以逆转病毒介导对人脐血CD34+造血细胞进行人MDR1基因转染的实验研究

目的：探讨逆转录病毒介导的MDR1基因转导入脐血CD34^ 细胞的最佳方法，为MDR1基因转导的临床应用打基础。方法：用磷酸钙沉淀法将含有人全长MDR1cDNA的逆转录病毒载体pHaMDR1/A转到包培育细胞PA317中，建立产病毒细胞系，以人脐血中分离的CD34^ 造血干/祖细胞为靶细胞，在体外进行基因转染，转导的条件为：与含病毒的上清液共培养12天，每天更换病毒上清液，上清液中加入IL-3，IL-6和SCF三种造血生长因子（HGF），转染后用集落培养法测定对COL的耐药性，用PCR检测14-17天所形成集落的MDR1 cDNA，计算转染率，用免疫组化法检测P170的阳性程度，并观察不同时间间隔加HGF对脐血CD34^ 细胞的扩增和转染的影响。结果：脐血CD34^ 细胞转染阳性为86.4％，P170的阳性率为77.0％，77.1％的集落对6ng/ml的COL耐受，57.4％的集落对7ng/ml的COL耐受。结论：此转染系统既能有较好的转导效果，也有较好的扩增效果，有一定的临床实用价值。     

不同方法转染人外周血淋巴细胞效率比较

目的 采用不同方法转染原代人外周血淋巴细胞,以寻找一种简便、高效的淋巴细胞转染方法.方法 采用聚蔗糖-泛影葡胺(Ficoll-Hypaque)分离法从健康人外周血中分离出单个核细胞,锥虫蓝检测细胞存活率.单个核细胞在6孔板内培养2h后吸出悬浮的淋巴细胞至24孔板中继续培养.分别采用电穿孔介导载体质粒(PEGFP-N1)转染活化的淋巴细胞、慢病毒(LVGFP)单次感染及慢病毒重复感染方法分别转染静息或活化的淋巴细胞,在荧光显微镜下观察感染后不同时间点细胞绿色荧光蛋白(GFP)表达情况,同时收集细胞用流式细胞术检测GFP阳性细胞的比例,比较不同条件下慢病毒感染淋巴细胞的效率.结果 通过Ficoll-Hypaque分离的单个核细胞的纯度可达95％,其活细胞率＞95％,获得的淋巴细胞占90％～ 95％,形态较均一.“2 100V、10 ms、1个脉冲”电穿孔淋巴细胞后可见散在荧光,但随着时间推移荧光逐渐减弱,72 h基本看不到荧光.慢病毒单次感染静息期淋巴细胞48 h后无绿色荧光,流式细胞术检测GFP阳性细胞＜1％.慢病毒单次感染增殖期淋巴细胞可见绿色荧光,且随着病毒浓度增大,荧光逐渐增强,GFP阳性细胞在1％～5％之间.慢病毒重复感染增殖期淋巴细胞可见较强绿色荧光,GFP阳性细胞在5％～10％左右,随着时间推移荧光逐渐增强.结论 慢病毒重复感染增殖期淋巴细胞,能有效地将外源基因导入淋巴细胞内稳定表达.     

OSM对黑色素瘤的抑制作用与基因-放射治疗效果

目的：通过细菌内同源重组,制备含绿荧光（GFP）基因的CD腺病毒,初步观察其体外杀瘤作用。方法：先构建含CD基因的腺病毒梭载体pAdTrack-CMV-CD,与骨架载体pAdEasy-1在细菌内重组为pAd-CD,经293细胞包装为增殖缺陷性腺病毒,体外感染人结肠癌细胞株LoVo,并给予前药5－FC,观察其体外杀瘤效果。结果：pAdTrack-CMV-CD和同源重组的pAd-CD经酶切鉴定正确,pAd-CD转染293细胞,包装扩散腺病毒后,感染LoVo细胞,予5－FC治疗,4d后在荧光温微镜下观察到转染LoVo和未转染细胞胞均被杀死,并且 这种旁观者效应不依赖于细胞间接解。结论：细菌内质粒同源重组法可以高效制备含绿荧光（GFP)基因的CD腺病毒,体外腺病毒介导CD／5－FC可以杀伤肿瘤细胞,其旁观者效应不依赖于细胞间接触。     

可调控重组腺相关病毒对MCF-7细胞基因转染效率及表达的研究

 目的 探讨Tet调控下自杀基因HSVtk重组腺相关病毒载体（rAAV/HSVtk/Tet-On）对人乳腺癌细胞株（MCF-7）的作用。方法 将HSVtk和Tet-On基因分别定向克隆入腺相关病毒质粒pAAV MCS中,构建重组腺相关病毒载体pAAV/TRE/HSVtk/Tet-On,与辅助质粒pAAV-RC、pHelper和包装细胞293进行包装、纯化。用β半乳糖苷酶原位染色法检测报告基因rAAV/LacZ在MCF-7中的表达,计算其基因转染效率。采用斑点杂交、RT-PCR检测MCF-7细胞基因组中HSVtk基因整合、病毒滴度及其表达。结果 rAAV/HSVtk/Tet-On病毒滴度为2.38×10^11particle/ml,LacZ基因在感染的MCF-7中能持续表达,感染效率为20%～30%。纯化后的重组腺相关病毒感染MCF-7后,能将目的基因转移到靶细胞中,并在Dox诱导下,使GCV对rAAV感染的MCF-7细胞具有明显的杀伤作用。结论 HSVtk/Tet-On自杀基因调控系统可通过重组腺相关病毒载体成功转染人乳腺癌细胞株MCF-7,使其HSVtkmRNA表达增加,在Dox诱导下,GCV对rAAV（≥10⁴v.p/cell）感染的MCF-7细胞具有明显的杀伤作用。     

腺相关病毒载体及其介导的自杀基因疗法

自杀基因疗法是肿瘤治疗的方法之一。腺相关病毒（AAV）载体介导的自杀基因疗法尤其备受关注。通过对AAV载体的选择改建，调控外源基因的有效表达，提高基因转染的效率，充分发挥自杀基因的杀伤作用及旁观者效应来达到治疗肿瘤的目的。本文就AAV载体及其介导的自杀基因疗法方面的进展作一综述。     

多药耐药基因转染人骨髓间充质干细胞对其化疗保护作用的研究

目的：慢病毒介导的多药耐药基因（mdr1）转染人骨髓间充质干细胞（hBMSCs）,探讨其对化疗药物的耐受性。方法：采用percoll密度离心法自骨髓中分离MSCs并进行纯化和扩增,流式细胞仪鉴定。克隆mdr1基因,构建慢病毒载体,命名为TG-005-mdr1／tap。脂质体转染法转染携带mdr1基因的逆转录病毒载体导入293T包装细胞,获得的病毒上清感染hMSCs,GTP荧光技术和western—blot检测mdr1基因的表达,MTT法检测hMSCs对化疗药物的耐受性。结果：成功构建携带mdr1基因的慢病毒载体,采用GTP荧光技术检测慢病毒转染率达83．44％,western—blot检测mdr1编码产物P—Pg蛋白高表达,转染后hMSCs对化疗药物的耐受性明显高于对照组。结论：转染mdr1基因后的hMSCs具有化疗耐受性,可为化疗骨髓防护提供新思路。     

siMDR1重组慢病毒载体的构建及其对A549和A549／DDP细胞的影响

背景与目的:肿瘤细胞的多药耐药性(multidrug resistance,MDR)是导致化疗失败的最主要原因,也是癌症术后复发及转移的主要原因.本研究旨在探讨MDR1基因RNA干扰(RNA interference,RNAi)重组慢病毒对人肺腺癌A549细胞和对顺铂耐药的A549/DDP细胞的MDR1基因的抑制作用.方法:设计获得针对MDR1的短发卡状RNA(small hair pin RNA,shRNA)的寡核苷酸序列,退火后插入线性化的pSUPER载体(H1启动子下游).把构建获得的载体转染A549细胞,RT-PCR法检测其对MDR1的干扰作用,筛选确定MDR1基因RNAi有效靶序列;合成靶序列的01igo DNA,与含启动子和绿色荧光蛋白(green fluorescent protein,GFP)的PTM载体连接产生PTM-siMDR1慢病毒载体;用重组慢病毒体外感染A549和A549/DDP细胞.结果:成功构建可以表达针对MDR1的发卡状RNAi,能有效的下调MDR1的表达.成功构建获得针对MDR1基因的慢病毒载体PTM-siMDR1,包装慢病毒,浓缩病毒悬液的滴度为0.5×109TU/mL.感染PTM-siMDRI的A549/DDP细胞对顺铂的敏感性明显提高,逆转倍数达3.81倍.结论:siiDR1重组慢病毒载体感染A549/DDP细胞,能明显改善其对顺铂的耐药性.     

腺相关病毒-CD40配体转导入肺癌细胞

目的 探讨重组腺相关病毒（rAAV）不同血清型载体对人肺癌细胞的转导效率,比较自身互补AAV（scAAV）与传统单链AAV（ssAAV）载体的转导效率,遴选理想载体携带CD40L配体（CD40L）转导入肺癌细胞,并观察转CD40L肺癌细胞对树突状细胞（DCs）的刺激作用。方法 以荧光显微镜和流式细胞术测定rAAV—GFP及rAAV—CD40L感染肺癌细胞的基因转导效率;转导CD40L的肺癌细胞与DCs共培养后,ELISA测定培养上清中IL-12水平。结果 AAV血清型5（AAV2／5）的转导效率明显高于AAV2／1、AAV2／2,AAV2／6、AAV2／7、AAV2／8、AAV2／9和AAV2／10,scAAV2／5高于ssAAV2／5。且化疗药卡铂能提高转导效率;转导CIMOL的肺癌细胞能促进DCs分泌IL-12。结论 scAAV2／5可能为肺癌基因治疗的理想载体,转CD40L的肺癌细胞对DCs的刺激作用明显增强,scAAV2／5-CD40L有望成为肺癌新的基因治疗药物。     

MDR1基因下调逆转人白血病阿霉素耐药细胞株K562/ADM的耐药性

目的：探讨RNA干扰（RNAi）人MDR1基因对人白血病阿霉素耐药细胞株K562/ADM耐药性的影响。方法：应用针对人MDR1基因的RNAi质粒pENTRTM/U6-MDR1转染人白血病阿霉素耐药细胞株K562/ADM和亲本细胞株K562,48 h后实时荧光定量PCR检测MDR1 mRNA表达,流式细胞术检测P-gp蛋白表达和P-gp功能,MTT法检测细胞对ADM的耐药性。结果：与未转染细胞相比,K562/ADM耐药细胞pENTRTM/U6-MDR1组的MDR1 mRNA和P-gp蛋白表达和功能均显著下降（ P <0.05）,对阿霉素的耐药性显著降低（ P <0.05）。结论：MDR1基因下调可逆转人白血病阿霉素耐药细胞株对阿霉素的耐药性。     

一种用流式细胞术检测基因转移效率的新方法

背景与目的：基因转移效率一直以来都是用表达报告基因的细胞（显色或发荧光）占细胞总数的百分比（即转染率）来表达,此百分比是由研究者借助显微镜（或荧光显微镜）计数得出来的。该方法（本文称为人工计数法）存在客观性、准确性不够和工作量大、且不能同时检测基因表达效率等问题。本研究拟寻找一种客观、准确、简便地检测基因转移和表达效率的新方法。方法：以绿色荧光蛋白（GFP）基因为目的基因、重组腺病毒（AdCMV/GFP)为基因载体,用人工计数法测定其对肝癌细胞株HepG2的转染率;用流式细胞术（flow cytometry,FCM)测定其对肝癌细胞株HepG2、Bel7402、Hep3B、SMMC7721和鼻咽癌细胞株CNE－2的转染效率和GFP（绿色荧光蛋白）基因的表达效率（以荧光指数表示）。结果：以FCM测得AdCMV/GFP对HepG2的转染率与载体量呈对数相关（与人工法相似,但前者略高）,荧光指数与载体量呈线性相关（γ＝0．9984）,其它细胞株的结果与HepG2相似。结论：采用FCM法测定基因转移效率克服了主观因素的影响,测定结果客观准确,可以用于测定基因载体对真核细胞的转染效率,或研究转录调控元件的功能。     

多药耐药基因增强骨髓细胞对抗癌药物毒性的抵御能力

目的　探讨多药耐药基因 (mdr1基因 )的骨髓细胞转染增强骨髓细胞对抗癌药的抵御能力。方法　我们用mdr1基因表达质粒pHaMDR1/A ,通过脂质体介导 ,转染小鼠和人骨髓细胞 ,并采用骨髓移植建立小鼠动物模型。结果　成功的将mdr1基因导入了小鼠和人骨髓细胞并获得了表达 ,其细胞已对阿霉素、足叶乙甙 (Vp 16 )、长春新碱和秋水仙碱等产生了明显的抗性。同时 ,转染mdr1基因小鼠骨髓细胞的移植 ,在受体小鼠重建造血功能 ,受体小鼠骨髓细胞亦对抗癌药具有抗性。结论　从小鼠骨髓细胞、人骨髓细胞和小鼠体内模型 3个水平证实了mdr1基因的骨髓细胞转染可增强骨髓细胞对抗癌药的抵御能力     

PRELIMINARY STUDY OF RETROVIRAL MEDIATED TRANSFER OF THE HUMAN mdr-1 GENE INTO MURINE AND HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS

ＰＲＥＬＩＭＩＮＡＲＹＳＴＵＤＹＯＦＲＥＴＲＯＶＩＲＡＬＭＥＤＩＡＴＥＤＴＲＡＮＳＦＥＲＯＦＴＨＥＨＵＭＡＮｍｄｒ１ＧＥＮＥＩＮＴＯＭＵＲＩＮＥＡＮＤＨＵＭＡＮＨＥＭＡＴＯＰＯＩＥＴＩＣＳＴＥＭ／ＰＲＯＧＥＮＩＴＯＲＣＥＬＬＳＦｅｎｇＫａｉ冯凯Ｐｅ...     

RNAi对白血病细胞mdr-1基因和多药耐药表型的影响

目的:探讨RNA干扰技术(RNAi)对慢性粒细胞白血病急变细胞系K562/AO2细胞mdr1基因的抑制和耐药表型的逆转作用。方法:选择合成封闭mdr-1基因的小干扰序列(si-MDR1),以1个碱基突变的si-MDR1-mut为对照序列,在脂质体介导下转染至K562/AO2细胞系。RT-PCR和Western blot检测mdr1 mRNA及P-gp蛋白水平,流式细胞术分析细胞内柔红霉素(daunorubicin,DNR)积累量,并以四甲基唑蓝快速比色法(MTT)反映K562/AO2对阿霉素、长春新碱、足叶乙甙药物敏感性的变化。结果:实验证实该序列能高效封闭K562/AO2细胞内mdr-1基因表达,增加细胞内化疗药物DNR积累量,增强K562/AO2细胞对阿霉素、长春新碱、足叶乙甙的敏感性。结论:RNAi可以通过抑制mdr1基因表达,逆转K562/AO2细胞耐药表型。     

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% +/- 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% +/- 6.53% of the infected T47D cells and 41.52% +/- 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100-500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.     

Improvement of Transduction Efficiency of Recombinant Adeno-associated Virus Vector by Entrapment in Multilamellar Liposomes

Recombinant adeno-associated virus (AAV) has attracted considerable interest as a potential vector for human gene therapy, but its transduction efficiency is quite low. The present study demonstrated AAV vector-associated liposomes to be more effective for in vitro gene transfer to human glioma cells than are liposomes containing plasmid DNA. Using vector-associated liposomes increased the transduction efficiency more than 10-fold compared to liposomes containing plasmid DNA and more than 6-fold compared to AAV alone. From these results, AAV vector-associated liposomes appear to be a good candidate for in vivo gene delivery to human gliomas.     

Directed evolution of adeno-associated virus for glioma cell transduction

Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10⁴ genome copies/cell), and at low doses it was 1.45–1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.     

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.     

Efficient gene transfer of CD40 ligand into ovarian carcinoma cells with a recombinant adeno-associated virus vector

Recombinant adeno-associated virus type 2 (rAAV) has many properties of an ideal vector for gene therapy: broad spectrum of susceptible cells, efficient gene transfer, persistent transgene expression in vivo, and no indiction of vector-related toxicity. Ovarian carcinoma cell lines, however, were previously reported to be quite resistant to rAAV transduction. Using an optimized adenovirus-free packaging system, highly purified rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and for mCD40 ligand (AAV/CD40L) were generated. Their transduction efficiency in ovarian carcinoma cell lines was assessed with and without irradiation prior to infection. As measured by flow cytometry, transgene expression in up to 92% of cells was achieved with AAV/EGFP. gamma-irradiation (20 Gy) significantly increased the transduction rates up to 3.5-fold in cell lines with low susceptibility to AAV infection. The aquired capability of AAV/CD40L transduced tumor cells to activate dendritic cells was demonstrated in a second step. Dendritic cells were generated from human peripheral blood monocytes and maturized by stimulation with IL-4 and GM-CSF. Co-cultivation of mCD40L transgenic tumor cells with these dendritic cells resulted in strong ELISA-determined expression of IL-12 as an indicator of dendritic cell activation. We conclude that transduction of tumor cells with rAAV encoding mCD40L is a promising strategy for tumor immunotherapy which may be further developed to a vaccination approach with transgenic ovarian carcinoma cells generated by ex vivo transduction.