可逆性永生化鼠源胰岛β细胞系的建立及生物学特性的研究

发布时间：2018-06-24 07:29

本文选题：Tet-on + advanced　；参考：《暨南大学》2011年硕士论文

【摘要】：目的利用可调控的Tet-on advanced系统和SV40T基因构建永生化小鼠胰岛p细胞系,观察回复后胰岛p细胞生物学特性,评估可逆性永生化小鼠胰岛β细胞系回复后的安全性和功能性,为解决胰岛移植的细胞来源提供实验依据。方法采用Gateway基因重组技术,构建Tet-on advanced系统调控表达的慢病毒载体pLV.EX2d.P/puro-EF1 ArtTA(M2)和pLV.EX3d.P/neo-TRESV40TIRES/eGFP。脂质体lipotamin-2000转染法,将重组的两种慢病毒载体分别转染入包装细胞293FT,获得高滴度的病毒颗粒；再将病毒颗粒共同转染纯化培养的原代小鼠胰岛细胞,转染24小时后加入强力霉素诱导SV40T表达,48小时后加入嘌呤霉素和G418进行抗生素筛选获得体外扩增能力的永生化小鼠胰岛细胞,对多个永生化细胞克隆进行ELISA检测基础胰岛素分泌量,RT-PCR检测胰岛p细胞相关因子,筛选出有生理功能的可逆性永生化小鼠胰岛p细胞系。选取Tet-on调控下的RIB细胞株第10代细胞,去掉DOX诱导使永生化基因SV40T沉默而获得回复后的RIB细胞。通过RT-PCR及细胞免疫荧光技术检测回复前后SV40T及胰岛p细胞相关基因在mRNA和蛋白水平的表达；染色体核型分析、软琼脂克隆形成实验、细胞增殖试验和葡萄糖刺激胰岛素释放实验监测可逆性永生化胰岛β细胞在DOX调控前后的安全性和功能性变化。结果重组慢病毒载体pLV.EX2d.P/puro-EF 1 ArtTA(M2)和pLV.EX3d.P/neo-TRESV40T IRES/eGFP经PCR电泳检测显示构建成功,并验证了重组载体的正确性；分别转染包装细胞293FT,24小时后可见到转染pLV.EX3d.P/neo-TRESV40TIRES/eGFP载体的293FT有绿色荧光表达,转染效率高达90%以上,48后小时收集两种病毒上清进行浓缩,经龙胆紫染色法检测病毒滴度分别能达到107和108；将获得的病毒颗粒同时转染小鼠原代胰岛细胞,转染后24小时加入DOX诱导,可见到处于终末分化状态的胰岛细胞有增殖现象,转染后48小时进行G418和嘌呤霉素的筛选,稳定转染的细胞继续增殖,余下细胞死亡；细胞克隆中经胰岛素ELISA检测阳性的有20株,再进行小鼠胰岛β细胞的相关基因RT-PCR检测,筛选到一株细胞克隆能表达Pdx、Nkx6.1、Pax6、Insulin胰岛β细胞相关基因,成功建立了可逆性永生化小鼠胰岛p细胞系(RIB系)。RIB系在有DOX诱导下增殖能力旺盛,去除DOX进行回复后的RIB细胞增殖速度减慢,5天后完全停止生长；RT-PCR及细胞免疫荧光检测RIB系在DOX诱导下SV40T基因和蛋白水平均有表达,去除DOX即在短期内停止增殖不表达SV40T,胰岛β细胞相关标志基因和蛋白在回复前后均有表达；回复前后的RIB细胞核型分析均为正常二倍体,平板克隆实验未见其致瘤性；RIB系葡萄糖刺激胰岛素释放实验胰岛素分泌能力较弱,回复后胰岛素分泌能力增强,但仍与原代胰岛细胞有差距,约为正常胰岛分泌量的1/3-1/2。结论利用Tet-on advanced系统调控的重组慢病毒载体pLV.EX2d.P/puro-EF1 ArtTA(M2)和PLV.EX3d.P/neo-TRESV40TIRES/eGFP成功建立的RIB系,其生存期延长,在体外增殖能力较强,且核型检测正常,胰岛β细胞相关基因和蛋白仍继续表达,回复后RIB细胞系停止增殖,不表达永生化基因SV40T,而胰岛素分泌能力增强,但分泌量仍与正常胰岛有差距。
[Abstract]:Objective To investigate the biological characteristics of islet 尾 - cell line after recovery and evaluate the safety and functionality of 尾 - cell line after recovery of pancreatic islet 尾 - cell line , and to provide experimental basis for solving the source of pancreatic islet transplantation .

Methods The recombinant plasmids pLV . EX22d . P / puro - EF1 and pLV . EX3d . P / neo - TRESV40TIRES / eGFP were constructed by Gateway gene recombination . The recombinant plasmids were transfected into the packed cells 293FT by lipotamin - 2000 transfection .
After 24 hours of transfection , the cultured primary mouse pancreatic islet cells were co - transfected and purified . After 24 hours of transfection , it was induced to express SV40T . After 48 hours , Puromycin and G418 were added to screen the islet cells .
Chromosome karyotype analysis , soft agar colony formation assay , cell proliferation assay , and glucose - stimulated insulin release assay were used to monitor the safety and functional changes of 尾 - cells in 尾 - cells before and after control .

Results The recombinant lentivirus vectors pLV . EX22d . P / puro - EF 1 and pLV . EX3d . P / neo - TRESV40T were constructed successfully by PCR electrophoresis , and the correctness of the recombinant vector was verified .
The transfected pLV . EX3d . P / neo - TRESV40TIRES / eGFP vector was transfected into 293 FT . The transfection efficiency was over 90 % . After 48 hours , two kinds of supernatant were collected and concentrated .
The virus particles were transfected into the mouse primary islet cells at the same time . After 24 hours after transfection , the cells were induced to proliferate . After transfection , the cells were screened by G418 and puromycin 48 hours after transfection , and the cells stably transfected continue to proliferate and the remaining cells die ;
In the cell clones , 20 strains were detected by ELISA and RT - PCR was performed on the 尾 - cells of mouse pancreatic islets .
RT - PCR and immunofluorescence were used to detect the expression of SV40T gene and protein in vitro .
The results showed that there was no tumor genicity in the normal diploid and plate clone experiments .
The results showed that the insulin secretion was weak and the insulin secretion increased after recovery , but it was still different from that of the primary islet cells , which was about 1 / 3 - 1 / 2 of the normal islet secretion .

Conclusion The recombinant lentivirus vector pLV . EX22d . P / puro - EF1 , TA ( M2 ) and PLV.EX3d . P / neo - TRESV40TIRES / eGFP have been successfully established . The survival time is prolonged , the ability to proliferate in vitro is strong , and the related genes and proteins of 尾 - cells in the pancreatic islets are still expressed .
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】