PDX-1、NeuroD1及MafA转导小鼠骨髓间充质干细胞及小鼠iPS细胞制备胰岛素分泌细胞的实验研究
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摘要
目的观察胰岛素转录关键调控基因PDX-1、NeuroD1及MafA转染小鼠骨髓间充质干细胞(mBMSCs)和小鼠诱导多能干细胞(iPSCs)后，能否定向分化为胰岛素分泌细胞，探索体外基因转染制备胰岛素分泌细胞用于移植治疗1型糖尿病的可行性。
方法
(1)全基因合成小鼠PDX-1、NeuroD1及MafA基因（以下简称三基因）编码区域序列并测序。将目的基因片段分别与内部核糖体进入位点序列-绿色荧光蛋白（IRES-GFP）进行PCR拼接，得到目的基因与GFP的共表达片断，并将含目的基因序列的质粒重组到腺病毒载体上。分别将含目的基因PDX-1、NeuroD1及MafA的腺病毒载体转染293A细胞，制备重组腺病毒Ad-mPDX-1-IRES-GFP、Ad–mNeuroD-IRES-GFP、Ad-mMafA-IRES-GFP，通过免疫法检测腺病毒液滴度。
(2)体外分离培养及鉴定mBMSCs；小鼠Oct4、Sox2、Klf4及cMyc经Tet-On慢病毒系统转导小鼠胚胎成纤维细胞（MEFs），挑选并扩增克隆。通过形态学鉴定，干性基因Nanog、Rex-1、SSEA-1检测，体内外三胚层分化等实验筛选鉴定iPSCs。
(3)重组腺病毒Ad-mPDX-1-IRES-GFP、 Ad–mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP体外联合转染mBMSCs及小鼠iPSCs。转染获得的细胞分别用RT-PCR检测胰岛β细胞功能基因的表达；免疫荧光检测胰岛素蛋白的表达及定位；ELISA检测不同浓度葡萄糖刺激下胰岛素的分泌量。
(4)三基因转染的mBMSCs及小鼠iPSCs定向分化为胰岛素分泌细胞后，移植到糖尿病小鼠模型肝脏，免疫组化检测其在肝内胰岛素的表达；空腹血糖监测检测移植细胞在糖尿病小鼠体内的功能发挥。
结果
(1)重组腺病毒Ad-mPDX-1-IRES-GFP、 Ad-mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP经Pac I单酶切后凝胶电泳显示会形成约2,000bp的小条带及腺病毒载体将近35,000bp的大条带，与预期及测序结果一致，表明含三基因的同源重组腺病毒载体构建正确。
(2) LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc转染MEFs后，成功获取iPSCs，能形成边缘光整的致密克隆；表达干性基因Nanog、Rex-1、SSEA-1；在体内外能分化为三胚层组织。
(3) Ad-mPDX-1-IRES-GFP、Ad–mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP体外联合转染的mBMSCs及小鼠iPSCs能分化为胰岛素分泌细胞，RT-PCR结果显示其胰岛β细胞功能基因的表达与小鼠胰岛β细胞株MIN6类似；免疫荧光检测见细胞胞浆内有胰岛素合成；ELISA检测结果显示细胞对不同浓度的葡萄糖有较好的反应性。
(4)免疫组化镜检发现移植细胞注射区域的肝实质内可见相对集中的棕黄色细胞，表明移植细胞胞浆内表达胰岛素蛋白；空腹血糖监测显示移植细胞能够控制糖尿病小鼠的高血糖，在体内发挥良好的治疗作用。
结论
(1) LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc能成功将MEFs重编程为iPS细胞。
(2)胰岛素转录关键调控基因PDX-1、NeuroD1和MafA三者能协同作用，促进mBMSCs及小鼠iPSCs定向分化为具有显著的胰岛素合成和分泌能力的胰岛素分泌细胞。
(3)胰岛素转录关键调控基因修饰的mBMSCs及小鼠iPSCs移植体内能发挥一定的治疗作用，有希望成为胰岛β细胞的替代细胞用于移植治疗1型糖尿病。
Objective To evaluate the effect of insulin gene transcription regulators PDX-1,NeuroD1and MafA on the differentiation of bone marrow mesenchymal stem cells（mBMSCs）and induced pluripotent stem cells (iPSCs) into insulin-producing cells.Andto confer the new approach of cell transplantation therapy for type I diabetes.
Methods
⑴Coding regional sequences of transcription factors PDX-1, NeuroD1and MafAwere obtained by total gene synthesis. Then made a confirm by sequencing. Splicingsegments of target genes were augmented and jointed together with the internal ribosomeentry site sequence-green fluorescent protein（IRES-GFP） respectively,and to obtain thegene fragments expressing target genes and GFP. The next the plasmids harboring targetgenes were restructuring to adenovirus vectors. The recombinant adenovirus vectors whichharbored target genes were transfected into packaging cell line293A. And the adenoviruswhich had aggression was harvested. The titer of the adenovirus was determined byimmune method.
(2)Mouse Embryonic Fibroblasts (MEFs) were infected with lentivirus(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. Toconfirm iPSCs pluripotency, Morphology of iPSCs, mES cell-specific cell surface markers,including Nanog, SSEA-1and Rex-1, differentiation experiments both in vitro and in vivowere detected.
⑶mBMSCs and iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad–mNeuroD1-IRES-GFP and Ad-mMafA-IRES-GFP), and then differentiated intoinsulin-producing cells in vitro. RT-PCR was applied to detecting target genes and insulingene expression, immunofluorescence to identifying the presence and location of insulinprotein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) for evaluatingthe secretory volume of insulin at different concentration of glucose.
⑷Diabetic mice were transplanted with infection mBMSCs and iPSCs under the liverparenchyma. The grafts were analyzed by Fasting plasma glucose were used to assess thefunctions exertion of engrafted cell in vivo and revealed the therapeutic effect.
Results
⑴Recombinant adenovirus vectors expressing PDX-1,NeuroD1and MafA werelinearized with Pac I, separated by agarose gel electrophoresis.2,000bp of small strap andnearly35,000bp a of big strap were appeared, with the expected results are consistent. Itproved the exactitude of homologous adenovirus vectors.
⑵MEFs were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. TheiPSCs derived from MEFs were able to grow into clones with clear borders and wereconfirmed the expression of mES cell-specific cell surface markers, including Nanog,SSEA-1and Rex-1. Differentiation experiments showed that suspension cultures of allclones formed EBs in vitro and all three layers in vivo.
⑶The β-cell-specific transcriptional regulators and the insulin gene were expressed inmBMSCs after infection. Immunofluorescence analyses documented the activation ofexpression of insulin in the cytoplasm of differentiated cells. The release of a significantinsulin content by these cells was detected in response to a certain concentrations ofglucose stimulation.
⑷Immunohistochemistry was performed to detect the expression of insulin in theliver tissue of diabetic mice and it exhibited positive staining of insulin. The results offasting plasma glucose demonstrated the ability of these insulin-producing cells-transplanted mice to dispose of a glucose load.
Conclusions
⑴MEFs which were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) can be reprogrammed into iPSCs.
⑵The combination of PDX-1, NeuroD1and MafA markedly induces insulinbiosynthesis and secretion in mBMSCs and iPSCs.
⑶Transplantation of mBMSCs and iPSCs modified by transcriptional factors hastherapeutical effect on type I diabetes and thereby is a novel approach efficiently induceinsulin-producing surrogateβ cells.
方法
(1)全基因合成小鼠PDX-1、NeuroD1及MafA基因（以下简称三基因）编码区域序列并测序。将目的基因片段分别与内部核糖体进入位点序列-绿色荧光蛋白（IRES-GFP）进行PCR拼接，得到目的基因与GFP的共表达片断，并将含目的基因序列的质粒重组到腺病毒载体上。分别将含目的基因PDX-1、NeuroD1及MafA的腺病毒载体转染293A细胞，制备重组腺病毒Ad-mPDX-1-IRES-GFP、Ad–mNeuroD-IRES-GFP、Ad-mMafA-IRES-GFP，通过免疫法检测腺病毒液滴度。
(2)体外分离培养及鉴定mBMSCs；小鼠Oct4、Sox2、Klf4及cMyc经Tet-On慢病毒系统转导小鼠胚胎成纤维细胞（MEFs），挑选并扩增克隆。通过形态学鉴定，干性基因Nanog、Rex-1、SSEA-1检测，体内外三胚层分化等实验筛选鉴定iPSCs。
(3)重组腺病毒Ad-mPDX-1-IRES-GFP、 Ad–mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP体外联合转染mBMSCs及小鼠iPSCs。转染获得的细胞分别用RT-PCR检测胰岛β细胞功能基因的表达；免疫荧光检测胰岛素蛋白的表达及定位；ELISA检测不同浓度葡萄糖刺激下胰岛素的分泌量。
(4)三基因转染的mBMSCs及小鼠iPSCs定向分化为胰岛素分泌细胞后，移植到糖尿病小鼠模型肝脏，免疫组化检测其在肝内胰岛素的表达；空腹血糖监测检测移植细胞在糖尿病小鼠体内的功能发挥。
结果
(1)重组腺病毒Ad-mPDX-1-IRES-GFP、 Ad-mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP经Pac I单酶切后凝胶电泳显示会形成约2,000bp的小条带及腺病毒载体将近35,000bp的大条带，与预期及测序结果一致，表明含三基因的同源重组腺病毒载体构建正确。
(2) LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc转染MEFs后，成功获取iPSCs，能形成边缘光整的致密克隆；表达干性基因Nanog、Rex-1、SSEA-1；在体内外能分化为三胚层组织。
(3) Ad-mPDX-1-IRES-GFP、Ad–mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP体外联合转染的mBMSCs及小鼠iPSCs能分化为胰岛素分泌细胞，RT-PCR结果显示其胰岛β细胞功能基因的表达与小鼠胰岛β细胞株MIN6类似；免疫荧光检测见细胞胞浆内有胰岛素合成；ELISA检测结果显示细胞对不同浓度的葡萄糖有较好的反应性。
(4)免疫组化镜检发现移植细胞注射区域的肝实质内可见相对集中的棕黄色细胞，表明移植细胞胞浆内表达胰岛素蛋白；空腹血糖监测显示移植细胞能够控制糖尿病小鼠的高血糖，在体内发挥良好的治疗作用。
结论
(1) LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc能成功将MEFs重编程为iPS细胞。
(2)胰岛素转录关键调控基因PDX-1、NeuroD1和MafA三者能协同作用，促进mBMSCs及小鼠iPSCs定向分化为具有显著的胰岛素合成和分泌能力的胰岛素分泌细胞。
(3)胰岛素转录关键调控基因修饰的mBMSCs及小鼠iPSCs移植体内能发挥一定的治疗作用，有希望成为胰岛β细胞的替代细胞用于移植治疗1型糖尿病。
Objective To evaluate the effect of insulin gene transcription regulators PDX-1,NeuroD1and MafA on the differentiation of bone marrow mesenchymal stem cells（mBMSCs）and induced pluripotent stem cells (iPSCs) into insulin-producing cells.Andto confer the new approach of cell transplantation therapy for type I diabetes.
Methods
⑴Coding regional sequences of transcription factors PDX-1, NeuroD1and MafAwere obtained by total gene synthesis. Then made a confirm by sequencing. Splicingsegments of target genes were augmented and jointed together with the internal ribosomeentry site sequence-green fluorescent protein（IRES-GFP） respectively,and to obtain thegene fragments expressing target genes and GFP. The next the plasmids harboring targetgenes were restructuring to adenovirus vectors. The recombinant adenovirus vectors whichharbored target genes were transfected into packaging cell line293A. And the adenoviruswhich had aggression was harvested. The titer of the adenovirus was determined byimmune method.
(2)Mouse Embryonic Fibroblasts (MEFs) were infected with lentivirus(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. Toconfirm iPSCs pluripotency, Morphology of iPSCs, mES cell-specific cell surface markers,including Nanog, SSEA-1and Rex-1, differentiation experiments both in vitro and in vivowere detected.
⑶mBMSCs and iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad–mNeuroD1-IRES-GFP and Ad-mMafA-IRES-GFP), and then differentiated intoinsulin-producing cells in vitro. RT-PCR was applied to detecting target genes and insulingene expression, immunofluorescence to identifying the presence and location of insulinprotein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) for evaluatingthe secretory volume of insulin at different concentration of glucose.
⑷Diabetic mice were transplanted with infection mBMSCs and iPSCs under the liverparenchyma. The grafts were analyzed by Fasting plasma glucose were used to assess thefunctions exertion of engrafted cell in vivo and revealed the therapeutic effect.
Results
⑴Recombinant adenovirus vectors expressing PDX-1,NeuroD1and MafA werelinearized with Pac I, separated by agarose gel electrophoresis.2,000bp of small strap andnearly35,000bp a of big strap were appeared, with the expected results are consistent. Itproved the exactitude of homologous adenovirus vectors.
⑵MEFs were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. TheiPSCs derived from MEFs were able to grow into clones with clear borders and wereconfirmed the expression of mES cell-specific cell surface markers, including Nanog,SSEA-1and Rex-1. Differentiation experiments showed that suspension cultures of allclones formed EBs in vitro and all three layers in vivo.
⑶The β-cell-specific transcriptional regulators and the insulin gene were expressed inmBMSCs after infection. Immunofluorescence analyses documented the activation ofexpression of insulin in the cytoplasm of differentiated cells. The release of a significantinsulin content by these cells was detected in response to a certain concentrations ofglucose stimulation.
⑷Immunohistochemistry was performed to detect the expression of insulin in theliver tissue of diabetic mice and it exhibited positive staining of insulin. The results offasting plasma glucose demonstrated the ability of these insulin-producing cells-transplanted mice to dispose of a glucose load.
Conclusions
⑴MEFs which were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) can be reprogrammed into iPSCs.
⑵The combination of PDX-1, NeuroD1and MafA markedly induces insulinbiosynthesis and secretion in mBMSCs and iPSCs.
⑶Transplantation of mBMSCs and iPSCs modified by transcriptional factors hastherapeutical effect on type I diabetes and thereby is a novel approach efficiently induceinsulin-producing surrogateβ cells.
引文
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