miRNA-210在人骨髓间充质干细胞诱导成骨分化中的作用及意义

发布时间：2018-09-04 06:20

【摘要】： 背景 miRNA(microRNA)是由约22个核苷酸组成的单链小RNA,并在真核生物细胞中广泛存在,miRNA基因约占整个基因组的1%。近些年来的各项研究表明miRNA一个非常庞大的家族,存在于是在植物、线虫、果蝇、小鼠、大鼠和人等多个物种中。miRNA对各种进行基因表达调控是通过在转录后水平与其靶mRNA的互补配对,导致mRNA的翻译、翻译抑制以及降解。一个异常复杂的调控网络由miRNA与靶mRNA分子组成,参与包括细胞繁殖与凋亡,细胞分化与发育,以及逆境应答等多种生物学过程。骨髓间充质干细胞(MSCs)可具有多向分化潜能,能分化成多种类型的结缔组织,如骨组织、软骨组织、骨骼肌组织、肌腱组织、韧带组织、真皮组织、脂肪组织,也可分化成神经系统的神经元细胞和神经胶质细胞等。骨髓间充质干细胞(MSCs)易于在体外扩增,来源又比较广泛,是目前组织工程中一种重要的种子细胞。各种生物体的生长发育、信号转导、组织分化以及疾病发生等miRNA均参与调控,其同时它众多生物体发育和行为等的变化miRNA同样起着决定作用。在干细胞增殖以及分化中miRNA的作用及意义这些年来开始受到关注。在许多种干细胞中miRNA都有表达,并且通过调节干细胞中某些关键基因的表达,对干细胞的保持自我更新以及多向分化等功能发挥作用。目的通过检测骨髓间充质干细胞及其诱导分化的成骨细胞中miRNA-210基因的表达高低,推测miRNA-210的功能,证明miRNA-210调控人骨髓间充质干细胞向成骨分化。从而,进一步验证了在骨髓间充质干细胞定向分化中miRNA的重要作用及意义。方法 1.对收集的标本中的人骨髓间充质干细胞采用全骨髓贴壁法(贴壁筛选法)、密度梯度离心法进行分离培养。人骨髓间充质干细胞的鉴定是通过观察细胞形态特点并对细胞表面标志物(CD29、CD44、CD34、CD45)采用流式细胞仪检测来完成。 2.采用文献报道的经典的方法:β-甘油磷酸钠、地塞米松和维生素C联合诱导人MSCs向成骨细胞方向分化。诱导分化的成骨细胞的鉴定是碱性磷酸酶(ALP)染色、茜素红S法染色来对比完成。 3.利用Realtime-PCR观察对比miRNA-210在人MSCs与其诱导成的成骨细胞的差异。 4.采用将miRNA-210模拟物及阻遏物转染至人MSCs中14天后,通过碱性磷酸酶(ALP)染色、茜素红S法染色对转染后的细胞进行鉴定。结果 1.经全骨髓贴壁法(贴壁筛选法)及密度梯度离心法分离贴壁培养所得到的细胞,采用流式细胞仪检测的结果为:细胞表面标志物CD34、CD45为阴性,细胞表面标志物CD29、CD44为阳性,符合骨髓间充质干细胞基本特征。 2.采用文献报道的经典的方法:β-甘油磷酸钠、地塞米松和维生素C联合诱导14天后得到的细胞:碱性磷酸酶染色(ALP)染色及茜素红S法染色均为阳性,符合成骨细胞的特征。 3.利用Realtime-PCR观察与对比miRNA-210在人MSCs与其诱导成的成骨细胞的差异。表明miRNA-210在人MSCs中高表达,在诱导成的成骨细胞低表达。 4.将miRNA-210模拟物(miRNA-210 mimics)及阻遏物(miRNA-210 inhibitors)转染至人MSCs中14天后,通过碱性磷酸酶(ALP)染色、茜素红S法染色对转染后的细胞进行鉴定。转染miRNA-210模拟物的细胞能够保持骨髓间充质干细胞基本特征;而转染miRNA-210阻遏物的细胞碱性磷酸酶(ALP)染色、茜素红S法染色均阳性。结论 1.用全骨髓贴壁法(贴壁筛选法)及密度梯度离心法可以从骨髓中分离得到人MSCs,人MSCs可在体外培养被诱导分化为成骨细胞。 2.通过Realtime-PCR的结果可以推测miRNA-210具有保持骨髓间充质干细胞特性的功能。 3.通过转染miRNA-210模拟物及阻遏物进一步证明miRNA-210具有保持骨髓间充质干细胞特性的功能。
[Abstract]:background
MicroRNA (microRNA) is a single-stranded small RNA composed of about 22 nucleotides and widely exists in eukaryotic cells. The microNA gene accounts for about 1% of the entire genome. Recent studies have shown that microNA is a very large family of plants, nematodes, fruit flies, mice, rats and humans. MicroNA is carried out in a variety of species. Gene expression regulation results in the translation, translation inhibition and degradation of mRNA by complementary pairing with its target mRNA at post-transcriptional level. An unusually complex regulatory network consists of microRNAs and target mRNA molecules involved in a variety of biological processes, including cell proliferation and apoptosis, cell differentiation and development, and stress response.
Bone marrow mesenchymal stem cells (MSCs) can differentiate into many types of connective tissue, such as bone tissue, cartilage tissue, skeletal muscle tissue, tendon tissue, ligament tissue, dermal tissue, adipose tissue, nerve cells and glial cells of the nervous system. Bone marrow mesenchymal stem cells (MSCs) are easy to differentiate into. In vitro expansion is a relatively important source of seed cells in tissue engineering.
MicroRNAs are involved in the regulation of growth and development, signal transduction, tissue differentiation and disease occurrence of various organisms. At the same time, microRNAs play a decisive role in the development and behavior of many other organisms. Both of them are expressed and play an important role in maintaining self-renewal and multidirectional differentiation of stem cells by regulating the expression of some key genes in stem cells.
objective
By detecting the expression of microRNA210 gene in bone marrow mesenchymal stem cells and osteoblasts induced by microRNA210, the function of microRNA210 was speculated, which proved that microRNA210 regulated the osteogenic differentiation of human bone marrow mesenchymal stem cells.
Method
1. Bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherence (adherence screening) and density gradient centrifugation.
2. Using the classical method reported in the literature, human MSCs were induced to differentiate into osteoblasts by sodium glycerophosphate, dexamethasone and vitamin C. The osteoblasts were identified by alkaline phosphatase (ALP) staining and alizarin red S staining.
3. Realtime-PCR was used to observe and compare the difference between miRNA-210 and MSCs induced osteoblasts.
4. The transfected cells were identified by Alizarin red S staining and alkaline phosphatase (ALP) staining after 14 days of transfection of mimic RNA-210 and repressor into human MSCs.
Result
1. The adherent cells were isolated by whole bone marrow adherent method (adherent screening) and density gradient centrifugation. The results of flow cytometry were as follows: cell surface markers CD34, CD45 were negative, cell surface markers CD29, CD44 were positive, which accorded with the basic characteristics of bone marrow mesenchymal stem cells.
2. Alkaline phosphatase staining (ALP) and alizarin red S staining were positive in the cells obtained 14 days after induction with beta-glycerophosphate, dexamethasone and vitamin C, which accorded with the characteristics of osteoblasts.
3. Realtime-PCR was used to observe and compare the difference between human MSCs and osteoblasts induced by microRNA-210. The results showed that microRNA-210 was highly expressed in human MSCs and low expressed in induced osteoblasts.
4. Fourteen days after transfection of human MSCs with microRNA210 mimics and microRNA210 inhibitors, the transfected cells were identified by alkaline phosphatase (ALP) staining and alizarin red S staining. Cell alkaline phosphatase (ALP) staining and alizarin red S staining were all positive.
conclusion
1. Human MSCs can be isolated from bone marrow by whole bone marrow adherence screening and density gradient centrifugation. Human MSCs can be induced to differentiate into osteoblasts in vitro.
2. The results of Realtime-PCR suggested that microRNA-210 could maintain the properties of bone marrow mesenchymal stem cells.
3. Transfection of mimics and repressors of microRNA210 further proved that microRNA210 has the function of maintaining the characteristics of bone marrow mesenchymal stem cells.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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