Growth inhibition of human glioma cells modulated by retrovirus gene transfection with antisense IL-8

The human glioma cell line, NP-1, expresses IL-8 mRNA and constitutively secretes IL-8 protein. Administration of recombinant IL-8 increased the proliferation of NP-1 cells in a dose-dependent manner. In the presence of anti-IL-8 antiserum, the IL-8 mediated proliferation of NP-1 cell growth was inhibited. Further, NP-1 cell growth was inhibited by transfection of retroviral constructs encoding antisense IL-8 bothin vitro andin vivo models. These results suggest that antisense IL-8 gene therapy could be beneficial in gliomas where autocrine stimulation by IL-8 is implicated.     

PA317产生高滴度带有人TNF-α基因逆转录病毒的研究

逆转录病毒介导的基因转移技术要过渡到临床应用，主要解决如何使病毒载体具有高滴度而不具有复制活性．本文旨在探索建立一种合适的方法，能产生高滴度而且是安全的逆转录病毒载体。采用带有人肿瘤坏死因子基因的LXSN载体转染PA317包装细胞，建立产病毒的包装细胞株，结果表明：病毒载体滴度受包装培养液体积和胎牛血清浓度影响：包装细胞在32℃产生的病毒滴度比37℃高，而且比较稳定。产病毒包装细胞和被感染的靶细胞经PCR分析显示．被转染和被转导的细胞都有病毒基因的整合．而且包装细胞不会产生具有复制活性的逆转录病毒，因此能进一步用于临床研究．     

人G-CSF基因的克隆及其重组逆转录病毒载体的构建与初步应用

活化骨髓是指在体外经IL-2激活后具有抗瘤活性和造血功能的一类骨髓细胞;与LAK细胞相比具有广谱,高效的杀伤活性及低IL-2依赖性等特征。文献报道活化骨髓移植能产生移植物抗白血病(GVL)效应。我们从1993年6月开始应用活化骨髓和LAK细胞体外双重净化自体骨髓移植(ABMT)和移植后应用低剂量IL-2和LAK细胞治疗白血病,10例白血病接受该方案治疗,其中急性非淋巴性白血病6例、淋巴瘤性白血病和慢性粒细胞性白血病经化疗和干扰素治疗未达CR者分别为3例和1例。结果表明:①3例对化疗和干扰素治疗无效的病人移植后CR;②10例病人除1例淋巴瘤性白血病病人移植CR10个月复发外,其余病人移植后持续CR为25-1个月,中位数为14个月;③活化骨髓和LAK细胞体外双重净化不影响CFU-GM的回收率,移植净化骨髓不影响造血功能重建;④活性骨髓和LAK细胞净化ABMT后机体细胞免疫功能恢复明显快于未净化移植病     

人白细胞介素2重组逆转录病毒载体的构建与表达

通过RT-PCR克隆到含有全部编码序列的人IL-2 cDNA,并经核苷酸测序加以证实。将其克隆至逆转录病毒载体pLXSN,构建成人IL-2的重组逆转录病毒表达载体pLXSN-hIL2,体外经CRIP细胞包装后病毒滴度达7.6×10~5CFU/ml。NIH3T3小鼠成纤维细胞感染hIL-2重组逆转录病毒后,分泌IL-2水平达118.2U/ml;PCR从其基因组DNA中扩增到NeoR基因片段,提示重组逆转录病毒载体己整合至宿主细胞的基因组DNA中。本研究为开展人IL-2基因治疗奠定了基础。     

Establishment and characterization of spontaneously immortalized Schwann cells from murine model of globoid cell leukodystrophy (twitcher)

The twitcher mouse is a murine model of human globoid cell leukodystrophy (GLD; Krabbe disease) caused by a genetic defect in the activity of galactosylceramidase (GALC). An accumulation of cytotoxic metabolite, galactosylsphingosine (psychosine), in myelin forming cells (oligodendrocytes and Schwann cells) of the twitcher mouse as well as patients with GLD has been suggested to cause dysfunction of these cells and subsequent demyelination in the central and peripheral nervous system. To investigate further the cellular pathomechanism of GLD, we established spontaneously immortalized Schwann cell lines from the twitcher mouse. Long-term cultures of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of 3-week-old twitcher mice were maintained for 6 months, and spontaneously developed colonies were expanded further and characterized. One of the cell lines, designated TwS1, showed distinct Schwann cell phenotypes, was passaged twice a week and maintained for over 10 months without phenotypic alterations. The TwS1 cells had a nonsense mutation in the GALC genome, and showed markedly reduced GALC activity and elevated psychosine levels. Ultrastructurally, varieties of cytoplasmic inclusions were demonstrated in TwS1 cells. When TwS1 cells were infected with a retrovirus vector encoding GALC, GALC activity was markedly increased and psychosine levels were significantly decreased. These immortalized Schwann cells can be useful in studies on the nervous system lesions in GLD.     

TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning

The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCR-negative Jurkat T-cell line was equipped with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct to allow measurement of TCR-mediated activation. To establish the feasibility of this tumor-specific TCR transduction, we cloned the TCR genes of a known T-cell clone specific for the tumor antigen CAMEL (CTL-recognized antigen on melanoma) into a retroviral construct. Jurkat reporter cells transduced with this construct, Jrt-TCRalpha3beta5, were tested for their reactivity against CAMEL-expressing melanoma cells, peptide-loaded T2 cells and CAMEL-transfected COS-1 cells. The melanoma cell lines were poorly recognized, but peptide-pulsed and -transfected cells effectively stimulated NFAT signaling. The activation of TCR(+) Jurkat reporter cells was shown to be dependent on the antigen density on the target cells and the expression level of the coreceptor CD8 on the Jurkat cells. To verify the benefit of this TCR reconstitution method for identification of novel antigens, pools of the cDNA library from which CAMEL was originally cloned were transfected in COS-1 cells and screened with Jrt-TCRalpha3beta5. Identical cDNA pools were found that were positive with these cells and with the CAMEL-specific CTL clone. Our results illustrate that TCR-reconstituted Jurkat reporter cells are a useful tool in the identification of novel tumor antigens by cDNA expression cloning.     

反义AFP增强子/TK基因重组逆转录病毒载体的构建及在肝癌细胞中的专一性表达

目的构建在肝癌细胞中特异性高表达的重组逆转录病毒载体,观察其对体外培养肝癌细胞生物学行为的影响。方法应用DNA重组技术将α-甲胎蛋白増强子(AFE)核心区DNA与单纯性疱疹病毒胸苷激酶(TK)基因融合,反向插入逆转录病毒载体pLXSN,重组体命名为pL/TK/AFE/SN;经PA317细胞包装、G418筛选、病毒滴度测定,挑选滴度高的抗性克隆细胞株扩增培养,收获病毒上清,感染体外培养的Hap3B肝癌细胞和Hela宫颈癌细胞。用Southernblot、Northernblot检测转染细胞的DNA、mRNA,以羟甲基无环鸟苷(GCV)对细胞的杀伤毒性检测外源基因在转染细胞中的表达。结果酶切及Southernblot证实插入pL/TK/AFE/SN的反义AFE/TK基因大小、方向及克隆位点正确。达到一定的产病毒滴度。Northernblot分析显示Hep3B肝癌细胞有TK基因mRNA水平表达;应用GCV后,导入反义AFE/TK基因的Hep3B肝癌细胞生长明显受抑制(P<0.01)。结论成功构建了含反义AFE/TK基因的重组逆转录病毒载体并包装出具有感染能力的假型逆转录病毒;重组载体所含的反义AFP増强子能调控外源性TK基因特异地在Hep3B肝癌细胞中高表达。     

白细胞介素2基因转染肿瘤细胞体外生物学特性的研究

将人IL-13基因导入肿瘤细胞观察其对小鼠体内肿瘤生长的抑制作用.P815是强致癌性的DBA/2小鼠肥大细胞瘤,将含人IL-13cDNA的质粒载体以磷酸钙共沉淀法导入P815K系,择出一分泌IL-13能力为40ng(每10~6细胞/每24小时)的亚克隆,同法制备分泌鼠IL-2能力为2.2ng的p815 IL-2细胞,及人IL-13分泌量为700ng的CHO IL-13细胞.p815 IL-13细胞一侧肋腹皮下接种同系DBA/2小鼠(每鼠5×10~5细胞),观察到肿瘤的短暂生长,其后出现完全性肿瘤排斥,第25～32天时,绝大多数小鼠肿瘤完全消失;p815 IL-2细胞接种小鼠出现相似过程,而亲本p815细胞接种后肿瘤持续生长至巨大肿瘤.60～70天后给     

Sequence heterogeneity of murine acquired immunodeficiency syndrome virus: the role of endogenous virus

A defective murine leukemia virus is the causative agent ofmurine acquired immunodeficiency syndrome (MAIDS). We have clonedcDNAs from both virus infected and non-infected cells usingthe PCR methods with primers corresponding to the franking sequenceof the unique p12 gag gene. Sequence analysis of these cDNAclones revealed: (i) the presence of endogenous virus relatedto MAIDS virus in C57BL/6 mice, (ii) B cell lineage specificexpression of endogenous virus and (iii) extensive heterogeneityof MAIDS virus recovered from virus infected cells due to therecombination of the related viruses (defective pathogenic virus,ecotropic virus and endogenous virus). These findings suggestthat the creation of virus variants in infected cells may playan important role in virus pathogenesis and escape from immuneattack during the development of MAIDS.     

Human T-cell leukemia virus type 1 molecular variants, Vanuatu, Melanesia

Four of 391 Ni-Vanuatu women were infected with variants of human T-cell leukemia virus type 1 (HTLV-1) Melanesian subtype C. These strains had env nucleotide sequences approximately 99% similar to each other and diverging from the main molecular subtypes of HTLV-1 by 6% to 9%. These strains were likely introduced during ancient human population movements in Melanesia.