胰岛素样生长因子诱导骨髓间充质干细胞向少突胶质细胞定向分化

发布时间：2018-05-16 10:19

本文选题：间充质干细胞 + IGF-1　；参考：《华中科技大学》2009年博士论文

【摘要】： 目的: 1.体外探讨分离与培养大鼠骨髓间充质干细胞(mesenchymal stem cells, MSCs)的方法,并观察其体外的多向分化与增殖能力以及其生物学特性,还对细胞表面干细胞相关标志物进行鉴定,为骨髓间充质干细胞的进一步研究奠定基础。 2.探讨胰岛素样生长因子和多种细胞因子联合诱导骨髓间充质干细胞向少突胶质细胞分化的可行性,实验条件。并用激光共聚焦显微镜观察分化细胞的阳性率,及形态学变化寻找最佳的诱导条件,为获得大量少突胶质细胞移植修复脊髓损伤奠定基础。 3.在实验二的基础上,观察骨髓间充质干细胞的增殖分化过程,判断IGF-1在少突胶质细胞分化中的那个环节起作用,寻找胰岛素样生长因子和多种细胞因子联合诱导骨髓间充质干细胞向少突胶质细胞分化的作用机制,并给予一定的干预措施,进一步予以证实。方法: 1.取原代细胞后,用贴壁筛选法和胰蛋白酶消化法分离纯化,倒置相差显微镜观察细胞形态及生长特征,研究其增殖过程,绘制出生长曲线,采用免疫组化法对细胞表面干细胞标志CD44进行鉴定,对第4代细胞分别用成骨,成软骨,成脂肪和成神经诱导剂培养,行碱性磷酸酶染色,Van kossa银染色法,Ⅱ型胶原免疫组化染色,油红O染色,NeuN抗体免疫组化染色以及形态学观察,证实其干细胞的多向分化潜能。 2.选取生长良好的第4代骨髓间充质干细胞,胰酶消化后,离心加入诱导液,转移至含有盖玻片的培养皿中,细胞数为1×10~4个/cm~2,放入体积分数为0.05的CO_2孵箱中培养48h后,诱导组细胞更换培养液,用含不同浓度IGF-1的分化培养液培养3d后,观察骨髓间充质干细胞向少突胶质细胞诱导分化过程中的形态学变化,收取细胞进行特异性标志物大鼠磷脂碱性蛋白(MBP)、半乳糖脑苷脂(Glac)、半定量RT-PCR(semi-quatitative reverse transctiption-polymerase chain reaction)检测,Western-blot测定大鼠磷脂碱性蛋白(MBP)含量,免疫细胞化学染色后激光共聚焦显微镜检测骨髓间充质干细胞向少突胶质细胞定向分化的阳性率。并筛选出最佳IGF-1(Insulin growthfactor-1)浓度剂量的分化培养液。 3.根据BMP2(bone morphogenetic proteins)在少突胶质细胞的形成中起抑制作用,少突胶质细胞的成熟需要BMP信号的抑制剂,为了获得IGF—1诱导少突胶质细胞分化的分子机制,我们推断IGF—1的影响是通过抑制BMP2信号通路。我将分化培养液中加入不同剂量的BMP2培养3天,收取细胞进行特异性标志物大鼠磷脂碱性蛋白(MBP)、半乳糖脑苷脂(Glac)、胶质纤维酸性蛋白(GFAP)、微管相关蛋白—2 (MAP-2)半定量RT-PCR检测,并进行统计学处理,免疫细胞化学染色后激光共聚焦显微镜检测骨髓间充质干细胞向少突胶质细胞定向分化的阳性细胞百分比。结果: 1.所分离培养的细胞形态呈长梭形或多边形,细胞生长曲线呈S形,群体倍增时间约为31小时,经成骨诱导剂诱导培养2周后,碱性磷酸酶染色成阳性,Van kossa银染色法阳性表明该细胞可向成骨方向分化,经成软骨诱导剂诱导培养2周后,Ⅱ型胶原免疫组化染色阳性,表明该细胞可向成软骨方向分化,而分离培养的细胞经成脂肪诱导剂诱导培养2周后,油红O染色可见显微镜下大量细胞内充满红色脂肪滴,表明该细胞可向脂肪细胞方向分化,所培养的细胞经化学性神经诱导剂诱导培养8小时后,NeuN抗体免疫组化染色阳性,细胞呈神经元样形态表现,表明该细胞可向神经细胞方向分化。MSCs抗原CD34免疫细胞化学染色呈阴性反应,CD44免疫细胞化学染色见镜下有棕黄色颗粒沉积。以上结果提示,用贴壁筛选法所分离培养的细胞为具有多向分化潜能的大鼠骨髓间充质干细胞。 2.①骨髓间充质干细胞向少突胶质细胞诱导分化过程中的形态学变化:经诱导分化后,大部分骨髓间充质干细胞表现出少突胶质细胞的形态学特征,胞质向细胞核回缩,细胞突起向外延伸,折光性增强,随时间延长多个细胞突起相互连接形成典型的网状结构。②少突胶质细胞特异性标志物mRNA的表达:细胞诱导分化后可检测到磷脂碱性蛋白mRNA、半乳糖脑苷脂mRNA的特异性条带。③少突胶质细胞阳性率:在诱导分化条件下,半乳糖脑苷脂阳性率为65%,磷脂碱性蛋白阳性率为45%,微管相关蛋白2阳性率为10%。比较含不同IGF-1浓度的分化培养液,发现含500μg/L IGF-1浓度的培养液少突胶质细胞标志物阳性率最高。④Western-blot检测磷脂碱性蛋白表达水平明显增高,以含500μg/L IGF-1浓度的培养液分化组最为明显。 3.在分化培养液中加入不同浓度的BMP2,发现向少突胶质细胞的分化明显受到抑制,仅部分骨髓间充质干细胞表现出少突胶质细胞的形态学特征,与BMP2的浓度成依赖关系,浓度越高(5μg/L)少突胶质细胞标志物(Glac,MBP)的阳性细胞越少,收取细胞进行特异性标志物大鼠磷脂碱性蛋白(MBP)、半乳糖脑苷脂(Glac)半定量RT-PCR检测,未检查出特异性条带。在低浓度(0.05μg/L—0.5μg/L)的情况下,可以观察到部份细胞向少突胶质细胞分化,这个结果与BMPs抑制少突胶质分化的特性相一致。结论: 1.通过取大鼠骨髓组织培养,用贴壁筛选法和胰蛋白酶消化法分离纯化,可以分离出稳定的含CD44抗原的有多向分化潜能的骨髓间充质干细胞,该细胞不断可以向中胚层成细胞分化,而且可以向外胚层神经细胞分化,取材方便,容易获得是体外研究干细胞定向分化的理想细胞。 2.合适的培养基和一定浓度的IGF-1有利于骨髓间充质干细胞向少突胶质细胞定向分化,该结果通过探讨诱导骨髓间充质干细胞向少突胶质细胞分化的可行性,实验条件,为以后的间充质干细胞向少突胶质细胞的定向分化移植治疗脊髓损伤打下基础。 3.胰岛素样生长因子诱导骨髓间充质干细胞向少突胶质细胞定向分化,其主要作用机理是通过抑制少突胶质细胞前体在脊髓中特异化的抑制性因子BMPs的信号通道,促进少突胶质细胞的产生。
[Abstract]:Objective:
1. in vitro, the method of separating and cultivating mesenchymal stem cells (MSCs) of rat bone marrow mesenchymal stem cells (MSCs) was studied in vitro, and the ability of multidirectional differentiation and proliferation in vitro and its biological characteristics were observed, and the markers of cell surface stem cells were identified, which lay the foundation for further study of bone marrow mesenchymal stem cells.
2. the feasibility of combining insulin-like growth factors and multiple cytokines to induce differentiation of bone marrow mesenchymal stem cells into oligodendrocytes, and the experimental conditions were investigated. The positive rates of differentiated cells were observed by laser confocal microscopy, and the optimal induction conditions were found by morphological changes, so as to obtain a large number of oligodendrocytes to repair the spinal cord. The pulp injury lays the foundation.
3. on the basis of experiment two, the proliferation and differentiation process of bone marrow mesenchymal stem cells (MSCs) was observed and the role of IGF-1 in oligodendrocyte differentiation was judged, and the mechanism of insulin like growth factor and multiple cytokines combined to induce bone marrow mesenchymal stem cells to differentiate into oligodendrocytes was found, and some intervention was given. Measures are further confirmed.
Method:
1. after the primary cells were extracted, the cell morphology and growth characteristics were observed by the adherent screening method and trypsin digestion method. The cell morphology and growth characteristics were observed by inverted phase contrast microscope. The proliferation process was studied, the birth length curve was plotted, and the cell surface stem cell marker CD44 was identified by immunohistochemistry. The fourth generation cells were formed into bone, cartilage, adipose tissue and fat, respectively. Neurogenic inducer culture, alkaline phosphatase staining, Van Kossa silver staining, type II collagen immunohistochemical staining, oil red O staining, NeuN antibody immuno histochemical staining and morphological observation, confirmed the multidirectional differentiation potential of the stem cells.
2. the fourth generation bone marrow mesenchymal stem cells with good growth were selected. After trypsin digestion, the cells were centrifuged and transferred into the culture dish containing the cover glass. The number of cells was 1 x 10~4 /cm~2, and the cells were incubated for 48h in the CO_2 incubator with the volume fraction of 0.05. After the culture, the cells were replaced by the culture solution containing different concentrations of IGF-1, and the culture medium was cultured for 3D, Morphological changes during differentiation of bone marrow mesenchymal stem cells to oligodendrocytes were observed, and cell phospholipid alkaline protein (MBP), galactose brain glycoside (Glac), semi quantitative RT-PCR (semi-quatitative reverse transctiption-polymerase chain reaction) and phosphorus in rats were collected for the determination of phosphorus in rats. Lipid alkaline protein (MBP) content, immunocytochemical staining and laser confocal microscopy were used to detect the positive rate of directional differentiation of bone marrow mesenchymal stem cells to oligodendrocytes, and the optimal IGF-1 (Insulin growthfactor-1) concentration of differentiation culture medium was screened.
3. according to the inhibition of BMP2 (bone morphogenetic proteins) in the formation of oligodendrocytes, the maturation of oligodendrocytes requires an inhibitor of BMP signal. In order to obtain the molecular mechanism of IGF - 1 induced oligodendrocyte differentiation, we infer that the effect of IGF - 1 is through the inhibition of BMP2 signaling pathway. In different doses of BMP2 culture for 3 days, the cells were collected for specific markers of phospholipid alkaline protein (MBP), galactose brain glycoside (Glac), glial fibrillary acidic protein (GFAP), microtubule related protein 2 (MAP-2) semi quantitative RT-PCR, and the statistics were carried out. After immunocytochemical staining, laser confocal microscopy was used to detect bone marrow The percentage of positive cells differentiated from mesenchymal stem cells to oligodendrocytes.
Result:
The cell morphology of the 1. isolated cultures was long shuttle shaped or polygon, the cell growth curve was S shaped, and the population doubling time was about 31 hours. After 2 weeks of induction of osteogenic inducer, alkaline phosphatase staining was positive. Van Kossa silver staining method showed that the cell could differentiate into osteogenic direction. After induction culture of cartilage inducer for 2 weeks, type II type was found. The collagen immunohistochemical staining was positive, indicating that the cells could differentiate into cartilage. After 2 weeks of induced culture, the isolated cells were filled with red fat droplets in a large number of cells under microscope, indicating that the cells could differentiate into adipocytes, and the cultured cells were induced by chemical nerve inducers. After 8 hours of induction and culture, the immunocytochemical staining of NeuN antibody was positive, and the cells showed neuron like morphology. It showed that the cells could differentiate into.MSCs antigen CD34 by immunocytochemical staining and negative reaction to nerve cells. CD44 immunocytochemical staining showed brown yellow granules under the microscope. The isolated cells are multipotent differentiation rat bone marrow mesenchymal stem cells.
2. (1) morphologic changes during differentiation of bone marrow mesenchymal stem cells into oligodendrocytes: after differentiation, most bone marrow mesenchymal stem cells show morphological characteristics of oligodendrocytes, the cytoplasm retracts to the nucleus, the cell protruding outwards, the refraction is enhanced, and the multiple cell protrusions are connected with time to connect with each other. A typical reticular formation. (2) the expression of oligodendrocyte specific marker mRNA: the specific bands of phospholipid alkaline protein mRNA and galactose brain glycoside mRNA were detected after induction of differentiation. (3) the positive rate of oligodendrocytes: the positive rate of galactose brain glycosides was 65% and the positive rate of phospholipid alkaline protein was 4 under the induced differentiation condition. 5%, the positive rate of microtubule related protein 2 was 10%. compared with the differentiation culture medium containing different IGF-1 concentrations. It was found that the positive rate of oligodendrocyte markers in the medium containing 500 mu g/L IGF-1 was the highest. 4. The expression level of phospholipid alkaline protein was significantly increased by Western-blot, and the most obvious differentiation group with the concentration of 500 mu g/L IGF-1 was the most obvious.
3. with the addition of different concentrations of BMP2 in the differentiated medium, it was found that the differentiation of oligodendrocytes was obviously inhibited. Only some bone marrow mesenchymal stem cells showed morphological characteristics of oligodendrocytes, which were dependent on the concentration of BMP2. The higher the concentration (5 g/L), the less positive cells of oligodendrocyte markers (Glac, MBP) were the less, the less the positive cells of the oligodendrocytes (Glac, MBP) were collected. The specific markers of phospholipid alkaline protein (MBP) and galactose brain glycoside (Glac) were detected by the cells, and the specific bands were not detected. In the case of low concentration (0.05 u g/L - 0.5 mu g/L), some cells could be observed to be divided into oligodendrocytes. This result is in accordance with the characteristics of BMPs inhibition of oligodendrocyte differentiation. Cause.
Conclusion:
1. through the culture of rat bone marrow tissue, we can isolate and purify the bone marrow mesenchymal stem cells with multidirectional differentiation potential with CD44 antigen, which can differentiate into mesoderm cells, and can be differentiated from the mesoderm cells, which can be easily obtained and easily obtained. It is an ideal cell for studying the directional differentiation of stem cells in vitro.
2. a suitable medium and a certain concentration of IGF-1 are beneficial to the differentiation of bone marrow mesenchymal stem cells into oligodendrocytes. The result is to explore the feasibility of inducing bone marrow mesenchymal stem cells to differentiate into oligodendrocytes, and the experimental conditions are for the treatment of spinal cord with oligodendrocyte differentiation and transplantation for the future. Damage lays the foundation.
3. insulin-like growth factors induce bone marrow mesenchymal stem cells to differentiate into oligodendrocytes, the main mechanism of which is to promote the production of oligodendrocytes by inhibiting the signal channel of the inhibitory factor BMPs of the oligodendrocyte precursor in the spinal cord.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【共引文献】