FF细胞、小鼠ES细胞、小鼠胚胎成纤维细胞（MEFs）或小鼠睾丸生殖细胞的培养板孔内，感染6—12h后，用相应培养基替换感染液，数天后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功感染不同哺乳类细胞。结果按标准程序生产的携带EGFP基因慢病毒（病毒上清或浓缩后的病毒）成功高效率感染293FF细胞、MEFs或小鼠睾丸生殖细胞；用浓缩后的病毒（携带EGFP基因）感染小鼠ES细胞，亦可获得EGFP阳性的ES细胞克隆。结论熟练掌握了慢病毒包装、浓缩及鉴定等技术，同时针对不同哺乳类细胞建立了相应的慢病毒介导的外源基因体外传递系统，这些为相关后续研究打下了良好的基础。     

MicroRNA-375表达对大肠癌细胞生物学特性的影响

目的:研究构建miRNA-375慢病毒表达载体并转染结直肠癌细胞,探讨其对结直肠癌细胞生物学特性的影响.方法:采用qRT-PCR法检测结直肠癌细胞株中miRNA-375表达水平.构建FUA-ZMCS-EF1-EGFP-miR-375/inhibitor慢病毒表达载体,建立稳定过表达miRNA-375及其抑制剂的HCT-116亚细胞系,体外观察细胞生长速度并绘制生长曲线,流式细胞仪检测细胞凋亡率,划痕实验检测miRNA-375对HCT-116细胞迁移能力的影响,Western blot法检测miRNA-375对细胞内ERK磷酸化水平的影响.结果:结直肠癌细胞株中miRNA-375低表达不但能促进细胞生长、抑制细胞凋亡,而且能促进细胞内ERK磷酸化水平、增加细胞迁移力和侵袭力.结论:结直肠癌miRNA-375发挥抑癌基因的作用,为miRNA-375在结直肠癌基因诊断及治疗中的应用提供了实验依据.     

慢病毒介导新型Tet-On系统调控大鼠GDNF和TH双基因表达对帕金森病大鼠的实验研究

目的观察改良Tet-On系统修饰慢病毒(Lv-TH-GDNF)目的基因的表达调控及纹状体内直接转移对帕金森病(PD)大鼠的作用。方法 1用Lv-TH-GDNF与rt TA2s-M2病毒感染He La细胞,免疫印迹法检测强力霉素(Dox)对TH、GDNF基因表达的调控。2用Lv-TH-GDNF与rt TA2sM2病毒共同注射到PD大鼠患侧纹状体,Dox诱导目的基因表达。通过观察阿扑吗啡(APO)诱导旋转行为、黑质多巴胺能神经元数量、患侧纹状体DA、DOPAC含量评估Lv-THGDNF治疗效应;通过移植侧纹状体内TH与GDNF蛋白量评估外源基因在体内的表达。结果 1在体外He La细胞实验,仅Dox阳性组见TH、GDNF蛋白条带。2在动物体内实验,病毒移植4周后,与PBS对照组相比,仅病毒+Dox组大鼠旋转行为明显改善(P<0.01),损伤侧黑质致密部TH阳性细胞数、纹状体DA、DOPAC含量及TH和GDNF蛋白量明显增高(P<0.01)。结论新型Tet-On系统修饰的Lv-THGDNF目的基因表达受四环素类抗生素调控,在体外实验未见基础活动,且纹状体内直接转移对PD大鼠有一定治疗作用。     

人乳腺癌MDR1基因稳定沉默细胞的筛选及其生物学特性

目的筛选MDR1沉默的人乳腺癌细胞并比较其生物学特性。方法采用BLOCK-iT Lentiviral RNAi Expres-sion System生产表达MDR1基因shRNA的慢病毒载体,转导敏感人乳腺癌细胞MCF-7和耐阿霉素人乳腺癌细胞MCF-7/ADM,杀稻瘟菌素(blasticidin)筛选获得MDR1稳定沉默细胞MCF-7/RNAi和MCF-7/ADM/RNAi。定量RT-PCR检测MDR1mRNA表达,流式细胞术检测P-GP蛋白表达和功能,MTT法检测药物敏感性。结果经10 mg/L blasticidin筛选12 d获得MCF-7/RNAi和MCF-7/ADM/RNAi细胞。MCF-7、MCF-7/RNAi、MCF-7/ADM和MCF-7/ADM/RNAi细胞的MDR1mRNA相对表达水平分别为1、0.13、17.14和2.01;P-GP表达分别为(1.5±0.3)%、(1.2±0.2)%、(89.4±3.6)%和(16.3±1.9)%;细胞内Rh123的潴留分别为(92.4±3.1)%、(90.6±4.0)%、(13.6±1.6)%、(72.4±2.8)%;IC50值分别为0.90、0.92、19.61和4.04 mg/L。结论应用慢病毒载体稳定沉默MDR1基因可有效逆转人乳腺癌细胞耐药性。     

PRL-3基因RNA干扰慢病毒载体的构建及在SW480细胞中的表达

目的 构建PRL-3基因慢病毒干扰载体及建立PRL-3基因稳定干扰的结直肠癌细胞亚系.为PRb-3基因功能研究奠定基础.方法 设计PRL-3基因特异性RNAi靶序列,与pENTRTM/U6线性载体定向连接,构建pENTRTM/U6真核入门表达载体.通过与pENTRTM/U6 entry clone与pLenti6/BLOCK-iTTM-DEST载体进行重组,获得pLenti6/BLOCK-iTTM-DEST真核表达慢病毒干扰载体.利用包装细胞293FT获得重组的慢病毒,感染大肠癌细胞株SW480细胞,杀稻瘟菌素筛选获得稳定干扰PRL-3基因的细胞亚系.结果 成功构建PRL-3 pLenti6/BLOCK-iTTM-DEST真核表达慢病毒干扰载体并获得相应的慢病毒,病毒悬液的滴度为6×105U/L.RT-PCR、Western blotting结果 表明,克隆1 PRL-3mRNA及蛋白显著降低.结论 成功构建人PRL-3基因特异性的慢病毒干扰载体,获得PRL-3基因稳定干扰的大肠癌细胞亚系.     

Biological and Genetic Differences Between Lung- and Brain-Derived Isolates of Maedi-Visna Virus

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Hypothalamic Corticotropin-Releasing Factor is Centrally Involved in Learning Under Moderate Stress

The corticotropin-releasing factor (CRF) neuropeptide is found to have a pivotal role in the regulation of the behavioral and neuroendocrine responses to stressful challenges. Here, we studied the involvement of the hypothalamic CRF in learning under stressful conditions. We have used a site-specific viral approach to knockdown (KD) CRF expression in the paraventricular nucleus of the hypothalamus (PVN). The two-way shuttle avoidance (TWSA) task was chosen to assess learning and memory under stressful conditions. Control animals learned to shuttle from one side to the other to avoid electrical foot shock by responding to a tone. Novel object and social recognition tasks were used to assess memory under less stressful conditions. KD of PVN-CRF expression decreased the number of avoidance responses in a TWSA session under moderate (0.8 mA), but not strong (1.5 mA), stimulus intensity compared to control rats. On the other hand, KD of PVN-CRF had no effect on memory performance in the less stressful novel object or social recognition tasks. Interestingly, basal or stress-induced corticosterone levels in CRF KD rats were not significantly different from controls. Taken together, the data suggest that the observed impairment was not a result of alteration in HPA axis activity, but rather due to reduced PVN-CRF activity on other brain areas. We propose that hypothalamic CRF is centrally involved in learning under moderate stressful challenge. Under ‘basal'' (less stressful) conditions or when the intensity of the stress is more demanding, central CRF ceases to be the determinant factor, as was indicated by performances in the TWSA with higher stimulus intensity or in the less stressful tasks of object and social recognition.     

Novel lentiviral vectors for human gene therapy

This review summarises an emerging viral vector system for use in human gene therapy - lentiviral vectors. Lentiviral vectors have several advantages over existing viral vectors. They can stably express transgenes in non-dividing cells in vivo without provoking a significant immune response. They can be produced to a high titre and can be pseudotyped with heterologous envelope proteins to confer broad tropism. Although not without safety concerns, the properties of lentiviral vectors makes them an attractive choice for human gene therapy.     

Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.