【作者】 刘鹏；

【导师】 金岩； 刘源；

【作者基本信息】 第四军医大学 ， 口腔基础医学， 2008， 博士

【摘要】 除了骨髓组织,成人外周血中也存在着如造血干细胞(HSCs)和间充质干细胞(MSCs)等多潜能干细胞成分。相对于骨髓的采集,外周血的干细胞采集具有供体创伤小、来源广泛和自体取材无免疫排斥反应等优点。近年来,循环间充质干细胞(Peripheral Blood Mesenchymal Stem Cells,PBMSCs)和循环成纤维细胞(Circulating Fibrocytes,CF)这两种新型的多潜能成体干细胞在人类以及其他哺乳动物外周血中被发现并鉴定,它们在机体组织器官的正常和非正常功能活动和创伤修复中发挥着重要的作用。本研究着眼于应用成体干细胞分离技术,在体外分离培养外周血来源的成体干细胞—循环成纤维细胞,观察它们在体内和体外的细胞增殖、分化、以及与多种皮肤组织细胞的相互作用;从细胞因子调控等多方面研究其作用机理,并将其应用于皮肤创伤的修复过程,为组织工程皮肤的研究开辟了新的思路,更有助于提高组织工程皮肤产品的临床应用效果。本研究主要分为四个部分1.人外周血来源成体干细胞的体外培养和鉴定目的:通过多种方法分离人外周血单个核细胞,在不同条件下获得CF,研究了其细胞增殖特点和对其进行了详细的鉴定。方法:采用淋巴细胞分离液(Percoll/ Ficoll)分离含有外周血单个核细胞的白细胞群,直接体外培养或磁珠分选法分离单个核细胞进行培养,从而获得CF细胞;通过不同细胞因子促进CF细胞增殖,比较效果;培养过程中描画CF细胞生长曲线;应用免疫组织化学方法和流式细胞仪对体外培养的细胞进行鉴定。结果:通过分离成人外周血单个核细胞的方法可以获得纯度较高的CF细胞;在培养过程中应用M-csf和TPO两种细胞因子可以促进CF细胞的成熟和增殖;细胞生长曲线显示经过细胞因子的促进作用,CF细胞在体外培养过程中增殖速度加快。其中合适浓度的TPO的促进作用更为明显且细胞转化率低,是理想的促进CF细胞体外增殖的作用因子;免疫组织化学鉴定结果显示,本实验中体外培养获得的贴壁纤维样细胞表达CollagenⅠ且有CollagenⅠ阳性的基质分泌。流式细胞仪鉴定结果显示体外培养的细胞其表面标志性抗原的表达随着时间改变而不断变化,但均符合CF细胞的特征。鉴定结果显示获得的细胞明确属于CF细胞。2.外周血来源成体干细胞在体外培养过程中的多向分化能力研究目的:探讨CF细胞作为一种多潜能成体干细胞其成骨、成脂和向神经细胞分化的能力。方法:采用与雪旺细胞(Schwann cells,SCs)共培养以及直接添加细胞因子NGF的方法,促进CF细胞向神经细胞分化,通过免疫组织化学、Western blot以及RT-PCR等方法对诱导的细胞进行鉴定;以添加地塞米松(dexamethasone,100 nM);β磷酸甘油钠(β-glycerophosphate,10 mM);维生素C/抗坏血酸(Ascorbic acid,50μM)等试剂的培养基对CF细胞进行成骨诱导,通过大体形态和茜素红染色鉴定诱导的细胞;以地塞米松(dexamethasone,1μM)、0.5 mM 3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine,IBMX)、胰岛素(insulin,10μg/ml)、吲哚美辛(Indomethacin,100 mM)等试剂为活性成分的成脂诱导液,对体外培养的CF细胞进行成脂诱导,通过大体形态和油红O染色鉴定诱导的细胞。结果:免疫组织化学染色结果显示在两种诱导条件下CF细胞均表达神经标志物NSE和NF-H。western blot和RT-PCR结果也显示同样的结果且共培养组的表达更强。成骨诱导结果细胞茜素红染色显示阳性,显示CF细胞在一定条件下具备成骨分化能力;油红O染色结果显示阳性说明CF细胞可以向脂肪细胞转化。3.体外观察循环成纤维细胞对皮肤组织细胞的作用影响目的:1)二维环境下通过共培养的方法观察CF细胞对皮肤组织细胞的作用影响;2)三维条件下通过体外构建复合CF细胞的组织工程人工皮肤,观察CF细胞对皮肤组织细胞的作用影响。方法:1)以丝裂霉素C处理体外培养的原代CF细胞,使其成为类滋养层细胞;将体外分离培养的真皮成纤维细胞(Fibroblasts,FBs)和皮肤角阮细胞(Keratinocytes,KCs)与制备的类滋养层CF直接接触共培养,通过直接光镜观察、MTT法、BrdU-ELISA掺入法、流式细胞仪分析细胞周期以及细胞运动(划痕)实验(Scratch assay)等手段观察两种皮肤组织细胞在体外的形态和增殖变化。2)体外构建具有复合CF细胞的真皮层的组织工程人工皮肤,通过大体观察,HE染色组织学观察,Pan-CK免疫组织化学染色等手段研究三维环境下CF细胞对皮肤组织细胞的作用影响。结果:1)丝裂霉素C处理后的CF细胞多次传代后基本停止增殖,经过与皮肤组织细胞的共培养后逐渐死亡;与类滋养层CF细胞共培养的FBs细胞形态基本没有明显变化,而KCs则形态变化明显,出现表皮去分化特征性的细胞形态;MTT、BrdU-ELISA和流式细胞仪分析细胞周期显示共培养后的FBs增殖加快,而KCs则增值速度减慢,提示CF细胞促进FBs的增殖作用和对KCs的抑制作用;细胞划痕试验则显示共培养后的FBs细胞移行能力加强,而KCs则呈现相反的趋势。2)与具有单纯复合成纤维细胞真皮的对照组相比,复合有CF细胞的真皮层的组织工程皮肤体外培养过程中体积收缩更为明显,提示成纤维细胞增殖加速;但与正常对照组相比,其上皮化过程不良,角化层结构较差。4.循环成纤维细胞参与裸鼠皮肤创伤修复的实验研究目的:将体外分离培养获得的人CF细胞通过循环途径移植入裸鼠体内,观察其参与皮肤组织创伤修复的效果。方法:建立裸鼠背部全层皮肤创伤模型;体外以PKH26荧光染料标记体外培养的人CF细胞;鼠尾静脉注射方法将标记好的人CF细胞移植入裸鼠创伤模型体内,设立空白对照;大体观察创面愈合情况,测量各组创面面积计算创面收缩率,3w左右采用抗人单克隆抗体(Pan-CK,α-SMA,Ⅷ等)对创伤部位皮肤组织进行取材免疫组织化学染色;定期取创面组织进行HE染色组织学观察,冰冻切片进行荧光显微镜检测PKH26阳性细胞。结果:实验组和对照组裸鼠创伤皮肤均出现愈合,通过大体观察和计算创面收缩率结果发现实验组的创面愈合快于对照组;实验组愈合创面的上皮化较对照组好,血管生成更丰富;实验组和对照组在各时间段均未在创面区域组织内发现PKH26阳性的细胞;抗人单抗免疫组织化学染色结果显示实验组创面区域在3w后各抗体均有表达,提示迁移至裸鼠皮肤创面参与修复的细胞可能来源于移植的人CF细胞。综上所述,CF细胞在体外来源广泛,体外较易分离培养,通过某些细胞因子的作用可以有效促进其增殖。CF细胞作为一种创伤修复过程中的重要效应细胞,能有效促进FBs的增殖和提高其迁移能力,但其对KCs细胞是否在体内和体外作用一致还需要进一步研究;作为一种AS细胞,CF细胞在体外一定条件下可向多个方向进行分化,在干细胞移植治疗应用中具有重要研究和应用价值;通过体内移植CF细胞可以有效促进皮肤创面的愈合,在急慢性以及大面积皮肤创伤的治疗上具有良好应用前景更多还原

【Abstract】 As in bone marrow, there are two kinds of multipotential stem cells in adult peripheral blood, including haemopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs),which have more abundant source and are easier to be separate in vitro. They also have the predominance of autoallergic origin without immunological rejection and minimal trauma of donator. For the past few years, as two new types of multipotential adult stem cells, peripheral blood mesenchymal stem cells(PBMSCs) and circulating fibrocytes(CF) were found and identified in peripheral blood of human and other mammalian. These cells play their important roles in normal and abnormal functions of organism and the repair of defects.In this research, we try to isolate circulating fibrocytes from human peripheral blood by a adult stem cell tech in vitro, and observe the cell proliferation and differentiation of CF and their influence upon multiple skin tissue cells. We also apply them to the repair of skin defects. The results develop a new path of application in tissue engineering skin and increase their possibility for clinic applications.There are four parts in this research as followsPart 1:Culture and identification of an adult stem cell from human peripheral blood in vitro:Objective: To isolate mononuclear cells from human peripheral blood by different methods and investigate the cell proliferation and identification of CF. Methods: Human mononuclear cells were separated by Percoll/Ficoll for direct culture or immunomagnetic magnetic beads sorting(MAC)in vitro. The CF cells were derived at last; Multiple cytokines were used to stimulate the proliferation of CF and growth curve of cultured cells was drawn; Immunohistochemistry and flow cytometry were utilized for identification of CF. Results: High purity of CF cells were cultured by mean of mononuclear cells separation from peripheral blood; M-csf and TPO both stimulated the cell proliferation of CF in vitro; Growth curve of cultured cells indicate that TPO performs a oppressive role in stimulating of cell proliferation and exhibited lower transdifferentiation efficiency between two kinds of cytokines. So we conclude that TPO is a ideal cytokine for amplification of CF cells; Immunohistochemistry results indicated that cultured cells and their ECM express CollagenⅠ; Flow cytometry indicate that the level of specified surface markers like CD34 and CD14 did change time to time. And this phenomenon does accord with the specification of CF cells in vitro.Part 2: Mutiple differentiation potency of an adult stem cell from peripheral bloodObjective: To investigate the multiple differentiation capacity of CF as an adult stem cell. Methods: Nerve growth factor and Co-culture with Schwann cells (SCs) were applied for neural induction of CF. Immunohistochemistry, western blot and RT-PCR were utilized for identification of differented cells; Dexamethasone(100 nM),β-glycerophosphate(10 mM) and Ascorbic acid(50μM) were added in the medium of cultured CF for Osteogenesis induction. The cells were incubated with adipogenic induction medium (DMEM containing 10% FBS, 1 _M dexamethasone, 0.5 mM methyl-isobutylxantine, 10μg/ml insulin, and 100 mM indomethacin; all from Sigma). After 72 h, the medium was changed to maintenance medium (DMEM containing 10% FBS and 10μg/ml insulin) for 24 h. The cells were treated three times with adipogenic induction medium and maintained in the maintenance medium for one additional week; And these cells at last were fixed for Oil-red staining and alizarin red staining.Results: Immunohistochemistry indicated that NSE and NF-H were both expressed after these two neural induction procedures. The alike situation did exist in the results of western blot and RT-PCR. And in latter two procedures ,Co-cultured groups did express a higher level of NSE and NF-H; CF cells treated with the osteogenic and adipogenesis induction procedure under went a change in their morphology from spindle-shaped to cuboidal or bigger and rounder one; Some parts of adherent cells formed calcium deposits, which stained with alizarin red and Oil-red staining.Part 3 : Investigation of effects of circulating fibrocytes on skin tissue cellsObjective: 1) To investigate the effects of CF cells on skin tissue cells by co-culture them together in 2- dimension in vitro. 2) To investigate the CF effects on skin tissue cells by preparation of tissue engineering skin in vitro. Methods: 1) CF cells were exposed to Mitomycin-C for preparation of trophocyte–like cells; Fibroblasts and keratinocytes were isolated and harvested to co-culture with exposed CF. Skin tissue cells proliferation and migration were investigated through direct light microscope observation, MTT assay, BrdU-ELISA assay, cell cycle assay and cell scratch assay. 2)Full thickness tissue engineering skin with or without (control) were prepareded in vitro in the method of our Labs. And these artificial skin films were investigated by macro observation, HE staining and Pan CK staining. Results: 1) CF treated with mitomycin-C did exhibit a trophoblast specification; Co-cultured FBs did change too much in there morphous but the KCs exhibit a dedifferentiation trend; MTT assay, BrdU-ELISA , cell cycle assay, scratch assay and 3-dimension investigation indicated that CFs did simulate the proliferation and migration of FBs but exhibit a inhibited one on KCs in vitro.Part 4. An investigation of skin repair in Nude mouse by circulating fibrocytesObjective: To investigate the effect of in vitro harvested and circulating transplanted CF on skin repair of Nude mouse. Methods: Full thickness skin defect module was prepared on the back of Nude mouse as an acute skin injury model. CF labeled with PKH26 were injected from tail vein of Nude mouse in experimental group, and PBS was for the blank control. Macro observation and contraction rate were estimated in the whole period of recovery.Immunohistochemistry (Anti human monoclonal Pan-CK,α-SMA,Ⅷ) and HE staining were carried out after 3 weeks. Successive fluorescence microscope observation was taken for PKH26 positive cells in frozen section. Results: Both skin defects in experimental group and blank control recovered, and experimental group exhibit a faster recovery than blank control. PKH26 positive cells were not observed in both groups. Epithelization procedure and vascularization were more active in experimental group. Anti human monoclonal antibody were detected in every section of experimental group. All of the above, CF is a open derived, easy to be isolated and cultured adult stem cells. They can be proliferated by several cytokins in vivo and in vitro. As an important cell type in repair of tissue defects, it will simulate the proliferation of FBs. But according to the results, we must take a advanced investigation to explain the diverse effect of CF on KCs in vitro and in vivo. In our study, CF can differentiate to different lineage including mesenchymal and non-mesenchymal cells. In transplanting research, we observed that CF can obviously improve the repair of skin defects. These results give us a hint that the application of CF for large dimension skin effects is a perspective one.更多还原