胰島聯(lián)合骨髓間充質(zhì)干細(xì)胞治療糖尿病大鼠的研究
發(fā)布時(shí)間：2018-12-26 15:55
【摘要】：以標(biāo)準(zhǔn)的糖尿病大鼠為模型,探索同種異體的SD大鼠胰島聯(lián)合骨髓間充質(zhì)干細(xì)胞(Bone Marrow Mesenchymal Cells,BMSCs)能否提高治療糖尿病的遠(yuǎn)期療效,為臨床開展胰島聯(lián)合BMSCs移植治療糖尿病的提供實(shí)驗(yàn)依據(jù),并觀察同種異體大鼠聯(lián)合BMSCs移植后細(xì)胞在體內(nèi)的分布、歸巢情況,為臨床細(xì)胞移植后細(xì)胞的定植、歸巢提供理論依據(jù)。鏈脲佐菌素(Streptozotocin,STZ)構(gòu)建大鼠糖尿病模型;貼壁法分離、培養(yǎng)BMSCs,免疫熒光法和流式細(xì)胞鑒定BMSCs的表面抗原(CD44、CD34、CD90、CD45、CD29);膠原酶法分離大鼠胰島,雙硫腙法(Dithizone,DTZ)鑒定胰島細(xì)胞;將細(xì)胞分為對照組;BMSCs培養(yǎng)組;單獨(dú)胰島組;胰島與BMSCs共培養(yǎng)組,于培養(yǎng)后的第1、3、7天取其上清液進(jìn)行ELISA法檢測各組的胰島素與C-肽的分泌;18F-FDG體外標(biāo)記胰島與BMSCs,隨機(jī)將糖尿病大鼠分成:對照(n=15)、單獨(dú)胰島移植組(n=15)、單獨(dú)BMSCs移植組(n=15)、胰島與BMSCs聯(lián)合移植(n=15),經(jīng)門靜脈移植分別移植(對照組0.2ml生理鹽水;胰島組0.2ml胰島500IEQ/100g;干細(xì)胞組0.2ml的3*10^5/100g個(gè)BMSCs;聯(lián)合組0.1ml胰島500IEQ/100g+0.1ml 3*10^5/100g個(gè)BMSCs)。移植后的第1、3、7、14、30天每組每次均隨機(jī)處死3只大鼠,取肝臟、血液,ELISA測量移植后各組大鼠血清的胰島素與C-肽的含量及檢測大鼠的血清的肝功能,免疫熒光法了解肝組織內(nèi)胰島素的表達(dá)情況,HE檢測肝臟的病理情況,PET-CT示蹤移植入體內(nèi)的細(xì)胞分布情況。經(jīng)STZ法誘導(dǎo)糖尿病大鼠期間有95%的大鼠血糖濃度在一周內(nèi)有3天連續(xù)高于16.8mmol/L的,伴隨體重下降,總膽紅素、谷丙轉(zhuǎn)氨酶、谷草轉(zhuǎn)氨酶均升高;經(jīng)全貼壁法培養(yǎng)的BMSCs大多是呈長梭形生長,免疫熒光法的檢測結(jié)果顯示BMSCs為CD44陽性,CD34陰性,流式細(xì)胞儀測量結(jié)果表明BMSCs是高表達(dá)CD90、CD29,低表達(dá)CD45的;膠原酶法能夠很有效的分離出胰島細(xì)胞,且被DTZ染成猩紅色的就是胰島細(xì)胞,未被染成猩紅色的就是外分泌腺;體外共培養(yǎng)中的胰島形態(tài)與單獨(dú)胰島的形態(tài)相比保存的更好,存活的時(shí)間也更長,胰島素與C-肽的分泌也較單獨(dú)胰島的高;移植后聯(lián)合組的血糖在正常血糖內(nèi)波動(dòng)一個(gè)月,單獨(dú)胰島組的血糖水平只能在正常血糖范圍波動(dòng)3天,單獨(dú)BMSCs組、對照組的大鼠在移植后的血糖水平未明顯降低。聯(lián)合組在移植后的第1、7、14、21、30天的血糖明顯低于單獨(dú)胰島組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);聯(lián)合組比胰島組能有效的修復(fù)肝臟的功能;聯(lián)合組的胰島素與C-肽的分泌也比單獨(dú)胰島組高;聯(lián)合組和單獨(dú)胰島組的炎細(xì)胞侵潤都不明顯。PET-CT結(jié)果顯示,BMSCs主要均勻分布在肝臟的右肝,胰島主要聚集在肝門靜脈末端分支,且BMSCs圍繞在胰島的周圍,對照組的肝臟無明顯的顯影。同種異體的胰島聯(lián)合BMSCs治療大鼠糖尿病的效果優(yōu)于單獨(dú)胰島移植。PET-CT能直觀的揭示胰島與BMSCs經(jīng)門靜脈移植入后在肝內(nèi)的分布。
[Abstract]:To explore whether islet of islets combined with bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Cells,BMSCs) of allogeneic SD rats can improve the long-term curative effect of diabetic rats. To provide the experimental basis for the clinical treatment of diabetes mellitus with islet and BMSCs transplantation, and to observe the distribution and homing of cells after allograft combined with BMSCs transplantation, and to provide the theoretical basis for the implantation and homing of the cells after clinical cell transplantation. Streptozotocin (Streptozotocin,STZ) was used to establish diabetic model in rats, and the surface antigen (CD44,CD34,CD90,CD45,CD29) of BMSCs was identified by BMSCs, immunofluorescence assay and flow cytometry. Rat islets were isolated by collagenase and identified by dithizone (Dithizone,DTZ). The cells were divided into control group, BMSCs culture group, isolated islet group; The supernatants of islet and BMSCs were taken from the supernatant on the 7th day after culture and the secretion of insulin and C-peptide was detected by ELISA method. 18F-FDG labeled islets and BMSCs, were randomly divided into three groups: control group (n = 15), islet transplantation group (n ~ 15), BMSCs transplantation group (n ~ 15), islet / BMSCs transplantation group (n ~ (15). Portal vein transplantation (control group, 0.2ml saline); Islet group 0.2ml islet 500 IEQ / 100g; stem cell group 0.2ml 3n10 ^ 5 / 100g BMSCs; + 0.1ml islet 500IEQ/100g 0.1ml 10 ^ 5100g BMSCs). Three rats were randomly killed in each group on the 1st day after transplantation. The liver and blood were taken. ELISA was used to measure the contents of insulin and C-peptide in serum and the liver function of the rats. The expression of insulin in liver tissue was detected by immunofluorescence, the pathological changes of liver were detected by HE and the distribution of cells transplanted into body by PET-CT tracer. During the period of diabetes induced by STZ, 95% of the diabetic rats had higher blood glucose concentration than that of 16.8mmol/L for 3 days in one week. With the decrease of body weight, total bilirubin, alanine aminotransferase and alanine aminotransferase were all increased. The results of immunofluorescence assay showed that BMSCs was CD44 positive and CD34 negative. Flow cytometry showed that BMSCs expressed high expression of CD90,CD29, and low expression of CD45. Collagenase method can effectively isolate islet cells, and the scarlet cells stained by DTZ are islet cells and exocrine glands that are not stained scarlet. The morphology of islets in vitro was better than that of isolated islets, the survival time was longer, and the secretion of insulin and C-peptide was higher than that of isolated islets. After transplantation, the blood glucose of the combined group fluctuated within the normal blood glucose for one month, the blood glucose level of the islet group only fluctuated within the normal blood glucose range for 3 days, the blood glucose level of the BMSCs group and the control group did not decrease significantly after transplantation. The blood glucose in the combined group was significantly lower than that in the islet group after transplantation on the 1st day after transplantation (P0.05), and the liver function in the combined group was more effective than that in the islet group. The secretion of insulin and C-peptide in the combined group was also higher than that in the islet group alone. The results of PET-CT showed that BMSCs mainly distributed in the right liver of liver, pancreatic islets mainly concentrated in the branches of portal vein of liver, and BMSCs was around the islets. There was no obvious development of liver in the control group. The effect of islet allograft combined with BMSCs in the treatment of diabetic rats was better than that of islet transplantation alone. PET-CT could intuitively reveal the distribution of islets and BMSCs in the liver after portal vein transplantation.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
本文編號：2392347
[Abstract]:To explore whether islet of islets combined with bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Cells,BMSCs) of allogeneic SD rats can improve the long-term curative effect of diabetic rats. To provide the experimental basis for the clinical treatment of diabetes mellitus with islet and BMSCs transplantation, and to observe the distribution and homing of cells after allograft combined with BMSCs transplantation, and to provide the theoretical basis for the implantation and homing of the cells after clinical cell transplantation. Streptozotocin (Streptozotocin,STZ) was used to establish diabetic model in rats, and the surface antigen (CD44,CD34,CD90,CD45,CD29) of BMSCs was identified by BMSCs, immunofluorescence assay and flow cytometry. Rat islets were isolated by collagenase and identified by dithizone (Dithizone,DTZ). The cells were divided into control group, BMSCs culture group, isolated islet group; The supernatants of islet and BMSCs were taken from the supernatant on the 7th day after culture and the secretion of insulin and C-peptide was detected by ELISA method. 18F-FDG labeled islets and BMSCs, were randomly divided into three groups: control group (n = 15), islet transplantation group (n ~ 15), BMSCs transplantation group (n ~ 15), islet / BMSCs transplantation group (n ~ (15). Portal vein transplantation (control group, 0.2ml saline); Islet group 0.2ml islet 500 IEQ / 100g; stem cell group 0.2ml 3n10 ^ 5 / 100g BMSCs; + 0.1ml islet 500IEQ/100g 0.1ml 10 ^ 5100g BMSCs). Three rats were randomly killed in each group on the 1st day after transplantation. The liver and blood were taken. ELISA was used to measure the contents of insulin and C-peptide in serum and the liver function of the rats. The expression of insulin in liver tissue was detected by immunofluorescence, the pathological changes of liver were detected by HE and the distribution of cells transplanted into body by PET-CT tracer. During the period of diabetes induced by STZ, 95% of the diabetic rats had higher blood glucose concentration than that of 16.8mmol/L for 3 days in one week. With the decrease of body weight, total bilirubin, alanine aminotransferase and alanine aminotransferase were all increased. The results of immunofluorescence assay showed that BMSCs was CD44 positive and CD34 negative. Flow cytometry showed that BMSCs expressed high expression of CD90,CD29, and low expression of CD45. Collagenase method can effectively isolate islet cells, and the scarlet cells stained by DTZ are islet cells and exocrine glands that are not stained scarlet. The morphology of islets in vitro was better than that of isolated islets, the survival time was longer, and the secretion of insulin and C-peptide was higher than that of isolated islets. After transplantation, the blood glucose of the combined group fluctuated within the normal blood glucose for one month, the blood glucose level of the islet group only fluctuated within the normal blood glucose range for 3 days, the blood glucose level of the BMSCs group and the control group did not decrease significantly after transplantation. The blood glucose in the combined group was significantly lower than that in the islet group after transplantation on the 1st day after transplantation (P0.05), and the liver function in the combined group was more effective than that in the islet group. The secretion of insulin and C-peptide in the combined group was also higher than that in the islet group alone. The results of PET-CT showed that BMSCs mainly distributed in the right liver of liver, pancreatic islets mainly concentrated in the branches of portal vein of liver, and BMSCs was around the islets. There was no obvious development of liver in the control group. The effect of islet allograft combined with BMSCs in the treatment of diabetic rats was better than that of islet transplantation alone. PET-CT could intuitively reveal the distribution of islets and BMSCs in the liver after portal vein transplantation.
【學(xué)位授予單位】：電子科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
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