精原干细胞途径建立hFIX转基因小鼠及Flt3配基乳腺生物反应器研究

【作者】 王宁；

【导师】 薛京伦；

【作者基本信息】 复旦大学 ， 遗传学， 2003， 博士

【摘要】 （一）精原干细胞是精子的前体细胞，是雄性成体动物中唯一可以自我增殖、更新并向下一代传递遗传信息的细胞类型。对精原干细胞的操作和修饰在建立转基因动物模型、男性不孕症的治疗等方面有重要意义，对于生殖细胞途径的基因治疗也有重要的参考价值。我们研究EffecteneTM转染试剂介导下，将基因在体外条件下导入小鼠精原干细胞及移植的可能性，并与其他一些方法的效率进行了比较。同时探讨了活体中转染精原干细胞的可能性，并在此基础上建立了hFIX转基因小鼠模型。1. 在离体条件下探讨和比较了不同转染方法EffecteneTM、电击、脂质体对精原干细胞的转染可能性和效率。结果表明EffecteneTM可以介导精原干细胞的转染。尽管其转染效率还低于其他二倍体细胞及电击方法，但要比其他途径的转染方法效率高且无明显的细胞损伤。2. 将EffecteneTM转染后的精原干细胞移植到Busulfan处理过的小鼠睾丸中，发现转染了外源基因的精原干细胞可以重新增殖、分化， 并表达外源基因。附睾总DNA的PCR结果验证了所观察到的结果3. 不同时间进行移植对阳性细胞克隆的数量有影响，最佳的移植时间应为转染后12小时以内。结合EffecteneTM转染方法及精原干细胞移植技术并进行优化可以应用于建立转基因动物模型。4. 活体中曲精细管注射方法的效果要比睾丸细胞间质直接注射方法效果好，精原干细胞的转染效率更高。5. 通过玻璃针将EffecteneTM包裹的人凝血因子IX表达载体注入小鼠的曲精细管。通过PCR和Southern对子代41只小鼠进行鉴定，有2只仔鼠的基因组中整合有人凝血因子IX基因表达框架，整合率为4％（2/41）。通过ELISA在其中一只小鼠的血浆中检测到hFIX的表达，表达量为4.6ng/ml。<WP=7>（二）FLT-3 配基（fms like tyrosine-3 ligand，FL）是继干细胞因子（SCF）、巨噬细胞集落刺激因子（GM-CSF）后发现的又一种膜结合型早期造血生长因子，它以多种同源异型蛋白形式存在、广泛分布于造血基质细胞以及肾、肝、胎盘等组织中，具有很强的协同刺激造血干、祖细胞增殖分化的能力。FL不仅对造血干细胞（HSC）和造血祖细胞（HPC）的增生、分化、动员、维持及长期重建具有重要作用，还能明显的促进淋巴细胞特别是树突状细胞（DC）、NK细胞等免疫系统组成细胞的增生。FL在某些感染性疾病、器官移植、肿瘤的基因治疗等方面有重要、广阔的临床应用前景。为了实现乳腺反应器转基因动物方式生产人Flt3配基，我们在克隆人FL基因胞外区片段，在离体、活体条件下表达了有功能活性的人FL。在此基础上构建了人FL乳腺特异性表达载体，并在人乳腺癌细胞中验证了载体表达人FL的有效性。1． 从人外周血单核细胞中用RT－PCR方法克隆了人573bp的FL基因胞外区片段，在此基础上构建了真核表达载体pCDNA3K3，并在BHK细胞中进行了表达检测。ELISA结果表明表达载体pCDNA3K3在体外BHK细胞中的表达量为：5.7ng/mL。Western检测结果表明所构建的pCDNA3K3在真核细胞中可以表达人FL基因。2． 将pCDNA3K3质粒通过大剂量稀释后注射小鼠尾静脉后，对小鼠血浆中人FL蛋白的含量进行了连续的检测，并用流式细胞仪对小鼠外周血中CD3＋、CD4＋、CD8＋、CD34＋细胞亚群的变化进行了检测。结果表明人FL基因可在小鼠中高效表达，其最高值为33.7ug/mL，表达持续2周左右。小鼠外周血中CD3＋、CD4＋、CD8＋、CD34＋细胞亚群的数量均有明显的增高。3． 为了探讨建立FL乳腺生物反应器的可能性，以人FL胞外区0.6kb cDNA片段分别构建了小鼠MAR元件／β-酪蛋白(β-casein)和绵羊β-乳球蛋白(β-Lactoglobulin BLG)启动子控制的hFL乳腺特异性表达载体：pMBCFL、pBCFL、pBFL 。表达载体瞬时转染人乳腺癌细胞ZR－7530，经ELISA方法检测，其表达量分别为：2.7ng/mL、1.8ng/mL、2.1ng/mL。更多还原

【Abstract】 （1）Spermatogenic cell （spermatogonial stem cell） is the only cell type in postnatal mammals which has the capability to self-renew and to contribute genetic information to the next generation. The manipulation of spermatogenic cells and modification of their genomes have great significance in treatment for male sterility, gene therapy by germ cells as well as building transgenic animal model. With this assay we analyzed the possibility of Effectene TM reagent mediated gene transferring into spermatogenic cells in vitro. The efficiency of it was compared with other methods. The effect of transplantation with different time schedules on the transfection efficiency and gene expression was also investigated. Based on the development of transfection of spermatogenic cells by Effectene TM reagent in vivo, the transgenic mice with human coagulation factor IX was established.1. We compared the possibility and efficiency of transfection of mice spermatogenic cells by different methods in vitro including Effectene TM reagent, electroporation, liposome, calcium phosphate etc. The results showed that Effectene TM reagent could help to introduce exogeous gene to spermatogenic cells in vitro. Although the efficiencies observed from all methods are lower than normal haploid cells, the Effectene TM reagent showed more efficiency and lower damage to cells.The spermatogenic cells transfected by Effectene TM reagent were then transplanted to seminiferous tubule of recipient mice treated by Busulfan.<WP=9>2. Spermatogenesis rebuilding, normal proliferation and differentiation and gene expression were found in seminiferous tubules. Exogenous gene was detected in the epididymis genomic DNA of the recipient mice by PCR.3. The different time schedule of transplantation has great impact on the number of the positive cell clones, optimal time for transplantation should be within 12 hours after transfection. Combined with optimized transfection technique mediated by Effectene TM reagent, it offers a promising tool for production of transgenic animal model. 4. The efficiency of transfection of spermatogenic cells in vivo by seminiferous tubule injection of EffecteneTM reagent: DNA was higher than the result from testis interstitial injection method. 5. Building of human coagulation factor IX transgenic mice by injecting Effectene TM reagent: DNA (hFIX) complex into the seminiferous tubules of mice. After detection by PCR and Southern, 2 mice whose genome integration with hFIX expression element were found within 41 progenies, the efficiency of integration was 4％（2/41）. One of the 2 mice expressed hFIX protein at 4.6ng/ml (detected by ELISA)（2）Flt3 ligand （fms like tyrosine-3 ligand，FL）is a membrane-bound type hemopoietic growth factor which discovered after SCF and GM-CSF. It widely distributed in hemopoietic interstitial cells, kidney, liver, placenta and other tissues with different isoforms. FL could greatly promote the proliferation,<WP=10>differentiation, mobilization and rebuilding of hemopoietic progenitor/stem cells when synergized with other growth factors. Meanwhile FL could significantly stimulate the proliferation of lymphocytes especially DCs and NK cells. FL has wide and promising potential in clinic therapy of some infectious disease, tissue transplantation as well as cancer therapy. To produce FL by the mammary gland bioreactor of transgenic animal, we cloned the fragment from outside-membrane region and expressed hFL both in vivo and in vitro. Mammary gland tissue specific expression vectors for human FL gene were constructed and tested in human mammary gland tumor cells.1. 537bp human FL gene fragment was cloned by RT-PCR from total RNA of peripheral blood mononuclear cells (PBMC) and eukaryotic expression vector was constructed. Detection of the expression of pCDNA3K3 in BHK cells showed that the transient expression level is 5.7ng/mL. Western result demonstrated that 537bp human FL gene fragment could express human FL correctly.2. After injection the plasmid pCDNA3K3更多还原