【作者】 刘柳；

【导师】 归来； 蔡国斌；

【作者基本信息】 中国协和医科大学 ， 整形外科， 2008， 博士

【摘要】 皮肤软组织缺损是整形和烧伤外科经常遇到的问题,临床上通常采用自体皮或异体皮肤移植进行修复。但由于供区不足、免疫排斥及传播疾病等缺点,寻找一种理想的皮肤替代物一直是临床上一个急需解决的难题。组织工程皮肤的出现为这个问题的解决开创了崭新的方法。目前此类的组织工程皮肤和生物敷料种类繁多,但仍不成熟,存在着皮肤耐磨性差、自体种子细胞来源缺乏、异体种子细胞诱发免疫排斥、培养时间长、治疗费用昂贵等缺点。本研究应用组织工程学原理,在前人研究的基础上,体外扩增同种异体胎儿成纤维细胞作为种子细胞构建三维培养,并用来修复鼠皮肤缺损,同时也在临床观察其治疗效果。为本领域提供了新的思路和方法。目的探索人胎儿成纤维细胞(human embryonic fibroblasts,HEF)的培养方法和特性,通过异体淋巴细胞混合试验,揭示培养后不同代的胎儿和普通成纤维细胞免疫原性的变化,探讨HEF作为皮肤组织工程种子细胞的可行性。探索应用HEF和鼠尾胶原(rat tail tendon collagen,RTT collagen)构建组织工程皮肤的方法,观察其组织结构和分泌特性。应用构建的组织工程皮肤修复实验动物皮肤缺损,观察治疗效果和修复后组织特点。最终通过临床应用证明其临床应用的可能性、可靠性和有效性。方法采用组织块法、酶消化法培养人胚胎和普通皮肤成纤维细胞,观察获得成纤维细胞数量、细胞形态、细胞活力、生长周期等指标。将不同代数人胚胎和普通成纤维细胞,分别与健康的同种异体志愿者的外周血淋巴细胞混合,建立异体细胞体外免疫排斥模型。通过H3-TdR标记,液体闪烁计数仪检测异体淋巴细胞增殖情况。将鼠尾胶原溶液、浓缩培养基、NaOH溶液和以血清重悬的普通和胚胎成纤维细胞溶液在适当的比例下混合后形成凝胶构建真皮类似物。HE染色观察其结构。用ELASA法测试三维真皮培养基中的TGF-β1和IL-6的含量。将胎儿成纤维细胞采用荧光染料Hoechst33342染色后,构建人工真皮,移植于裸鼠背部的全层皮肤缺损,观察其愈合过程。术后示踪胎儿成纤维细胞,并取材做光镜下组织观察和基底膜PAS特殊染色。分离培养SD大鼠胚胎成纤维细胞用上述实验的方法制备三维构建。将培养的人工真皮修复大鼠皮肤全层缺损并用空白鼠尾胶原做对照,观察其愈合过程和病理变化,用免疫组化方法测试创面CD31和波形蛋白的表达。选取扩张术后皮瓣边缘坏死造成全层皮损的患者12例。部分创面采用制备的胚胎成纤维细胞组织工程皮肤覆盖,选取邻近创面作为对照,对照区采用空白的鼠尾胶原或凡士林油纱布覆盖。治疗期间观察创面有无炎症反应、有无渗出等情况;对患者创面进行有效性及安全性评估;观察创面的愈合情况及不良反应;计算应用部位与对照的愈合时间。结果采用酶消化法所获得的细胞数量最大,为7×10~7个/g组织,并且需时短,但细胞形态及活力较差。采用组织块法培养细胞,细胞形态和活力好,适合本研究。与普通成纤维细胞相比,人胚胎成纤维细胞具有生长速度快,分裂指数高,代谢迅速和分泌功能合理的特点。在评价细胞免疫原性的实验中,我们发现普通和胚胎成纤维细胞培养后各代和原代相比,对异体淋巴细胞刺激的增殖作用显著降低,而普通成纤维细胞下降的趋势更明显。胚胎成纤维细胞培养后几乎对异体淋巴细胞无刺激增殖作用。利用鼠尾胶原做真皮支架形成的人工真皮中成纤维细胞生长状态良好,并且具有一定的组织强度。检测其培养液中的TGF-β1和IL-6,胎儿成纤维细胞组在各个时相上分别显著低于和高于普通成纤维细胞组。裸鼠动物实验中,术后20日用胎儿成纤维细胞构建的组织工程皮肤覆盖的裸鼠背部创面已愈合,表皮较薄弱。而单纯用空白鼠尾胶原覆盖的创面仍留有痂皮。基底膜PAS染色见实验组表皮真皮之间有连续的基底膜结构。创面荧光标记的细胞量随着时间减少。SD大鼠实验中,病理学检查表明移植TEF skin的大鼠创面上皮化良好,成纤维细胞和胶原排列规则,血管化满意。临床实验中,患者术后病情平稳,创面无明显炎性反应及渗出。创面愈合时间与应用单纯鼠尾胶原的对照组相比明显缩短2—4天,与应用凡士林纱布的对照组相比缩短8—10天。未见排斥等不良反应。结论人胚胎成纤维细胞易于获取,容易培养,性质稳定,培养后几乎无抗原性,是组织工程种子细胞的良好选择。利用鼠尾胶原可以构建真皮类似物,该成纤维细胞胶原凝胶复合物有着良好的生物学特性,成纤维细胞保持着相当的活力,它是继续做应用研究的良好模型。此人工真皮可以满意的修复动物皮肤全层缺损。满意的临床疗效提示,利用胎儿成纤维细胞构建的人工真皮,在未来有较大的研究和应用潜力。更多还原

【Abstract】 Construction of tissue engineered skin using fetal fibroblastsSkin defects are main problems in plastic and burns surgery.Usually, autograft of skin or allograft of skin is used to repair skin defects. However,these therapies have many disadvantages,such as insufficient autografts,a severe morbidity of donor site,immune elimination towards allografts,etc.So it is necessary to invent a new method for constructing tissue-engineered skin which will be useful in clinical practice.Since Rheinwald and Green(1975) established the culture method of the keratinocytes,tissue-engineered skin became approached.In the present time,a lot of biological dressing,like Apligraft,Dermograft,Intergra, and Allodone etc,are widely used in clinical for wound healing treatments. But most of these tissue-engineered skin products have their disadvantages,such as poor physical performances,scarce of autologous seed cells,immune exclusive reaction towards xenogenous seed cells,long culturing time,expensive cost,etc.In this study,based on the previous researches,we amplify allogenic fetal fibroblasts in vitro and construct tissue engineering skin to repair mice skin deficiency and simultaneously observe clinical effects on patients.So it provides a new method to find ideal seed cells of tissue engineering skin and to treat skin deficiency.ObjectiveTo establish a strategy for amplify human embryonic fibroblasts in vitro,then to analyse if cultured fetal fibroblasts haves ome advantages to be the seed cells and to reveal how immunogenicity of cultured fetal fibroblasts changed,those were mixed with allogenic lymphocytes. To establish a method for construction of tissue-engineered skin using human fetal fibroblasts and rat tail collagen,then to observe structures and secretion characteristics of this TE skin.To repair experimental animal skin defects using TE skin,then to observe therapeutic effectiveness and its structure after wound healing.The final aim is to testify the possibility,effectiveness of clinical repairment of skin defects with the tissue engineered skin substitute.MethodsHuman embryonic and common fibroblasts were cultured with the method of tissue clump and enzyme digestion.The cell shape,cell activity, growth cycle were observed.The different generations of the two types of fibroblasts were individually mixed with allogenic lymphocytes in vitro to observe how the allogenic lymphocytes proliferate.Rat tail collagen was prepared and mixedwith the cultured human fetal or common fibroblasts suspension in calf serum,concentrated culture medium and sodium hydroxide to form dermis equivalent.The cell collagen gel was stained with HE staining to observe.The concentration of TGF-β1 and IL-6 in the supernatant fluid of the culture medium were measured by ELISA method.HEFs were stained with fluorescent dye Hoechst33342,these HEFs were seeded into rat tail collagen to form dermis equivalent and implanted into nude mice with dorsal skin defects.Effectiveness was observed postoperation and equivalent skin was excised and observed under light microscope.PAS staining was also performed on the basement membrane of living TE skin.Fibroblasts were isolated and cultured from the dorsum of SD rat embryo.Tissue-engineered skin was prepared using the cultured SD rat embryonic fibroblasts.We transplanted the TE skin to adult SD rats and compared them with simple rat tail collagen graft to observe their developments and their histological changes.The expressions of CD31 and vimentin of wound healing were also detected with immunohistochemisty staining.Twelve cases of full-thickness skin defects after soft tissue expansion were repaired with tissue-engineered fetal skin.The other near wound were selected as control group.The simple rat tail collagen and the petrolatum gauze cover the control region respectively.The inflammatory reaction,bleeding were observed during the treatment period. The effectiveness and the safety of TE skin had been assessed in patients. The healing condition and the bad reaction of the wound were observed, and the time to complete closure between application and control were calculated.ResultsWe obtained 7×10~7 cells by method of enzyme digestion,and needed very short time,but the cell shape and activity were not good.Using the tissue clump,the shape and activity were both good.Compared with common fibroblasts,human embryonic fibroblast had the characters such as fast growing rate,high mitosis index and rapidly and strong metabolism.In explore of the cells’ immunogenicity,we found both the common and the fetal fibroblasts,with the passages of fibroblasts increasing,the cpm gradually decreased.The cpm of initial passage and first passage descended sharply,especially common fibroblasts.In the fetal fibroblasts,the cpm of the second passage,the third passage,the fourth passage and the fifth passage are approximation to the blank control.The rat tail collagen gel made fibroblasts have a suitable condition and the fibroblasts can be stretched slightly without being torn or deformed.The concentration of TGF-β1 and IL-6 in HEF group were lower and higher individually than in common fibroblasts group on each time point.(P＜0.05) 20days postopration,the wounds on the back of nude mice covered by TEF skin had been healed with a thin layer of epithelium.But the wound covered with simple rat tail collagen was still crust.There was a continuous blue stained basement membrane fluctuating between the epithelium and the dermis.These marked HEFs were visible in the implant by fluorescent microscopy ten days after implantation and then gradually disappeared.In the experiment on SD rats,histological examination revealed good epithelization,regularly arranged collagenous fibers and fibroblast,and satisfactory vascularization in the TEF skin grafting group.During the clinical application,the patients’ condition after operation were stable without significant inflammatory reaction and bleeding on the wound.The healing time were significantly shortened 2-4 days compared with that of control group using rat tail collagen and 8-10 days compared with that of control group using petrolatum gauze.Safety assessment showed no clinical or laboratory evidence of rejection during the trial.There was not any ill effect after healing.ConclusionsHuman embryonic fibroblast is a promising source for tissue engineering for their characteristics such as easy acquirability, extensive proliferation,etc.The method of tissue clump is more suitable to our investigation.Human embryonic fibroblasts nearly have no antigenicity after being cultrued.Using the rat tail collagen can form dermis equivalent with the favorable biological traits of the fibroblasts interacting with extracellular matrix,which was the key to provide the artificial skin. The TEF skin we constructed can induce wounds healing with satisfactory effectiveness when grafted into experimental animals.In view of the therapeutic effects of this technique along with the simplicity in application,fetal skin cells could have great potential in tissue engineering.更多还原