【作者】 杨应斌；

【导师】 蔡绍皙；

【作者基本信息】 重庆大学 ， 生物医学工程， 2010， 博士

【摘要】 背景:人类骨髓干细胞(human bone marrow stem cells, hBMSC)移植给许多病人带来了希望的同时也带来了风险。干细胞治疗所带来的所有风险中,最严重而且最引人注目的是干细胞植入体内后发生恶性转化而致肿瘤的形成。因而制定一个干细胞植入后的可控制策略就成了各干细胞研究团队和临床医学研究者迫在眉睫要解决的问题。然而,制定这样的策略却不可避免地面临着许多挑战,包括高效肿瘤组织特特异性启动子的设计、获取大量hBMSC源性恶转细胞用于启动子的功能验证及体内外细胞毒性实验、恶性转化细胞异种移植成活率较低等。迄今为止仍然没有一个切实可行的方法来预防移入hBMSC发生致瘤性转化。目的:1.提高端粒酶启动子在hBMSC源性恶性转化细胞靶向转录活性的顺式修饰探讨,获取一种在端粒酶阳性细胞中具有高特异性和较强转录活性的hTERT启动子,为下一步预防干细胞发生恶性转化及靶向清除植入hBMSC源性恶性转化细胞打下基础;2.探寻一个在体外获取足够数量hBMSC源性恶性转化细胞的方法;3.建立一个为预防植入hBMSC发生恶性转化以及靶向清除发生恶性转化研究模型。方法:1. c-Myc结合元件对hTERT启动子的修饰:用化学合成方法在野生型hTERT(WThTERT)核心启动子片段(编码蛋白起始子ATG上游-268 bp～-10 bp)的3′端接入三个E-box序列,构建成修饰型hTERT(Mod1hTERT)启动子;分别用WThTERT和Mod1hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS(端粒酶阴性)、以及人骨髓干细胞(hBMSC)中表达,比较并分析不同启动子在端粒酶阳性与阴性细胞中的表达强度。2.源于hBMSC的恶性转化细胞的体外扩增:从骨髓中分离培养人骨髓干细胞,并用流式细胞仪进行表型鉴定;然后用慢病毒转染和致瘤化学诱导剂BPDE处理共同诱导骨髓干细胞发生恶性转化;用PALA进行IC50筛选纯化瘤变细胞克隆;最后再用PCR、RT-PCR、Western blot等方法对慢病毒转染、细胞周期调控相关基因表达进行分析。3.下调hTERT启动子在hBMSC中转录活性的修饰:在Mod1hTERT启动子的上游接入4个骨髓锌指蛋白-2(MZF-2)转录因子结合元件,形成新的修饰型启动子命名为Mod2hTERT启动子;分别从大肠杆菌K12和质粒pEGFP-N3中通过聚合酶链式反应合成胞嘧啶转氨酶(CD)基因和绿色荧光蛋白(EGFP)基因;用化学合成法分别制备野生型hTERT、Mod1hTERT及Mod2hTERT启动子;将EGFP和荧光素酶(luciferase)报告基因分别亚克隆入慢病毒载体pLenti6/V5-D-TOP中;将各启动子片段取代pLenti6/V5-D-TOPO中的CMV启动子片段;将重组慢病毒载体包装成病毒颗粒后转染正常人骨髓间充质干细胞(hBMSC)、慢病毒转染及化学致瘤剂BPDE处理后的人骨髓间充质干细胞(BR-hBMSC) ,以及PALA抗性的BR-hBMSC(PBR-hBMSC);通过荧光相差显微镜观察EGFP表达和荧光素酶分析kit检测荧光素酶活性(luciferase activity)。4. 5-FC对hBMSC源性恶性转化细胞的细胞毒性实验:分别用不同浓度梯度的5-FC来处理体外培养的转入WThTERT-luciferase-IRES-CD、Mod1hTERT-luciferase-IRES-CD、Mod2hTERT-luciferase-IRES-CD、CMV-luciferase-IRES-CD及prmoter(lack)-luciferase-IRES-CD融合基因的各组PBR-hBMSC细胞;用MTT法在体外测定不同浓度梯度5-FC对各组PBR-hBMSC的细胞毒性;转入WThTERT-luciferase-IRES-CD、Mod2hTERT-luciferase-IRES-CD、以及CMV-luciferase-IRES-CD的转基因PBR-hBMSC植入裸鼠的皮下一周后,腹腔注射5-FC 5㎎/天,并用活体荧光成像系统对植入细胞的生长状况进行观测。结果:1. c-Myc结合元件对hTERT启动子的修饰:在Mod1hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及hBMSC细胞组中没有观测到EGFP的表达;进一步用荧光素酶活性对启动子的转录活性进行定量分析表明,在端粒酶阳性的293FT、HepGⅡ、SGC7901和U2OS细胞株中,Mod1hTERT启动子调控下的荧光素酶活性要高于WThTERT启动子组(P<0.01),而在端粒酶阴性的原代人成纤维细胞hBMSC及骨肉瘤细胞U2OS细胞中,Mod1hTERT启动子与WThTERT启动子的转录活性无显著性差别(P>0.05)。2.源于hBMSC恶性转化细胞的体外扩增: PCR结果表明,hBMSC在慢病毒介导下可以获得外源的遗传性表型;RT-PCR检测结果表明,细胞周期调控相关基因c-myc、bcl-2表达显著上调(p<0.01),p53及mzf-2则显著下调(p<0.01)。3.下调hTERT启动子在hBMSC中转录活性的修饰:通过荧光相差显微镜观察结果表明,启动子Mod1hTERT和Mod2hTERT在PBR-hBMSC(慢病毒转染、BPDE处理后,具有PALA抗性的hBMSC)中的表达较强而在hBMSC和BR-hBMSC(慢病毒转染hBMSC)中只有微弱的表达;启动子CMV驱动的EGFP基因在所有的细胞中都有很强的表达; Luciferase activity分析结果表明,在293FT及PBR-hBMSC细胞中,Mod1hTERT及Mod2hTERT启动子的转录活性要显著高于WT-hTERT启动子的转录活性(Sig.<0.05),而在hBMSC及BR-hBMSC(慢病毒转染hBMSC)中,Mod2hTERT的转录活性得到了有效的抑制(Sig.<0.05)。4. 5-FC对hBMSC源性恶性转化细胞的细胞毒性实验: MTT实验结果表明,当5-FC浓度100μM时,在Mod1hTERT细胞组中存活细胞占5.79%,Mod2hTERT细胞组中占6.53%, CMV细胞组中只有1.97%的细胞存活;而在相同条件下实验阴性对照组prmoter(lack)-luciferase-IRES-CD中,存活的细胞达到96.27%;活体荧光成像系统检测结果表明,在给药时间为O周时WThTERT-luciferase-IRES-CD细胞组的最大荧光信号比Mod2-luciferase-IRES-CD细胞组及CMV-luciferase-IRES-CD细胞组低4.33及5.29倍;而给药2至此周后,各组间的荧光信号强度差异不显著。给药4至周后,Mod2-luciferase-IRES-CD细胞组及CMV-luciferase-IRES-CD细胞组的荧光信号基本消失,WThTERT-luciferase-IRES-CD细胞组的荧光信号仍然存在。结论:1.在hTERT核心启动子的3′端增加E-box元件的数量可以提高hTERT核心启动子序列的肿瘤靶向转录活性。2. hBMSC在BPDE和慢病毒共同诱导后,用PALA的IC50筛选可以得到瘤变细胞克隆。3.野生型hTERT启动子经过c-Myc作用元件E-box进行修饰后,可以有效提高hTERT启动子在端粒酶阳性细胞中的转录活性.而经过修饰MZF-2结合位点修饰后,可以有效降低hTERT启动子在MZF-2表达阳性的正常hBMSC中的转录活性。4. c-Myc及MZF-2结合元件修饰的Mod2hTERT启动子在恶性转化的骨髓干细胞中有较强的靶向转录活性,同时该启动子驱动CD及luciferase表达的PBR-hBMSC在体内外能被5-FC特异性清除。终上所述,本研究不仅为骨髓干细胞源性的恶性转化细胞清除提供了一个实验方法,而且也为以后的干细胞临床移植研究提供了一个安全性研究策略。更多还原

【Abstract】 Background: To many patients, transplantation of human bone marrow stem cells not only brings hopes, but also brings the risks. Of all of the risks of stem cell therapy, the most serious, and the risk that has gotten the most attention, is the risk for tumor formation. Human bone marrow stem cells do not exhibit the telomerase activation, but tumor cells will do. It is the responsibility of the community of stem cell scientists and clinicians to develop strategies to control the therapies after transplantation. However, this strategy must be faced many challenges, including high efficiency of tumor-targeting promoter design of mechanisms, obtaining a number of hBMSC-derived malignant cells for functional identification of designed promoters and tests of cytotoxicity in vitro and in vivo, and low survival rate of malignant cells after heterogenic implantation, etc. Until now, a reliable method has not been found to prevent neoplastic transformation of engrafted hBMSC.Purpose: 1. Investigate the improvement of targeting transcriptional activities in hBMSC-derived malignant cells; 2. Seek a method for obtaining enough number of hBMSC-derived malignant cells; 3. In order to establish a research model for prevention of malignant transformation and targeting eradication of malignant cells derived from hBMSC.Methods:1. Core hTERT promoter modified by c-Myc binding element: wild-type core hTERT (WThTERT) promoter fragment (from -268 to -10 upstream of the initiating ATG start codon) and the modified hTERT (Mod1hTERT) promoter fragment (a triplet of E-boxes were ligated to the 3′terminal of the WThTERT promoter) were generated through synthesis. The two reporter genes of enhanced green fluorescent protein (EGFP) and luciferase were expressed and assayed in 293FT, HepGⅡ, SGC7901, U2OS, and the primary culture human bone marrow stem cells (hBMSC) under the control of the WThTERT promoter and Mod1hTERT promoter, respectively.2. In vitro amplification of PALA resistant malignant cells derived from human bone marrow stem cells: isolated culture hBMSC from human bone marrow and assess their phaenotype by flow cytometer, FCM; after the treatment by carcinogenic reagent of BPDE and lentivirus infection, malignant cells could be sieved by selection of PALA; detect expression of lentivirus infection、cell cycle regulation related gene and telomerase activation by PCR, RT-PCT and west blot.3. Modification of hTERT promoter for down-regulation of transcriptional activities in human bone marrow stem cells: in this section, a tetrad of MZF-2 binding elements were ligated to the 5′terminal of the Mod1hTERT promoter, the new generated promoter was named as Mod2hTERT prmoter; Cytosine deaminase (CD) gene and enhanced green fluorescent protein (EGFP) gene from E.coli K12 and plasmid of pEGFP-N3 respectively are subcloned into plasmid of pLenti6/V5-D-TOP; The wild hTERT promoter fragment(WT-hTERT), Mod1hTERT, and Mod2hTERT promoters were generated by chemical synthesized; EGFP and luciferase gene code sequence into the lentivirus pLenti6/V5-D-TOP; CMV promoter in pLenti6/V5-D-TOP was replaced by above hTERT promoters, respectively; The hBMSC, BR-hBMSC and PBR-hBMSC were transfected by lentivirus; 6.The expression of EGFP under the controls of modified hTERT promoters were observed by fluorescence contrast phase microscope and the luciferase activity was detected by Kit.4. In vitro and in vivo cytotoxicity test: PBR-hBMSCs with fusion genes of WThTERT-luciferase-IRES-CD, Mod1hTERT-luciferase-IRES-CD, Mod2hTERT-luciferase-IRES-CD, CMV-luciferase-IRES-CD and prmoter(lack)-luciferase-IRES-CD were treated in the mediums with different concentration gradient of 5-FC, respectively; 2.MTT reduction assay were used for cytotoxicity detections of PBR-hBMSCs with different fusion genes in different concentration gradient of 5-FC; PBR-hBMSCs with fusion genes of WThTERT-luciferase-IRES-CD, Mod2hTERT-luciferase-IRES-CD, and CMV-luci-ferase-IRES-CD were transplanted into hypo of athymic mice; After one week, each mouse were detected with animal in vivo fluorescence imaging system were given a injection after given a injection of 5㎎ 5-FC once a day. The signal of survive engraft cells were detected once a week.Results:1. Core hTERT promoter modified by c-Myc cis element: in the Mod1hTERT groups, EGFP could be observed in the hTERT-positive groups of 293FT, HepGⅡ, and SGC7901 cells, but not in the hTERT-negative groups of U2OS and hBMSC. The luciferase activity of the Mod1hTERT promoter groups was significantly higher than that of the WThTERT promoter groups in the hTERT positive cell lines of 293FT, HepGⅡ, and SGC7901(P<0.01), however, no significant difference was found in U2OS and hBMSC (P>0.05). 2. In vitro amplification of PALA resistant malignant cells derived from human bone marrow stem cells:3. Modification of hTERT promoter for down-regulation of transcriptional activities in human bone marrow stem cells: in the Mod1hTERT groups, EGFP could be observed in the hTERT-positive groups of 293FT, HepGⅡ, and SGC7901 cells, but not in the hTERT-negative groups of U2OS and hBMSC. The luciferase activity of the Mod1hTERT promoter groups was significantly higher than that of the WThTERT promoter groups in the hTERT positive cell lines of 293FT, HepGⅡ, and SGC7901(P<0.01), however, no significant difference was found in U2OS and hBMSC (P>0.05).4. In vitro and in vivo cytotoxicity test: MTT assays results shown that the relative survival rates of cells were only 5.79% in the Mod1hTERT group, 6.53% in the Mod2hTERT group, and 1.97% in the CMV group, when treated with 100μM 5-FC; 2. Bioluminescence imaging system test showed that after the administration of 4-5 weeks, in grope of malignant cells derived from hBMSC Mod2hTERT promoter and malignant cells derived from hBMSC with CMV mice bioluminescence signal was vanished. It is suggested that the malignant cell neoplasia be cleared.Conclusions:1. 3’terminal extra E-boxes can improve the tumor-targeting transcription activity of the core hTERT promoter.2. Malignant transformation of hBMSC can be co-induced by transfection of lentivirus and treatment of BPDE. The malignant cells derived from hBMSC could be amplification in vitro with IC50 selection culture of PALA.3. 5′terminal extra MZF-2 binding sites of core hTERT promoter can effectively degrade transcriptional activity of hTERT promoter in MZF-2-positive cells of hBMSC.4. Transcriptional activity of modification with c-Myc and MZF-2 binding elements is stronger than the normal cell. While in vitro and in vivo, the expression of the CD promoter and luciferase gene in PBR-hBMSC has been cleared by 5-FC.In briefly, this study has not just provided a method for the research of targeting eradication of malignant cells derived hBMSC, but may also provide a strategy for bio-safety research in future clinic stem cell therapy.更多还原