【作者】 许永涛；

【导师】 李锋；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【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1×10⁴个/cm²,放入体积分数为0.05的CO₂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。更多还原

【Abstract】 Objective: 1.Investigate the method of isolation and cultivation of rat bone marrowmesenchymal stem cells (mesenchymal stem cells, MSCs) in vitro, and observe the ability ofproliferation and differentiation in vitro as well as their biological characteristics, but alsostem cell-related markers on the cell surface were identified for laying the foundation forfurther study on bone marrow-derived mesenchymal stem cells.2.Investigate the feasibility of experimental conditions of bone marrow-derived mesenchymalstem cells induced by the insulin-like growth factor and cytokines differentiated tooligodendrocyte . Observe the positive rate of differentiated cells by laser scanning confocalmicroscope and select the optimal conditions for inducing cells.3.On the basis of two experiments, to observe the bone marrow-derived mesenchymal stemcells proliferation and differentiation process, to determine the role of IGF-1 inoligodendrocyte differentiation and give a certain degree of intervention measures to confirmfurther.Methods: 1 From the original cells, adherent screening method and trypsin digestion wereused for isolation and purification of mesenchymal stem cells .Inverted phase contrastmicroscope were used to observe cell morphology and growth characteristics in the process ofits proliferation, MTT method to map out the growth curve, immunohistochemical methodwere used to identify cell surface markers CD44 of stem cells. the fourth passage cells were respectively cultured in osteogenic、adipogenic and neuro—differentiation induction medium, the alkaline Phosphatase staining, oil red O staining and morphological observation, multi-differentiation potential of stem cells were confirmed.2.At passage 4, BMSCs were incubated in serum-free medium, supplemented with N2, 20 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor for 48 hours, and incubated in medium containing 500 ng/mL insulin-like growth factor 1 and N2 for 3 days. Morphological changes were observed using an phase contrast microscope. Semiquantitative RT-PCR was utilized to detect specific marker mRNA expression of oligodendrocytes. Western-blot Determination was utilized to detect basic protein in rat phospholipid (MBP).Using neuron marker anti-microtubule-associated protein, astrocyte marker anti-glial fibrillary acidic protein, oligodendrocyte marker anti-galactocerebroside, anti-myelin basic protein antibody, immunocytochemical staining was performed to detect the positive rate of the differentiation of BMSCs into oligodendrocytes. Select the best IGF-1 (Insulin growth factor-1) concentration in the culture medium divided doses.3.in accordance with BMP2 (bone morphogenetic proteins) inhibiting oligodendrocyte formation , in order to obtain the molecular mechanisms of IGF-1 inducing oligodendrocyte differentiation, we infer the effects of IGF-1 are the inhibition of BMP signaling pathway. To determine whether BMP2 inhibit MSCs differentiate to oligodendrocyte, The differentiation culture medium with different doses of BMP2 were used for 3 days and to collect cell-specific markers for rat phospholipid basic protein (MBP), galactose cerebroside (Glac) and take statistical treatment ,semi-quantitative RT-PCR detection, immunocytochemical staining、laser scanning confocal microscope .Then to detect the percentage of positive cells of bone marrow-derived mesenchymal stem cells to oligodendrocyte differentiation.Results: After isolation and culture , cells arranged spindle or polygonal.cells were S-shaped growth curve, population doubling time were 20 hours of osteoblasts. induced by the agent 14 days after induction, alkaline Phosphatase staining is positive results , suggesting that the cells differentiated into osteogenic direction, the isolation and culture of the cells were induced into a fat-induced cultivate 14, and the oil red O staining shows that a large number of red cells with lipid droplets, suggesting that the cells can divide to the direction of fat cells, the cultured cells into neurons induced by the agent 6 hours after induction, cells were typical neuron-like form of performance, suggesting that the cells can divide to the direction of nerve cells. CD44 antigen MSCs immunocytochemical staining showed positive results, the above results, the isolated and cultured the cells comply with the requirements of experiments with multi-differentiation potential of bone marrow-derived mesenchymal stem cells.2①bone marrow-derived mesenchymal stem cells to the oligodendrocyte-induced morphological differentiation in the process of change: After induction of differentiation, the majority of bone marrow-derived mesenchymal stem cells showed oligodendrocyte morphological characteristics of the cytoplasm to the nucleus to shrinkage, cell processes extend outward, the refractive index increased, with the number of cell processes extend to form a typical interconnected network structure.②oligodendrocyte-specific expression of mRNA markers: cells can be detected after differentiation phospholipid basic protein mRNA, cerebroside galactose specific mRNA bands.③oligodendrocyte positive rate: conditions in inducing differentiation, galactose cerebroside positive rate was 65%, phospholipid basic protein-positive rate of 45%, microtubule-associated protein 2-positive rate was 10%. Comparison with different concentrations of IGF-1 in differentiation medium,we found that culture medium contain 500μg / L IGF-1 concentration was the highest positive rate in oligodendrocyte markers.④Western-blot detection of alkaline phospholipid significantly increased the level of protein expression in the culture medium o contain 500μg / L IGF-1 concentration.3 the differentiation culture medium by adding different concentrations of BMP2, was found that oligodendrocyte differentiation were inhibited, only part of bone marrow-derived mesenchymal stem cells showed oligodendrocyte morphological characteristics, and as dependent on the concentration of BMP2. the relationship between the higher concentration (5μg/L-50μg/L), the less of oligodendrocyte marker (Glac, MBP)-positive cells .charged cell-specific markers for rat phospholipid basic protein (MBP), semi - galactose cerebroside (Glac) semi-quantitative RT-PCR detection,we did not detect specific bands. At low concentrations (0.05μg/L-0.5μg/L) circumstances, it can be observed in some cells to oligodendrocyte differentiation, the results consistent with BMPs inhibitting oligodendrocyte differentiation characteristics.Conclusions: take tissue culture of rat bone marrow, adherent with screening, trypsin digestion and purification, MSCs can be isolated with a stable number of CD44 antigens to thedifferentiation potential of bone marrow-derived mesenchymal stem cells, the cells continuedto mesodermal cell differentiation into, but also to the ectodermal neural cell differentiation, itwas an ideal cell to study directed differentiation of stem cells in vitro.2.The combination of epidermal growth factor, basic fibroblast growth factor and insulin-likegrowth factor can effectively promote the directional differentiation of BMSCs intooligodendrocytes. We grasp the experiment conditions of bone marrow-derived mesenchymalstem cells to oligodendrocyte differentiation and lay the foundation for the treatment ofspinal cord injury by subsequent mesenchymal stem cells to the oligodendrocytedifferentiation transplantation.3.the main mechanism of insulin-like growth factor-induced bone marrow-derivedmesenchymal stem cells to oligodendrocyte differentiation, are to inhibit the signal path ofspecific BMPs inhibitory factor of oligodendrocyte precursors in the spinal cord .更多还原