PDX1聯(lián)合細(xì)胞因子體外誘導(dǎo)人臍帶MSCs分化為胰島素分泌細(xì)胞
發(fā)布時(shí)間：2018-09-08 19:09
【摘要】： 目的： 探討胰十二指腸同源框-1基因(pancreatic and duodenal homeobox factor 1, Pdx1)聯(lián)合細(xì)胞因子體外誘導(dǎo)人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)分化為胰島β樣細(xì)胞。 方法： 1.臍帶間充質(zhì)干細(xì)胞的分離、培養(yǎng)和鑒定 采用膠原酶消化法體外分離臍帶MSCs;用MTT檢測(cè)細(xì)胞活性,并繪制生長(zhǎng)曲線；用流式細(xì)胞術(shù)檢測(cè)MSCs表面標(biāo)志和測(cè)定細(xì)胞周期；RT-PCR檢測(cè)OCT-3的表達(dá)；用地塞米松、胰島素誘導(dǎo)MSCs向脂肪細(xì)胞分化,用地塞米松溶液、β-甘油磷酸、維生素C誘導(dǎo)MSCs向成骨細(xì)胞分化。 2.PDX1聯(lián)合細(xì)胞因子誘導(dǎo)臍帶間充質(zhì)干細(xì)胞向胰島p樣細(xì)胞分化 用攜帶PDX1基因的重組腺病毒(Adxsi-CMV-PDX1,為本所構(gòu)建包裝并保存)感染MSCs7d后,聯(lián)合以下細(xì)胞因子------人表皮生長(zhǎng)因子(epidermai growth factor,EGF)、B27、胰高血糖素樣肽-1(glucagons-like peptide-1, GLP-1)、人β細(xì)胞調(diào)節(jié)素(betacelluin)、肝細(xì)胞生長(zhǎng)因子(hepatocye growth factor, HGF)、煙酰胺(nicotinamide,NIC)、β-巰基乙醇(β-Mercaptoethanol)誘導(dǎo)分化。RT-PCR檢測(cè)誘導(dǎo)后細(xì)胞PDX1、神經(jīng)元素3(Neurogenin, NGN3),胰島素(insulin),葡萄糖轉(zhuǎn)運(yùn)體2(glucose transporter-2, Glut2)和NK轉(zhuǎn)錄因子6.1(NK6 transcription factor related, locus 1, NKX6.1)基因表達(dá)的變化；Western blot、免疫細(xì)胞化學(xué)染色、免疫熒光染色等檢測(cè)誘導(dǎo)后PDX1、NKX6.1和insulin蛋白表達(dá)變化；化學(xué)發(fā)光法檢測(cè)誘導(dǎo)后細(xì)胞培養(yǎng)液上清中胰島素和C肽的分泌水平；再用25mmol/L葡萄糖刺激1h后,檢測(cè)胰島素與C-肽的分泌水平變化。用流式細(xì)胞術(shù)檢測(cè)誘導(dǎo)后細(xì)胞insulin(+)細(xì)胞百分率。 結(jié)果： 1.臍帶間充質(zhì)干細(xì)胞的分離、培養(yǎng)和鑒定 分離培養(yǎng)所得到的原代細(xì)胞24-48h貼壁,細(xì)胞呈梭形。傳到第4代時(shí),細(xì)胞形態(tài)均一,呈長(zhǎng)梭形,成漩渦狀排列生長(zhǎng)。臍帶MSCs強(qiáng)表達(dá)CD44、CD29,不表達(dá)CD106,CD34、CD45、、CD14、CD31和HLA-DR。細(xì)胞周期分析顯示處在G0／G1期的細(xì)胞比例占89.3%。向脂肪細(xì)胞誘導(dǎo)14d后,油紅O染色見胞漿中有桔紅色脂滴形成。向成骨誘導(dǎo)3w,堿性磷酸酶染色見細(xì)胞呈立方狀,細(xì)胞被染成褐色,為陽(yáng)性反應(yīng)。而且臍帶MSCs能表達(dá)OCT-3。 2.胰島p樣細(xì)胞鑒定 經(jīng)Adxsi-CMV-Pdxl感染臍帶MSCs7d并聯(lián)合細(xì)胞因子誘導(dǎo)3d,梭形細(xì)胞聚集形成島樣細(xì)胞團(tuán),用雙硫腙(Dithizone,DTZ)染色胞漿呈亮紅色,為陽(yáng)性反應(yīng)。誘導(dǎo)10-17d,RT-PCR檢測(cè)顯示誘導(dǎo)后的細(xì)胞表達(dá)PDX1、ngn3、NKX6.1、insulin和glut-2胰島相關(guān)基因,免疫細(xì)胞化學(xué)染色、免疫熒光染色和Western blot均表明誘導(dǎo)后的細(xì)胞表達(dá)PDX-1、NKX6.1和胰島素蛋白。在誘導(dǎo)第17d,培養(yǎng)液上清中胰島素分泌量為(473.11±51.52)mU/L, C肽分泌量為(1.61±0.41) ng/mL,在25mmol/L高糖協(xié)同作用1h后,胰島素含量高達(dá)(964.42±68.19)mU/L, C肽含量高達(dá)(3.72±1.52) ng/mL。實(shí)驗(yàn)組不同階段間差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。用流式細(xì)胞術(shù)檢測(cè)誘導(dǎo)后insulin(+)細(xì)胞百分率為(11.61±4.83)%。 結(jié)論： 1.臍帶中能分離出MSCs,而且能在體外培養(yǎng)、擴(kuò)增。臍帶MSCs體外具有向脂肪細(xì)胞、成骨細(xì)胞分化的能力。 2.PDX1聯(lián)合細(xì)胞因子誘導(dǎo),在體外能分化為胰島β樣細(xì)胞,分化的胰島β樣細(xì)胞能分泌胰島素和C肽,且能夠響應(yīng)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
本文編號(hào)：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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
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