【作者】 陈斌；

【导师】 郭永章；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 目的:外周血液来源的循环纤维细胞(circulating fibrocytes)是新近发现的一个具有成纤维细胞特性的白细胞亚群。循环纤维细胞在创伤后早期与炎性细胞同时进入创伤局部组织提示它们在创伤修复中具有重要的作用。由于它具有合成多种细胞外基质蛋白、细胞因子以及递呈抗原、收缩创面、促进新生血管形成的能力,因此被认为可以促进创伤的修复。但尚未见应用其进行皮肤创伤修复,尤其是慢性创面,如糖尿病创面的修复的相关研究。目前,通过体外培养的方法,循环纤维细胞可以从外周血液单核细胞中分离出来。但其纯化需要进行免疫磁珠或流式细胞分选以去除其它细胞类型的污染。此方法需要特殊的设备条件、获得的循环纤维细胞量较低且费用高昂,不利于临床应用研究的开展。为进一步开展临床应用研究,改良循环纤维细胞的纯化方法是必要的。本研究的目的是:(1)探索更为简便易行、高效的循环纤维细胞体外培养、纯化的方法。(2)对所获得细胞进行生物学鉴定。(3)观察循环纤维细胞对糖尿病创面愈合的影响并探索其机制。方法:(1)以绿色荧光蛋白(GFP)基因转染小鼠和同系野生型小鼠构建新型连体小鼠全层皮肤缺损模型,观察伤后第3、7天循环细胞在创面组织中的迁移情况,并采用免疫荧光组织染色的方法了解伤后第3天外周血液来源的循环纤维细胞、血管内皮细胞在创面的迁移情况,寻找循环纤维细胞在伤后早期及参与创伤修复的证据。(2)采用密度梯度离心法分离小鼠外周血液单核细胞,接种于含20%胎牛血清的DMEM培养液中进行持续培养46天,除给予换液前PBS漂洗外,不作任何免疫筛选。采用免疫荧光化学染色的方法对分离后5、10、14、21、28天的贴壁生长的细胞进行表型鉴定并观察其动态变化。并检测培养21天的cFb的增殖活性。(3)以实时荧光定量PCR方法检测分离培养14,28,35,45天的贴壁细胞表达生长因子TGF-β1、bFGF、PDGF-A、VEGF-A、趋化因子MIP-1α、MIP-2α、MCP-1及趋化因子受体CXCR4、细胞外基质蛋白Col-Ⅰ和α-SMA mRNA的能力,并与小鼠皮肤成纤维细胞、PBMCs进行对比,从而对其进行功能鉴定。采用体外培养的成纤维细胞划痕实验观察培养21天的cFb条件培养基对成纤维细胞增殖活性的影响。(4)在糖尿病小鼠背部复制全层皮肤缺损创面后,即刻经尾静脉注射培养28天的PKH26标记的cFb(cFb治疗组)或等体积PBS(PBS对照组),伤后第3、7、10、14、17、21天观测创面愈合情况。伤后第3、10天切取创面组织,观测PKH26标记的cFb在创面组织、肺脏、正常皮肤组织中的迁移情况。并通过免疫组织荧光染色观测两组创面组织中白细胞、巨噬细胞浸润率、新生血管形成率及细胞增殖指数。同时通过实时荧光定量PCR检测生长因子TGF-β1、PDGF-A、VEGF-A、趋化因子MIP-1α、MCP-1、细胞外基质蛋白Col-Ⅰ和α-SMA mRNA的能力。(5)数据以(?)±s表示。采用SPSS for Windows 11.0进行统计分析,多个样本均数间的比较应用One Way ANOVA检验及t检验,P＜0.05有显著性差异。Microsoft Excel2004 for Mac绘制统计图。结果:(1)本研究成功构建了连体小鼠全层皮肤缺损模型,并观察到在伤后第3、7天均有大量外周血来源的细胞(表达GFP~+)迁移进入创面组织或创缘皮下组织,但第7天浸润的GFP~+细胞已经开始减少。在伤后第3天浸润进入创面的GFP~+细胞主要是白细胞(21.4±4.3%)。此外,创面组织中也可检到7.6±4.4%的α-SMA~+/GFP~+细胞和4.8±3.1%的PECAM-1~+细胞。(2)在从外周血液分离出来3天后,大量贴壁细胞出现于培养皿中。这些细胞具有典型的梭形外形并可增殖形成克隆。以CD45/Col-Ⅰ和CD11b/Col-Ⅰ双色免疫荧光染色鉴定,在分离后第5天CD45~+/Col-Ⅰ~+细胞和CD11b~+/Col-Ⅰ~+细胞分别为7.5±2.7%和6.7±3.1%。但在培养过程中这些细胞的表型迅速变化。它们很快失去表达CD11b和CD45的能力,但却持续表达Col-Ⅰ。到分离培养第14天,大部分细胞均不在表达造血系细胞标记,但表达Col-Ⅰ的细胞却上升到56.8±18.5%。至第28天,96.5%±11.5%的细胞为Col-Ⅰ~+。整个培养过程中,α-SMA阳性细胞持续增加,至第28天达95.0±9.1%。(3)以实时定量PCR检测培养14、28、35、46天cFb表达生长因子、趋化因子及趋化因子受体和细胞外基质蛋白mRNA的能力,结果发现,与皮肤成纤维细胞相比较,cFb表达高水平的生长因子、趋化因子,同时特征性的表达趋化因子受体CXCR4(P＜0.05)。而它们表达Col-Ⅰ和α-SMA的能力却低于皮肤成纤维细胞(P＜0.05)。体外成纤维细胞划痕实验也证实,cFb条件培养基可以显著促进体外划痕伤口的愈合(P＜0.01)。(4)经尾静脉注射培养28天的cFb后,通过荧光显微镜观察,PKH26标记的cFb可靶向性的迁移至创面组织,而在肺脏、正常皮肤却未检出。与PBS对照组相比较,cFb治疗组创面组织白细胞、巨噬细胞浸润增加(P＜0005),新生血管形成及细胞增殖加快(P＜0.05)。而通过检测生长因子TGF-β1、PDGF-A、VEGF-A、趋化因子MIP-1α、MCP-1和细胞外基质蛋白Col-Ⅰ和α-SMA mRNA的表达水平,也发现在伤后第3天,cFb治疗组创面组织MIP-1α、MCP-1 mRNA的表达量显著高于对照组(P＜0.05),而生长因子的表达却在伤后第10天显著高于对照组(P＜0.01)。而从创面愈合率来看,cFb治疗组伤口愈合在伤后第3、7、10天较对照组快(P＜0.05)。到伤后21天,两组动物伤口均完全愈合。结论:(1)连体小鼠全层皮肤缺损模型可以用以观察循环细胞参与创伤修复的相关研究,包括cFb、血管内皮细胞在内的循环源性细胞均可在模型中通过免疫荧光染色检测到。(2)通过单纯贴壁筛选延时培养的方法,可以获得更多较高纯度的、具有典型功能特点的cFb。尽管这些细胞在表型上已失去表达造血系细胞标记的能力,至分离培养46天,这些细胞依旧具有较强的产生生长因子、趋化因子的能力,并表达趋化因子受体CXCR4。(3)培养的cFb可以在创伤后早期促进糖尿病小鼠全层皮肤缺损创面的愈合进展,其机制与增加创伤局部炎性细胞浸润和生长因子、趋化因子的水平有关。更多还原

【Abstract】 Objectives:Peripheral blood derived circulating fibrocytes are a newly indentified leukocyte subpopulation that displays fibroblast-like properties.These blood-borne cells rapidly enter sites of tissue injury at the same time as inflammatory cells,suggesting an important role for these cells in wound repair.Circulating fibrocytes are thought to play a role of accelerating wound repair by several mechanisms such as the scretion of extracellular matrix（ECM）, cytokine production,antigen presentation,wound constraction,and angiogenesis.But,until now,although its role in wound healing has been described in a number of papers,no any study has provided evidence that circulating fibrocytes could accelerat skin wound repair, especially chronic wound healing as diabitic ulcer.Presently,circulating fibrocytes can be isolated from periheral blood mononuclear cells（PBMCs）via cell culture in vitro.To avoid contamination of other cell types,after 10-14 days of initial isolation,circulating fibrocytes need to be purified by immunomagnetic negative selection.This method could to be improved due to it need special technology and instrument,while the cell yield is lower.For clinic studies on chronic wound healing using this novel cell type,it’s necessary to develop a simple and efficient method for cell purification.The aims of this study were（1）to develop a simple and efficient method for circulating fibrocytes purification and enrichment,（2）to identify circulating fibrocytes yielded from above method and（3）to investigate the effects and potential mechanisms of circulating fibrocytes in diabetic mouse full-thickness wound healing.Methods:（1）Mouse parabiotic skin wound healing model that consisted of an unirradiated transgenic GFP⁺ mouse（C57BL/Ka-eGFP）and an unirradiated wild-type mouse originated from same strian were generated by surgical conjunction and wounded on 14 days post parabiosis operation.The blood-borne cells migration in wound sites were observed at 3 days and 7 days after wounding.For harvest the evidence of circulating fibrocytes participated in the early stage of wound healing,immonofluorescence staining was performed to count the migration of blood-derived circulating fibrocytes,endothelia cells at 3 days after wounding. （2）Total mouse peripheral blood mononuclear cells were isolated by density gradient centrifugate with Ficoll/paque and cultured in DMEM supplement with 20%fetal bovine serum for 46 days.No any immunological selection methods were performed except PBS wash before medium replacement.At 5,10,14,21,and 28 days after initial isolation,the dynamic change of cell markers and the percentage of each cell subpopulation were analysised by dual immunofluorescent staing.（3）To identify cultured circulating fibrocytes with their function,at 14,28,35,and 46 days after initial isolation,the relative expression of growth factors（TGF-β1,bFGF,PDGF-A and VEGF-A）,chemokines （MIP-1α,MIP-2α,MCP-1）,chemokine receptor CXCR4 and ECM protein Col-Ⅰ,α-SMA mRNA were detected by real time PCR and data were compared with the expression of above gene in mouse dermal fibroblasts and PBMCs.Additionally,the effect of cultured cFb on the proliferation of dermal fibroblasts was determined via the in vitro scrape wound repair model. （4）Immediately following the made of dosal full-thickness wound on diabetic mouse,100μl （5.0×10⁵ cells）PKH26 labeled circulating fibrocytes（cFb group）or 100μl PBS were injected into the tail vein.The open wound area was measured at 3,7,10,14,17,and 21 days post wounding.For histology assay,tissue samples were harvested at 3,10 days post wounding, and the labled cFb were be traced under fluorescent microscope.The neutrophil and macrophage recruitment,angiogenesis,and cell proliferation were detected by immunofluorescence staining.Real time PeR was employed to investigate the relative expression of growth factors（TGF-β1,PDGF-A and VEGF-A）,chemokines （MIP-1α,MCP-1）,and ECM protein Col-Ⅰ,α-SMA mRNA in wound tissue.（5）Data are expressed as（?）±s.Statistical analysis was performed using SPSS for Windows 11.0.Results were determined to be stastically significant if P＜0.05.All stastical diagrams were graphed using Microsoft^?Excel 2004 for Mac.Results:（1）Using the parabiotic mouse full-thickness wound healing model,we determined a numerous blood-derived cells（express GFP⁺）migrated into wound site or wound edge,and the amount of these cells was decreased at 7 days post wounding.After staining with CD45, a-SMA or PECAM-1 antidodies,at 3 days post wound,the major population of these GFP⁺ cell was neutrophils（21.4±4.3%/HPF）.Additionally,α-SMA⁺/GFP⁺ cells（7.6±4.4%/HFP） and PECAM-1⁺ cells（4.8±3.1%/HPF）also detected in wound sites at 3 days post wounding. （2）After initial isolation from peripheral blood 3 days,a lot of adherent cells appeared in culture disk.These cells expressed typical spindle shape and formed clone.Identification with CD45/Col-Ⅰand CD11b/Col-Ⅰantibodies,around 6.7±3.1%of CD45/Col-Ⅰdouble postive cells and 7.5±2.7%of CD11b/Col-Ⅰdouble postive cells were detected at 5 days after isolation.But the cell markers changed dynamically during cultured in vitro.Rapidly,these cells lost hematopoietic cell marker,CD11b and CD45,but continually expressed Col-Ⅰ.At 14 days after isolation,most cells lost hematopoietic markers and the percentage of double positive cells decreased sharply while the percentage of Col-Ⅰ⁺ cells increased（56.8±18.5%）. At 28 days after isolation,a higher Col-Ⅰpositive cells percentage,96.5±11.5%,was determined by cell count under fluorescent microscope.During the period of cell culture,an increasing percentage ofα-SMA positive cells were detected.（3）Compared with mouse dermal fibroblasts,cultured circulating fibrocytes expressed higher mRNA of various growth factors and chemokins（P＜0.05）.As a typical feature of circulating fibrocytes,a higher relative expression of CXCR4（P＜0.05）also was detected in our study by real time PCR assay at 14,28,35,46 days after initial isolation.In contrast,the level of expression of Col-Ⅰandα-SMA were lower than dermal fibroblasts（P＜0.05）.（4）Using fluorescent microscope,PKH26 labled circulating fibrocytes were observed on the sections of cFb group wound site,but didn’t appear in lung and normal skin.Compared with PBS group,the recruitment of netrophils and macrophages,angiogenesis and cell proliferation were increassed（P＜0.05）.The expression of MIP-1α,MCP-1 mRNA in wound tissue also increased at 3 days post wounding（P＜0.05）,and the expression of TGF-β1, PDGF-A and VEGF-A mRNA increased at 10 days（P＜0.01）.At 3,7,10 days post wounding, wounds in the cFb group had closed fastly than those in PBS group（P＜0.05）.By day 21,all the wounds had closed in both groups.Conclusion:According to our studies,we concluded that,（1）Parabiotic mouse skin full-thickness wound model could be employed in the relative studys which explore circulating cells participate in wound repair.Including circulating fibrocytes,endothelial cells could be detected in parabiosis mouse wound tissue via immunofluoresence staining.（2）Using a simple and convenient method,as natural selection by extended culture period in vitro,more admissible purification of circulating fibrocytes which with typical function could be yielded.Although lost the markers of hematopoietic cell,these cells still express higher level of the production of growth factors,chemokines,and the chemokine receptor,CXCR4.（3）Cultured circulating fibrocytes may accelerat the early process of diabetic skin full-thickness wound healing.The most likely mechanism is relat to increasing the inflammatory cells infiltration and the synthesis of growth factors and chemokines.更多还原