人臍帶間充質(zhì)干細(xì)胞向胰島素分泌細(xì)胞分化的初步研究
發(fā)布時(shí)間：2018-10-20 13:13
【摘要】： 目的:探索人臍帶間充質(zhì)干細(xì)胞(Human Umbilical cord Wharton’s Jelly-derived Mesenchymal Stem Cells ,HUMSCs)在大鼠胰島細(xì)胞生長(zhǎng)的微環(huán)境中及在糖尿病大鼠體內(nèi)向胰島素分泌細(xì)胞分化的潛能。 方法:①實(shí)驗(yàn)材料: SD大鼠由廣州中醫(yī)藥大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。對(duì)動(dòng)物處置符合動(dòng)物倫理學(xué)標(biāo)準(zhǔn)。臍帶取自汕頭大學(xué)醫(yī)學(xué)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(duì)實(shí)驗(yàn)知情同意,并經(jīng)醫(yī)院倫理委員會(huì)批準(zhǔn)。②實(shí)驗(yàn)方法:分離培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,并采用膠原酶消化法分離大鼠胰島細(xì)胞。將HUMSCs與大鼠胰島細(xì)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(cè)HUMSCs分泌的胰島素水平,RT-PCR方法檢測(cè)共培養(yǎng)的HUMSCs中的Pdx-1基因的表達(dá),以未共培養(yǎng)的HUMSCs做對(duì)照。用熒光染料Hoechst33258標(biāo)記HUMSCs,經(jīng)尾靜脈將HUMSCs移植入糖尿病模型大鼠體內(nèi),一個(gè)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(cè)人insulin基因的表達(dá)。 結(jié)果:體外研究:①共培養(yǎng)條件下的HUMSCs由長(zhǎng)梭形逐漸變圓,并聚集成團(tuán)。②放射免疫法結(jié)果表明,共培養(yǎng)組HUMSCs隨著共培養(yǎng)時(shí)間的延長(zhǎng)分泌的胰島素量明顯增多,當(dāng)HUMSCs有聚集成團(tuán)傾向時(shí)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(duì)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(dá)Pdx-1 ,共培養(yǎng)3d的HUMSCs開始表達(dá)Pdx-1,共培養(yǎng)7d的HUMSCs仍表達(dá)Pdx-1,共培養(yǎng)14d的HUMSCs未表達(dá)Pdx-1。體內(nèi)研究:④熒光顯微鏡下看到移植標(biāo)記Hoechst33258的HUMSCs在大鼠的胰腺中呈均勻的點(diǎn)狀分部,發(fā)出藍(lán)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(shí)能夠檢測(cè)出人Insulin基因,而未經(jīng)移植HUMSCs的大鼠胰腺不表達(dá)人Insulin基因。⑥移植HUMSCs的糖尿病大鼠與對(duì)照組相比能明顯延長(zhǎng)生存期,P0.05。 結(jié)論:人臍帶間充質(zhì)干細(xì)胞具有在體內(nèi)外向胰島素分泌細(xì)胞分化的潛能,人臍帶間充質(zhì)干細(xì)胞可以作為胰島細(xì)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(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R587.1;R329
本文編號(hào)：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(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R587.1;R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 林曉波;何紅燕;羅敏潔;崔冰琳;馮學(xué)永;蔣學(xué)武;謝慶東;黃天華;應(yīng)文娟;馬廉;;人臍帶間充質(zhì)干細(xì)胞向心肌樣細(xì)胞分化的研究[J];實(shí)用兒科臨床雜志;2007年13期
2 ;In vitro cultivation and differentiation of fetal liver stem cells from mice[J];Cell Research;2005年05期
3 龍愛梅;李富榮;李路;;糖尿病細(xì)胞治療的研究進(jìn)展[J];醫(yī)學(xué)綜述;2006年01期
4 徐剛,袁敏生,Gordon C.Weir;胰島細(xì)胞移植的現(xiàn)狀[J];中國(guó)糖尿病雜志;2001年03期
5 馬廉;崔冰琳;馮學(xué)永;羅敏潔;蔣學(xué)武;楊立業(yè);謝慶東;黃天華;;人臍帶間充質(zhì)干細(xì)胞的生物學(xué)特性及向神經(jīng)樣細(xì)胞分化的研究[J];中華兒科雜志;2006年07期
，本文編號(hào)：2283288
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2283288.html
最近更新
教材專著
