|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|
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；
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。
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.
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.
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.
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.
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.
|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.|