双功能逆转录病毒载体介导耐药基因转染人脐血CD34+细胞能增强联合化疗抗性

目的　为探讨转染醛脱氢酶基因 (ALDH1)和多药耐药基因 (MDR1)的人脐血CD34 细胞能否同吮增强对活性环磷酰胺 ( 4 HC)和P gp转运泵靶药的抗性。方法　构建了同时含ALDH1和MDR1双耐药基因的逆转录病毒表达质粒G1Na ALDH1 IRES MDR1,经LipofectAMINE介导转染GP E86和PA317包装细胞 ,采用含长春新碱 (VCR)和 4 HC的培养基克隆选择后 ,收集重组病毒上清于单向型与双嗜型包装细胞行乒乓交互感染 ,获得PA317重组病毒生产细胞 (最高滴度达 5 6× 10 5CFU ml) ,将含ALDH1和MDR1耐药基因重组病毒上清在细胞生长因子刺激下重复感染人脐血CD34 细胞。结果　经PCR ,RT PCR ,Southernblot,Northernblot,FACS和MTT方法检测显示外源ALDH1与MDR1基因已经整合入转染靶细胞中基因组并获得有效表达 ,同时传递不同的耐药表型。经耐药基因修饰的脐血CD34 细胞对 4 HC和P gp转运泵靶药同时产生抗性 ,其IC50值分别比未转染细胞高 4倍 ( 4 HC) ,5 5倍 (柔红霉素 DNR)和 7 2倍 (VCR)。结论　双功能逆转录病毒载体介导两种不同耐药基因转染人脐血CD34 细胞能增强联合化疗抗性 ,本基因转移系统的建立为开展肿瘤基因治疗的临床研究奠定了实验基础。     

Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists

Objective To explore the role of endothelin (ET) in the pathogenesis of exercise-induced asthma (EIA), we investigated the effects of ET(B) receptor antagonists, ET-1 (11-21)fragment and N-cis-2,6-dimethylpi-peridinocardonyl-L-γ-methylleucyl-D-1-methoxycarbonyl tryptophanyl-D-norleucine (BQ788) on broncho-constriction elicited by isocapnic hyperpnea in guinea pigs. Methods Eighteen pathogen-free Hartley guinea pigs were randomly divided into three groups. A: normal saline (NS) inhalation control group (n=6), B: BQ788 group (n=6), and C: ET-1(11-21) fragment group (n=6). Guinea pigs were anesthetized with pentobarbital sodium. After measuring the basal value of lung resistance (R[L]) and dynamic compliance of the respiratory system (Cdyn), NS (0.96 ml), BQ788 (9 nmol) and ET-1(11-21)fragment (9 nmol) were inhaled. A rodent respirator with a dry 5%CO(2)-95%O(2) mixture at room temperature provided mechanical ventilation (V[T] 8 ml/animal, 100 breaths/min) for 5 min. R[L] and Cdyn of the 3 groups were measured again after isocapnic hyperpnea challenge. Results In the control group, isocapnic hyperpnea of dry gas elicited a marked increase in R[L] and decrease in Cdyn. R[L] and Cdyn of the guinea pigs from BQ788 group and ET-1(11-21)fragment group did not change significantly. Conclusion It was demonstrated that selective ET(B) receptor antagonists, ET-1(11-21) fragment and BQ788, inhibited the bronchoconstriction induced by isocapnic hyperpnea in guinea pigs. The data showed that ETs are potent constrictors of guinea pig airway smooth muscle via a direct effect on ET receptors. It was suggested that ET receptor antagonists, especially ET(B) receptor antagonist, might be beneficial in preventing EIA.     

Liposome-mediated Functional Expression of Multiple Drug Resistance Gene in Human Bone Marrow CD34＋ Cells

Myelosuppressionisanimportantdose limitingfactorformostofchemotherapeuticagentsinclinicalpractice.OverexpressionofP glycoprotein (P gp)encodedbyMDR1geneisaccompaniedbyfunctionaldrugresistance.TheexpressionofP gpisconsistent lylowinnormalbonemarrowcells,an…     

人胰岛素样生长因子-1逆转录病毒载体的构建及其在骨髓基质干细胞中的表达

目的构建人胰岛素样生长因子-1的逆转录表达载体,建立逆病毒介导的IGF-1基因转移系统。方法用逆转录-多聚酶链反应（RT-PCR）从人肝细胞克隆IGF-1的基因;经DNA测序分析证实后将目的基因插入逆转录病毒载体LXSN,制备pLXSN-IGF-1表达载体;借助阳离子脂质体转染包装细胞PA317,G418筛选阳性克隆,获取病毒上清;培养人骨髓基质干细胞,用病毒上清感染骨髓基质干细胞;采用RT-PCR及Western blot法检测目的基因在靶细胞的表达。结果经酶切电泳和DNA测序表明成功构建了IGF-1逆转录病毒表达载体,转染包装细胞后可以产生IGF-1逆转录病毒,病毒感染人骨髓基质干细胞后能够表达IGF1-1重组蛋白。结论成功建立逆转录病毒载体介导的IGF-1体外表达体系,能够快速、稳定地将IGF-1基因转入骨髓基质干细胞。     

双基因导入人脐血CD34^＋细胞共表达的调控

目的 观察携带双基因的逆转录病毒载体转导人脐血ＣＤ３４＾＋细胞后,猴病毒４０（ＳＶ４０）早启动子和内部核糖体进入位点（ＩＲＥＳ）对载体上,下游双基因共表达的影响。方法 分别构建含ＳＶ４０早启动子和ＩＲＥＳ的双基因逆转录病毒载体ｐＬＥＳＮ和ｐＬＥＩＮ,两者均携带增强型绿色荧光蛋白和新霉素抗性ｎｅｏ基因。重组载体经包装以其病毒上清感染经磁性细胞分离仪富集的人脐血ＣＤ３４＾＋细胞,而后进行集落形成细胞测     

MDR1基因修饰的自体骨髓移植后体内表达及时限的研究

目的 研究多药耐药基因1(multidrug resistanle gene 1, MDR1)转染的骨髓单个核细胞自体移植后,外源性MDR1基因在骨髓中的功能性表达及时限.方法 体外浓缩病毒上清转染法将MDR1基因导入兔骨髓单个核细胞;经大剂量环磷酰胺化疗预处理后,将转染的骨髓单个核细胞行自体骨髓移植;采用PCR法、免疫组织化学法和柔红霉素(daunorubicin, DNR)排出试验检测MDR1基因在受体骨髓中的整合及功能性表达.结果 自体骨髓移植后1～4个月,PCR法检测到外源性MDR1基因在骨髓细胞基因组中的整合;免疫组化法测得骨髓单个核细胞中P-gp阳性率分别为9.5%、8.5%、6.0%、3.5%;柔红霉素排除试验检测到定植的MDR1基因能功能性的表达.结论 转染MDR1基因的骨髓单个核细胞自体移植后,MDR1基因能定植于骨髓中并功能性的表达4个月.     

多药耐药mdr-1基因体外转染兔骨髓造血细胞及其表达与功能的研究

目的：探讨浓缩病毒上清转染法在体外将多药耐药mdr—1基因转入兔骨髓造血细胞的条件及检测转染后mdr—1基因在兔骨髓造血细胞中的表达及功能。方法：用秋水仙碱(90ng／ml)筛选含人类全长mdr—1cDNA的产病毒包装细胞PA317—HaMDR1／A1后进行细胞培养并制备浓缩病毒上清。采集新西兰大白兔骨髓并分离富含造血细胞的单个核细胞。骨髓造血细胞与浓缩病毒上清及细胞因子组合共培养。采用免疫组织化学法检测转染率，PCR法检测外源性mdr—1基因的整合，柔红霉素(daunorubicin DNR)泵出试验检测导入的mdr—1基因的功能。结果：秋水仙碱筛选后，产病毒包装细胞P糖蛋白(P-g;ucp[rpteom P-gp)表达增强：外源性mdr—1基因能成功的导入兔骨髓造血细胞，转染2日、4日、6日的转染率分别为22％、37％、39％，但转染4日组细胞生长状态最好；转入的mdr—1基因能发挥药物外排泵的功能。结论：采用浓缩病毒上清转染法能成功的将外源性mdr—1基因导人兔骨髓造血细胞中并获得稳定的功能性表达，为进一步研究mdr—1基因转染骨髓造血细胞后自体回输在大剂量化疗中对骨髓保护作用的研究提供了依据。     

Influence of recombinant retroviral vector expressing antisense TGFα on malignant phenotype of human pancreatic carcinoma cell line

ＩｎｆｌｕｅｎｃｅｏｆｒｅｃｏｍｂｉｎａｎｔｒｅｔｒｏｖｉｒａｌｖｅｃｔｏｒｅｘｐｒｅｓｉｎｇａｎｔｉｓｅｎｓｅＴＧＦαｏｎｍａｌｉｇｎａｎｔｐｈｅｎｏｔｙｐｅｏｆｈｕｍａｎｐａｎｃｒｅａｔｉｃｃａｒｃｉｎｏｍａｃｅｌｌｉｎｅＸｕＹａ许雅，ＬｉｕＴｏ...     

Influence of recombinant retroviral vector expressing antisense TGF alpha on malignant phenotype of human pancreatic carcinoma cell line.

OBJECTIVE To observe the effects of antisense TGF alpha on the growth of human pancreatic carcinoma cell line cells.

METHODS A recombinant retroviral vector expressing antisense TGF alpha was constructed, and transfected the ecotropic packaging cell line psi-2 with lipofectin. After the amphotropic packaging cell line PA317 was transfected with the virus supernatant of psi-2, the replication-defective, amphotropic retroviral supernatant was used to infect human pancreatic carcinoma cell line PC-7. Following puromycin selection, puromycin-resistant colonies were pooled and expanded to a cell line PC-7/AS-TGF alpha.

RESULTS The retroviral integration in the genomes of psi-2, PA317 and transformant PC-7 cells was confirmed by Southern blot hybridization. Northern blot hybridization showed a down regulation of endogenous TGF alpha in PC-7/AS-TGF alpha cell line. The high levels of growth inhibition and reduction of 3H-TdR incorporation in PC-7/AS-TGF alpha were evident. Also, the soft agar colony-formation and tumorigenicity in nude mice were significantly suppressed by antisense TGF alpha.

CONCLUSIONS The antisense TGF alpha expressing vector can block the target gene expression, suppress the cell growth and partially reverse the malignant phenotype of pancreatic carcinoma cells.

     

研究慢性粒细胞性白血病病理发生中下游信号传导和分子调控的干细胞模型

获得慢性粒细胞白血病人原代干祖细胞模型 ,旨在研究慢粒白血病病理发生中信号传导的分子机制。方法　转导b3a2bcr/ablcDNA到正常人CD34 细胞中构建人原代慢性粒细胞性白血病模型。采用细胞免疫组化测定了p2 10 BCR/ABL在CD34 细胞中的表达 ;细胞粘附、迁移实验进行模型鉴定 ;FACS法测定了p2 10 BCR/ABL转导及对照CD34 细胞p2 7kip和MDR 1Pgp的表达。结果　相对于对照的CD34 细胞 ,转导了bcr/abl的CD34 细胞对纤粘连蛋白 (fibronectin ,FN)的粘附性下降 ;而在FN上的迁移能力增强 ;在低浓度细胞因子或血清条件下表现为凋亡延迟 ;细胞的粒系克隆形成单位数量明显增多 ,这一模型再现了原代慢粒白血病的异常表型特征 ,并以此模型发现p2 10 BCR/ABL转染CD34 细胞上调了p2 7Kip水平并可诱导MDR 1Pgp异常表达。结论　该模型适用于慢粒病理发生中的信号分子传导及分子调控研究 ,提示了慢粒耐药的分子机制。     

Construction of Retroviral Vectors Containing Rat Fas Ligand Gene and FasL Expression Mediated by the Vectors in Hepatocellular Carcinoma Cells

Apoptosis is a form of cell death differentfrom necrosis.It is believed to be the essentialphysiologic process underlying controlled or pro-grammed celldeletion during phenomena such asembryonic development,cell differentiation,ortissue turnover[1] .Fas antigen or Apo- 1 ( recent-ly designated as CD95 ) is now known to belongto the TNF/ nerve growth factor receptor fami-ly.Itis a48- ku type transmembrane glycopro-tein.Fasreceptormakesan importantcontribu-tion to determining the lymphocyte l…     

VIP对人脐血CD34+细胞向肝系分化的影响初探

目的探讨血管活性肠肽(VIP)对人脐血造血干细胞(HSC)向肝系分化的影响.方法采用免疫磁分选技术纯化人脐血CD34+细胞,流式细胞术鉴定纯度;VIP和混合因子(包括VIP、EGF、HGF)作用CD34+细胞后,酶联免疫化学法测定CD34+细胞及上清AFP水平,免疫细胞化学法检测CD34+细胞上肝系标志AFP、白蛋白(ALB)、细胞角质素19(CK-19)的表达,Western blot方法检测CD34+细胞上ALB表达;巢式RT-PCR方法检测CD34+细胞上AFP、ALB的mRNA表达,随机选送ALB产物测序.结果免疫组化结果显示CD34+细胞上不表达CK-19蛋白,但有AFP和ALB蛋白表达;VIP作用CD34+细胞14 d后, 细胞内的AFP质量浓度(165.00±8.51 pg/mL)较对照组(270.00±11.37 pg/mL)显著下降(P<0.05).Western blot显示VIP作用后CD34+细胞内ALB蛋白表达有减弱趋势.人脐血CD34+细胞表达了AFP mRNA和ALB mRNA,随机选取ALB产物测序与GeneBank中ALB基因序列完全相同.结论人脐血CD34+细胞表达肝系标志AFP、ALB,虽未见CK-19表达,但仍提示造血干细胞有向肝细胞横向分化的可能.VIP降低了人脐血CD34+细胞AFP及ALB表达.这种对造血干细胞内胚层细胞标志物表达的抑制,提示VIP对HSC向肝细胞横向分化具有抑制作用.     

Liposome-mediated functional expression of multiple drug resistance gene in human bone marrow CD34<Superscript>+</Superscript> cells

Summary The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells. CAO Wenjing, female, born in 1968, Doctor in Charge     

OK-432对脐带血初始CD4~+T细胞向Th1细胞分化的诱导作用

目的 :探讨链球菌 OK- 432 ( OK- 432 )的抗肿瘤机理。方法 :采用磁珠法分离脐带血初始CD4+ T细胞 ,经 OK- 432诱导培养后 ,应用酶联免疫吸附测定 ( ELISA)法测定培养细胞上清中细胞因子的水平。结果 :OK- 432刺激脐带初始 CD4+ T细胞分泌 IFN-γ的数量明显增加 ,而 IL- 4产生明显减少。结论 :OK- 432可诱导脐带血初始 CD4+ T细胞向 Th1细胞分化     

血管内皮抑素基因转染人脐带血CD34+造血干细胞的实验研究

目的：探讨人血管内皮抑素(内抑素)基因转染人脐带血CD34^ 造血干细胞的方法及检测。方法：用逆转录病毒载体pLNCX，将人内抑素基因导人人脐带血CD34^ 造血干细胞。应用PCR及Western bolt方法检测人内抑素基因的转染和表达。结果：PCR证实，转染内抑素基因的人脐带血CD34^ 造血干细胞基因组中有550bp人内抑素特异性片段，Western bolt分析示人内抑素基因在转染细胞中获得稳定表达和分泌。结论：人内抑素基因可转染到人脐带血造血干细胞中并稳定表达。     

成人脂肪源间充质干细胞培养上清对人脐血间充质干细胞体外培养及扩增的支持作用

目的：研究人脂肪间充质干细胞培养上清对人脐带血（HUCB）中所含有间充质干细胞（MSCs）的影响。方法：取脐带血,肝素抗凝,Percoll淋巴细胞分离液分离出单个核细胞,低糖DMEM培养获得贴壁细胞层。与人脂肪源间充质干细胞培养上清共孵育。流式细胞仪检测表面抗原。结果：脐带血的单个核细胞与人脂肪源间充质干细胞培养上清共孵育经体外培养贴壁后出现形似纤维状细胞形态并表达与MSCs相关的抗原（CD13,CD44,CD71,CD166）,但不表达造血细胞抗原（CD34,CD45）,这与源于骨髓的MSCs一致。结论：人脐血间充质干细胞与脂肪源间充质干细胞培养上清共孵育,对脐血间充质干细胞体外分离培养及扩增有支持作用。     

IL-11基因修饰的基质细胞对造血细胞体外扩增的影响

目的　研究造血因子白介素 11(IL 11)基因修饰的基质细胞对造血细胞体外扩增的影响。方法　采用逆转录病毒载体将IL 11基因转入基质细胞HFCL ,用Northernblot检测基质细胞HFCLIL 11基因的表达 ,用流式细胞仪检测IL 11基因修饰的HFCL支持的脐血CD3 4 + 造血细胞体外扩增中表型为CD3 4 + CD3 8- 早期祖细胞和表型为CD3 4 + CD41+ 巨核系定向祖细胞的比例。结果　基质细胞HFCL能够表达逆转录病毒介导的IL 11基因 ,并且在这种IL 11基因修饰的基质细胞支持下 ,脐血CD3 4 + 造血细胞经过7d扩增 ,扩增细胞中表型为CD3 4 + CD3 8- 的早期祖细胞和表型为CD3 4 + CD41+ 巨核系祖细胞的比例分别为 (1.62± 0 .2 3 ) %、(9.9±1.1) % ,高于未转基因HFCL的 (0 .8± 0 .2 3 ) %、(6.5± 1.8) % ,而在相同条件下细胞因子支持的扩增细胞中则分别为 (0 .19±0 .14 ) %、(6.0± 1.1) %。结论　基质细胞能够表达经逆转录病毒载体介导的IL 11基因 ,而且经IL 11基因修饰的基质细胞能显著促进CD3 4 + CD3 8- 的早期祖细胞和CD3 4 + CD41+ 巨核系祖细胞扩增。     

Transwell培养体系中人AGM区基质细胞支持脐血CD34+细胞造血

[目的]探讨Transwell系统中人胚主动脉-性腺-中肾(AGM)区基质细胞对脐带血造血干/祖细胞的造血能力长期维持及扩增的作用.[方法]采用免疫磁珠方法分离人脐带血CD34 细胞,接种于底层铺有人AGM区基质细胞的Transwell培养板的Inserts中,非接触共培养7～35 d,每星期取样检测细胞总数,用半固体培养基分析CFU-C及HPP-CFU集落形成数,流式细胞术检测CD34 、CD34 CD38-细胞百分率.[结果]在Transwell中非接触共培养条件下,人AGM区基质细胞培养体系较胚胎躯干基质细胞和无饲养层培养体系对有核细胞总数、CFC和CD34 细胞均具有明显的扩增作用,共培养14 d的CD34 、CD34 CD38-造血干/祖细胞均获得峰值扩增(2.62±0.8和2.15±1.04,P<0.05),而MNC总数和CFC均在21 d获得最大扩增(32.5±4.3和4.2±0.6倍,P<0.05).克隆分析发现CFU-Mix、CFU-GM、BFU-E在共培养4～5星期后均仍然可见.原始祖细胞HPP-CFC在3星期也得到2.23倍的扩增,较空白及hFT对照组均有显著性差异(P<0.05),并在共培养35 d后仍可见HPP-CFU集落形成.[结论]人AGM区基质细胞hAGM-S3/hAGM-S4均具有造血支持作用,在非接触共培养条件下中可长期维持脐血中造血干/祖细胞的多系造血能力和高增殖潜能达21～35 d,对脐血CD34 /CD34 CD38-细胞数也有一定程度的扩增作用.     

转hLIF基因逆转录病毒载体饲养层细胞的建立及其对脐血CD34＋造血干/祖细胞的扩增作用

目的建立转人白血病抑制因子（hLIF）基因逆转录病毒载体的饲养层细胞,并观察其对CD34＋造血干/祖细胞（HSPC）的扩增作用。方法建立转hLIF基因逆转录病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因的表达;采用免疫磁珠法分离脐带血CD34＋HSPC,流式细胞术检测其纯度;将CD34＋HSPC与饲养层细胞共培养,流式细胞术检测各组增殖效果。结果建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实均有目的基因表达,免疫磁珠法分离的CD34＋HSPC纯度可达（95.6±2.58）%,与饲养层细胞共培养后CD34＋HSPC可扩增8.74倍,表面黏附分子CXCR4和CD54表达量仍较高。结论建立的转hLIF基因饲养层细胞对CD34＋HSPC有一定的扩增作用,且延缓其分化。     

mdr—1基因转移可提高肺癌细胞对阿霉素的抗性

以逆转录病毒介导，将人全长mdr-1cDNA导入人肺腺癌细胞系GLC82中，经G418和阿霉素筛选，挑选出3个阳性克隆。用mdr-1cDNA的一种特异引物在3个克隆中都扩增到1条167bp的带，表明mdr-1基因cDNA已稳定地整合在染色体基因组中。原位杂交显示转染细胞的mdr-1转录升高，免疫细胞化学发现转染细胞中P170糖蛋白呈弱阳性。MTT药敏实验表明3个克隆对阿霉素的抗药性分别是GLC细胞的6.4、7.0和8.8倍。提示在mdr-1基因cDNA转染的细胞中，mdr-1基因低表达足以大大提高细胞对药物的耐受性。