PDX1联合细胞因子体外诱导人脐带MSCs分化为胰岛素分泌细胞
发布时间：2018-09-08 19:09
【摘要】： 目的： 探讨胰十二指肠同源框-1基因(pancreatic and duodenal homeobox factor 1, Pdx1)联合细胞因子体外诱导人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)分化为胰岛β样细胞。 方法： 1.脐带间充质干细胞的分离、培养和鉴定 采用胶原酶消化法体外分离脐带MSCs;用MTT检测细胞活性,并绘制生长曲线；用流式细胞术检测MSCs表面标志和测定细胞周期；RT-PCR检测OCT-3的表达；用地塞米松、胰岛素诱导MSCs向脂肪细胞分化,用地塞米松溶液、β-甘油磷酸、维生素C诱导MSCs向成骨细胞分化。 2.PDX1联合细胞因子诱导脐带间充质干细胞向胰岛p样细胞分化 用携带PDX1基因的重组腺病毒(Adxsi-CMV-PDX1,为本所构建包装并保存)感染MSCs7d后,联合以下细胞因子------人表皮生长因子(epidermai growth factor,EGF)、B27、胰高血糖素样肽-1(glucagons-like peptide-1, GLP-1)、人β细胞调节素(betacelluin)、肝细胞生长因子(hepatocye growth factor, HGF)、烟酰胺(nicotinamide,NIC)、β-巯基乙醇(β-Mercaptoethanol)诱导分化。RT-PCR检测诱导后细胞PDX1、神经元素3(Neurogenin, NGN3),胰岛素(insulin),葡萄糖转运体2(glucose transporter-2, Glut2)和NK转录因子6.1(NK6 transcription factor related, locus 1, NKX6.1)基因表达的变化；Western blot、免疫细胞化学染色、免疫荧光染色等检测诱导后PDX1、NKX6.1和insulin蛋白表达变化；化学发光法检测诱导后细胞培养液上清中胰岛素和C肽的分泌水平；再用25mmol/L葡萄糖刺激1h后,检测胰岛素与C-肽的分泌水平变化。用流式细胞术检测诱导后细胞insulin(+)细胞百分率。 结果： 1.脐带间充质干细胞的分离、培养和鉴定 分离培养所得到的原代细胞24-48h贴壁,细胞呈梭形。传到第4代时,细胞形态均一,呈长梭形,成漩涡状排列生长。脐带MSCs强表达CD44、CD29,不表达CD106,CD34、CD45、、CD14、CD31和HLA-DR。细胞周期分析显示处在G0／G1期的细胞比例占89.3%。向脂肪细胞诱导14d后,油红O染色见胞浆中有桔红色脂滴形成。向成骨诱导3w,碱性磷酸酶染色见细胞呈立方状,细胞被染成褐色,为阳性反应。而且脐带MSCs能表达OCT-3。 2.胰岛p样细胞鉴定 经Adxsi-CMV-Pdxl感染脐带MSCs7d并联合细胞因子诱导3d,梭形细胞聚集形成岛样细胞团,用双硫腙(Dithizone,DTZ)染色胞浆呈亮红色,为阳性反应。诱导10-17d,RT-PCR检测显示诱导后的细胞表达PDX1、ngn3、NKX6.1、insulin和glut-2胰岛相关基因,免疫细胞化学染色、免疫荧光染色和Western blot均表明诱导后的细胞表达PDX-1、NKX6.1和胰岛素蛋白。在诱导第17d,培养液上清中胰岛素分泌量为(473.11±51.52)mU/L, C肽分泌量为(1.61±0.41) ng/mL,在25mmol/L高糖协同作用1h后,胰岛素含量高达(964.42±68.19)mU/L, C肽含量高达(3.72±1.52) ng/mL。实验组不同阶段间差异具有统计学意义(P0.05)。用流式细胞术检测诱导后insulin(+)细胞百分率为(11.61±4.83)%。 结论： 1.脐带中能分离出MSCs,而且能在体外培养、扩增。脐带MSCs体外具有向脂肪细胞、成骨细胞分化的能力。 2.PDX1联合细胞因子诱导,在体外能分化为胰岛β样细胞,分化的胰岛β样细胞能分泌胰岛素和C肽,且能够响应高糖的刺激。
[Abstract]:Objective:
Objective To investigate the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into islet beta-like cells induced by pancreatic and duodenal homeobox factor 1 (Pdx1) and cytokines in vitro.
Method:
1. isolation, culture and identification of umbilical cord mesenchymal stem cells
Collagenase digestion was used to isolate umbilical cord MSCs in vitro; MTT was used to detect cell activity and plot the growth curve; flow cytometry was used to detect the surface markers and cell cycle of MSCs; RT-PCR was used to detect the expression of OCT-3; dexamethasone and insulin induced MSCs to differentiate into adipocytes; dexamethasone solution, beta-glycerophosphate and vitamin C induced MSCs to differentiate into adipocytes. MSCs differentiated into osteoblasts.
2.PDX1 and cytokines induce differentiation of umbilical cord mesenchymal stem cells into islet P like cells
MSCs were infected with recombinant adenovirus carrying PDX1 gene (Adxsi-CMV-PDX1, packaged and preserved in our laboratory) for 7 days, and then combined with the following cytokines: epidermal growth factor (EGF), B27, glucagons-like peptide-1 (GLP-1), human beta-cell regulator (betacelluin), hepatocyte growth factor (h). Differentiation was induced by epatocye growth factor, HGF, nicotinamide (NIC), and beta-mercaptoethanol. PDX1, NGN3, insulin, glucose transporter-2 (Glut2) and NK transcription factor 6.1 (NK6 transcription factor, locus 1) were detected by RT-PCR. Western blot, immunocytochemical staining and immunofluorescence staining were used to detect the expression of PDX1, NKX6.1 and Insulin protein after induction; the secretion levels of insulin and C-peptide in the supernatant of the cultured cells were detected by chemiluminescence method; insulin and C-peptide were detected after stimulation with 25 mmol/L glucose for 1 hour. Changes in secretion level. The percentage of insulin (+) cells after induction was detected by flow cytometry.
Result:
1. isolation, culture and identification of umbilical cord mesenchymal stem cells
The primary cells adhered to the wall 24-48 hours after culture, and the cells were spindle-shaped. At the fourth generation, the cells were uniform in shape, spindle-shaped and arranged in a vortex. MSCs strongly expressed CD44, CD29, but not CD106, CD34, CD45, CD14, CD31 and HLA-DR. Cell cycle analysis showed that 89.3% of the cells were in G0/G1 phase. Four days later, orange-red lipid droplets were observed in cytoplasm by oil red O staining. After 3 weeks of osteogenic induction, the cells were cubic in shape and brown in color by alkaline phosphatase staining.
2. identification of islet P like cells
Adxsi-CMV-Pdxl infected umbilical cord MSCs for 7 days and combined with cytokines for 3 days. Spindle cells aggregated to form islet-like cell clusters. The cytoplasm was bright red with DTZ staining. After induction for 10-17 days, the cells expressed PDX1, ngn3, NKX6.1, insulin and GLUT-2 islet-related genes and immunocytochemistry. The results of staining, immunofluorescence staining and Western blot showed that the induced cells expressed PDX-1, NKX6.1 and insulin protein. On the seventeenth day of induction, the insulin secretion in the supernatant of culture medium was (473.11 (51.52) mU/L, the secretion of C peptide was (1.61 (0.41) ng/mL, and the insulin content was (964.42 (68.19) mU/L, and the content of C peptide was (964.42) mU/L) at 25 mol/ The amount of insulin (+) was as high as (3.72 (1.52) ng/mL. The difference was statistically significant (P 0.05). The percentage of insulin (+) cells was (11.61 (4.83)) after induction by flow cytometry.
Conclusion:
1. MSCs can be isolated from umbilical cord and cultured and amplified in vitro. Umbilical cord MSCs have the ability to differentiate into adipocytes and osteoblasts in vitro.
2. PDX1 combined with cytokines can differentiate into islet beta-like cells in vitro. Differentiated islet beta-like cells can secrete insulin and C-peptide, and can respond to high glucose stimulation.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2231429
[Abstract]:Objective:
Objective To investigate the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into islet beta-like cells induced by pancreatic and duodenal homeobox factor 1 (Pdx1) and cytokines in vitro.
Method:
1. isolation, culture and identification of umbilical cord mesenchymal stem cells
Collagenase digestion was used to isolate umbilical cord MSCs in vitro; MTT was used to detect cell activity and plot the growth curve; flow cytometry was used to detect the surface markers and cell cycle of MSCs; RT-PCR was used to detect the expression of OCT-3; dexamethasone and insulin induced MSCs to differentiate into adipocytes; dexamethasone solution, beta-glycerophosphate and vitamin C induced MSCs to differentiate into adipocytes. MSCs differentiated into osteoblasts.
2.PDX1 and cytokines induce differentiation of umbilical cord mesenchymal stem cells into islet P like cells
MSCs were infected with recombinant adenovirus carrying PDX1 gene (Adxsi-CMV-PDX1, packaged and preserved in our laboratory) for 7 days, and then combined with the following cytokines: epidermal growth factor (EGF), B27, glucagons-like peptide-1 (GLP-1), human beta-cell regulator (betacelluin), hepatocyte growth factor (h). Differentiation was induced by epatocye growth factor, HGF, nicotinamide (NIC), and beta-mercaptoethanol. PDX1, NGN3, insulin, glucose transporter-2 (Glut2) and NK transcription factor 6.1 (NK6 transcription factor, locus 1) were detected by RT-PCR. Western blot, immunocytochemical staining and immunofluorescence staining were used to detect the expression of PDX1, NKX6.1 and Insulin protein after induction; the secretion levels of insulin and C-peptide in the supernatant of the cultured cells were detected by chemiluminescence method; insulin and C-peptide were detected after stimulation with 25 mmol/L glucose for 1 hour. Changes in secretion level. The percentage of insulin (+) cells after induction was detected by flow cytometry.
Result:
1. isolation, culture and identification of umbilical cord mesenchymal stem cells
The primary cells adhered to the wall 24-48 hours after culture, and the cells were spindle-shaped. At the fourth generation, the cells were uniform in shape, spindle-shaped and arranged in a vortex. MSCs strongly expressed CD44, CD29, but not CD106, CD34, CD45, CD14, CD31 and HLA-DR. Cell cycle analysis showed that 89.3% of the cells were in G0/G1 phase. Four days later, orange-red lipid droplets were observed in cytoplasm by oil red O staining. After 3 weeks of osteogenic induction, the cells were cubic in shape and brown in color by alkaline phosphatase staining.
2. identification of islet P like cells
Adxsi-CMV-Pdxl infected umbilical cord MSCs for 7 days and combined with cytokines for 3 days. Spindle cells aggregated to form islet-like cell clusters. The cytoplasm was bright red with DTZ staining. After induction for 10-17 days, the cells expressed PDX1, ngn3, NKX6.1, insulin and GLUT-2 islet-related genes and immunocytochemistry. The results of staining, immunofluorescence staining and Western blot showed that the induced cells expressed PDX-1, NKX6.1 and insulin protein. On the seventeenth day of induction, the insulin secretion in the supernatant of culture medium was (473.11 (51.52) mU/L, the secretion of C peptide was (1.61 (0.41) ng/mL, and the insulin content was (964.42 (68.19) mU/L, and the content of C peptide was (964.42) mU/L) at 25 mol/ The amount of insulin (+) was as high as (3.72 (1.52) ng/mL. The difference was statistically significant (P 0.05). The percentage of insulin (+) cells was (11.61 (4.83)) after induction by flow cytometry.
Conclusion:
1. MSCs can be isolated from umbilical cord and cultured and amplified in vitro. Umbilical cord MSCs have the ability to differentiate into adipocytes and osteoblasts in vitro.
2. PDX1 combined with cytokines can differentiate into islet beta-like cells in vitro. Differentiated islet beta-like cells can secrete insulin and C-peptide, and can respond to high glucose stimulation.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
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