人多药耐药基因-1及二氢叶酸还原酶基因共转导小鼠造血细胞

耐药基因转导骨髓细胞对化疗病人造血系统的保护作用已受到人们的重视.本研究利用多药耐药基因-1(mdr-1)及二氢叶酸还原酶(dhfr)双基因转染小鼠骨髓细胞,观察造血细胞对两种耐药谱化疗药物的抵抗能力.结果表明,所用逆转录病毒载体转染率达到15%左右,转基因骨髓细胞CFU-GM对taxo1及MTX的耐药能力明显增强,说明外源基因在造血祖细胞中的正确表达;转基因骨髓细胞回输给同系小鼠7个月后,FCM技术仍可检测到外周血白细胞gp170的表达,且其基因组DNA中可检测到mdr-1及dhfr基因特异序列,表明两种基因可能已稳定整合至造血干/祖细胞中.上述结果为以骨髓细胞为靶体的耐药基因治疗提供了有用的实验室资料.     

mdr—1和DHFR双基因共转染入CD^＋34细胞拓宽造血细胞耐药谱的体外研究

目的：探讨将多药耐药基因（mdr-1)和二氢叶酸还原酶基因（DHFR)同时导入人CD^ 34细胞,以拓宽造血细胞耐药谱,改善骨髓耐受联合化疗的可行性,方法：将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架,通过引入IRES序列构建获得含mdr-1和DHFR（L22Y）双耐药基因的塑转录病毒载体pSF－DIM,通过脂质体介导包装,单嗜性和双 包装细胞上清交叉感染提高病毒滴度,低温离心病毒上清转染人脐血CD^ 34细胞,用流式细胞仪检测P－gp的表达,基因组PCR检测外源性耐药基因的整合,CFU－GM培养药性变化,结果：逆转录病毒载体pSF-DIM转人脐血CD^ 34细胞后,P-gp的表达较未转基因组增加了10．98％,基因组PCR同时检测到两种外源性药基因的整合,与未转基因组比较,在48nmol/L甲氢蝶呤和10ng/ml及12ng/ml紫杉醇（商品名Taxol）浓度水平,CFU－GM集落形成显著培养（P＜0．05）。结论：重组双耐药基因逆转录病毒载体pSF-DIM可度水平,CFU－GM集落形成显著增加（P＜0．05）,结论：重组双耐药基因逆转录病毒载体pSF-DIM可以有效介导mdr-1和DHFR双耐药基因进入人脐血CD34^ 细胞并获得共表达,拓宽了造血细胞药谱。     

mdr-1和DHFR双基因共转染人CD34+细胞拓宽造血细胞耐药谱的体外研究

目的　探讨将多药耐药基因 (mdr 1)和二氢叶酸还原酶基因 (DHFR)同时导入人CD3 4 细胞 ,以拓宽造血细胞耐药谱 ,改善骨髓耐受联合化疗的可行性。方法　将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架 ,通过引入IRES序列构建获得含mdr 1和DHFR(L2 2Y)双耐药基因的逆转录病毒载体pSF DIM ,通过脂质体介导包装 ,单嗜性和双嗜性包装细胞上清交叉感染提高病毒滴度。低温离心病毒上清转染人脐血CD3 4 细胞 ,用流式细胞仪检测P gp的表达 ,基因组PCR检测外源性耐药基因的整合 ,CFU GM培养观察耐药性变化。结果　逆转录病毒载体pSF DIM转导人脐血CD3 4 细胞后 ,P gp的表达较未转基因组增加了 10 .98% ;基因组PCR同时检测到两种外源性耐药基因的整合 ;与未转基因组比较 ,在 48nmol/L甲氨蝶呤和 10ng/ml及 12ng/ml紫杉醇 (商品名Taxol)浓度水平 ,CFU GM集落形成显著增加 (P <0 .0 5 )。结论　重组双耐药基因逆转录病毒载体pSF DIM可以有效介导mdr 1和DHFR双耐药基因进入人脐血CD3 4 细胞并且获得共表达 ,拓宽了造血细胞耐药谱     

内部核糖体进入位点介导多药耐药基因1在人CD34^＋细胞中的表达

为了达到双耐药基因共转染以拓宽造血细胞耐药谱的目的,初步观察了内部核糖体进入位点(IRES)介导多药耐药基因1(MDR1)在人早期造血细胞中的表达效率.由脑心肌炎病毒IRES调控MDR1基因翻译的双耐药基因逆转录病毒载体pSF-DIM和相同启动子调控的MDR1单耐药基因逆转录病毒载体pSF-MDR1,包装后获得的双嗜性包装细胞PA317/pSF-DIM和PA317/pSF-MDR1病毒滴度分别为8×104和1.3×105cfu/ml.用其上清感染人脐血CD34+细胞,经FCM检测表明基因转导后单基因转导组和双基因转导组P-gp表达均有提高,分别为28.82%(荧光强度25.49)和10.92%(荧光强度9.48),但单基因转导组高于双基因转导组.因此,IRES成功地介导了MDR1基因在人早期造血细胞中的表达,为后续的试验奠定了一定的基础.但是,双基因转导组和单基因转导组MDR1表达差异的原因还需进一步研究.     

原代骨髓基质细胞层对逆转录病毒载体介导的造血干/祖细胞基因转导的支持效应

为探讨建立原代骨髓基质细胞层的方法并观察基质接触对造血干／祖细胞(HSC／HPC)基因转移的影响，采用Ficoll—Hypaque分离成人骨髓单个核细胞(MNC)，用基质细胞培养液培养，传代4次以建立原代骨髓基质细胞层。将经细胞因子预刺激的造血干／祖细胞接种到经辐照处理的基质细胞层上，进行逆转录病毒介导的多药耐药基因(mdr1)转导，以半固体集落培养和聚合酶锭反应法(PCR)检测长春新碱(VCR)抗性集落数和mdr1基因扩增片段以测定转导效率。结果表明：原代骨髓基质细胞培养传代4—6周形成混合贴壁细胞层，主要由成纤维细胞组成。集落法和PCR法检测显示基质接触能提高骨髓造血干／祖细胞基因转导效率2.1—3.3倍，对逆转录病毒介导的造血干／祖细胞基因转导具有明显的支持效应。结论：基质细胞接触联合细胞因子作用可提高逆转录病毒介导的造血干／祖细胞基因转导效率。     

异基因骨髓移植小鼠联合输注IL-3/IL-6双基因转导的骨髓基质细胞促进造血恢复

探讨用逆转录病毒载体转入IL 3和IL 6双基因的骨髓基质细胞系QXMSC1IL 3/IL 6对异基因骨髓移植小鼠造血功能的促进作用。用逆转录病毒载体 (含小鼠IL 3cDNA ,人IL 6cDNA)分别转导到骨髓基质细胞系QXMSC1(H 2 d) ,构建骨髓基质细胞系QXMSC1IL 3/IL 6。供体小鼠BALB/c(H 2 d)骨髓去除骨髓中T细胞 ,受体小鼠C57BL/6(H 2 b)经γ射线致死量照射后 ,输入去除T细胞的供体骨髓 (1× 1 0 7/鼠 )的同时输入骨髓基质细胞QXMSC1IL 3/IL 6(5× 1 0 5/鼠 )。在骨髓移植后 2 0和 40天 ,分别检测骨髓移植小鼠外周血RBC ,WBC和骨髓有核细胞数及骨髓中CFU S ,CFU GM ,CFU E和CFU GEMM数。结果 :异基因骨髓移植共输入QXMSC1IL 3/IL 6基质细胞系可使异基因骨髓移植小鼠外周血RBC ,WBC数明显恢复 ,骨髓中有核细胞数 ,CFU S ,CFU GM ,CFU E和CFU GEMM明显增加。基质细胞系QXMSC1可作为有效的基因载体促进骨髓移植后造血功能重建。结论 :基质细胞转入细胞因子IL 3或IL 6基因可以进一步促进骨髓移植后造血功能重建 ,联合转导IL 3和IL 6有协同作用     

人多药耐药基因—1及二氢叶酸还原酶基因共转导小鼠造？…

耐药基因转导骨髓细胞对化疗病人造血系统的保护作用已受到人们的重视。本研究利用多药耐药基因－１（ｍｄｒ－１）及二氢叶酸还原酶（ｄｈｆｒ）双基因转染小鼠骨髓细胞,观察造血细胞对两种耐药谱化疗药物的抵抗能力。结果表明,所用逆转录病毒载体转染率达到１５％左右,转基因骨髓细胞ＣＦＵ－ＧＭ对ｔａｘｏｌ及ＭＴＸ的耐药能力明显增强,说明外源基因在祖细胞中的正确表达;转基因骨髓细胞回输给同系小鼠７个月后,ＦＣＭ技术     

造血干细胞的基因转移及其表达的实验研究

为探索一种安全、有效、简易的非病毒基因转移策略用于介导造血干细胞基因转移的可行性,本研究利用商品化的脂质体将两个标志基因(Neo R和Lac Z)共转导造血细胞。通过G418筛选、富集转导阳性细胞,观察了外源基因在小鼠原代骨髓细胞的基因转移率及其表达的稳定性。结果显示:通过脂质体介导,外源基因在小鼠骨髓细胞可获得有效的瞬时和稳定的表达。同时,经G418筛选,转导阳性细胞可得到明显的富集。提示利用脂质体这一非病毒载体个导造血干细胞基因转移的可行性。     

提高多药耐药基因导入人CD_(34)~ 细胞转导效率的探讨

目的　探讨逆转录病毒介导的多药耐药基因 (mdr1)导入人CD34 细胞的影响因素 ,以提高基因转导效率。方法　用流式细胞术 (FCM)检测不同组合细胞因子及人骨髓基质细胞 细胞因子支持的基因转导效率 ;用造血祖细胞集落培养观察耐药性 ;用FCM检测不同浓度紫杉醇素对基因转导细胞的作用。结果　细胞因子SCF Flt配体 (FL) IL 3组合支持的基因转导效率高于其它组合 (SCF IL 6 IL 3,SCF IL 6 IL 3 Tpo ,SCF IL 3)。骨髓基质细胞 细胞因子 (SCF FL IL 3)支持的基因转导效率 (2 0 .5 % )又高于单纯用该组细胞因子的转导效率 (15 .2 % ) ,并且前者形成的抗性集落形成细胞数多于后者。在 10ng ml紫杉醇作用下基因转导CD34 细胞的阳性率可达 38.5 %。结论　骨髓基质细胞 细胞因子 (SCF FL IL 3)对基因转导有较强的促进作用 ;一定浓度的紫杉醇具有富集基因转导细胞的作用     

骨髓基质细胞对人造血CD_34~+细胞体外扩增及基因转导的作用

目的：探讨造血基质细胞对人外周血干细胞体外培养扩增及基因转导的促进作用。方法：应用基质细胞支持培养扩增体系进行人造血干细胞体外培养扩增及基因转导。结果：骨髓基质细胞和造血生长因子共同支持组的造血ＣＤ＋３４细胞在体外培养３周后,其造血祖克隆形成能力较单纯造血生长因子支持组高３０．７％（Ｐ＜０．０５）。在有基质细胞支持时,逆转录病毒载体上清转导人造血ＣＤ＋３４细胞后,其造血细胞克隆中Ｎｅｏ基因阳性克隆是无基质支持对照组的２倍。结论：基质细胞的支持有维持造血干细胞原始造血活性及促进基因转导的双重好处。     

Toward gene therapy for Gaucher disease

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells

Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.     

多药耐药基因在骨髓移植模型中的表达

目的 探讨多药耐药基因（mdrl)在骨髓移植小鼠模型中表达情况，及转染mdrl的骨髓细胞对化疗中骨髓保护的可行性。方法 以小鼠骨髓细胞为靶细胞，通过逆转录病毒介导，将mdrl转入骨髓细胞，通过将转染基因的骨髓细胞回输同种小鼠体内的骨髓移植动物模型，观察不同时期移植小鼠骨髓细胞中mdrl的表达及功能，结果 （1）成功地将mdrl导入小鼠骨髓细胞中，转染率达到35%；（2）采用程序性移植方法成功建立了mdrl转基因鼠骨髓移植模型；（3）mdrl在移植小鼠细胞内稳定，有效地表达，观察1-5个月mdrl转染的细胞在受体鼠骨髓单个核细胞中所占比例分别为8.0%，8.0%,7.5%,4.0%,3.0%;(4)mdrl转染的骨髓细胞在化疗中有明显的骨髓保护作用。结论 从一个侧面证实了通过mdrl转染骨髓细胞在化疗中进行骨髓保护是一种长期，稳定，有效的方法。     

醛脱氢酶基因转导介导的K562细胞对环磷酰胺的耐受性

本研究的目的是观察经醛脱氢酶基因（ALDH1）转导的造血细胞对环磷酰胺的耐受性，以逆转录病毒载体pLXSN-ALDH1将醛脱氢酶基因是导入人造血细胞系K562，应用PCR证实原病毒的整合，用RT－PCR检测ALDH1基因表达和MTT法分析ALDH1过表达导致的4－氢过氧环磷酰胺的耐药表型。结果发现，逆转录病毒成功地介导了醛脱氢酶基因转移，全长ALDH1cDNA以原病毒形式整合入受体K562细胞基因组，RT－PCR检测到ALDH1基因转录表达。ALDH1过表达的基因转移细胞对环磷酰胺的耐受性明显提高（4倍），IC50≈10μmol／L。结论提示，体外ALDH1过表达足以引起对环磷酰胺耐受，为体内ALDH1基因转移以保护骨髓细胞免受环磷酰胺的毒性作用提供了实验依据。     

增强型绿色荧光蛋白逆转录病毒载体的构造和表达

逆转录病毒载体被广泛用作对造血细胞进行基因转移的工具,转导方法的改进有赖于应用可快速分析并被高效选择的基因标志,为此,我们克隆了增强型绿色荧光蛋白（ＥＧＦＰ）基因,并构建可表达ＥＧＦＰ的逆转录病毒载体ＬＧＳＮ,通过脂质体转梁和交互感染方法建立高滴度的逆转录产病毒细胞,用以分析对造血细胞标记ＥＧＦＰ基因的可行性,流式细胞术和荧光显微镜检测发现,ＥＧＦＰ病毒转录的ＧＰ＋ｅｖｎＡｍ１２细胞和Ｋ５６２细胞均可发出稳定的绿色荧光信号,阳性率可分别达９７％和８６％,聚合酶链反应分析显示ＬＧＳＮ转子细胞内有原病毒的整合,上述结果提示,作为新一代选择性标志,ＥＧＦＰ是适于研究对造血细胞基因转移与报告基因,对促进人类基因治疗的研究有重要意义。     

Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells

The effects of cytokine stimulation during retroviral transduction on in vivo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distinguish donor and recipient red blood cells (RBCs) and the enhanced green fluorescent protein (EGFP) as a marker for gene transfer. After transplantation, EGFP was detected in up to 90% of circulating RBCs, platelets, and leukocytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 months. Large quantitative differences in reconstitution were observed after transplantation with graded numbers (1000-30, 000) of EGFP(+) cells preconditioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulated BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stimulation, but approximately 30-fold reduced after KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not caused by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative transplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of their in vivo hematopoietic potential, and also emphasize that overall in vitro transduction frequency does not predict gene transfer to repopulating stem cells.     

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.     

Transduction of rhesus macaque hematopoietic stem and progenitor cells with avian sarcoma and leukosis virus vectors

Genome-wide integration site analyses showed that Moloney murine leukemia virus (MoMLV)- and lentivirus-derived vectors integrate preferentially into the coding regions of genes, posing a risk of insertional mutagenesis. Avian sarcoma and leukosis viruses (ASLVs) were previously reported to have a weak preference for gene-coding regions in a cell line study as compared with human immunodeficiency virus and MoMLV; however, thus far these vectors have not been studied for their potential efficacy in transduction of hematopoietic progenitor and stem cells. In this study we investigated for the first time the ability of ASLV-derived RCAS (replication-competent ALV LTR [avian leukosis virus long terminal repeat] with a splice acceptor) vectors to transduce rhesus macaque hematopoietic progenitors and long-term repopulating cells, in an autologous transplantation model. RCAS vectors can efficiently and stably transduce rhesus macaque CD34+ hematopoietic progenitor cells with an efficiency of transduction of up to 34% ex vivo. In two animals transplanted with RCAS vector-transduced autologous CD34+ cells, highly polyclonal hematopoietic reconstitution with sustained gene-marking levels in myeloid and lymphoid lineages was observed up to 18 months post-transplantation. These findings are encouraging and suggest that this vector system should be explored and further optimized for gene therapy applications targeting hematopoietic stem and progenitor cells.