MDR1基因转染小鼠骨髓干细胞在化疗中骨髓保护作用的研究

多药耐药基因 (ＭＤＲ1 )属于人类正常防御性基因 ,其编码产物Ｐ 糖蛋白 (Ｐ ｇｐ)在部分肿瘤细胞的高表达是肿瘤细胞对化疗药物产生多药耐药的主要原因。实验应用本课题组所构建的含有人全序列ＭＤＲ1ｃＤＮＡ逆转录病毒载体ＰＨａＭＤＲ1 /Ａ1的产病毒包装细胞ＰＡ3 1 7 ＨａＭＤＲ1 /Ａ1 ,其产生病毒颗粒的滴度约为 3 3 1× 1 0 5 ＣＦＵ/ｍｌ,上清液感染法将ＭＤＲ1基因转染Ｂａｌｂ/ｃ小鼠骨髓干细胞 ,转染率达 40 %左右 ,经ＰＣＲ、免疫组化、流式细胞术检测 ,在基因、蛋白质及细胞水平证实ＭＤＲ1基因已经转入小鼠骨髓细胞并具有药…     

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。     

IFN-α逆转白血病细胞耐药性的实验研究

目的：了解干扰素－α（ＩＦＮ－α）逆转白血病细胞多药耐药性（ＭＤＲ）的作用和可能机制。方法：采用链亲和素－胶体金原位杂交（ＳＡＧ－ＩＳＨ）检测了ＩＦＮ－α孵育前后的５２例白血病患者骨髓细胞的多药耐药基因（ＭＤＲ１）表达,用荧光分光光度法测定了ＩＦＮ－α孵育前后的５２例骨髓细胞内柔红霉素（ＤＮＲ）浓度。结果：全部５２例患者中２４例（４６．２％）ＭＤＲ１阳性,初治组与复发难治组相比相差显著。ＭＤＲ１阳性者骨髓细胞内柔红霉素浓度与阴性者相差显著。ＩＦＮ－α孵育后的ＭＤＲ１阳性率与孵育前相差不显著,但经ＩＦＮ－α孵育前后的ＭＤＲ１阳性者的骨髓细胞内ＤＮＲ浓度相差显著,阴性者亦然。结论：ＩＦＮ－α能增加白血病细胞内ＤＮＲ浓度,其逆转ＭＤＲ环节不在ＭＤＲ１基因水平上,而在其他耐药途径上     

急性淋巴细胞白血病微量残留病的PCR监测

①目的探讨急性淋巴细胞白血病（ＡＬＬ）微量残留病（ＭＲＤ）检测的临床意义。②方法应用筑巢式多聚酶链反应（ｎｅｓｔｅｄＰＣＲ）对３２例ＡＬＬ进行Ｔ细胞受体（ＴＣＲ）Ｖδ２Ｄδ３基因重排检测。③结果１８例Ｂ系ＡＬＬ中有１５例（７２．２％）、４例Ｔ系ＡＬＬ中有１例（２５．０％）存在ＴＣＲＶδ２Ｄδ３基因重排。同时对１６例进行３９例次微量残留病动态监测，其中６例ＭＲＤ－ＰＣＲ阴性病人随访５．１７～１６．１７年，无１例复发；１０例ＭＲＤ－ＰＣＲ阳性者，２例分别于阳性后０．２５，１．００年骨髓复发，１例有复发倾向，余７例随访４．３３～６．２５年无复发。④结论ＭＲＤ－ＰＣＲ阴性者预后良好，可望长期生存，治疗时应以ＭＲＤ－ＰＣＲ转阴为停止化疗的可靠指标，对ＭＲＤ－ＰＣＲ阳性者应定期监测ＭＲＤ变化，结合病人情况进行综合评价。     

粒—巨噬细胞集落刺激因子cDNA在造血祖细胞中的表达

目的；探讨逆转录病毒介导的ＧＭ－ＣＳＦｃＤＮＡ在造血母细胞中转移与表达。方法：采用电穿孔法将小鼠ＧＭ－ＣＳＦｃＤＮＡ重组逆转录病毒ｐＬＸＳＮ／ＧＭ和空载体ｐＬＸＳＮ转染病毒包装细胞ＰＡ３１７，再将含有ＧＭ－ＣＳＦ重组逆转录病毒的细胞培养上清液感染富含造血干祖细胞群体，采用ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析培养细胞基因组中外源基因的整合，用Ｄｅｘｔｅｒ培养体系检测培养３周的各组细胞增殖数，结果：在     

RT—PCR技术检测原发上皮性卵巢癌MDR1基因表达

目的建立适于临床检测原发上皮性卵巢癌ＭＤＲ１ｍＲＮＡ表达方法。方法应用半定量ＲＴ－ＰＣＲ技术对３７例原发上皮性卵巢癌、１０例正常卵巢和２０例外周血新鲜标本ＭＤＲ１基因进行了检测。结果原发上皮性卵巢癌标本中ＭＤＲ１基因阳性表达率为３０％,而正常卵巢和外周血标本则无ＭＤＲ１基因表达。结论ＲＴ－ＰＣＲ技术检测原发上皮性卵巢癌ＭＤＲ１基因表达具有灵敏度高、相对定量和结果可靠的优点,适于临床应用。     

IFN—α逆转白血病细胞耐药性的实验研究

目的：了解干扰素－α（ＩＦＮ－α）逆转白血病细胞多药耐药性（ＭＤＲ）的作用和可能机制。方法：采用链亲和素－胶体金原位杂交（ＳＡＧ－ＩＳＨ）检测了ＩＦＮ－α孵育前后的５２例白血病患者骨髓细胞的多药耐药基因（ＭＤＲ１）的表达,用荧光分光光度法测定了ＩＦＮ－α孵育前后的５２例骨髓细胞内柔红霉素（ＤＮＲ）浓度,结果：全部５２例患者中２４例（４６．２％）ＭＤＲ１阳性,初治组与复发难治组相比相差显著。ＭＤＲ１     

多发性骨髓瘤骨髓和外周血IgH基因重排的研究

多发性骨髓瘤（ＭＭ）的恶性克隆缺乏特异免疫表型和基因标志，其克隆起源尚未明确。为了探讨ＭＭ恶性克隆及其前体细胞检测方法，我们采用ＰＣＲ技术分析了ＭＭ骨髓和外周血ＩｇＨ基因重排。１．研究对象：确诊ＭＭ患者４２例，其中ＩｇＧ型２０例，ＩｇＡ型８例，ＩｇＤ型４例，ＩｇＭ型１例，轻链病９例。ＤｕｒＩｅ临床分期：Ⅰ期９例，Ⅱ期１６例，Ⅲ期１７例。另选１０例反应性浆细胞增多症和１２例正常人做对照。２．ＰＣＲ检测：骨髓涂片和外周血基因组ＤＮＡ抽提按常规进行。引物扩增片段为ＩｇＨ第三互补决定区（ＣＤＲ３）序列，…     

急性髓性白血病降钙素基因甲基化定量研究

为探讨降钙素（ＣＴ）基因高甲基化能否作为急性髓性白血病（ＡＭＬ）微小残留病（ＭＲＤ）的检测指标。采用设有内、外参照的聚合酶链反应（ＰＣＲ），结合限制内切酶和激光扫描技术，检测了４０例ＡＭＬ患者骨髓细胞的ＣＴ基因５′端甲基化率（ＣＴＭＲ）。结果：初发ＡＭＬ患者ＣＴＭＲ为５２５％±１９３７％，显著高于对照组（８１％±３７３％；Ｐ＜００００５），部分缓解（ＰＲ）组显著高于对照组而显著低于初发组；完全缓解（ＣＲ）组显著高于对照组而与ＰＲ组差别无显著性；ＡＭＬ患者的ＣＴＭＲ与骨髓白血病细胞数呈显著正相关（ｒ＝０．７１５，Ｐ＜０００１），ＣＲ组１６例中４例ＣＴＭＲ在５０％以上，分别于２、４、４５和９个月后复发。结果提示：ＣＴＭＲ有可能成为检测ＡＭＬ微小残留病的有用指标     

喉癌患者多药耐药基因,多药耐药相关蛋白基因的表达及临床意义

应用逆转录－多聚酶链式反应（ＲＴ－ＰＣＲ）技术,检测了３５例喉癌患者ＭＤＲ－１ｍＲＮＡ、ＭＲＰｍＲＮＡ基因的表达,同时,应用免疫组化ＳＡＢＣ法检测了６１例患者ＭＤＲ－１基因产物Ｐ－ｇｐ。喉癌组织中ＭＤＲ－１ｍＲＮＡ及Ｐ－ｇｐ的阳性表达率分别为３７．１％（１３／３５）和３４．４％（２１／６１）;３５例ＲＴ－ＰＣＲ检测标本中,两项指标间有一定的相关性（Ｐ〈０．０１）。晚期患者（Ｔ３－４,１８／１０）,     

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.     

Gene Transfer into Hematopoietic Cells of Mouse and Its in vivo Expression after Transplantation

Genetransferintohematopoieticcellshasfoundmanyapplicationinexperimentalresearchesaswellasclinicalpractice['1.Atpresent,thestrategyofgenetransferusedmostcommonlyistheretrovirusvectorsystem[2].However,theuseofretrovirusvectorssuffersfromseveraldrawbacks:(1)thegenerationandmanufacturingofretrovirusvectorsarecomplexandnotinexpensive;(2)generalsafetyconcernsareassociatedwiththeuseofanyviral--basevectorsystem,includingreplicationdefectivemurineretrovirusvectorsj(3)retrovirusvectorscurrentlyusedareab…     

二氢叶酸还原酶基因在肿瘤生物治疗中的作用

目的 :为了保护骨髓免受或少受化疗药物的损伤 ,用转染人类突变二氢叶酸还原酶 (DHFR)基因反转录病毒载体 ,研究其耐药特性及在小鼠造血细胞的表达和对造血细胞的保护作用。方法 :将含有DHFR基因的病毒上清转染小鼠骨髓造血细胞 ,将转染后的骨髓细胞从尾静脉回输给经致死量 (9Gy)照射的小鼠 ,用MTX和G4 18筛选。结果 :经G4 18和MTX筛选均可得到阳性克隆 ,实验组小鼠白细胞计数和红细胞压积逐渐恢复正常 ,而对照组小鼠 30d内全部死亡。结论 :经初步研究表明 ,该反转录病毒载体能成功转染小鼠造血细胞 ,并表达目的基因 ,使在MTX筛选下重建小鼠造血功能。     

Gene transfer into hematopoietic cells of mouse and itsin vivo expression after transplantation

Summary We have shown previously that high-efficient gene transfer can be attained in primary hematopoietic cells using liposome-mediated gene transfer strategy. In order to examine the stability of gene expression mediated by this gene transduction protocol, we observed the expression of marker genein vivo by using bone marrow transplantation (BMT) to engraft lethally irradiated mouse with the genetically modified hematopoietic cells. The results showed that the mouse transplanted with appropriated number of transduced cells remained alive and healthy. The PCR analysis and G418 selection of the spleen colonies and bone marrow cells isolated from lethally irradiated animals 15 days and 30 days after injection of genetically modified bone marrow cells showed that the progeny cells of the transduced hematopoietic stem cells still contained and expressed the transduced genes, suggesting that the hematopoietic system is at least partially re-constructed by the stem cells with marker gene and that the stable expression of foreign genesin vivo can be attained by using this easy and harmless transduction protocol. These findings provide experimental basis for clinician to further investigate the biology of marrow reconstruction and the mechanism of leukemia relapse after BMT. This project was supported by the grant of Scientific Research Foundation of Public Health Ministry (320. 2430-330).     

反转录病毒载体介导的人β—珠蛋白基因在小鼠体内整合和表达的？…

OBJECTIVE: To examine the in vivo properties of retroviral recombinants carrying partially deleted human beta-globin gene (delta beta) and truncated erythroid enhancer (292 bp and 341 bp of 5'HS2) at the mRNA levels following short- and long-term reconstitution in mice with infected marrow cells. METHODS: First ecotropic virus producer cell lines with higher virus titers were isolated using "ping-pong" procedures. Then the human beta-globin gene was transferred into murine hematopoietic progenitor cells and the integration and expression of transferred gene were analyzed by southern blot and RNase protection assay or RT-PCR. RESULTS: The virus titers of both recombinants increased obviously after "ping-pong" procedures. The transferred human beta-globin gene was detected in murine CFU-S12 and the expression level was about 0.5%-5% of endogenous mouse alpha-globin gene. In 3 of 14 mice surviving long-term transplanted with bone marrow cells transduced with high-titer virus, bone marrow, spleen and thymus from two mice and bone marrow and spleen from another mouse contained the intact proviral genome. Long-term expression of the transferred gene was seen in one mouse at level of 7% of endogenous murine alpha-globin gene. CONCLUSIONS: The transferred human beta-globin gene can stably integrate into murine hematopoietic stem cells mediated by retroviral vectors and express in an erythroid-specific manner.     

Sry监测体外扩增造血细胞植入效果的实验研究

目的：探讨Y染色体性别决定基因(Sry)作为评价和追踪造血细胞移植后植入状态检测指标的可行性。方法：根据Sry DNA序列设计引物及制备探针，然后进行PCR检测、斑点杂交和原位杂交．动态追踪经不同条件下体外扩增的纯系雄性小鼠BMMNC回输至雌性小鼠后的植入状态。结果：PCR结果显示在各移植组小鼠的骨髓有核细胞、脾细胞及外周血白细胞中均有Y染色体特异性序列的存在；斑点杂交结果显示在各回输组间并无明显差别，但用脾脏组织作原位杂交的结果则发现基质细胞支持扩增回输组的阳性颗粒数目与输注新鲜细胞组相似，但略少于雄性小鼠；而输注细胞因子扩增细胞实验组小鼠则明显少于雄性动物的阳性对照标本和基质细胞支持扩增回输组。结论：(1)经重复检测表明上述方法的重复性好，结果可信．可作为检测性别不同时造血干细胞移植效果的一种检测手段；(2)证实基质细胞支持下的体外扩增的造血细胞具有较好的植入能力。     

Experiments on Gene Transferring to Primary Hematopoietic Cells by Liposome

GenetransferintohematopoieticcelIshasmanyexperimentalaswellasclinicalapplications['].Atpresenttime,thestrategyofgenetransferusedmostlyistheretroviralvectorsystem['].However,theuseofretroviralvectorssuffersfromseveraldrawbacks:(l)thegenerationandmanufacturingofretroviralvectorsarecomplexandnotinexpensive;(2)generalsafetyconcernsareassociatedwiththeuseofanyviral--basevectorssystem,includingrepli-cationdefectivemurineretroviraIvectors;(3)retro-viralvectorscurrent1yusedareabletotransduceon1ydividi…     

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.     

Effect of Ligustrazine on Bone Marrow Microenvironment and Signal Transducti on Path in Irradiation Injured Mice

Ｗｅｈａｖｅｒｅｐｏｒｔｅｄ ｐｒｅｖｉｏｕｓｌｙｔｈａｔｌｉｇｕｓｔｒａｚｉｎｅｃｏｕｌｄｐｒｏｍｏｔｅｈｅｍａｔｏｐｏｉｅｓｉｓｔｈｒｏｕｇｈｉｍｐｒｏｖｉｎｇｂｏｎｅｍａｒｒｏｗｍｉｃｒｏｅｎｖｉｒｏｎｍｅｎｔａｎｄｅｎｈａｎｃｉｎｇａｄｈｅｓｉｖｅ