脂质体介导骨髓细胞双标志基因转移与体外扩增研究

目的：探讨脂质体介导骨髓细胞基因转移的效率及其表达的稳定性，为基因治疗与基因标志研究提供实验依据。方法：利用脂质体介导，将ＮｅｏＲ和ＬａｃＺ两个标志基因共转导骨髓细胞，通过Ｇ４１８筛选、富集转导阳性细胞，然后扩增这些细胞，观察基因转移率及扩增对转导细胞基因表达稳定性的影响。结果：脂质体介导的骨髓细胞基因转移率为１３．３３％±２．６８％，经Ｇ４１８筛选、富集后，转导阳性细胞可达到４６．０６％±３．４７％。经体外扩增处理，扩增前后无显著性差异。结论：利用脂质体介导的基因转移方法介导骨髓细胞基因转移可获得高效、稳定的表达。     

新型逆转录病毒载体介导的人多药耐药基因转移小鼠造血干／祖细胞

为探讨逆转录病毒介导的外源基因转导对骨髓造血重建的影响,我们以人多药耐药基因(mdr-1)为报告基因,采用包含Friend脾灶形成病毒(spleen focus-forming vims)和鼠胚胎干细胞病毒序列的新型逆转录病毒载体SF-MDR,以病毒包装细胞与小鼠造血细胞体外共培养法进行基因转染,并对基因转导后造血细胞的体外耐药能力及体内造血重建能力进行了观测。结果表明,该载体可有效地介导mdr-1基因转导,明显提高小鼠骨髓造血细胞对秋水仙素及紫杉醇的耐受能力,对细胞体内造血重建能力没有影响,体内移植8个月后经紫杉醇体内筛选,7/7的小鼠可于外周血细胞基因组DNA中检出外源mdr-1基因的存在。     

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.     

造血干细胞基因转移研究进展

造血干细胞具有的自我更新能力和移植后重建造血的特性使其成为外源基因导入的良好靶细胞。骨髓细胞培养、干细胞的纯化及体外转化中应用造血因子可明显提高干细胞目的基因的转移效率，造血干细胞的基因转移已成为基因治疗的重要方法。     

逆转录病毒载体介导Neo~R基因转入正常人造血祖细胞

本文应用逆转录病毒载体介导基因转移法将含有新霉素抗性(neo~R)基因的双拷贝逆转录病毒载体pN2A转入正常人造血祖细胞。应用Ficoll分层液(比重1.064)富集人造血干、祖细胞,预培养后与已经照射的生产重组病毒包装细胞株共同培养24小时,经含G418(1000μg/ml)体外半固体培养,可见具有抗性的CFU-GM集落生长。应用PCR方法检测转染细胞中neo~R基因的转移和表达,在生产逆转录病毒的辅助细胞株PA317/N2及体外液体培养的骨髓细胞中均可扩增出neo~R基因的DNA片段,进一步证实了neo~R基因在正常人造血干、祖细胞的有效转移和表达。     

非病毒载体介导脑源性神经营养因子基因转染骨髓间充质干细胞:脂质体及电穿孔转染法的比较

背景:基因转染细胞有病毒及非病毒载体法,因病毒载体面临安全性、免疫排斥等问题,实验探讨脂质体及电穿孔转染法.目的:比较脂质体及电穿孔法介导人脑源性神经营养因子基因转染骨髓间允质干细胞的细胞转染特性和体外表达情况,建立基因工程细胞.方法:①脂质体法:取体外分离、培养的第3代豚鼠骨髓间充质干细胞,将质粒-脂质体混合物加入含细胞的培养基中培养6 h,再加入胎牛血清的培养基,孵育48 h后行免疫组织化学检测,即为瞬时表达.48 h后加入含G418培养基筛选.②电穿孔法:取骨髓间充质干细胞,胰酶消化,用无血清培养基重悬细胞,将细胞悬液加入电转化池中,加入质粒,将电转化池移至电极间放电转导.转染48 h后,检测目的基因瞬时表达.48 h后加入含G418培养基筛选.用免疫组织化学及RT-PCR检测两种方法脑源性神经营养因子基因表达情况.结果与结论:免疫组织化学显示脂质体介导转染的脑源性神经营养因了瞬时表达率约为5.80%,电穿孔法约为24.29%.脂质体法转染筛选14 d后细胞几乎全部死亡;电穿孔法转染后筛选并扩大培养建立工程细胞,免疫组织化学显示工程细胞脑源性神经营养因子阳性表达率达90%以上,RT-PCR扩增产物电泳证实目的基因阳性条带.结果表明,用电穿孔法可成功建立脑源性神经营养因子基因修饰骨髓间充质干细胞的工程细胞,并用免疫组织化学和RT-PCR办法证实细胞体外表达目的基因.     

骨髓间充质干细胞基因工程改造方法的比较

目的:对脂质体介导、反转录病毒载体介导、腺相关病毒载体介导这3种常用的基因转染方法进行比较,寻找一种适合于骨髓间充质干细胞的基因转移方法。方法:实验于2004-10/2005-06在首都医科大学北京神经科学研究所完成。①在脂质体介导下,将增强的绿色荧光蛋白基因导入骨髓间充质干细胞,然后通过G418抗性筛选,观察转染效率。②反转录病毒介导的基因转染,首先利用LipofectAMINETM 2000转染包装细胞系PT67,获得重组反转录病毒上清,然后用病毒上清直接感染骨髓间充质干细胞。转染后的细胞同样用G418筛选,得到阳性克隆后进行定点消化、扩增。③在腺相关病毒介导的基因转染过程中,首先通过磷酸钙沉淀法转染包装细胞系HEK293,得到重组AAV-LacZ病毒颗粒直接感染骨髓间充质干细胞,1周后进行β-gal染色,计算转染效率。结果:①脂质体介导的基因转染24h后在荧光显微镜下观察,可见少量细胞呈现绿色荧光蛋白阳性,阳性率大约为5%。抗生素筛选3周后细胞全部死亡,经过多次试验均未得到阳性细胞克隆。②反转录病毒感染后,在荧光显微镜下观察可见少量骨髓间充质干细胞表达增强的绿色荧光蛋白。当加入G418筛选后,大量细胞死亡,1周后仅有极少数细胞存活。继续培养3～4周后,细胞形成克隆,定点消化细胞克隆,扩增后95%的细胞表达绿色荧光蛋白。③腺相关病毒上清感染骨髓基质细胞1周后,用β-gal染色估计骨髓间充质干细胞转染效率大约为75%。但传代后阳性细胞所占比例明显下降,大约2%。结论:骨髓间充质干细胞易于接受外援基因。3种基因转移方法相比:脂质体转染法转染效率最低,不适合骨髓间充质干细胞;反转录病毒载体法感染效率最高,最适用于骨髓间充质干细胞;而腺相关病毒载体法感染效率较高,但该载体系统没有抗生素筛选,所以无法使阳性细胞得到纯化和扩增,其可能更适合于体内基因直接感染。     

多西环素诱导表达下IL-3基因转染小鼠骨髓基质细胞促进造血

本研究探讨用多西环素调控IL-3基因表达的小鼠骨髓基质细胞系对小鼠造血干细胞增殖分化的促进作用。构建含小鼠IL-3基因逆转录病毒载体系统，转染小鼠骨髓基质细胞系，获得QXMSClTet-on-IL-3；体外加入多西环素诱导IL-3基因表达并检测IL-3表达活性；观察细胞培养条件上清液对造血祖细胞集落形成单位的作用及QXMSClTet-on-IL-3与骨髓细胞共培养对造血干细胞增殖分化的影响。结果表明：多西环素提高了QXMSClTet-on-IL-3细胞系IL-3的表达，促进骨髓造血祖细胞克隆形成数；与骨髓细胞共培养可促进造血干细胞的增殖分化。结论：应用多西环素诱导外源基因转染的骨髓基质细胞IL-3的表达，可促进造血。     

携带绿色荧光蛋白基因的逆转录病毒载体的构建及其介导的T细胞基因转移研究

为了构建含绿色荧光蛋白（green fluorescent protein，GFP）基因的逆转录病毒载体和研究逆转录病毒对T细胞的感染能力，利用亚克隆技术将磷酸甘油酸激酶启动子（phosphoglycerate kinase promoter，PGK）基因和GFP全长cDNA插入逆转录病毒载体pLXSN，采用磷酸钙沉淀法将重组载体转染PA317包装细胞，G418筛选出抗性克隆，收集滴度最高的病毒上清感染NIH3T3和T细胞，在倒置荧光显微镜下观察GFP表达情况。结果表明；重组逆转录病毒载体转染PA317包装细胞后，可在荧光显微镜下观察到GFP的表达。G418筛选后，含GFP的逆转录病毒可感染原代培养的T细胞。结论：逆转录病毒载体能够快速、稳定地将外源基因转移至T细胞，可作为介导T细胞基因转移的重要工具。     

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     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral- mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

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)     

Bone marrow stromal cells as a vehicle for gene transfer.

Adoptive transfer of genetically modified somatic cells is playing an increasingly important role in the management of a wide spectrum of human diseases. Hematopoietic stem cells and lymphocytes have been used to transfer a variety of genes, however, they have limitations. In this study, the feasibility of retroviral gene transduction of bone marrow stromal cells, and the engraftment characteristics of these cells following infusion, was investigated in a murine transplantation model. Stromal cells derived from Balb/c mouse bone marrow were transduced with a replication-defective retrovirus containing the LacZ gene. Following three rounds of transduction, between 5 and 40% of the cells were positive for the LacZ gene. A total of 2 x 106 cells were infused into the same mouse strain. After the infusion, the LacZ gene was detected by PCR in the bone marrow, spleen, liver, kidney and lung; however, only the spleen and bone marrow samples were strongly positive. Quantitative PCR demonstrated that between 3 and 5% of spleen and bone marrow cells, and 1% of liver cells contained the LacZ gene at 3 weeks after infusion; <0.2% transduced cells were found in other organs. No difference was noted in engraftment between mice with or without irradiation before transplantation, suggesting that engraftment occurred without myeloablation. The infused transduced cells persisted for up to 24 weeks. Self-renewal of transplanted stromal cells was demonstrated in secondary transplant studies. Ease of culture and gene transduction and tissue specificity to hematopoietic organs (bone marrow, spleen, liver) is demonstrated, indicating that stromal cells may be an ideal vehicle for gene transfer.     

逆转录病毒介导的GM-CSF在骨髓基质细胞中基因转移与表达

为了探索逆转录病毒介导的GM-CSF在骨髓基质细胞(BMSC)中的基因转移与表达,采用电穿孔法将重组质粒pLXSN/GM和空载体pLXSN转染PA317细胞,经G418筛选,用抗性克隆培养上清液成功地感染了BMSC。经PCR和Southern blot分析证实BMSC基因组中整合有外源NeoR和GM-CSF cDNA,原位杂交显示有较强的GM-CSFmRNA的表达,经增殖法和集落形成法表明转染的BMSC分泌较多的GM-CSF。据此表明,转染的BMSC较未转染BMSC具有更强的支持造血功能,提示BMSC可作为造血功能障碍基因治疗的靶细胞,为GM-CSF基因治疗血液病奠定基础。     

High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood

Umbilical cord blood is rich in hematopoietic stem and progenitor cells and has recently been used successfully in the clinic as an alternative source of engrafting and marrow repopulating cells. With the likelihood that cord blood stem/progenitor cells will be used for gene therapy to correct genetic disorders, we evaluated if a TK-neo gene could be directly transduced in a stable manner into single isolated subsets of purified immature hematopoietic cells that demonstrate self-renewed ability as estimated by colony replating capacity. Sorted CD34(3+) cells from cord blood were prestimulated with erythropoietin (Epo), steel factor (SLF), interleukin (IL)-3, and granulocyte-macrophage colony stimulating factor (GM-CSF) and transduced with the gene in two ways. CD34(3+) cells were incubated with retroviral-containing supernatant from TK-neo vector-producing cells, washed, and plated directly or resorted as CD34(3+) cells into single wells containing a single cell or 10 cells. Alternatively, CD34(3+) cells were sorted as a single cell/well and then incubated with viral supernatant. These cells were cultured with Epo, SLF, IL-3, and GM-CSF +/- G418. The TK-neo gene was introduced at very high efficiency into low numbers of or isolated single purified CD34(3+) immature hematopoietic cells without stromal cells as a source of virus or accessory cells. Proviral integration was detected in primary G418-resistant(R) colonies derived from single immature hematopoietic cells, and in cells from replated colonies derived from G418R-colony forming unit-granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) and -high proliferative potential colony forming cells (HPP-CFC). This demonstrates stable expression of the transduced gene into single purified stem/progenitor cells with replating capacity, results that should be applicable for future clinical studies that may utilize selected subsets of stem/progenitor cells for gene therapy.     

Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons

Efficient transduction of hematopoietic stem cells is a prerequisite for successful hematopoietic stem cell gene therapy. Oncoretroviral vectors are the most widely used vectors for hematopoietic gene therapy studies. However, these vectors require cell division, and thus efficient transduction of quiescent stem cells has been difficult to achieve. Lentiviral vectors can transduce non-dividing cells and therefore may be more efficient in transducing quiescent hematopoietic stem cells. We have used a competitive repopulation assay in the baboon to compare transduction of hematopoietic repopulating cells by lentiviral and oncoretroviral vectors. Baboon CD34-enriched marrow cells were transduced in the presence or absence of multiple hematopoietic growth factors using a short, 2-day, transduction protocol. Here, we show that efficient lentiviral transduction of hematopoietic repopulating cells was only achieved when cells were transduced in the presence of multiple growth factors. Using these conditions, up to 8.6% of hematopoietic repopulating cells were genetically modified by the lentiviral vector more than 1 year after transplant. Interestingly, the number of lentivirally marked cells increased over time in three of four animals. In conclusion, these results suggest that lentiviral vectors are able to tranduce multilineage hematopoietic stem cells, and thus, may provide an alternative vector system for clinical stem cell gene therapy applications.     

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。     

Drug selection of mutant methylguanine methyltransferase from different oncoretroviral backbones results in multilineage hematopoietic transgene expression in primary and secondary recipients

Optimized hematopoietic gene therapy requires vectors with strong expression in the desired target cell population and the ability to select for the expressing transduced cells. In the context of drug resistance selection of repopulating hematopoietic stem cells in the mouse, we examined tissue expression after transduced marrow transplantation of the drug selection gene, G156A mutant O6-methylguanine-DNA methyltransferase (G156A MGMT). To gain more experience with the rigor of the impact of selection on tissue-specific gene expression, we also asked whether there are expression differences between three different onco-retroviral backbones--MPSV, SF, and MFG. MGMT expression was compared after O6-benzylguanine (BG) and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) drug selection in vivo. After mice were transplanted with cells transduced with MPSV, MFG, or SF retroviral vectors expressing G156A MGMT and drug treated, nearly complete replacement by transduced progenitors was observed in the marrow. Each backbone supported MGMT expression in all four hematopoietic lineages in vivo indicating that MGMT-mediated selection is indeed robust. Expression in marrow, spleen, and thymus was very similar between the vectors and differences were most likely due to differences in gene copy number per selected cell. In primary and secondary recipients, the highest expression was observed in MFG and this was the vector that transduced at the greatest proviral copy number per cell. These data indicate that strong selection pressure using the MGMT gene to protect primary and secondary repopulating murine stem cells from the toxicity of BCNU. Regardless of the vector backbone used, multiorgan expression was observed without evidence of gene silencing. These data help establish mutant, BG-resistant MGMT as a potent selection gene for stem cell selection in vivo.     

Molecular analysis of retroviral transduction in chronic myelogenous leukemia.

We have developed a polymerase chain reaction (PCR) assay for detection of integrated retroviral transgenomes containing the neo G418 resistance gene in colonies (40 cells or more) grown in G418 selection after exposure to the neo-positive retrovirus LNL6. This assay also provides for simultaneous characterization of these colonies as belonging to a chronic myelogenous leukemic (bcr-abl positive) or nonleukemic population (bcr-abl negative). Using these techniques, we assessed transduction of the LNL6 retrovirus into the normal and leukemic cells of a blast-crisis chronic myelogenous leukemia (CML) patient. This work was designed to support the use of the LNL6 retroviral marker to help identify the origin of relapse after autologous marrow infusion. The data from these experiments show that the majority of CML blast crisis cells that, following exposure to the LNL6 virus, produce colonies under rigorous G418 selection are indeed transduced by the virus, as shown by the presence of the neo retroviral gene. Most of these colonies are also shown to be leukemic by PCR detection of the bcr-abl RNA. This demonstrates the feasibility of the study of CML marrow for retroviral marking. These procedures will be of use in establishing if relapse arises from leukemic blasts which contaminate purged autologous bone marrow infused following intensive therapy for leukemia.