靶基因调控的脐血干/祖细胞体外长期扩增与调控
파기인조공적제혈간/조세포체외장기확증여조공
Targeted expansion and regulation of genetically modified cord blood stem/progenitor cells in vitro
  • 万方数据
    中国组织工程研究与临床康复 중국조직공정연구여림상강복
    JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
    2008年 8期 1563-1567 ,共5页

    赵声明%彭明婷%顾惜春%常乃柏

    조성명%팽명정%고석춘%상내백

    酪氨酸激酶JAK2%基因治疗%脐血CD34+细胞%扩增 酪氨痠激酶JAK2%基因治療%臍血CD34+細胞%擴增
    락안산격매JAK2%기인치료%제혈CD34+세포%확증
    背景:脐血干细胞是基因治疗最理想的靶细胞之一,但基因转移率低下是目前面临的主要障碍.酪氨酸激酶JAK2在造血干/祖细胞自我更新中扮演着重要的作用,为了克服脐血基因转移率低下的障碍,根据基因调控表达技术原理,是否可开发一个可以靶向扩增JAK2基因修饰的脐血CD34+细胞体系.目的:探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和安全性. 单位:卫生部北京医院血液科.材料:实验于2003-06/2006-04在卫生部北京医院血液科实验室完成.脐血取自健康、足月、自然分娩后立即断脐的脐血.脐血由北京医院妇产科提供,产妇及家属均知情同意,实验经医学伦理委员会批准.MiniMACS磁性分离仪、免疫磁珠吸附CD34单抗购自德国Miltenyi Biotec公司, 流式细胞仪购自美国FACScalibur,人重组干细胞因子、Flt3配体、人白介素-6、粒细胞-巨噬细胞集落刺激因子、粒细胞集落刺激因子、血小板生成素为PeproTec产品,SPF级裸鼠购自北京医科大学动物中心.方法:构建逆转录病毒载体MGI-F2JAK2,内含有JAK2基因的功能催化区和两个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(2xF36v,F2)组成.AP20187可与F36v特异结合引起JAK2二聚化而激活细胞内信号传导.该载体同时含有绿色荧光蛋白报告基因,用作检测细胞增殖的标记.应用MiniMACS免疫磁珠分选系统纯化分离脐血CD34+ 细胞,用含JAK2的逆转录病毒上清转染脐血CD34+细胞.转导后的CD34+ 细胞在集落刺激因子、Flt3配体、血小板生成素、白介素-6细胞因子的联合培养条件下,以不加或加入AP20187分别作为对照组和实验组.主要观察指标:①应用流式细胞仪测定两组CD34+细胞中所含绿色荧光蛋白细胞的百分率,确定基因转移率.②扩增后的脐血祖细胞集落培养结果.③取培养10周的脐血CD34+细胞于裸鼠的胁部皮下注射,30 d后观察成瘤情况.结果:①实验组与对照组均可获得CD34+ 细胞大量扩增.随着培养时间的延长,实验组扩增的CD34+细胞GFP阳性率由基线水平逐渐上升于第11周时达到95%以上,而对照组绿色荧光蛋白报告基因阳性率逐渐下降到基线水平以下并逐渐消失.②实验组转基因CD34+ 细胞于12周后仍可产生造血祖细胞集落红系祖细胞、粒单系祖细胞、脐血多向造血祖细胞,所形成的造血祖细胞集落以粒单系祖细胞为主.③裸鼠实验无致瘤特性.结论:转染JAK2 基因的人脐血CD34+ 细胞协同其他细胞因子可以体外长期扩增脐血干祖细胞,对今后开展干细胞治疗某些遗传性血液病有潜在的应用价值.
    배경:제혈간세포시기인치료최이상적파세포지일,단기인전이솔저하시목전면림적주요장애.락안산격매JAK2재조혈간/조세포자아경신중분연착중요적작용,위료극복제혈기인전이솔저하적장애,근거기인조공표체기술원리,시부가개발일개가이파향확증JAK2기인수식적제혈CD34+세포체계.목적:탐토전기인JAK2개도적제혈간조세포장기확증조공적가행성화안전성. 단위:위생부북경의원혈액과.재료:실험우2003-06/2006-04재위생부북경의원혈액과실험실완성.제혈취자건강、족월、자연분면후립즉단제적제혈.제혈유북경의원부산과제공,산부급가속균지정동의,실험경의학윤리위원회비준.MiniMACS자성분리의、면역자주흡부CD34단항구자덕국Miltenyi Biotec공사, 류식세포의구자미국FACScalibur,인중조간세포인자、Flt3배체、인백개소-6、립세포-거서세포집락자격인자、립세포집락자격인자、혈소판생성소위PeproTec산품,SPF급라서구자북경의과대학동물중심.방법:구건역전록병독재체MGI-F2JAK2,내함유JAK2기인적공능최화구화량개여소분자파향기인합성약물(AP20187)결합적위점단백(2xF36v,F2)조성.AP20187가여F36v특이결합인기JAK2이취화이격활세포내신호전도.해재체동시함유록색형광단백보고기인,용작검측세포증식적표기.응용MiniMACS면역자주분선계통순화분리제혈CD34+ 세포,용함JAK2적역전록병독상청전염제혈CD34+세포.전도후적CD34+ 세포재집락자격인자、Flt3배체、혈소판생성소、백개소-6세포인자적연합배양조건하,이불가혹가입AP20187분별작위대조조화실험조.주요관찰지표:①응용류식세포의측정량조CD34+세포중소함록색형광단백세포적백분솔,학정기인전이솔.②확증후적제혈조세포집락배양결과.③취배양10주적제혈CD34+세포우라서적협부피하주사,30 d후관찰성류정황.결과:①실험조여대조조균가획득CD34+ 세포대량확증.수착배양시간적연장,실험조확증적CD34+세포GFP양성솔유기선수평축점상승우제11주시체도95%이상,이대조조록색형광단백보고기인양성솔축점하강도기선수평이하병축점소실.②실험조전기인CD34+ 세포우12주후잉가산생조혈조세포집락홍계조세포、립단계조세포、제혈다향조혈조세포,소형성적조혈조세포집락이립단계조세포위주.③라서실험무치류특성.결론:전염JAK2 기인적인제혈CD34+ 세포협동기타세포인자가이체외장기확증제혈간조세포,대금후개전간세포치료모사유전성혈액병유잠재적응용개치.
    BACKGROUND: Cord blood stem cells are one of ideal target cells for gene therapy, but low gene transferring rate is the main difficulty at recent. Janus kinase tyrosine 2 (JAK2) plays an important role in self-renewing of cord blood stem/progenitor cell12s. Therefore, cord blood CD34+ cell line modified by target-amplified JAK2 genes has been developed yet by using gene regulating expression technique in order to overcome low transferring rate of cord blood genes.OBJECTIVE: To investigate the feasibility and reliability of a long-term amplified regulation for cord blood stem/progenitor cells mediated by transgene JAK2. SETTING: Department of Hematology, Beijing Hospital, Ministry of Health.MATERIALS: The experiment was carried out in the Laboratory of Hematological Department, Beijing Hospital, Ministry of Health from June 2003 to April 2006. Cord blood was derived from umbilical cord which was immediately cut from healthy, full-term and natural-parturition infants and was provided by Department of Obstetrics & Gynecology, Beijing Hospital. The experiment was approved by the local ethical committee, and informed consent was obtained from expectant mothers and their relatives for the use of cord blood cells. MiniMACS magnetic separation apparatus and immunomagnetic beads adsorbing CD34 single antibody were provided by Miltenyi Biotec Company, Germany; flow cytometer by FACScalibur, USA; recombinant human stem cell factor (rhSCF), Flt3 ligand (FL), human interleukin-6 (hIL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and thrombopoeitin (TPO) by PeproTec Company; nude mice of the SPF level by Animal Center of Beijing Medical University.METHODS: Retroviral vector MGI-F2JAK2, which was composed of functional catalytic domain of JAK2 genes and two site proteins (2xF36v, F2) combined with synthetic drug (AP20187) of target gene of small molecules, was constructed. AP20187 might specially combine with F36v to cause dimerization of JAK2 so as to activate signal conduction in cells. In addition, the vector included green fluorescence protein reporter gene, which was regarded as a label to detect proliferation. MiniMACS magnetic separation apparatus was used to purify and separate cord blood CD34+ cells. While, retrovirus supernatant including JAK2 was used to transfer cord blood CD34+ cells. After transduction, CD34+ cells were cultured with stem cell factor (SCF), Flt3 ligand, TPO and IL-6 and divided into control group (not adding AP20187) and experimental group (AP20187).MAIN OUTCOME MEASURES: ① Flow cytometer was used to detect percentage of green fluorescence protein reporter gene in the CD34+ cells and to determine gene transferring rate. ② Colony culture results of cord blood stem/progenitor cells after amplification. ③ Nude mice were given subcutaneous injection of ten-week cultured cord blood CD34+ cells at costa and neoplasia was observed after 30 days. RESULTS: ① Plentiful amplification of CD34+ cells was observed in both experimental group and control group. With the culture time passing by, positive rate of gel-filtered platelet of amplified CD34+ cells in the experimental group was gradually increased based on the basic level and more than 95% in the 11th week; however, positive rate of green fluorescence protein reporter gene in the control group was gradually decreased below the basic level and disappeared finally. ② Transgenic CD34+ cells in the experimental group still could generate brust forming unit-erythroid (BFU-E), colony-forming units granulocute/monocyte (CFU-GM) and multipotential hematopoietic progenitors (CFU-Mix); especially, CFU-GM was the main cell in hemopoietic progenitor cell (HPC). ③ Nude mice did not have neoplasia. CONCLUSION: Human cord blood CD34+ cells of transferring JAK2 genes may cooperate with other cytokines to amplify cord blood stem/progenitor cells in vitro for long. Therefore, this is potentially valuable for stem cells to treat some hereditary hematologic disease.
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