本文選題：1型糖尿病 + 人臍帶間充質(zhì)干細(xì)胞�。� 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的評價hUMSCs在體外誘導(dǎo)為胰島前體細(xì)胞后治療T1D大鼠的效果。方法1.分離、培養(yǎng)及鑒定hUMSCs;2.取第4代hUMSCs采用分階段聯(lián)合誘導(dǎo)方法誘導(dǎo)并觀察誘導(dǎo)后細(xì)胞形態(tài)變化;3.分別取誘導(dǎo)后第7d、14d和21d細(xì)胞進(jìn)行免疫熒光技術(shù)檢測Ngn3、胰島素和MafA表達(dá);Western-blot法檢測誘導(dǎo)后第7d、14d和21d細(xì)胞MafA和Glut2蛋白表達(dá);4.選擇正常雄性SD大鼠44只,隨機(jī)分為誘導(dǎo)細(xì)胞組、干細(xì)胞組、模型組和正常組,正常組給予生理鹽水,余均給予鏈脲佐菌素腹腔注射建立T1D模型;5.將經(jīng)CM-DIL標(biāo)記的胰島前體細(xì)胞移植到糖尿病大鼠胰腺被膜下,實驗周期為60天,分為2W、4W、6W、8W四個時間段,觀察各組血糖和體重變化情況;HE染色觀察大鼠胰島形態(tài);Western-blot法檢測各時期各組MafA表達(dá)改變;分析移植后MafA和大鼠血糖相關(guān)性;分析MafA在干細(xì)胞組和誘導(dǎo)細(xì)胞組之間的表達(dá)差異性。結(jié)果1.成功分離培養(yǎng)出hUMSCs,誘導(dǎo)后細(xì)胞形態(tài)寬大、出現(xiàn)集落;2.免疫熒光技術(shù)檢測Ngn3、胰島素和MafA蛋白均為陽性表達(dá);Western-blot檢測誘導(dǎo)后細(xì)胞MafA在正常組和7d時幾乎無表達(dá),14d和21d組稍增高,Glut2在7d增高,14d時達(dá)高峰,21天時稍弱;3.共有38只大鼠成模,實驗組大鼠血糖于第7天時升高;細(xì)胞移植后,干細(xì)胞組和誘導(dǎo)細(xì)胞組血糖均不同程度下降,但誘導(dǎo)細(xì)胞組降低更明顯,模型組仍維持在高血糖狀態(tài);模型組體重在整個實驗過程中呈現(xiàn)持續(xù)降低,誘導(dǎo)細(xì)胞組和干細(xì)胞組體重均在第4周后呈升高趨勢;4.Western-blot法顯示移植后各組均能檢測到MafA表達(dá)且均呈升高趨勢,干細(xì)胞組在第6W時明顯升高,誘導(dǎo)細(xì)胞組在第4W時明顯升高;移植后MafA和血糖相關(guān)性兩組均呈負(fù)性相關(guān);MafA在干細(xì)胞組和誘導(dǎo)細(xì)胞組之間的表達(dá)無差異性。結(jié)論人臍帶間充質(zhì)干細(xì)胞在微環(huán)境下誘導(dǎo)可誘導(dǎo)分化為胰島前體細(xì)胞,干細(xì)胞和胰島前體細(xì)胞移植后對T1D鼠有降血糖和升高體重的作用。
[Abstract]:Objective to evaluate the effect of hUMSCs on T 1D rats induced by islet progenitor cells in vitro. Method 1. Isolation, culture and identification of hue MSCs2. The fourth passage of hUMSCs was induced and observed the morphological changes of the cells by the method of stepwise combined induction. Ngn3 was detected by immunofluorescence on day 14 and 21 after induction, and the expression of MafA and Glut2 protein was detected by Western-blot on day 14 and 21 respectively. Forty-four male Sprague-Dawley rats were randomly divided into induced cell group, stem cell group, model group, normal group and normal group. The rest rats were given streptozotocin intraperitoneal injection to establish T1D model. The islet precursor cells labeled with CM-DIL were transplanted under the pancreatic capsule of diabetic rats. The experimental period was 60 days, which was divided into four periods: 2WN 4W + 6W + 8W. The changes of blood glucose and body weight in each group were observed by HE staining. The expression of MafA in each group was detected by Western-blot method. The correlation between MafA and blood glucose after transplantation was analyzed. The difference of MafA expression between stem cell group and induced cell group was analyzed. Result 1. HU MSCs were isolated and cultured successfully. Ngn3 was detected by immunofluorescence technique, and the expression of insulin and MafA protein was detected by Western-blot. The expression of MafA in the normal group and the 7 day group was almost no higher than that in the 21 day group. The expression of Glut2 was slightly higher in the control group and at the 21st day group. The expression of Glut2 was slightly higher than that of the control group on the 7th day and reached the peak on the 14th day and reached the peak on the 21st day. After transplantation, blood glucose in stem cell group and induced cell group decreased in varying degrees, but decreased more obviously in induced cell group, and remained in hyperglycemia state in model group. The body weight of the model group decreased continuously during the whole experiment, and the body weight of the induced cell group and stem cell group increased after 4 weeks. Western-blot analysis showed that the expression of MafA was detected in all groups after transplantation and showed an increasing trend. In stem cell group, the expression of MafA in stem cell group was significantly higher than that in induced cell group at the 4th week, and there was no difference in the expression of MafA between stem cell group and induced cell group after transplantation. Conclusion Human umbilical cord mesenchymal stem cells induced by microenvironment can be induced to differentiate into islet precursor cells. Stem cells and islet precursor cells can reduce blood sugar and increase body weight of T 1D mice after transplantation.
【學(xué)位授予單位】：貴州醫(yī)科大學(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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