Expression of MDR1 by Normal Bone Marrow Cells and its Implication for Leukemic Hematopoiesis

Expression of MDR1 is a well-characterized mechanism leading to resistance of tumor cells to drugs like vinca-alkaloids, anthracyclines, and epipodophyllotoxins. In hematopoiesis, recent data indicate that not only leukemic cells, but also some populations of normal hematopoietic cells, particularly CD34+ progenitor cells as well as peripheral blood lymphocytes, express a functional multidrug-resistant phenotype. Among CD34+ cells, we found evidence that myeloid committed precursor cells (CD34+/CD33+) have lower levels of MDR1 expression than earlier CD34+ cell populations, but there was no difference in MDR 1 expression between CD34+/HLA-DR- and CD34+/HLA-DR+ sub-populations. During normal myeloid differentiation, MDR1 expression is down-regulated, which is similar to our observations in acute myelogenous leukemia (AML): MDR 1 expression was only rarely detected in acute promyelocytic leukemia, which was in contrast to other subtypes of AML; also, within leukemic subpopulations of the same patient, higher MDR 1 levels were correlated with a more immature immunophenotype. Regarding regulation of MDR 1 expression, we did not observe changes of MDR 1 expression in normal CD34+ cells in response to various cytokines. However, in 2 patients with AML treated with interleukin-3 and granulocyte-colony stimulating factor, respectively, a significant down-regulation of MDR1 expression was found after 24 hours. In conclusion, there is evidence that the pattern of MDR 1 expression observed in leukemias reflects the distribution of MDR1 in normal hematopoiesis. In contrast to normal CD34+ cells, leukemic cells from some AML patients can respond to cytokines with a down-regulation of MDR 1, which may contribute to response to cytokine/chemotherapy combinations.     

Current Status of Retroviral Vector Mediated Gene Transfer into Human Hematopoietic Stem Cells

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs has not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.     

Current Status of Retroviral Vector Mediated Gene Transfer into Human Hematopoietic Stem Cells

Genetic modification of hematopoietic stem cells (HSCs) has been proposed as a treatment strategy for a variety of hematologic diseases, tracking marked cells or conferring resistance to chemotherapeutic agents. Despite early enthusiasm, the results of clinical studies involving gene transfer into HSCs have not resulted in therapeutic benefits for the vast majority of treated patients. This review describes the limitations and advances that have been made in the areas of gene transfer vectors, identification of the appropriate HSCs to target for genetic modifications and the methods used to perform gene transfer.     

TC-1基质细胞系促进逆转录病毒介导的小鼠骨髓造血细胞基因转移的实验研究

目的:摸索逆转录病毒介导的理想的造血细胞基因转染体系.方法:以人多药耐药基因1(mdr-1)及增强型绿色荧光蛋白基因(EGFP)为报告基因,比较了小鼠骨髓细胞与病毒包装细胞共培养、与骨髓基质细胞系TC-l共培养及单纯上清感染等不同体系的转染效率.结果:与单纯上清转染法相比,TC-1基质细胞可明显提高外源基因的转染效率.3组中,以与病毒包装细胞共培养组转染效率最高.结论:骨髓基质细胞介导基因转染的方法,既可促进靶基因的转染效率,又可避免重组辅助病毒引起的感染,具有较好的临床应用前景.     

Multipotent and Committed CD34+ Cells in Bone Marrow Transplantation

In order to study the role of CD34⁺ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34⁺ cells were also performed to evaluate their in vitro hematopoietic function. CD34⁺ cells were detectable in bone marrow cells on day 14. More than 80% of CD34⁺ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34⁺ cells. In normal bone marrow cells, CD34⁺, CD33⁺ cells amounted to about 40% of CD34⁺ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34⁺, CD33⁻ cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34⁺, CD33⁺ cells as committed progenitors and CD34⁺, CD33⁻ cells as multipotent stem cells have distinctive biological behaviors in BMT.     

Ad-EGFP-MDR1转染对小鼠骨髓细胞的影响

目的：通过研究Ad-EGFP-MDR1转染的骨髓细胞的细胞周期,凋亡相关蛋白表达和增殖分化有无改变,探讨基因修饰的骨髓细胞用于治疗的可行性。方法：用重组腺病毒Ad-EGFP-MDR1感染小鼠骨髓细胞,采用体外集落培养观察转染骨髓细胞与未转染细胞的增殖能力。采用免疫印迹检测两组细胞有无凋亡相关蛋白Bax、Caspase-9、p53的表达差异。使用流式细胞仪检测两组细胞的细胞周期有无差异。通过锥虫蓝拒染率判断细胞存活情况。结果：与对照组骨髓细胞比较,转染Ad-EGFP-MDR1的骨髓细胞体外集落形成差异无统计学意义。在转染的骨髓细胞和对照细胞中均未检测到凋亡相关基因Bax,Caspase-9,p53的表达,转染骨髓细胞与未转染对照骨髓细胞比较细胞周期差异无统计学意义（P>0.05）。锥虫蓝染色两组细胞的拒染率差异无统计学意义（P>0.05）。结论：骨髓细胞转染重组腺病毒Ad-EGFP-MDR1后其细胞凋亡相关基因,细胞增殖分化能力及细胞活性无明显变化,有利于进一步开展基因修饰骨髓细胞体内回输和体内安全性检测。     

IL-3基因修饰的骨髓基质细胞辅助大剂量化疗后造血功能恢复

为研究基因治疗在造血功能损伤后恢复中的应用,本课题以携带小鼠IL-3基因的复制缺陷型重组腺病毒载体转染骨髓基质细胞,对大剂量化疗后的小鼠进行脾内移植观察造血功能的恢复情况.结果表明,缺陷型腺病毒载体能有效地转染小鼠原代骨髓基质细胞,转染效率在80%以上(MOI=10);基因修饰的骨髓基质细胞体外分泌IL-3的水平可达110U/ml/10~6细胞/24小时;在大剂量环磷酰胺治疗后脾内移植IL-3基因修饰的基质细胞能有效地升高实验小鼠外周血白细胞总数;病理检测发现IL-3基因修饰的基质细胞治疗组小鼠脾脏和骨髓中细胞增生较其它组明显活跃;经IL-3基因修饰的基质细胞治疗组小鼠脾淋巴细胞对ConA反应明显增强.结果提示IL-3基因修饰的骨髓基质细胞体内移植对大剂量化疗后机体造血与免疫功能的恢复都有较好的促进作用.     

腺病毒介导的KDR启动子-胸苷激酶系统对血管内皮细胞的选择性杀伤作用

目的研究腺病毒介导的KDR启动子-胸苷激酶系统对血管内皮细胞的选择性杀伤作用.方法应用PCR克隆出人KDR基因启动子序列-225 bp～+127 bp,以AdEasy system为载体,构建携带受KDR启动子或CMV启动子调控tk基因表达的2种重组腺病毒质粒pAdKDR-tk和pAdCMV-tk,在293细胞中包装、扩增后,体外感染表达KDR的脐静脉血管内皮细胞系HUVEC和不表达KDR的肝癌细胞系HepG2,并给予不同浓度的丙氧鸟苷(GCV)处理,5 d后收集存活细胞并计数.结果产生的病毒滴度均为5×109pfu/ml.在MOI为100、GCV为50μg/ml条件下,AdKDR-tk转染HUVEC后细胞生存率下降(31.49%±6.42%),AdKDR-tk转染HepG2后细胞生存率为76.57%±3.49%,而转染AdCMV-tk的2细胞系生存率均显著下降,细胞生存率分别为22.24%±3.77%(HUVEC)和26.53%±6.84%(HepG2).结论KDR基因启动子可调控HSV-tk在血管内皮细胞中特异性表达,为进一步开展靶向肿瘤血管内皮的自杀基因治疗研究奠定了基础.     

重组腺病毒介导mIL-4基因转染的G422胶质母细胞瘤体外生物学特性及体内致瘤性的改变

以重组的复制缺陷性腺病毒为载体将鼠IL-4基因转染G422小鼠脑胶质母细胞瘤细胞,观察其体外生物学特性和体内致瘤性的变化。结果显示:基因转染的肿瘤细胞有目的基因mRNA的表达,分泌高水平的mIL-4,和野生型G422细胞相比,其体外的细胞增殖能力无显著差异,而接种皮下后肿瘤生长延缓、小鼠的存活期延长。     

Ultrastructural Features of CD34+ Hematopoietic Progenitor Cells from Bone Marrow, Peripheral Blood and Umbilical Cord Blood

Hematopoietic progenitor cells from different sources have been widely characterized, but their ultrastructural morphology has never been described in detail. In this study, imunomag-netically separated CD34⁺ cells from normal bone marrow (BM), mobilized peripheral blood (PBSC) and human umbilical cord blood (CB) were studied by transmission electron microscopy (TEM) using a cytochemical method which reveals endogenous myelo-peroxidase (MPO) activity. This technique is particularly suited for detecting early signs of the myeloid commitment. The CD34⁺ cells from PBSC were morphologically very homogeneous and 94.7 ± 4.5% of these cells were MPO^-: these ultrastructural features are generally considered typical of immature cells. The CD34⁺ BM cells were instead more heterogeneous, with 24.6 ± 7.4% showing intense MPO activity. The ultrastructural characteristics of CB cells fell between those observed in PBSC and BM, but there was a high percentage of morphologically immature cells with no evidence of MPO activity (about 83%). The number of apoptotic cells within samples from different sources was also examined both by TEM and flow cytometry. The percentage of apoptotic cells was 0.7% in PBSC, 2.3% in BM, 2.9% in CB from vaginal delivery and 11.6% in CB from cesarean section. These observations confirm the relative phenotypic immaturity of CB in comparison with BM cells; they also suggest that CB collected after cesarean section may be associated with reduced stem cells viability.     

腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究

编委会 Editorial Board名誉主编 Honorary Editor-in-chief吴孟超 Wu Meng chao(Second Military Medical University,Shanghai 200433学术顾问 Academic Advisers巴德年 Ba Denian(Chinese Academy of Medical Sciences,Beijing 100730)刘新垣 Liu Xinyuan(Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031)吴旻 WuMin(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)汤钊猷 Tang Zhaoyou(Shanghai Medical University,Shanghai 200032)主编 Editor-in-chief张友会 Zhang Youhui(Cancer Institute,Chinese Academy of Medical Sciences,Beijing,100021)副主编 Associate Editor-in-chief崔正言 Cul Zhenyan(Department of Immunology,Shandong Academy of Medical Sciences,Jinan 250001)钱振超 Qian Zhenchao(Department of Patho-physiology,Dalian Medical University,Dalian 116027)何球藻 He Qiuzao(Department of Immunology,Shanghai Medical University,Shanghai 200032)董志伟 Dong Zhiwei(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)常务副主编 Managing Editor-in-chief     

Immunomagnetic Purging of T8 Cells from Peripheral Blood and Bone Marrow: A Preclinical Study for Allogeneic Bone Marrow Transplantation

Methodology for T-cell subset depletion in allogeneic BMT was developed using anti-T8 antibody and magnetic microspheres. Anti-T8 antibodies bound effectively to the cytotoxic/suppressor T-cell subset and were subsequently removed by anti-mouse IgG coated magnetic microspheres. Following optimization of antibodyxell binding, microsphere:cell ratios and flow rate, T8-cells in peripheral blood were effectively removed (95-99% depletion), with an overall recovery of 37.8% ± 5.8%, and with little, if any, differential damage to either stem cells or to NK-cells. When T8 cells were removed from bone marrow mononuclear cells, an average recovery of 76% ± 8.5% was observed, with 90% ± 4.3% removal of T8 cells. This method was developed both for peripheral blood and bone marrow and could therefore be readily used in a clinical purge setting for allogeneic BMT.     

转染人突变dhfr基因的小鼠骨髓细胞对小鼠造血功能的长期保护作用

目的:将转染了人突变dhfr基因的第二代小鼠骨髓,移植给经致死剂量照射的第三代小鼠,观察该基因对小鼠造血功能的长期保护作用.方法:分离存活的第二代小鼠骨髓有核细胞,直接移植给经致死剂量照射的同系小鼠,以MTX筛选,观察小鼠血象和生存率变化,PCR和Southem印迹杂交分析目的基因在小鼠染色体DNA中的整合与表达情况.结果:在大剂量MTX筛选下,实验组小鼠造血功能逐渐恢复,对照组3周内全部死亡.实验组生存率和生存期明显高于对照组,但较前两代生存率低.PCR和Southern印迹分析结果提示,实验组脾脏和肝脏组织中均检测到前病毒标志基因neo~R和dhfr基因的特异务带.结论:转染了人突变dhfr基因的第二代小鼠骨髓,能有效地重建经致死剂量照射的第三代小鼠造血功能.保护骨髓免遭大剂量MTX所致的严重骨髓抑制,dhfr基因在小鼠基因组DNA中的稳定整合是这种长期保护作用的物质基础.     

E1A基因对人肺腺癌细胞化疗敏感性的影响 *

目的：探讨Ｅ１Ａ基因对人肺腺癌细胞的化疗增敏作用。方法：通过脂质体介导将Ｅ１Ａ基因导入人肺腺癌细胞系Ａ－ｎｉｐ９７３,经Ｇ４１８筛选获得稳定表达Ｅ１Ａ的转染细胞（Ａｎｉｐ９７３－Ｅ１Ａ）。用不同浓度顺铂、泰素及ＶＰ１６等化疗药物处理细胞,观察Ｅ１Ａ基因对人肺腺癌细胞的化疗增敏作用。结果：Ａｎｉｐ９７３－Ｅ１Ａ细胞对顺铂、泰素的敏感性显著增加,顺铂的ＩＣ５０值减少了７倍,并且敏感性随时间的延长而增加。但对ＶＰ１６,Ｅ１Ａ基因的稳定表达在该细胞系未显示增敏作用。免疫细胞化学染色显示,Ｅ１Ａ基因抑制了ＨＥＲ－２／ｎｅｕ的表达。结论：Ｅ１Ａ基因能增加人肺腺癌细胞对顺铂、泰素等化疗药物的敏感性。该作用可能与Ｅ１Ａ基因抑制ＨＥＲ－２／ｎｅｕ的表达有关。为在恶性肿瘤治疗上化疗和基因治疗联合应用的可能性提供了实验依据。     

基因工程人催乳素(rhPRL)促进骨髓移植小鼠的造血重建

选用BACB/c鼠经致死放疗后行同基因骨髓移植(SBMT),在小鼠SBMT后腹腔注射rhPRL(10μg/次,隔日进行,共10次).结果显示,小鼠骨髓和脾脏中造血前体细胞(CFU-C和BFU-E)比例增加,外周血中WBC、RBC和PLT亦均有明显的时相提前,该剂量rhPRL无任何病理损伤和增长体重的作用,提示rhPRL在促进BMT造血重建有应用前景.     

双特异抗体导向骨髓中活化T细胞清除骨髓中肿瘤细胞

先用抗CD3单抗和IL-2激活肺癌患者骨髓中的T淋巴细胞,然后按不同比例将人肺癌细胞株PC84045细胞掺入到骨髓细胞中,再加入能同时识别CD3抗原和人肺癌细胞表面抗原的双特异抗体CD3-ALTO4,在IL-2和IL-3存在下共同培养3d后测试.在T细胞与PC84045癌细胞之比为8:1时,对PC84045肺癌细胞净化效率为4Logs,16;1时为5Logs,并在NC裸鼠体内证实清除效果是确切的.净化后的骨髓CFU-GM、BFU-E收获率均>85%(P>0.05;P>0.05);而且净化后的骨髓中的T细胞仍保持着对PC84045肺癌细胞很强的杀伤能力(P<0.01).双特异抗体CD3-ALTO4能有效地导向骨髓中活化T细胞清除污染的肿瘤细胞,造血干细胞无明显损伤.本文方法处理的骨髓细胞还保持着高度特异的细胞毒活性,移植这样的骨髓细胞将会在体内进一步清除化疗残存的肿瘤细胞,加速移植后免疫功能再建,降低复发.     

In situ Gene Transfer and Suicide Gene Therapy of Gastric Cancer Induced by N-Ethyl-N'-nitro-N-nitrosoguanidine in Dogs

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV-TK) gene was introduced in situ into cancer tissues in the stomach of three dog, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus-producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus-producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAG lacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV-TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV-TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAG lacZ gene transfer, but was found after high-titer Ad.CAGHSV-TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.     

腺病毒介导的肝癌自杀基因疗法的实验研究

本文观察了胞嘧啶脱氨酶(CD)基因/5-氟胞嘧啶(5FC)自杀基因疗法对肝癌模型的治疗作用。以CMV启动子调控CD基因的重组腺病毒载体AdexCMV.CD在体外能有效转染人肝癌细胞株SMMC-7721和HepG2,转染后的细胞体外生长能力无明显变化,对5FC的敏感性明显增高。当以AFP上游调控序列驱动CD基因的重组腺病毒载体AdexAFP.CD分别转染SMMC-7721和HepG2时,CD基因能在HepG2中表达,使HepG2对5FC的敏感性增高,但不能使SMMC-7721的生长受到5FC的抑制。[~3H]TdR掺入法观察体外旁观者效应时发现,当细胞总数中转染细胞数超过20％时,其生长明显被5FC抑制。将AdexCMV.CD直接注射入裸鼠接种SMMC-7721细胞形成的皮下肿瘤中,并全身应用5FC后,与对照组相比,肿瘤大小在开始治疗后第8天约缩小3倍,第18天约缩小4倍,以上结果表明,腺病毒介导的CD/5FC自杀基因系统能在体内、外有效地抑制肝癌的生长。以AFP上游调控序列驱动CD基因的腺病毒载体介导的基因转染能在AFP阳性的肝癌细胞中特异表达。     

人骨髓间充质干细胞在新生小鼠脑中的植入和分化

背景与目的:探讨人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)在体内向神经细胞分化的可能性.材料与方法:将标记绿色荧光蛋白的人骨髓间充质干细胞植入到新生3 d的小鼠侧脑室中,分别于移植后0 d,9 d和14 d处死受体鼠,取其脑组织,4%多聚甲醛固定后行冠状面冰冻切片,荧光显微镜下观察hMSCs的植入,激光共聚焦显微镜检测植入细胞神经特异性蛋白的表达,定位双重标记的植入细胞.结果:受体小鼠脑中均可检出植入的细胞,此类细胞表达神经元细胞特异的微管相关蛋白β-Ⅲ-Tubulin(Tuj1)、微管相关蛋白2(MAP2),一些细胞表达神经胶质细胞特异的胶质原纤维酸性蛋白(GFAP).结论:hMSCs植入后受到脑组织特定微环境的影响,在体内可以向神经细胞分化并参与到发育的神经系统中.     

共刺激分子B7和肿瘤免疫

为了更好地使IL-3在体内发挥其造血促进作用和免疫增强作用，于本实验中建立了IL-3基因疗法的小鼠模型，并动态观察了其外周血细胞数量的变化．通过基因转染、G418抗性筛选、有限稀释及IL-3活性测定，从16株NIH3T3成纤维细胞克隆中选出一株分泌IL-3高达2416U／ml的克隆株。将其移植入小鼠腹腔后，实验小鼠血清可检测出一定水平的IL-3并维持至半个月之久。实验小鼠外周血自细胞，特别是中性粒细胞数量显著增加，血小板也有不同程度的升高。该实验结果表明成纤维细胞途径能将IL-3基因携至体内进行有效表达并发挥显著的生物学作用。