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学科主题: 内科学
题名:
人脐带间充质干细胞定向诱导分化为胰岛样细胞的实验研究
作者: 申义
答辩日期: 2016-05-11
导师: 王意忠
专业: 内科学
授予单位: 北京大学
授予地点: 北京大学航天临床医学院
学位: 硕士
关键词: 人脐带间充质干细胞 ; 胰岛样细胞 ; 诱导分化 ; 胰岛素 ; 巢蛋白
其他题名: An Experimental Study of Induced Directional Differentiation of HuMSCs into Islet-like Cells
分类号: R329
摘要:

背景:

糖尿病是世界上最常见的慢性疾病之一,随着人们生活水平的提高,老龄化与肥胖症发病率的增长,糖尿病的患病率也在逐年增加。根据国际糖尿病联盟2015年发布的最新数据显示,全球共有4.15亿名成年糖尿病患者,即世界每11个人中就有1个患有糖尿病。然而,糖尿病的发病机理尚未被完全阐明,临床治疗糖尿病的方法包括糖尿病教育、饮食控制、运动疗法、药物疗法,血糖监测和其它常规疗法,但这些仅仅是对症治疗,患者的胰岛功能在逐渐下降,治疗效果大多数并不理想。因此,寻找新的方法来治疗糖尿病是一个非常重大并且紧迫的研究课题。胰岛细胞移植技术治疗糖尿病是一种新方法,但临床上同种异体胰腺移植由于手术复杂、死亡率高、免疫排斥反应强烈及供体的严重不足等诸多因素阻止了其广泛的应用。随着干细胞的深入研究及转化医学的进展,利用干细胞诱导分化的胰岛细胞治疗糖尿病成为广大医务人员和糖尿病患者新的希望。国内外研究证实人脐带间充质干细胞可定向诱导分化为胰岛样细胞,但分化过程中胰岛素与巢蛋白表达的变化等相关研究甚少。

目的:

建立分离培养人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, HuMSCs)的方法,对其表面标志进行鉴定。

体外间充质干细胞(mesenchymal stem cells, MSCs)向胰岛样细胞诱导分化,探讨其分化过程中胰岛素和巢蛋白的表达,为临床选择最佳的细胞移植时机提供一定的参考价值;

体外诱导HuMSCs分化为胰岛样细胞,评估其生物学功能。

方法:

人脐带间充质干细胞的培养:采用UltraCULTURETM培养基在GMP标准的实验室进行人脐带间充质干细胞的培养。选择直接贴壁法进行干细胞的扩增培养,在人脐带间充质干细胞长至80%时,用0.25 %胰蛋白酶将其消化、终止消化、离心并制成细胞悬液,按照1:3分瓶继续培养,传至3代后,将部分细胞用于诱导分化,一部分作为对照继续培养。将盛有细胞和培养液的培养瓶置于倒置显微镜下,观察细胞的形态及密度等。

人脐带间充质干细胞的鉴定:取培养的第3代细胞,胰蛋白酶消化后制成细胞悬液,适量PBS液洗涤细胞,调整细胞数量为5×105/150 ul后分装6个检测管中。分别加入抗体CD44、CD90、CD105、CD14、CD34、HLA-DR等各10ul,4℃孵育30 min,流式细胞仪检测其表型。

人脐带间充质干细胞成骨分化检测:细胞以1×104个/孔的密度接种于6孔板,每孔加入2ml培养基,待细胞长至80%时,更换为成骨诱导培养液,每3天换液1次。诱导9天后,进行茜素红染色。

人脐带间充质干细胞成脂分化检测:细胞以2×104个/孔的密度接种于6孔板,每孔加入2ml培养基,待细胞长至80%时,更换为成脂诱导培养液,每3天换液1次。诱导2-3周后,进行油红O染色。

人脐带间充质干细胞的诱导分化为胰岛样细胞:按照两阶段诱导方案将干细胞定向诱导为胰岛样细胞。第一阶段:在UltraCULTURE培养基中加入4 nmol/L 活化素A,25μg/L表皮生长因子,100μg/L β-神经生长因子,10 mmol/L尼克酰胺,诱导培养至14天;第二阶段,在UltraCULTURE的培养基中加入1%胰岛素-转铁蛋白-硒,10 mmol/L尼克酰胺,10μg/L碱性成纤维细胞生长因子,继续诱导培养14天。在诱导分化过程中观察细胞形态变化,流式细胞技术检测分化细胞巢蛋白及胰岛素的表达,双硫腙染色鉴定胰岛样细胞团中锌离子的表达。

结果:

流式细胞仪检测人脐带间充质干细胞表面抗原,阴性表达CD14-FITC(0.15%)、CD34-PE(0.20%)、HLA-DR-APC(0.04%),阳性表达CD44-FITC(99.86%)、CD90-PE(99.02%)、CD105-APC(99.8%),符合干细胞表面标志表达。

人脐带间充质干细胞成功诱导分化为成骨细胞与成脂细胞,茜素红与油红O染色均呈阳性。

诱导组分化过程中胰岛素水平逐渐增高,至28天达到较高水平,对照组胰岛素表达呈阴性。另外,诱导分化第14天,巢蛋白表达水平达到较高水平,随着诱导时间延长表达水平逐渐下降。

人脐带间充质干细胞诱导28天形成胰岛样细胞团,双硫腙染色阳性。本实验将人脐带间充质干细胞成功诱导分化为胰岛样细胞,并且检测了诱导过程中胰岛素与巢蛋白的表达情况。

结论:

该研究证实采用直接贴壁法培养的人脐带间充质干细胞,具有很高的增殖活性,流式细胞仪鉴定符合间充质干细胞的生物学特性。

成功将人脐带间充质干细胞诱导分化为骨细胞、脂肪细胞,证实了人脐带间充质干细胞具有体外诱导分化的潜能。

在体外利用多种诱导因子可以将人脐带间充质干细胞分化为胰岛样细胞。在28天的诱导分化过程中, 胰岛素表达水平随着体外诱导培养时间的延长呈现逐渐递增的趋势,而巢蛋白表达水平呈现先递增,然后逐渐递减的变化。

英文摘要:

Background: Diabetes is one of the most common onic diseases around the world. With the improvement of people’s living standard and increase of aging and incidence of obesity, the incidence of diabetes tends to grow year by year. According to the latest data released by International Diabetes Federation in 2015, a total of 415 million adults suffered from diabetes in the global context, i.e., one of 11 people suffer from diabetes. However, the pathogenesis of diabetes has not been fully elucidated. Clinical treatments of diabetes include diabetes education, diet therapy, exercise therapy, drug treatment, blood sugar monitoring and other routine therapies, but all of these are just symptomatic treatments. The patient’s pancreatic islet function gradually declines. Most treatments don’t have an ideal effect. Thus, looking for a new way to treat diabetes is a very important and urgent research topic. Islet cell transplantation is a new technology for treating diabetes. However, clinically allogeneic pancreas transplantation has complex operation, high mortality, strong immune rejection response and seriously inadequate donors, which prevent its widespread application. As studies on stem cells and clinical translation deepen, using differentiated islet cells induced by stem cells to treat diabetes becomes a new promise for vast medical staff and patients with diabetes. Domestic and foreign research confirms that human umbilical cord mesenchymal stem cells (HuMSCs) can be directionally induced and differentiated into islet-like cells, but there are few studies on the changes of insulin and nestin expressions, etc. during differentiation.

Objective:

To establish a way to separate HuMSCs, and identify their surface marker.

Mesenchymal stem cells (MSCs) are induced and differentiated into islet-like cells. So the present study attempts to explore the expressions of insulin and nestin during differentiation and provide a certain reference value for choosing an optimal timing for cell transplantation clinically.

To induce and differentiate HuMSCs into islet cells in vitro and evaluate their biological functions.

Methods:

The culture of HuMSCs: HuMSCs were cultured in a GMP standard laboratory using UltraCULTURETM . Direct adhesion method was ed to multiply stem cells. When HuMSCs grew to 80%, they were digested with 0.25% trypsin, centrifuged, made into cell suspension and cultured in 1:3 separating flask. Three generations later, some cells were used to induce differentiation and others were cultured as control. The culture flask containing cells and culture solution was placed under inverted microscope, to observe the morphology and density, etc. of cells.

The identification of HuMSCs: the third generation of cultured cells was garnered and made into cell suspension after trypsin digestion. The cells were flushed with a proper amount of PBS and separated into 6 test tubes after the cell number was adjusted as 5×105/150 ul. After that 10ul of CD44, CD90, CD105, CD14, CD34 and HLA-DR antibodies etc. were added and incubated at 4℃ for 30 min. Their phenotype was detected with a flow cytometry.

The osteogenic differentiation testing of HuMSCs: the cells were seeded into a 6-well plate at a density of 1×104 cells per well and 2 ml culture medium was added to each well. When cells grew to 80%, replaced with osteogenic induction culture medium once every 3 days. After inducting for 9 days, alizarin red staining was conducted.

The adipogenic differentiation testing of HuMSCs: the cells were seeded into a 6-well plate at a density of 2×104 cells per well and 2 ml culture medium was added to each well. When cells grew to 80%, replaced with adipogenic induction culture medium once every 3 days. After inducting for 2 to 3weeks, oil red O staining was conducted.

The induced differentiation of HuMSCs into islet-like cells: according to a 2-stage induction scheme, stem cells were directionally induced into islet cells. State 1: 4 nmol/L activing A, 25μg/L epidermal growth factor, 100μg/L β-nerve growth factor and 10 mmol/L niacinae were added to UltraCULTURE, induced and cultured for 14 days. Stage 2: 1% insulin- transferrin-selenium, 10 mmol/L niacinae and 10μg/L alkaline fibroblast growth factor were added to UltraCULTURE, induced and cultured for 14 days. During the induced differentiation, the morphologic changes of cells were observed, the expressions of nestin and insulin were tested using flow cytometry and the expression of zinc ions in islet-like cell clusters was identified using dithizone staining.

Results:

After the surface antigen of HuMSCs was detected using flow cytometry, the negative expressions of CD14-FITC (0.15%), CD34-PE (0.20%) and HLA-DR-APC (0.04%) and positive expressions of CD44-FITC (99.86%), CD90-PE (99.02%) and CD105 and APC (99.8%) conform to surface marker expressions of stem cells.

HuMSCs were induced and differentiated into osteoblasts and lipoblasts successfully. Both alizarin red and oil red O staining had positive results.

During the differentiation, the insulin level in induced group gradually increased and reached a high level on 28d. The insulin expression in control group was negative. In addition, 14d after the induced differentiation, the expression level of nestin reached a high level. As induction time prolonged, the expression level gradually declined.

After inducing HuMSCs for 28 days, islet-like cell clusters were formed. The dithizone staining showed positive results. This experiment induced and differentiated HuMSCs into islet-like cells successfully and detected the expressions of insulin and nestin during induction.

Conclusion:

The present study proves that HuMSCs cultured with direct adherence method have high proliferation ability. Flow cytometry detection shows that the cells conform to biological acteristics of mesenchymal stem cells.

HuMSCs are induced and differentiated into osteoblasts and lipoblasts successfully, which proves that HuMSCs have the potential of in vitro induction and differentiation.

Using a variety of inducing factors in vitro, HuMSCs can be differentiated into islet-like cells. In the process of 28-day induced differentiation, the expression level of insulin tends to grow gradually with the prolongation of in vitro induced culture time, while the expression level of nestin tends to first rise and then drop with the prolongation of induced culture time.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125279
Appears in Collections:北京大学航天临床医学院_学位论文

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作者单位: 北京大学航天临床医学院

Recommended Citation:
申义. 人脐带间充质干细胞定向诱导分化为胰岛样细胞的实验研究[D]. 北京大学航天临床医学院. 北京大学. 2016.
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