本文關(guān)鍵詞：Ⅱ型膠原GAGs支架對HUCMSCs成軟骨誘導(dǎo)的實驗研究　出處：《昆明醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人臍帶間充質(zhì)干細(xì)胞 種子細(xì)胞 糖胺聚糖 支架 組織工程軟骨

【摘要】：實驗?zāi)康模罕敬窝芯康闹饕康氖窃u估人臍帶間充質(zhì)干細(xì)胞(human umbilical cord derived mesenchymal stem cells HUCMSCs)作為軟骨組織工程的種子細(xì)胞的可行性,同時評估II型膠原復(fù)合糖胺聚糖支架材料作為軟骨組織工程種子細(xì)胞載體的可行性。主要包括了三部內(nèi)容：一.人臍帶間充質(zhì)干細(xì)胞的培養(yǎng)、傳代、凍存、復(fù)蘇以及鑒定,對其生物學(xué)特性進行初步的研究,為組織工程學(xué)種子細(xì)胞的研究提供實驗基礎(chǔ)；二.II型膠原復(fù)合糖胺聚糖支架的制作及其理化性質(zhì)的鑒定；三.利用人臍帶間充質(zhì)干細(xì)胞在Ⅱ型膠原復(fù)合糖胺聚糖支架體外培養(yǎng)成軟骨組織,并對培養(yǎng)出的軟骨組織進行鑒定。實驗方法：1.利用昆明總醫(yī)院臨床試驗科細(xì)胞室提供的原代人臍帶間充質(zhì)干細(xì)胞,用含10%的胎牛血清、100U/mL青霉素、100mg/L鏈霉素的DMEM/F-12培養(yǎng)基進行培養(yǎng),待其生長至貼壁覆蓋約80%-90%時按1：3比例進行傳代,并在倒置顯微鏡下觀察HUCMSCs的生長、增殖情況及其形態(tài)變化。取原代、第三代細(xì)胞進行流式細(xì)胞儀檢測。用胰蛋白酶/EDTA消化液將傳代的細(xì)胞消化,1000轉(zhuǎn)/分鐘下離心5分鐘,將離心的最下層細(xì)胞以FBS配制的10%二甲基亞砜重置,-80℃冰箱保存24小時后將其轉(zhuǎn)入液氮罐內(nèi)保存,3個月后取出進行復(fù)蘇、培養(yǎng)、傳代及流式細(xì)胞術(shù)。2.通過將軟骨粉碎后行脫細(xì)胞處理,制備II型膠原復(fù)合糖胺聚糖多孔支架,在掃描電鏡下進行觀察,測定支架材料的孔徑、孔隙率,并測試其親水性,對支架材料進行相應(yīng)的組織學(xué)及生化分析。3.分離培養(yǎng)P3代的人臍帶間充質(zhì)干細(xì)胞,并將配制好的人臍帶間充質(zhì)干細(xì)胞混懸液均勻的移植到經(jīng)輻照消毒后的II型膠原復(fù)合糖胺聚糖支架上,不加誘導(dǎo)劑,用含10%的胎牛血清的DMEM/F-12培養(yǎng)基進行培養(yǎng),3周后取出部分樣品進行甲苯胺藍和番紅精O染色,并行II型膠原免疫組化染色。實驗結(jié)果：1.在細(xì)胞接種后約3小時,即可見少量的臍帶間充質(zhì)干細(xì)胞開始在培養(yǎng)瓶中貼壁生長,細(xì)胞形態(tài)呈梭形、紡錘形,少部分可呈集落生長；在接種24小時后可見大多數(shù)細(xì)胞已完成貼壁生長；第3天至第4天左右可見細(xì)胞貼壁覆蓋程度達到80-90%,顯微鏡下觀察見：細(xì)胞呈均一的梭形生長,類似于成纖維細(xì)胞,呈典型的漩渦狀排列生長。細(xì)胞在傳代過程中形態(tài)并無明顯變化,在細(xì)胞傳至第3代時即剩下形態(tài)較為單一的細(xì)胞。流式細(xì)胞檢測分析顯示：原代培養(yǎng)的HUCMSCs已經(jīng)開始高表達CD44、CD90、CD105,但是仍有一定程度的表達CD34、CD45,而第三代的HUCMSCs則高度表達CD29、CD44、CD90、CD105等間充質(zhì)細(xì)胞標(biāo)志,幾乎不表達CD34、CD45等內(nèi)皮細(xì)胞、造血細(xì)胞標(biāo)志及主要組織相容性抗原人白細(xì)胞DR抗原。HUCMSCs凍存3個月后復(fù)蘇,顯微鏡下觀察顯示細(xì)胞形態(tài)飽滿,細(xì)胞活性良好,傳代生長良好,與凍存前基本一致,流式細(xì)胞檢測其表面標(biāo)志物的測定結(jié)果與未凍存的HUCMSCs類似。2.Ⅱ型膠原復(fù)合糖胺聚糖支架材料為白色、多孔的泡沫狀圓柱體,表面孔隙較多,掃描電鏡下可見：支架材料呈多孔泡沫狀,孔隙分布均勻,孔隙之間相互貫通,支架孔隙率為：(91.8%±2.17)%,孔徑平均,孔徑值在110--230μm之間,支架材料親水性良好,吸水膨脹率為(213.71%±1.31)%。甲苯胺藍、番紅精O及Ⅱ型膠原免疫組化染色呈陽性。3.Ⅱ型膠原復(fù)合糖胺聚糖支架材料隨著細(xì)胞培養(yǎng)的時間逐漸變的表面光滑,質(zhì)地有韌性,可觀察到有軟骨樣組織形成,并逐步變多,甲苯胺藍、番紅精O及Ⅱ型膠原免疫組化染色均呈陽性。結(jié)論：1.人臍帶間充質(zhì)干細(xì)胞經(jīng)檢測顯示符合間充質(zhì)干細(xì)胞的基本生物學(xué)特性,可以作為軟骨組織工程良好的種子細(xì)胞來源；2.Ⅱ型膠原復(fù)合糖胺聚糖支架材料具備適合細(xì)胞生長、增殖的孔徑和孔隙率,親水性及生物相容性良好,可以作為軟骨組織工程良好的細(xì)胞載體；3.人臍帶間充質(zhì)干細(xì)胞在Ⅱ型膠原復(fù)合糖胺聚糖支架材料上體外可以不經(jīng)誘導(dǎo)而初步構(gòu)建組織工程軟骨,為軟骨損傷的修復(fù)奠定了一定的實驗基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R318.08;R687

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