Ⅱ型胶原GAGs支架对HUCMSCs成软骨诱导的实验研究

发布时间：2017-12-28 18:39

本文关键词：Ⅱ型胶原GAGs支架对HUCMSCs成软骨诱导的实验研究　出处：《昆明医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：实验目的：本次研究的主要目的是评估人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells HUCMSCs)作为软骨组织工程的种子细胞的可行性,同时评估II型胶原复合糖胺聚糖支架材料作为软骨组织工程种子细胞载体的可行性。主要包括了三部内容：一.人脐带间充质干细胞的培养、传代、冻存、复苏以及鉴定,对其生物学特性进行初步的研究,为组织工程学种子细胞的研究提供实验基础；二.II型胶原复合糖胺聚糖支架的制作及其理化性质的鉴定；三.利用人脐带间充质干细胞在Ⅱ型胶原复合糖胺聚糖支架体外培养成软骨组织,并对培养出的软骨组织进行鉴定。实验方法：1.利用昆明总医院临床试验科细胞室提供的原代人脐带间充质干细胞,用含10%的胎牛血清、100U/mL青霉素、100mg/L链霉素的DMEM/F-12培养基进行培养,待其生长至贴壁覆盖约80%-90%时按1：3比例进行传代,并在倒置显微镜下观察HUCMSCs的生长、增殖情况及其形态变化。取原代、第三代细胞进行流式细胞仪检测。用胰蛋白酶/EDTA消化液将传代的细胞消化,1000转/分钟下离心5分钟,将离心的最下层细胞以FBS配制的10%二甲基亚砜重置,-80℃冰箱保存24小时后将其转入液氮罐内保存,3个月后取出进行复苏、培养、传代及流式细胞术。2.通过将软骨粉碎后行脱细胞处理,制备II型胶原复合糖胺聚糖多孔支架,在扫描电镜下进行观察,测定支架材料的孔径、孔隙率,并测试其亲水性,对支架材料进行相应的组织学及生化分析。3.分离培养P3代的人脐带间充质干细胞,并将配制好的人脐带间充质干细胞混悬液均匀的移植到经辐照消毒后的II型胶原复合糖胺聚糖支架上,不加诱导剂,用含10%的胎牛血清的DMEM/F-12培养基进行培养,3周后取出部分样品进行甲苯胺蓝和番红精O染色,并行II型胶原免疫组化染色。实验结果：1.在细胞接种后约3小时,即可见少量的脐带间充质干细胞开始在培养瓶中贴壁生长,细胞形态呈梭形、纺锤形,少部分可呈集落生长；在接种24小时后可见大多数细胞已完成贴壁生长；第3天至第4天左右可见细胞贴壁覆盖程度达到80-90%,显微镜下观察见：细胞呈均一的梭形生长,类似于成纤维细胞,呈典型的漩涡状排列生长。细胞在传代过程中形态并无明显变化,在细胞传至第3代时即剩下形态较为单一的细胞。流式细胞检测分析显示：原代培养的HUCMSCs已经开始高表达CD44、CD90、CD105,但是仍有一定程度的表达CD34、CD45,而第三代的HUCMSCs则高度表达CD29、CD44、CD90、CD105等间充质细胞标志,几乎不表达CD34、CD45等内皮细胞、造血细胞标志及主要组织相容性抗原人白细胞DR抗原。HUCMSCs冻存3个月后复苏,显微镜下观察显示细胞形态饱满,细胞活性良好,传代生长良好,与冻存前基本一致,流式细胞检测其表面标志物的测定结果与未冻存的HUCMSCs类似。2.Ⅱ型胶原复合糖胺聚糖支架材料为白色、多孔的泡沫状圆柱体,表面孔隙较多,扫描电镜下可见：支架材料呈多孔泡沫状,孔隙分布均匀,孔隙之间相互贯通,支架孔隙率为：(91.8%±2.17)%,孔径平均,孔径值在110--230μm之间,支架材料亲水性良好,吸水膨胀率为(213.71%±1.31)%。甲苯胺蓝、番红精O及Ⅱ型胶原免疫组化染色呈阳性。3.Ⅱ型胶原复合糖胺聚糖支架材料随着细胞培养的时间逐渐变的表面光滑,质地有韧性,可观察到有软骨样组织形成,并逐步变多,甲苯胺蓝、番红精O及Ⅱ型胶原免疫组化染色均呈阳性。结论：1.人脐带间充质干细胞经检测显示符合间充质干细胞的基本生物学特性,可以作为软骨组织工程良好的种子细胞来源；2.Ⅱ型胶原复合糖胺聚糖支架材料具备适合细胞生长、增殖的孔径和孔隙率,亲水性及生物相容性良好,可以作为软骨组织工程良好的细胞载体；3.人脐带间充质干细胞在Ⅱ型胶原复合糖胺聚糖支架材料上体外可以不经诱导而初步构建组织工程软骨,为软骨损伤的修复奠定了一定的实验基础。
[Abstract]:Objective: the aim of this study was to evaluate the human umbilical cord mesenchymal stem cells (human umbilical cord derived mesenchymal stem cells HUCMSCs) feasibility as the seed cells for cartilage tissue engineering, and evaluation of type II collagen composite scaffold as the carrier for the feasibility of glycosaminoglycan seed cells in cartilage tissue engineering. Mainly includes three contents: 1. Human umbilical cord mesenchymal stem cell culture, subculture, cryopreservation and resuscitation, identification and preliminary study of its biological characteristics and provide experimental basis for the research of tissue engineered seed cells; identification of chemical properties of two type.II collagen glycosaminoglycan composite support production and management three. Use; human umbilical cord mesenchymal stem cells in type II collagen glycosaminoglycan scaffolds in vitro cartilage tissue, and to identify the cartilage tissue culture. Methods: 1. clinical trials by Kunming general hospital cell room, the primary human umbilical cord mesenchymal stem cells were cultured with 10% fetal bovine serum, penicillin, streptomycin 100mg/L 100U/mL DMEM/F-12 medium, the growth of adherent to cover about 80%-90% according to 1:3 than were patients, and observe the HUCMSCs the growth, proliferation and morphological changes under inverted microscope. The primary and third generation cells were detected by flow cytometry. By trypsin digestion. The cells of /EDTA digestion, 1000 rpm centrifugal 5 minutes, most of the cells will reset the centrifugal FBS prepared 10% two dimethyl sulfoxide, -80 C refrigerator 24 hours after transferred into liquid nitrogen preservation, removed after 3 months of recovery, training, passage and flow cytometry. 2., II collagen and glycosaminoglycan porous scaffolds were prepared by removing the cells after comminution of the cartilage. Scanning electron microscopy was used to observe the pore size and porosity of the scaffolds, and to test their hydrophilicity, and to make corresponding histological and biochemical analysis of the scaffolds. The 3. generation of P3 were isolated and cultured human umbilical cord mesenchymal stem cells, and the prepared human umbilical cord mesenchymal stem cell suspension transplantation to the uniform type II collagen glycosaminoglycan scaffolds after irradiation sterilization, without inducer, were cultured with 10% fetal bovine serum DMEM/F-12 medium after 3 weeks, some samples were removed by toluidine blue and red O staining, parallel II collagen immunohistochemical staining. Results: 1. cells in about 3 hours after inoculation, which shows that a small amount of umbilical cord mesenchymal stem cells in culture flask adherent growth, cell morphology was spindle, spindle, a small part of a colony growth; in 24 hours after inoculation of most cells visible has completed the adherent growth of third to fourth; days visible adherent cell coverage reached 80-90%, microscope: cells were spindle shaped and uniform growth, similar to fibroblast cells showed typical whorls of growth. There is no obvious change in the morphology of the cells during the passage of the cells, and the more single cells are left in the third generation of cells. Flow cytometric analysis showed that primary cultured HUCMSCs have high expression of CD44, CD90 and CD105, but there is still a certain degree of expression of CD34, CD45, and the third generation of the HUCMSCs, CD44, CD29 high expression of CD90, CD105 and other mesenchymal cell marker, CD34 and CD45 almost no expression in endothelial cells and hematopoietic cell markers and major histocompatibility antigen of human leukocyte antigen DR. HUCMSCs was cryopreserved for 3 months, and it was resuscitated. Microscopic observation showed that the cell morphology was full, the cell viability was good, and the passage grew well, which was basically consistent with that before cryopreservation. The surface markers detected by flow cytometry were similar to those of unfrozen HUCMSCs. 2. type II collagen composite scaffold material for foam cylinder from white, porous surface, more porous, under a scanning electron microscope: the scaffold had a porous, uniform pore distribution, pore connectivity between the porosity of the scaffold is: (91.8% + 2.17)%, the average aperture, aperture value between 110--230 m. A good scaffold hydrophilicity, water swelling rate (213.71% + 1.31)%. Toluidine blue and safranin O and type II collagen immunohistochemical staining. 3. type II collagen glycosaminoglycan scaffolds with composite surface cell culture time gradually become smooth, texture, toughness, can be observed in the formation of cartilage like tissue, and gradually become more, toluidine blue and safranin O and type II collagen immunohistochemical staining were positive. Conclusion: 1. human umbilical cord mesenchymal stem cells after testing to demonstrate compliance with the basic biological characteristics of mesenchymal stem cells, cartilage tissue engineering as a good source of seed cells; 2. type II collagen composite scaffold materials have glycosaminoglycan suitable for cell growth and proliferation of the aperture and porosity, hydrophilicity and biocompatibility, can be used as cell carrier good cartilage tissue engineering; 3. human umbilical cord mesenchymal stem cells in vitro in type II collagen glycosaminoglycan scaffolds material can be induced by construction of tissue engineering cartilage, which provided the experimental basis for repairing articular cartilage injury.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08;R687

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