骨髓间充质干细胞促进合并局部放射损伤创伤愈合作用的实验研究

【作者】 艾国平；

【导师】 粟永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2001， 博士

【摘要】 放射损伤复合创伤多见于战时核爆炸与平时核事故，在现代战争中，如贫铀武器可引起合并放射损伤的创伤。就合并放射损伤的创伤修复而言，一方面射线对全身损伤效应可影响局部创面的愈合，另一方面射线可直接作用于创面的修复细胞，导致创伤局部修复细胞凋亡、坏死致数量减少，同时严重地抑制修复细胞的增殖功能，难于生成健康的肉芽组织，形成难愈性创面和经久不愈的溃疡。因此，合并放射损伤的创伤难愈是防原医学和创伤医学的重要课题之一。目前促进创伤愈合的主要措施有药物促愈、生物敷料覆盖、细胞因子应用、皮肤移植以及转基因治疗等方法。应用细胞因子可以使一般创伤愈合速度提高，但对难愈性创伤则疗效不肯定；基因疗法虽然克服了细胞因子半衰期短的缺点，但仍存在基因转染的靶向性和可调控性问题；目前皮肤移植仍是临床最有效的治疗手段之一，但自体皮来源有限，异体皮或人工合成皮肤代用品还受到免疫排斥反应的困扰。随着干细胞组织工程学的发展，针对创面修复细胞数量减少和功能降低是导致放创复合伤难愈的根本问题，设想能否利用干细胞的可获得性、可扩增性和可定向诱导分化性，为组织修复提供新的思路、途径和措施。 关于应用干细胞于组织修复分化诱导为骨、软骨、肌腱等的文献报道较多，而对于软组织创伤的修复未见报道，更未见于合并放射损伤创伤的修复。本实验集中研究骨髓间充质干细胞（Mesenchymal stem cells，MSC）及其直接应用于创伤局部的促愈作用是基于以下设想，来源于中胚层的间充质干可分化诱导为组织修复细胞，将MSC直接植入在创伤局部可以直接观察其在创伤局部的演变过程，并研究其是否对创伤具有直接促愈作用，为此本课题分三部分进行研究，首先建立MSC的分离、培养和鉴定的方法；然后将其直接植入到局部放射损伤合并创伤的创面，观察其是否具有促愈作用；最后对其植入创面促愈效应机理进行探讨，主要结果如下： 1．成功建立了人和小香猪 MSC分离、培养、扩增条件。以选用 12h贴壁的有核细胞、加入 7．5％胎牛血清、种植密度 6X 10‘／ml的培养条件最适宜细胞生长，利用该条件已培养了14代，细胞具有快速增殖的能力，3—4天达到对数生长期，各代细胞形态均一，呈梭状或类成纤维状。 2．分离培养的细胞不表达1、Ill型胶原、Vlll因子和CD34分子，负性筛选证实培养细胞不是成纤维细胞、内皮细胞和造血干细胞；超微结构显示培养细胞表现为早期幼稚细胞形态发育的特征；成功诱导所筛选细胞向成骨细胞和成软骨细胞分化，进一步证实该细胞为中胚层干细胞，并为MSC在骨和软骨组织修复中的应用奠定了一定基础。 3．局部照射20Gy，可导致皮肤组织中MSC的数量减少和增殖能力下降，此为创伤难愈的重要原因之一。 4．针对这样的创伤，将培养的MSC直接植入到创面，荧光示踪显示＊盯能在创面留存较长的时间：创面残留面积百分比显示＊sC能加速创面愈合；肉芽组织中成纤维细胞和毛细血管数量明显多于对照组：愈合过程中羟脯氨酸的含量明显升高，胶原合成和沉积增加；电镜观察也显示MSC组局部成纤维母细胞合成分泌胶原的能力增加；表明MSC对创面有明显的肯定的促愈效应。 5．无细胞羊膜基质是MSC附载的良好载体，附载MSC的羊膜基质对创面具有更好的促愈作用，为皮肤组织工程的开展展示了良好的应用前景。 6．将MSC直接植入到创伤局部的促愈机理可能为：（）移植健康的MSC到创面可增加局部干细胞的数量；（二）体外培养检测 MSC可分泌IL七、IL七、G（SF等细胞因子促进成纤维细胞增殖和迁移；认为将MSC植入到创面后可分泌有关的因子吸引和趋化炎症细胞和修复细胞向创面迁移，启动修复阶段，增加伤口中修复细胞（成纤维细胞，血管内皮细胞）的数量；（）创伤局部微环境对植入的MSC有促进增殖作用，并影响 ·Vll· ＆。MSC基因表达变化，尤其是1型胶原基因表达和蛋白合成增加，提示在局部微环境作用下，MSC可分泌细胞外基质或／和直接分化为成纤维细胞参与组织的修复；综上认为MSC的促愈作用是在干细胞与创伤局部微环境相互作用下实现的。 本实验首先建立了MSC分离培养扩增的条件，并对扩增的细胞进行鉴定，明确为骨髓间充质干细胞；在此基础上将MSC直接移植到合并局部放射损伤的创面，观察到MSC对创面有明显的肯定的促愈效应，并对其促愈机理进行了初步探讨，旨在为创伤治疗提供新的思路、新的途径和新的措施，同时为干细胞组织工程的研究奠定基础。更多还原

【Abstract】 The experimental study of bone marrow mesenchymal stem cells on the healing of wound combined withlocal radiation injuryPh D Student: Guoping AiTutor: Prof.Yongping SuAbstractWound combined with radiation injury occur in nuclear attacks as well as in the nuclear accidents, especially caused by the depleted uranium weapons in modern war. The depletion and dysfunction of repairing cells numbers in wound are mainly responsible for the healing deficits. Thus, how to promote the healing processes of these injures are still one of the important issues in surgical fields. Recently, some methods to promote the healing of the wounds were reported. Such as the application of medicines, bio-dressings, cytokines, skin graft, transgene treatment, etc, but some shortcomings still existed. Cyctokines can promote the speed of healing. However, whether it can treat delayed wound remains unknown. Although the transgene method can overcome these shortcomings of applying cytokines, but it’s hard to control the target direction and regulation of gene expression. The skin graft is the most effective treatment in the impaired wound, but it’s very difficult to get enough allograft skin, the xenograft skin and artifical skin substitutes exist immune reactions. With the development of the stem cell engineering, we consider that we could use the characteristics of stem cells, which are highly proliferative and differentiated and could be easily obtained, to treat the skin wound combined with radiation injury which is caused by the depletion of repairing cells and dysfunction and try to develop a new treatment and method for curing woundhea1ing delay. It has been proposed that the stem cells will augrnent local repairor regeneration of bone, canilage or tCndon, bllt no reports abollt its aPPliCationin soft tissue injuries, not mention to aPply in wound combined radiation injwr.The present investigation was focused on the bone marrow mesenchymal stemcells (MSC) derived from mesoderm, Which can be induced to differentiate intorepairing cells in certain conditions, we directly trgnsPlanted MSC into thewounds in order to observe directly the effects of healing and to eXPlore themechanism of healing. The main resultS are as follows:l. TO iso1ate, cultUre, induce proliferation of hUInan and swine MSC. Thebest culture conditions in vitro fOr MSC groWth were to use the adhereni ceIlsin l2 hours, 7.5% fetal bovine serurn and the 6 xl0’/ml celIs density in theculture system. In this condition, cells could be maintalned for l4 passages andhad strong ability of proliferation and the duPlication time of cells was 3 to 4days. Cultured ce1ls had homogeneous spindle-shape.2. Cultured ce1ls were not fibroblast, endothelial cells and hematopoieticstem cells, and didn’t exPress the anigens of collagen I, III and CD34. Theultrastructure of cells showed the characteristics of infantile cells. At the sametime, we successfully induced the culfored cel1s to differentiate into osteoblasts,chondrocytes and codried that cultured ce1ls were stem cells. It’s the basicstep of MSC aPplication to repair bone and cedilage.3. The major mechanisms of the skin healing delay followed by local20Gy gamma-ray irradiation is the major factor of the depletion of stem cellsnumbers and depression of stem cells proliferation in wound.4. Therefore, MSC were directly transp1amed into the skin wound.Fluorescently labeled cells were visualized and photograPhed using afluorescent microscoPe. The cells of fluorescently labe1ed nuclei could beenseen in skin for a long time and could augment the healing of wound. The. III.numbers of fibroblasts and caPillary vessels were increased and the contents ofhydroxyProline and the synthesis of collagen wer eIhaed in MSC grouPs. TheHsis and secretion of collagen in fibroblaStS were increased under the eleCtrnAncroscope. It’s very sure and sigIilfican tha tranSPlantaon of MSC couldpromote the heaiing of wound.5. The amnion matrix wt更多还原