【作者】 孙士锦；

【导师】 麻晓林；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 皮肤是人体面积最大的器官,也是机体自我更新速度最快的组织之一,它是机体免于脱水、损伤、感染的第一道防线,是维持内环境稳定和阻止微生物、化学物质等侵入的屏障。尽早覆盖创面,恢复皮肤的屏障功能,十分重要。常规的治疗方法采用自体皮肤或同种异体、异种皮肤的移植和人工替代物覆盖等,由于存在着供皮来源有限,同种异体、异种皮肤移植有免疫排斥反应或传染疾病的危险,人工皮肤费用昂贵等问题,常规方法难以满足临床应用的需要。因此,促进皮肤创伤修复已成为组织修复领域亟待解决的难题之一,研究皮肤创伤愈合修复具有重要的理论和实际意义。成体干细胞(adult stem cells,ASCs)是一类具有多向分化潜能的细胞群体,因其存在于各种组织及器官中,来源广泛,不涉及伦理问题,在临床救治中具有广泛的应用前景,已成为近年医学研究和应用的热点之一。目前,ASCs已经从大多数组织中被分离和鉴定,并且能够诱导分化为同一胚层或者不同胚层的细胞类型,显示了其多向分化的潜能。很多研究表明,将ASCs特别是骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)应用于组织损伤修复,取得很好的效果。创伤形成的创面从根本上说是组织缺损,而干细胞独特的多向分化潜能、自我更新能力和增殖分化能力强的重要特性,正好满足皮肤修复的需要。大量实验研究发现,BMSCs在体内、体外可以分化为表皮细胞、成纤维细胞以及血管内皮细胞,这都为干细胞治疗皮肤创伤后缺损提供了理论支持。因此,针对皮肤创伤修复,本研究将BMSCs引入皮肤创伤后的创面治疗,围绕干细胞在创伤部位募集这一主题,明确BMSCs可以通过动员、募集作用参与创伤后组织修复,进一步作深入细致的研究,并初步探讨干细胞在创伤部位募集的机制,为临床治疗各种皮肤缺损提供了新的治疗思路。本研究首先对小鼠的BMSCs进行分离、培养,并对其进行诱导分化、鉴定,在此基础上观察了移植的BMSCs在皮肤创伤修复过程中募集现象,重点明确移植的BMSCs可以向创面募集,促进皮肤创伤的修复;同时,我们进一步探讨了其可能的机制,根据已知的趋化原理,采用基因转染的方法修饰BMSCs,促进BMSCs向创面的迁移,增加BMSCs向创面募集的数量,进一步提高BMSCs修复皮肤创伤的效果。主要结果和结论:1.本实验采用全骨髓贴壁法分离纯化BALB/C雄性小鼠的BMSCs,体外培养可大量扩增和传代,获得了体外培养扩增BMSCs的简单、稳定、高效的方法;通过实验鉴定及观察,BMSCs体外具有成骨、成脂肪诱导分化的潜能,已经符合种子细胞的基本条件,无论其“质和量”均能够满足进一步研究和应用的需要。2.本实验中采用Co60全身照射(照射剂量3.5Gy),使骨髓产生放射性损伤以造成受体骨髓灭活,为移植的BMSCs留出空间,减少了动物自体骨髓干细胞的影响,移植培养的BMSCs后,成功建立骨髓移植嵌合体模型,以模拟自体BMSCs的作用,满足实验需求。本实验骨髓移植嵌合体皮肤创伤动物模型的成功建立,为各种免疫功能不同小鼠放创复合伤模型建立提供了参考。3.本实验中,利用了三种不同细胞标记方式:CFDA-SE荧光标记、Y染色体标记、携带GFP基因的病毒载体转染标记BMSCs,通过给受体雌性BALB/C小鼠尾静脉注射来源于供体雄性BALB/C小鼠的标记BMSCs,分别利用组织切片后普通荧光显微镜观察,定量Real-timePCR检测创面组织中Y染色体的表达,以及冰冻切片后DAPI复染激光共聚焦显微镜观察,结果显示,无论从直观的现象观察,还是从创面组织Y染色体检测,都清楚地证实了BMSCs可以募集到创面,并参与了皮肤创伤的修复。4.创伤后免疫功能正常的BALB/C鼠创面的愈合时间为14.00±1.41d,而免疫功能缺陷的裸鼠、SCID鼠创面的愈合时间分别为17.16±1.17d和19.83±0.76d,与BALB/C鼠比较存在显著差异(P<0.01)。5.本实验通过RT-PCR检测创面组织中SDF-1的基因表达发现,创面愈合过程中BALB/C鼠SDF-1基因表达于伤后1d即有增高, 5d达峰值(P<0.01) ,然后逐渐下降,但仍明显高于对照组,14d创面基本愈合时接近对照组。而裸鼠、SCID鼠创面中SDF-1基因表达于伤后逐渐增高,所测时相点中伤后7d达峰值(P<0.01),与BALB/C鼠相比,SDF-1基因表达峰值延后,然后逐渐下降, 14d创面未愈合时,SDF-1基因表达仍高于对照组。本研究结果表明,SDF-1既是调节局部炎症的重要趋化因子,也是调节组织器官损伤修复的关键细胞因子。正常皮肤基础性表达SDF-1;创伤后,创面SDF-1表达增加,BMSCs募集到达创面,参与修复。另外机体的免疫功能也将影响BMSCs募集,从而影响创面愈合。这为临床上治疗各种创面,加速创面的尽早愈合提供了新的研究思路和方法。6.本实验成功地构建了CXCR4的腺病毒表达载体Adv-CXCR4,并转染BMSCs,尾静脉回输BMSCs检测创面组织中Y染色体的表达,结果显示:无论未转染Adv-CXCR4的BMSCs还是Adv-CXCR4转染的BMSCs移植,伤后1d,创面即检测到Y染色体,最高峰出现在伤后5d,持续明显表达14d以上;与未转染Adv-CXCR4的BMSCs相比,Adv-CXCR4转染的BMSCs移植后能更多地分布于BALB/C鼠的创面,二者相差显著(P<0.01)。同时,将Adv-CXCR4转染的BMSCs移植到BALB/C鼠的体内后,其创面平均愈合时间为12.46±1.17d,较移植未转染Adv-CXCR4的BMSCs的创面平均愈合时间14.00±1.41d,提前了1.5~2d。更多还原

【Abstract】 As the biggest organ of the body and one of tissues with the fastest self-renewal, the skin is the first line of defense against dehydration, injury and infection,and plays an important role in maintaining homeostasis and preventing invasion of microorganisms and chemical substances. Therefore, it is critically important to recover barrier function after skin injury. Conventional therapies for skin injury included autogenous skin transplantation, auto-skin graft, hetero-skin graft as well as artificial surrogate coverage. However, because of limited resources of donor skin, auto-skin graft and hetero-skin graft may result in high cost and have risks of immunological rejection or disease infection, which adds difficulties to clinical application. Therefore, how to promote repair of skin wound is now one of tough problems and studies on repair of skin wound and wound healing are of theoretical and practical significance.Adult stem cells (ASCs) are a kind of cell colony with potential of multi-directional differentiation. In the meantime, ASCs can be obtained from all kinds of organs and tissues, without involvement in ethics problem, and has wide prospect of clinical application, which contributes to the fact that ASCs have been one of research hot spots in recent years. At present, ASCs have been separated from most tissues and identified, which shows that ASCs can be induced to differentiate into cell types at same or different embryonic layers, indicating its potential of multi-directional differentiation. Several studies reported that ASCs especially BMSCs could obtain satisfactory results for repair of tissue injury.Wound is tissue injury in itself. Stem cells have potential of multi-directional differentiation, self-renewal and good proliferation and differentiation and can satisfy requirement of skin repair. Large number of experiments found that BMSCs could in vivo and in vitro differentiate into epidermal cells, fibroblasts and vascular endothelial cells, which provides theoretical basis for skin wound repair with stem cells.The aim of the study was to ascertain whether BMSCs participated in repair of skin wound by means of mobilization and recruitment and preliminarily explore mechanism of stem cells recruiting at wound site so as to cater fresh treatment methods for all kinds of skin defects.In the study, BMSCs were first separated and cultured and then were induced to differentiate and identified. Recruitment of the transplanted BMSCs in repair of skin wound was observed to make sure whether the transplanted BMSCs could recruit towards wound to facilitate repair of skin wound. Meanwhile, the possible mechanism was further explored. Based current chemotaxis theory, BMSCs were modified by means of gene transfection to promote their migration to the wound skin and further boost effect of BMSCs in repair of skin wound.Main results and conclusions are as follows:1.We used variability adherence method for separation and purification of BMSCs from male BALB/C mice and found that in vitro culture could obtain large quantity of amplification and passages, which contributes to a simple, stable and effective method for in vitor culture and amplification of BMSCs. Experimental assessment and investigation showed that in vitro BMSCs had potential of inducing osteogenesis and adipogenesis, which accorded with pacing factor of seed cells and could meet demand of further studies.2.Systemic irradiation with Co60 (irradiation dose at 3.5Gy) was used in this study to induce radiation damage of bone marrow in order to inactivate host bone marrow, which may keep space for transplanted BMSCs and reduce influence of autogeneic bone marrow stem cells. After transplantation of cultured BMSCs, a bone marrow transplantation chimera model was successfully established to simulate function of autogeneic BMSCs, which caters reference for establishment of combined injury model of mice with different immune function.3.The study involved three kinds of cell labeling methods, ie, CFDA-SE fluorescence labeling, Y chromosomal marker and BMSCs labeling with adenovirus vector carrying GFP gene. After the labeled BMSCs from male BALB/C mice were injected into female BALB/C mice via vena caudalis, fluorescence microscope was used detect the labeled BMSCs and real-time PCR were employed to detect expression of Y chromosome in wound tissues after histological section; DAPI counterstain confocal microscopy was used detect the labeled BMSCs in wound tissues after frozen section. The results could clearly proved that BMSCs could be recruited on the wound and participate in repair of skin wound.4.The wound healing time for BALB/C mice with normal immune function after injury was (14.00±1.41) days, while that for nude mice and SCID mice with immune defects were (17.16±1.17) days and (19.83±0.76) days respectively, with statistical difference compared with BALB/C mice (P<0.01).5.Fluorescent quantitation RT-PCR was used to detect gene expression of SDF-1 in wound tissues, which showed that gene expression of SDF-1 was elevated in BALB/C mice at day 1 after injury and reached peak at day 5 (P<0.01). Gene expression of SDF-1 was then gradually decreased (but still remained higher than control group) and reached similar level to control group on primary wound healing at day 14. Gene expression of SDF-1in skin wound of nude mice and SCID mice was gradually increased after injury and reached peak at day 7 after injury (P<0.01), which was delayed compared with BALB/C mice. Gene expression of SDF-1was then gradually decreased and maintained at higher level than control group before wound healing at day 14. The results indicated that SDF-1 is either important chemotatic factor regulating regional inflammation or key cytokine regulating repair of tissue or organ injury. SDF-1 is expressed in normal skins. After skin wound, expression of SDF-1 is increased, when BMSCs are recruited on the wound for repair of the wound. In addition, immune function of the organism will affect recruitment of BMSCs and result in late wound healing, which provides novel method for clinical treatment of different kinds of wounds and promotion of wound healing.6.We successfully constructed adenovirus expression vector Adv-CXCR4 of CXCR4. BMSCs were transfected and retransfused via vena caudalis to detect expression of chromosome Y in wound skin tissues, which showed that chromosome Y was detected in wound skin tissues at day 1 after injury either for BMSCs free from Adv-CXCR4 tranfection or transplanted BMSCs transfected with Adv-CXCR4. The expression peak emerged at day 5, which lasted for over 14 days. In the meantime, compared with BMSCs free from Adv-CXCR4 transfection, transplanted BMSCs transfected with Adv-CXCR4 could distribute on the wound of BALB/C mice, with statistical difference (P<0.01). After BMSCs transfected with Adv-CXCR4 were transplanted into BALB/C mice, the wound healing time were (12.46±1.17) days, with 1.5~2 days shorter than (14.00±1.41) days for skin wound treated with transplantation of BMSCs free from transfection with Adv-CXCR4.更多还原