人脐源性胰岛前体细胞移植治疗1型糖尿病的实验研究

发布时间：2018-05-01 06:31

  本文选题：1型糖尿病 + 人脐带间充质干细胞　； 参考：《贵州医科大学》2017年硕士论文

【摘要】：目的评价hUMSCs在体外诱导为胰岛前体细胞后治疗T1D大鼠的效果。方法1.分离、培养及鉴定hUMSCs;2.取第4代hUMSCs采用分阶段联合诱导方法诱导并观察诱导后细胞形态变化;3.分别取诱导后第7d、14d和21d细胞进行免疫荧光技术检测Ngn3、胰岛素和MafA表达;Western-blot法检测诱导后第7d、14d和21d细胞MafA和Glut2蛋白表达;4.选择正常雄性SD大鼠44只,随机分为诱导细胞组、干细胞组、模型组和正常组,正常组给予生理盐水,余均给予链脲佐菌素腹腔注射建立T1D模型;5.将经CM-DIL标记的胰岛前体细胞移植到糖尿病大鼠胰腺被膜下,实验周期为60天,分为2W、4W、6W、8W四个时间段,观察各组血糖和体重变化情况;HE染色观察大鼠胰岛形态;Western-blot法检测各时期各组MafA表达改变;分析移植后MafA和大鼠血糖相关性;分析MafA在干细胞组和诱导细胞组之间的表达差异性。结果1.成功分离培养出hUMSCs,诱导后细胞形态宽大、出现集落;2.免疫荧光技术检测Ngn3、胰岛素和MafA蛋白均为阳性表达;Western-blot检测诱导后细胞MafA在正常组和7d时几乎无表达,14d和21d组稍增高,Glut2在7d增高,14d时达高峰,21天时稍弱;3.共有38只大鼠成模,实验组大鼠血糖于第7天时升高;细胞移植后,干细胞组和诱导细胞组血糖均不同程度下降,但诱导细胞组降低更明显,模型组仍维持在高血糖状态;模型组体重在整个实验过程中呈现持续降低,诱导细胞组和干细胞组体重均在第4周后呈升高趋势;4.Western-blot法显示移植后各组均能检测到MafA表达且均呈升高趋势,干细胞组在第6W时明显升高,诱导细胞组在第4W时明显升高;移植后MafA和血糖相关性两组均呈负性相关;MafA在干细胞组和诱导细胞组之间的表达无差异性。结论人脐带间充质干细胞在微环境下诱导可诱导分化为胰岛前体细胞,干细胞和胰岛前体细胞移植后对T1D鼠有降血糖和升高体重的作用。
[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.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

【参考文献】

相关期刊论文 前8条

1 檀梦天;洪艳;韩晶;蒋鑫;;Hes1在hUMSCs向胰岛前体细胞诱导过程中的表达改变[J];世界华人消化杂志;2016年09期

2 高勇;喻萍;蔡涛;陈翔;;Ngn3基因及其转录调控网络在胰腺内分泌发育与功能调控中的作用[J];中国细胞生物学学报;2013年07期

3 邢小娟;效梅;丹慧国;安立龙;;胰岛发育相关的转录因子[J];中国细胞生物学学报;2013年06期

4 张芬熙;洪艳;梁文妹;;人脐带间充质干细胞的分离培养及超微结构特点研究[J];贵阳医学院学报;2013年01期

5 武京国;谢方南;马慧雨;王黔;曹谊林;肖苒;;EGFP和CM-Dil示踪骨髓间充质干细胞构建组织工程骨的体内研究[J];中国美容医学;2012年03期

6 王广宇;赵芳;郝永蕾;朱旅云;李晓玲;胡丽叶;马利成;单巍;杨少玲;;脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞分化及移植治疗1型糖尿病(英文)[J];中国组织工程研究与临床康复;2011年40期

7 袁庆新;滕丽萍;徐宽枫;德伟;刘超;;大鼠胰腺发育过程中胰岛素释放功能完善的研究[J];南京医科大学学报(自然科学版);2010年11期

8 吕璐璐;宋永平;魏旭东;房伯俊;张艳莉;李玉富;;人脐带和骨髓源间充质干细胞生物学特征的对比研究[J];中国实验血液学杂志;2008年01期

相关博士学位论文 前1条

1 曲惠廷;间充质干细胞向胰岛素分泌细胞分化的调控及应用基础研究[D];山东大学;2014年

相关硕士学位论文 前1条

1 祁兵;不同剂量人脐带间充质干细胞对实验性糖尿病大鼠血糖的影响[D];新乡医学院;2014年

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