人臍帶間充質(zhì)干細胞向胰島素分泌細胞分化的初步研究
發(fā)布時間：2018-10-20 13:13
【摘要】： 目的:探索人臍帶間充質(zhì)干細胞(Human Umbilical cord Wharton’s Jelly-derived Mesenchymal Stem Cells ,HUMSCs)在大鼠胰島細胞生長的微環(huán)境中及在糖尿病大鼠體內(nèi)向胰島素分泌細胞分化的潛能。 方法:①實驗材料: SD大鼠由廣州中醫(yī)藥大學實驗動物中心提供。對動物處置符合動物倫理學標準。臍帶取自汕頭大學醫(yī)學院第二附屬醫(yī)院婦產(chǎn)科健康足月妊娠剖宮產(chǎn)的胎兒,產(chǎn)婦及家屬對實驗知情同意,并經(jīng)醫(yī)院倫理委員會批準。②實驗方法:分離培養(yǎng)人臍帶間充質(zhì)干細胞,并采用膠原酶消化法分離大鼠胰島細胞。將HUMSCs與大鼠胰島細胞以半透膜相隔共同培養(yǎng),觀察HUMSCs的形態(tài)變化,在共培養(yǎng)的第3,7,14天采用放射免疫法檢測HUMSCs分泌的胰島素水平,RT-PCR方法檢測共培養(yǎng)的HUMSCs中的Pdx-1基因的表達,以未共培養(yǎng)的HUMSCs做對照。用熒光染料Hoechst33258標記HUMSCs,經(jīng)尾靜脈將HUMSCs移植入糖尿病模型大鼠體內(nèi),一個月后取大鼠胰腺制作快速冰凍切片,熒光顯微鏡下觀察HUMSCs在胰腺組織中的定植情況。60天后取移植鼠的胰腺組織,采用半定量RT-PCR方法檢測人insulin基因的表達。 結(jié)果:體外研究:①共培養(yǎng)條件下的HUMSCs由長梭形逐漸變圓,并聚集成團。②放射免疫法結(jié)果表明,共培養(yǎng)組HUMSCs隨著共培養(yǎng)時間的延長分泌的胰島素量明顯增多,當HUMSCs有聚集成團傾向時可以隨葡萄糖刺激濃度的增高而分泌的胰島素量也增高,與對照組相比差異顯著(P0.05)。③Pdx-1基因的RT-PCR結(jié)果表明未經(jīng)共培養(yǎng)的HUMSCs不表達Pdx-1 ,共培養(yǎng)3d的HUMSCs開始表達Pdx-1,共培養(yǎng)7d的HUMSCs仍表達Pdx-1,共培養(yǎng)14d的HUMSCs未表達Pdx-1。體內(nèi)研究:④熒光顯微鏡下看到移植標記Hoechst33258的HUMSCs在大鼠的胰腺中呈均勻的點狀分部,發(fā)出藍色熒光,表明移植入體內(nèi)的HUMSCs可以在大鼠胰腺內(nèi)定植。⑤Insulin的RT-PCR結(jié)果表明移植HUMSCs的大鼠胰腺在第60天時能夠檢測出人Insulin基因,而未經(jīng)移植HUMSCs的大鼠胰腺不表達人Insulin基因。⑥移植HUMSCs的糖尿病大鼠與對照組相比能明顯延長生存期,P0.05。 結(jié)論:人臍帶間充質(zhì)干細胞具有在體內(nèi)外向胰島素分泌細胞分化的潛能,人臍帶間充質(zhì)干細胞可以作為胰島細胞的來源。
[Abstract]:Aim: to explore the potential of human umbilical cord mesenchymal stem cells (Human Umbilical cord Wharton's Jelly-derived Mesenchymal Stem Cells, HUMSCs) to differentiate into insulin-secreting cells in the microenvironment of rat islet cell growth and in diabetic rats. Methods: 1 Experimental materials: SD rats were provided by Experimental Animal Center of Guangzhou University of traditional Chinese Medicine. The disposal of animals meets the standards of animal ethics. The umbilical cord was taken from the fetus of the second affiliated Hospital of Shantou University School of Medicine, Obstetrics and Gynecology, healthy full-term pregnancy and caesarean section. The pregnant women and their families gave informed consent to the experiment. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured, and rat islet cells were isolated by collagenase digestion. HUMSCs and rat islet cells were cultured separately by semi-permeable membrane to observe the morphological changes of HUMSCs. The insulin secreted by HUMSCs was detected by radioimmunoassay and the expression of Pdx-1 gene in co-cultured HUMSCs was detected by RT-PCR method on the 7th and 14th day of co-culture. Unco-cultured HUMSCs was used as control. HUMSCs was transplanted into diabetic model rats with fluorescent dye Hoechst33258 labeled HUMSCs, through tail vein. The pancreas of rats was harvested one month later to make rapid frozen sections. The colonization of HUMSCs in pancreatic tissue was observed under fluorescence microscope, and the expression of human insulin gene was detected by semi-quantitative RT-PCR after 60 days. Results: (1) HUMSCs in co-culture condition gradually became round from long spindle shape and gathered into clusters. 2 the results of radioimmunoassay showed that the amount of insulin secreted by HUMSCs in co-culture group increased obviously with the prolongation of co-culture time. The amount of insulin secreted by HUMSCs increased with the increase of glucose-stimulated concentration (P0.05). The RT-PCR results of 3Pdx-1 gene showed that the uncultured HUMSCs did not express Pdx-1. Expression of Pdx-1, in HUMSCs from 3 days after co-culture, HUMSCs from 7 days of co-culture and Pdx-1, from 14 days to 14 days, HUMSCs did not express Pdx-1.. In vivo study: (4) under fluorescence microscope, HUMSCs labeled with Hoechst33258 was seen as a uniform dot in the pancreas of rats, emitting blue fluorescence. The results of RT-PCR of 5Insulin showed that the pancreas of rats transplanted with HUMSCs could detect human Insulin gene at day 60. However, the pancreas of untransplanted HUMSCs rats did not express human Insulin gene. 6 the survival time of diabetic rats transplanted with HUMSCs was significantly longer than that of the control group (P0.05). Conclusion: human umbilical cord mesenchymal stem cells have the potential to differentiate into extroverted insulin-secreting cells in vivo, and human umbilical cord mesenchymal stem cells can be used as a source of islet cells.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R587.1;R329
本文編號：2283288
[Abstract]:Aim: to explore the potential of human umbilical cord mesenchymal stem cells (Human Umbilical cord Wharton's Jelly-derived Mesenchymal Stem Cells, HUMSCs) to differentiate into insulin-secreting cells in the microenvironment of rat islet cell growth and in diabetic rats. Methods: 1 Experimental materials: SD rats were provided by Experimental Animal Center of Guangzhou University of traditional Chinese Medicine. The disposal of animals meets the standards of animal ethics. The umbilical cord was taken from the fetus of the second affiliated Hospital of Shantou University School of Medicine, Obstetrics and Gynecology, healthy full-term pregnancy and caesarean section. The pregnant women and their families gave informed consent to the experiment. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured, and rat islet cells were isolated by collagenase digestion. HUMSCs and rat islet cells were cultured separately by semi-permeable membrane to observe the morphological changes of HUMSCs. The insulin secreted by HUMSCs was detected by radioimmunoassay and the expression of Pdx-1 gene in co-cultured HUMSCs was detected by RT-PCR method on the 7th and 14th day of co-culture. Unco-cultured HUMSCs was used as control. HUMSCs was transplanted into diabetic model rats with fluorescent dye Hoechst33258 labeled HUMSCs, through tail vein. The pancreas of rats was harvested one month later to make rapid frozen sections. The colonization of HUMSCs in pancreatic tissue was observed under fluorescence microscope, and the expression of human insulin gene was detected by semi-quantitative RT-PCR after 60 days. Results: (1) HUMSCs in co-culture condition gradually became round from long spindle shape and gathered into clusters. 2 the results of radioimmunoassay showed that the amount of insulin secreted by HUMSCs in co-culture group increased obviously with the prolongation of co-culture time. The amount of insulin secreted by HUMSCs increased with the increase of glucose-stimulated concentration (P0.05). The RT-PCR results of 3Pdx-1 gene showed that the uncultured HUMSCs did not express Pdx-1. Expression of Pdx-1, in HUMSCs from 3 days after co-culture, HUMSCs from 7 days of co-culture and Pdx-1, from 14 days to 14 days, HUMSCs did not express Pdx-1.. In vivo study: (4) under fluorescence microscope, HUMSCs labeled with Hoechst33258 was seen as a uniform dot in the pancreas of rats, emitting blue fluorescence. The results of RT-PCR of 5Insulin showed that the pancreas of rats transplanted with HUMSCs could detect human Insulin gene at day 60. However, the pancreas of untransplanted HUMSCs rats did not express human Insulin gene. 6 the survival time of diabetic rats transplanted with HUMSCs was significantly longer than that of the control group (P0.05). Conclusion: human umbilical cord mesenchymal stem cells have the potential to differentiate into extroverted insulin-secreting cells in vivo, and human umbilical cord mesenchymal stem cells can be used as a source of islet cells.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R587.1;R329
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