【作者】 杨欢；

【导师】 叶章群； 陈志强；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分：中国人产甲酸草酸杆菌中甲酰辅酶A转移酶基因FRC和草酰辅酶A脱羧酶基因OXC慢病毒过表达载体的构建及慢病毒制备目的：产甲酸草酸杆菌(Oxalobacter fomigenes, Ox.F)是一种能降解草酸的肠道厌氧菌,其中降解草酸的关键酶是甲酰辅酶A转移酶(Formyl-CoA transferase, FCoAT)和草酰辅酶脱羧酶(Oxalyl-CoA Decarboxylase, OcoAD)。从中国人肠道产甲酸草酸杆菌Ox.F中克隆编码FCoAT的FRC基因和OCoAD的OXC基因,并将FRC基因和OXC基因分别与过表达慢病毒载体重组,制备能表达FCoAT和FCoAT的慢病毒,为下一步转染肝干细胞治疗高草酸尿症奠定基础。方法：用细菌基因组提取试剂盒提取中国人Ox.F的基因组DNA作为模板,用Taq高保真DNA聚合酶分别扩增FRC基因和OXC基因片段,用DNA凝胶回收试剂盒切胶回收扩增片段,用DNA连接试剂盒将回收产物分别与pMD18-T Simple载体连接,分别获得FRC基因和OXC基因的克隆载体,并转化大肠杆菌DH5α感受态细胞,挑取阳性克隆并进行测序,筛选出携带完整目的基因的质粒pMD18T simple- FRC和pMD18T simple-OXC,质粒提取,用BamHⅠ限制性内切酶分别对pMD18T simple-FRC和pMD18T simple-OXC进行酶切获得目的基因,连接目的基因片段FRC和携带绿色荧光蛋白的pLenti6.3/v5 DEST-IRES-EGFP,连接基因片段OXC与表达红色荧光蛋白的pLenti6.3/v5 DEST-IRES-DsRED,分别转化大肠杆菌DH5α感受态细胞,挑取阳性克隆,质粒小量提取并测序鉴定。重组慢病毒过表达载体pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED转染HEK293T细胞,包装收集并测定慢病毒滴度。结果：PCR扩增分别获得含有大小约为1.3 kb和1.7 kb的DNA片段,大小与Genebank中FRC和OXC基因序列相符。测序鉴定证实pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED分别含有正向的FRC cDNA和OXC cDNA,转染HEK293T细胞实验24小时后,在荧光显微镜下分别可见大量绿色荧光和红色荧光,病毒滴度分别为1.15×108TU/ml和9.75×107TU/ml。结论：成功构建表达中国人肠道产甲酸草酸杆菌FCoAT基因FRC的慢病毒载体pLenti6.3-FRC-IRES-EGFP和OcoAD基因OXC的慢病毒pLenti6.3-OXC-IRES-DsRED,并制备了高滴度的过表达慢病毒载体。第二部分：胚胎来源大鼠肝干细胞的分离培养鉴定及绿色荧光转染目的：研究大鼠胚胎来源的肝干细胞分离培养鉴定,将绿色荧光蛋白转入原代肝干细胞为后续转基因肝干细胞移植动物实验提供示踪条件。方法：体外联合机械分离和胶原酶消化法分离孕13.5 d SD大鼠胚胎来源肝干细胞,随后通过半量换液法纯化细胞,采用间接免疫荧光法、Real—time PCR和Western Blot对培养原代5天的细胞进行鉴定,应用特异性培养基培养传代纯化的细胞。利用电转染技术将绿色荧光蛋白转入传至第三代的细胞。结果：原代培养的胎肝干细胞24 h后显微镜下可见细胞半贴壁状态,形态大致相同,呈圆形或卵圆形；第2天观察细胞为大小几乎一致的圆形；培养第3天出现一些由3、5个形态一致的细胞集落,细胞直径6～10μm；细胞集落不断增大,至第5天集落中细胞数量增至10个左右,集落由大小不等的致密圆形细胞组成,界限清楚；第8天后细胞呈上皮样铺展；第12天时,细胞变大呈煎蛋样摊开,形态不规则,细胞浆内可见颗粒,生长变得缓慢；传代后细胞扩增速度无明显变化,至第3代仍保持较均一的上皮细胞状。通过间接免疫荧光法和Western Blot从蛋白水平检测出纯化细胞表达干细胞标志物CD34和胆管细胞标志物CK19, Real-time PCR检测出甲胎蛋白AFP和白蛋白ALB在基因水平表达,可认为分离纯化细胞为肝干细胞；绿色荧光能在电转后的肝干细胞中稳定表达。结论：联合机械分离法和酶消化法分离纯化出孕鼠胎肝干细胞是一种简单、经济、有效的方法,稳定表达绿色荧光的肝干细胞为下一步草酸分解基因转染的肝干细胞移植治疗PH提供相应基础。第三部分：慢病毒介导甲酰辅酶A转移酶FRC基因和草酰辅酶A脱羧酶OXC基因转染肝干细胞的实验研究目的：观察甲酰辅酶A转移酶FRC基因和草酰辅酶A脱羧酶OXC基因慢病毒表达载体转染后的肝干细胞的目的基因表达情况和草酸分解功能分析。方法：用前期构建的慢病毒过表达载体pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED转染分离纯化的肝干细胞,然后流式分选,并用Real—time PCR和Western Blot检测阳性细胞中目的基因的表达情况。最后用离子色谱仪检测转染目的基因、转染空质粒后细胞对于含草酸培养液中的草酸分解功能。结果：pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED两种慢病毒过表达载体成功转染肝干细胞,分别可见绿色荧光和红色荧光,流式细胞仪分选能提高阳性表达率。Real—time PCR和Western Blot分别检测到FRC和OXC在基因水平和蛋白水平表达。离子色谱仪检测出pLenti6.3-FRC-IRES-EGFP稳转的细胞培养一周后各组草酸浓度分别为空白组[(1.58±0.03)g/L]、转染空质粒细胞组[(1.57±0.01)g/L]、转染细胞组[(1.37±0.02)g/L],三组相比有统计学显著差异(P<0.01)结论：慢病毒转染草酸分解基因是肝干细胞持续获得草酸分解能力的一种有效途径,具有草酸分解功能的肝干细胞为细胞移植治疗高草酸尿奠定基础。更多还原

【Abstract】 PartⅠConstruction and Identification of Recombinant Lentiviral Expressing Vectors FRC and OXC From Chinese Oxalobacter FormigenesOBJECTIVE:Oxalobacter formigenes(Ox.F) is an obligate anaerobe which can degrades oxalates using oxalyl-CoA decarboxylase and formyl-CoA transferase encoded by the OXC and FRC genes, respectively. To clone FRC and OXC from Oxalobacter formigenes in Chinese people intestines, then construct and manufacture recombinant lentiviral expressing vectors pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED,providing a basis for further study on transgenic hepatic stem cells for treatment for hyperoxalurias.MATERIALS AND METHODS:The Oxalobacter formigenes were isolated from Chinese fecal specimens previously and stored in our laboratory. Then template DNA from Chinese Oxalobacter formigenes was isolated by bacterial genome extraction kit, the cDNA of FRC and OXC were cloned by PCR with Taq DNA polymerases and then ligated with pMD18T simple vectors after retrieve and purification. The ligation products were transformed into competent E.coli DH5α.The positive recombinant clones were selected and identified by a complementation, restriction endonuclease digestion. The cloning vector first digested with BamHⅠand the lentivirus vectors pLenti6.3/v5 DEST-IRES-EGFP and pLenti6.3/v5 DEST-IRES- DsRED were ligated and transformed respectivly. The DNA sequence analysis were performed to confirm the recombinant lentiviral vector and the titer of constucted lentivirus vectors were detected.RESULTS:Two fragments of 1287 bp and 1707bp was obtained by PCR respectivly. The enzyme and PCR analyses revealed that the correct FRC and OXC cDNA was cloned. The sequence of pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED was identical to the sequence of cloned cDNA respectivly.HEK293T cells were observed green and red fluorescent after 24h transfection, Two type of virus titers were 1.15×108TU/ml and 9.75×10’TU/ml respectively.CONCLUSIONS:FRC and OXC was cloned correctly and the recombinant lentiviral vector pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED was constructed successfully, then high titer of Lentivirus was prepared PartⅡThe identification and GFP transfection of fetal hepatic stem cells with mechanical separation and enzymatic digestion methodsOBJECTIVE:To isolate and identificate hepatic stem cells by mechanical separation and enzymatic digestion methods from rat fetal liver and observe green fluorescent protein (GFP) expression after tranfection with pEGFP-C1 plasmid.MATERIALS AND METHODS:Mechanical separation and enzymatic digestion method were used to isolate fetal hepatic stem cells from fetal Sprague Dawley rat liver tissues following pregnant for 13.5 days. Half-amount medium replacement was used for purify isolated fetal liver cells following cultured in specific medium. Immunofluorescence technique, Real-time PCR and Western Blot was used to identify adherent cells.The fetal hepatic stem cells of 3 passage were tranfected by electroporation with pEGFP-C1 plasmid.RESULTS:The isolated fetal hepatic stem cells adhered to the culture plastic and presented with round or oval shape after 24-hours cultivation in vitro. Isolated cells were almost equal circular after 2 days cultivation. Cells grew into a colony which was constructed by 3 or 5 cells at 3 days, with cell diameter of 6-10μm; cell colony became more bigger and constructed by 10 cells; the colony was composed of dense round cells of various sizes with clear boundary at 5 days. At 8 days, they grew like epithelium cell. At 12 days, cells became big and extended like fried egg with irregular forms, especially in cytoplasm. Following passage, there were no significant changes in cell amplification speed. Cells still presented epithelium-like shape at the passage 3. The adhered cells at 5 days following primary incubation were positively for human stem cell factor receptor CD34 and cytokeratin 19 using immunofluorescence technique and Western Blot. Real-time PCR detected the AFP and ALB expressed in the adhered cells. GFP can stably express in transgenic cells after pEGFP-C1 plasmid transfection.CONCLUSIONS:The mechanical separation and enzymatic digestion methods is a simple, economic and effective method to isolate hepatic stem cells from rat fetal liver. The transgenic fetal hepatic stem cell with GFP is the basis of hepatic stem cell transplantation for curation of PHs. PartⅢFunctional Analysis of transgenic fetal hepatic stem cells with PLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsREDOBJECTIVE:To investigate the oxalate-degrading effects of fetal hepatic stem cells with lentivirul vector transfection of genes encoding oxalate-degrading enzyme from Chinese Ox.F in vitro.MATERIALS AND METHODS:We applied the pre-constucted over-expression lentiviral vector pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED to transfect fetal hepatic stem cells, and then sort positive cells by flow Cytometry.Then detect the expression of oxalate-degrading enzyme genes used Real—time PCR and western Blot. Finally, The oxalate-degrading function of transgenic cells was analyzed by ion chromatography.RESULTS:The hepatic stem cells were successfully transfected with Lentiviral overexpression vector pLenti6.3-OXC-IRES-DsRED and pLenti6.3-FRC-IRES-EGFP. Real—time PCR and Western Blot respectively detected in the expression of FRC and the OXC in gene and protein levels. Compared with the three cell groups,The concentration of oxalate in culture medium of pLenti6.3-FRC-IRES-EGFP transgenic cells[(1.37±0.02)g/L] was lower than that of blank vector transgenic cells control group [(1.57±0.01)g/L]and blank control group [(1.58±0.03)g/L] (P< 0.01).CONCLUSIONS:Lentiviral vector transfection of oxalate-degrading enzyme genes is an effective way which make fetal heptic stem cells have potency of oxalate-degrading sustainably, It lay a good foundation for cell transplantation in the management of Hyperoxaluria.更多还原