【作者】 张云松；

【导师】 高建华；

【作者基本信息】 第一军医大学 ， 整形外科学， 2007， 博士

【摘要】 [背景及目的]随着修复重建外科的发展，自体组织移植及人工材料替代修复软组织缺损成为重要的治疗手段，尽管这能有效地改善受区的组织缺损状况，但自体移植物的来源，血供及其供区的继发畸形都限制了它的使用。此外，还有其它多种多样的方法也被尝试，包括自体真皮移植，自体脂肪细胞游离移植，胶原注射，人工材料填充等等。但这些方法的治疗效果也不十分理想：例如人工材料的移植排斥反应，游离脂肪移植后的吸收且吸收率难以预测等。尽管修复软组织缺损的方法较多，却难以完全满足临床的需要。组织工程技术的出现为临床上更好的解决组织缺损等难题提供了一个新的思路。组织工程技术能以少量种子细胞，经体外扩增后与生物材料复合，修复较大的组织或器官缺损，重建生理功能，为真正实现无损伤修复组织缺损和真正意义上的形态、结构与功能重建开辟了新的途径。组织工程学的发展需要几个必备的因素，即：(1)可获得的种子细胞，并能控制其生长和组织形成；(2)结构精确的三维支架，以适合再造需要的组织量和形状，而且与细胞具有良好的组织相容性；(3)适宜的微环境，能提供充分的血供和营养，维持细胞增殖和组织功能。目前已证实脂肪组织中存在类似骨髓基质细胞的间充质干细胞，且被命名为脂肪组织来源的干细胞(ASC_S)。这种干细胞经证实同样具有体内外多向分化诱导潜力，如能向脂肪细胞、成骨细胞、软骨细胞、肌肉细胞及心肌细胞定向转化，对多种组织的损伤具有良好的修复作用，而且取材方便，获取量大，不易造成供区继发畸形等，已作为组织工程研究中的重要种子细胞。组织工程常用的支架材料分为天然材料和人工合成材料，天然材料包括：天然多糖类材料有纤维素、甲壳素、透明质酸等；天然蛋白质类材料有胶原和纤维蛋白等；人工合成的材料有聚乳酸(PLA)、聚乙醇酸(PGA)或两者聚合物聚乳酸-聚乙醇酸(PLGA)、聚四氟乙烯、羟基磷灰石、聚乙烯醇、海藻酸盐等。依据他们各自特点，被不同程度地应用于组织工程的研究。由于应用脂肪细胞组织工程技术修复软组织缺损近期才引起学者们的关注，故而用于构建脂肪组织工程有效的生物材料支架研究不多，主要为可降解的多孔泡沫，如PLGA、透明质酸、膨体聚四氟乙烯等，然而，究竟何种材料能成为脂肪组织工程种子细胞的理想载体尚不十分明确，且关于脂肪组织工程的研究国内报道甚少，对这一课题进行广泛深入的探讨具有深远意义。本实验研究了人ASC_S向脂肪组织的体外定向诱导分化能力，同时判断种子细胞ASC_S与I型固态胶原支架及纤维蛋白胶可注射支架复合后在体内构建组织工程化脂肪组织的可能性，以寻找适宜的脂肪组织工程种子细胞载体，探索构建组织工程化脂肪组织的有效方法；另外，对ASCs进行DiI体外荧光标记，观察该标记物对细胞生物性状的影响，为脂肪干细胞研究寻找理想的细胞标记方法。[材料与方法]一、成人脂肪来源干细胞(ASCs)的分离培养及鉴定取人吸脂术抽吸物的脂质部分，使用酶消化法进行原代培养，观察细胞形态及功能变化，细胞传至第3代后供实验用。利用流式细胞仪检测细胞表面部分与干细胞相关的分子如CD29、CD34、CD44，确定细胞类型；同时对细胞进行体外定向成脂、成骨诱导分化，并以油红O染色及茜素红染色进行鉴定。二、脂肪干细胞体外DiI荧光标记及观察标记物对细胞生物性状的影响荧光染料DiI体外标记ASCs，倒置显微镜及荧光显微镜观察细胞生长情况及传代后荧光强度的变化；检测细胞标记后成脂能力。使用XTT比色分析法检测接种细胞增殖率，同时将培养细胞上清液进行乳酸脱氢酶(LDH)含量测定，检测荧光染料的细胞毒性。三、以I型胶原固态支架为载体材料构建组织工程化脂肪组织的实验研究1．细胞-支架复合物体外生物相容性检测将脂肪干细胞接种于支架材料形成复合物，显微镜下观察细胞生长情况，进行XTT比色分析检测接种细胞增殖率；同时将培养细胞上清液进行乳酸脱氢酶(LDH)含量测定，检测材料的细胞毒性；DiI标记前后细胞-支架粘附率检测；最后扫描电镜观察DiI标记前后细胞与载体的黏附性。2．体内构建组织工程化脂肪组织将I型胶原支架与DiI标记的成脂诱导后ASCs混合体外培养三天，设空白支架为对照组，同体双侧植入裸鼠背部皮下，12周后取出植入物。进行组织工程新生物大体观察和湿重测定后荧光显微镜观察大体组织标本，最后组织学检测、油红O染色定性。四、以纤维蛋白胶可注射支架为载体材料构建组织工程化脂肪组织的实验研究1．细胞-支架复合物体外生物相容性检测纤维蛋白胶由A(纤维蛋白原)、B(凝血酶)两液组成，将A液与脂肪干细胞混合，A、B两液分开抽入双联注射器中，通过一混合管使两液混合，体外形成细胞-凝胶复合物，显微镜下观察细胞生长情况，进行XTT比色分析检测接种细胞增殖率；同时将培养细胞上清液进行乳酸脱氢酶(LDH)含量测定，检测材料的细胞毒性；检测DiI标记前后细胞-支架粘附率；扫描电镜观察DiI标记前后细胞与载体的黏附性。2．体内构建组织工程化脂肪组织将A液与标记DiI的成脂诱导后ASCs混合制成细胞悬液，双联注射器将A、B两液混合后同体双侧注入裸鼠背部皮下，设空白支架为对照组，12周后取出注入物。进行组织工程新生物大体观察和湿重测定后荧光显微镜观察大体组织标本，最后组织学检测、油红O染色定性。[结果]1．原代培养的ASCs形态类似于成纤维细胞，具有很强的增殖及多向分化能力。在脂肪及成骨分化培养基的作用下，分化出成熟的脂肪细胞和成骨细胞，油红O、茜素红染色阳性。实验检测到细胞表面抗原分子CD29、CD34、CD44持续表达，说明其具有干细胞特性。2．DiI标记脂肪干细胞阳性率为100％，该荧光染料不破坏细胞，不干扰细胞增殖，体外培养一周内未见荧光减弱，当细胞传至第三代后，荧光有所减弱，第六代细胞荧光明显降低，荧光标记阳性率不到30％。3．两种支架均与ASCs有良好的相容性及黏附性，对细胞无毒性，不影响细胞增殖；两组实验组均在裸鼠皮下发现新生组织块，胶原支架组新生物平均湿重为0.019g，纤维蛋白胶支架组新生物平均湿重为0.028g，常规病理切片及油红O染色均证实其为成熟脂肪组织，荧光显色阳性证实其为外源性。两组空白支架对照组未见脂肪组织形成。[讨论]软组织缺损的修复重建是整形外科面临的重大挑战。针对小范围软组织缺损，临床多采用游离脂肪移植技术。但游离移植的脂肪颗粒由于缺乏有效的血液供应，仅仅只有40％-60％体积的能存活，而且这种存活率术前还难以估计。组织工程技术的出现为解决上述难题提供了一种革命性的思路。通过这项技术的应用，我们就有可能获得足够体积的自体软组织来源。成人的间充质干细胞被证实能被诱导分化为多种细胞系。在脂肪组织中也被证实包含这种具有多向分化潜力的细胞。脂肪组织类似于人类骨髓，同样来自于中胚层而且有研究已经证实在脂肪基质中包含有多能干细胞。这种干细胞被命名为脂肪组织来源干细胞，缩写为ASCs。ASCs作为种子细胞与其他细胞相比具有相当的的优越性，首先，ASCs容易获得，对患者造成的痛苦小。对于肥胖者又需组织工程种子细胞进行整形治疗的患者尤其适合。其次，这种细胞在体外增殖速度快，传代培养可获得大量有分化能力的细胞。在这个实验中，ASCs能在体外分化为成熟的脂肪细胞，而且脂肪分化诱导后的ASCs(adipo-ASCs)与I型胶原支架混合培养后能在裸鼠皮下新生结构功能完整的脂肪组织块。该研究提示，ASCs作为种子细胞的确可以应用于脂肪组织的组织工程研究，并且能合成具有三维结构及功能的脂肪组织。特别值得一题的是，相对整形重建外科而言，术后瘢痕是需要重点考虑的因素。传统的片状或海绵状支架材料都不可避免要在受区做切口方可植入，这不可避免会残留瘢痕。在我们实验中，我们使用纤维蛋白胶可注射支架构建组织工程化脂肪组织也获得了非常令人鼓舞的结果。这样，注射性支架就能很好地解决安全性和美观的要求，在将来可能的临床应用中将具有重大的优势。[结论]用酶消化法从人吸脂术吸出的脂质部分提取的细胞为脂肪组织来源干细胞ASCs。该细胞能作为种子细胞与I型胶原固体支架及纤维蛋白胶可注射支架在体内成功构建脂肪组织。可注射性纤维蛋白胶支架因其应用时操作简单、可塑、微创，无疤痕，显示出明显的优势。更多还原

【Abstract】 [Background]In recent years following the development of rehabilitation and reconstruction surgery, artificial substitutes and autologous transplantation have become important methods for treating soft tissue defects. Although with promising prospects and encouraging results, there’s limits such as short of autologous tissue, lack of blood supply and possibility of donor site morbidity, which greatly restrict the application of these new techniques. Moreover, other methods including autologous dermis transplantation, autologous fat transplantation and collagen injection, filled with artificial material et al. However each method carries considerable disadvantages: for example, synthetic materials invariably result in foreign body reactions, and free adipose tissue grafts shrink to an unpredictable extent. Althogh kinds of methods were used for the repair of soft tissue deformities, No methods was proved to be ideal. Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges.Tissue engineering involves there items: (1) seed cells, whose growth is needed to be under strict control and high proliferation is alsoessential. (2) three-dimensional scaffold structure, which is needed to be degradable and to maintain stable volume and shape. Moreover, good compatibility with seed cells is also indispensabile; (3) appropriate micro-environment, which can provide adequate blood supply and nutrition, ensure cell proliferation and functions.Previous studies have identified a putative stem cell population within adipose stromal compartment. This cell population, termed adipose-derived stromal cells (ASCs), can be isolated from adipose tissue. ASCs can differentiate toward the multiple lineages like adipocyte, Cartilage cells, muscle cells and cardiomyocyte. ASCs are easier to obtain, carry a relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Thus, the use of adult ASCs as the seed cell of to engineer adipose tissue seems to be more inspiring.In general, Scaffolds include synthetic materials and natural materials. Several popular scaffolds like polylactic acid (PLA). polyglycolic acid (PGA), or both poly lactic-co-glycolic acid (PLGA), PTFE, Hydroxyapatite, polyvinyl alcohol, alginate etal were used to perform multiple tissue engineering. Adipose tissue engineering is a new field so only a few scaffolds were tried including the porous biodegradable materials like PLGA, hyaluronic acid, ePTFE. But no consensus was made of which materials is most ideal for adipose tissue engineering. Considering the lack of study on this field, further researches on it may be of great importance.[purpose]To induce the adipogenic differentiation in vitro by ASCs harvested from human being and to assess the possibility of constructing adipose tissue via the attachment of ASCs to typeⅠcollagen scaffold and injectable fibrin glue scaffold, on the other hand, DiI was used for labeling, the effect of this kind of fluorescent dye on ASCs is also need to be detected.[Material and Methods]1. ASCs Isolation and identificationUsing the lipid coming from liposuction surgery and using enzyme digestion method for primary culture and observe changes in cell morphology and function of cells. The cells are used for experiments after the generation three. Flow cytometry was used to detect the molecular expressionwhich is associated with the surface of the stem cells such as CD29,CD34, CD44; making cells in vitro to adipogenic, osteogenic differentiation. Oil Red O staining and Alizarin red staining to identify the success of differentiation.2. Changes of biological properties of ASCs which is labeled with DiI fluorescent marker in vitroASCs was labelled with Fluorescent dyes DiI labeled in vitro, then we use inverted microscope and fluorescence microscopy to observe changes of cell growth and cellular fluorescence intensity. Then we compare the difference of Adipogenic capability before and after DiI labeling. XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.3. Construction of tissue-engineered adipose tissue using solid typeⅠcollagen as scaffold1. Detection of ASCs-Scaffold in vitro biocompatibilityASC_S inoculate to a scaffold to form complexes; Discrepancy microscopy and laser scanning confocal microscope are usedto observe cell growth; XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.Adhesion rate changes and SEM observation of ASCs are detected before and after marked by DiI.2. Construction of tissue engineered adipose tissue in vivoCollagen typeⅠand dipogenic differentiated ASC_S which is marked with DiI and are mixed cultured in vitro for three days.The mixed materials are implated subcutaneously under the back of nude mice, at the same time,blank scaffold is used for control, implants are taken out for examination after 12 weeks. Weights of newly constructed tissue are calculated. In order to detect the origin of the newly constructed tissue, fluorescent observation is performed. Histological observation and Oil Red O staining are carried out to identify the newly constructive tissues.4. Construction of tissue-engineered adipose tissue using injectable fibrin glue as scaffold1. The observation of biocompatibility of Scaffold fibrin glue in vitroA (fibrinogen). B (prothrombin) comprising fibrin glue, ASC_S mixed with A, A and B into double-pumped syringe separately. mixed the two liquid through a mixture tube to form cell-gel complex in vitro.Discrepancy microscopy and laser scanning confocal microscope are used to observe cell growth. XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.Adhesion rate changes and SEM observation of ASCs are detected before and after marked by DiI.2. Construction of tissue engineered adipose tissue in vivoA Solution is mixed with adipogenic differentiated ASC_S marked with DiI, mixe A with B into double-pumped syringe. The mixed materials are injected subcutaneously under the back of nude mice, at the same time, blank scaffold is used for control on the contrary side, implants are taken out for examination after 12 weeks. Weights of newly constructed tissue are calculated. In order to detect the origin of the newly constructed tissue, fluorescent observation is performed. Histological observation and Oil Red O staining are carried out to identify the newly constructive tissues.[Results]1. The original cultured ASCs looks like tofibroblasts, but it has strong tendency for high proliferation and multiple differentiation. With the effect of adiposic and osteogenic differentiation medium, it is proved to be able to differentiate into mature adipocyte and bone cells. The differentiation can be proved by Oil Red O, Alizarin red positive staining respectively. These kind of cell (ASCs) is also proved to be CD29, CD34 and CD44 positive expression which are one of the main proof for stem cells.2. Almost all ASCs is labelled with DiI, a kind of fluorescent dye, It is proved not to destroy cells and interfere with cell proliferation. No changes of fluorescence is detected during the first week cultured in vitro, But as it was passaged to the third generation cells, intercellualr fluorescence weakens, After six passage, fluorescence was decreased to less than 30%.3. Both collagen and fibrin glue represent good compatibility and adhesion with ASCS without little toxicity; newly constructed tissue are found in both experiments. The average weight of newly constructed tissue is about 0.019g mixed with collagen scaffold and 0.028g mixed with fibrin glue respecitively. Results of HE staining and Oil Red O staining testify that the newly constructed tissue is mature adipose tissue, the positive fluorescence staining make it sure that the tissue was construted with implated human ASCs. No new tissue was found in the control side.[Discussion]The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery. In patients suffering from soft tissue defects, free adipose tissue grafts transplantation has been popularly applied clinically. But it is reported that there will be 40-60% volume loss during conventional grafting procedures with autologous fat tissue transplantation.Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges. Perhaps sufficient supply of autologous soft tissue can be obtained by this technique.Adult mesenchymal stem cells have been proven to be a well-established source for multiple mesenchymal tissue-forming cell lineages, including adipocytes. Fat tissues have been reported to contain such multipotent cells. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Previous studies have identified a putative stem cell population within the adipose stromal compartment. This cell population is termed adipose-derived stromal cells (ASCs).ASCs are easier to obtain, carry a relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Thus, the use of adult ASCs to engineer adipose tissue implants seems to be more inspiring.Adipose derived stem cells can successfully differentiated into mature adipocyte exhibiting a adipose-like morphology and expressing of intracytoplasmic lipid droplet. While attached to collagen I scaffold and transplanted under the skin of nude mouse, new formed adipose tissue is found at a fairly satisfactory volume. These results indicate that only adipo-ASCs can be used as the cell source for future adipose tissue engineering.In plastic and reconstructive surgery, minimal post-operation scars are ideal. The conventional sheet- or sponge-type scaffolds leave scars when transplanted via incision. We successflully use fibrin glue as scaffolds, which is injectable and also provide appropriate rigidity in our last part. The newly formed tissue is more inspiring. Thus, injectable fibrin glue scaffold is more valuable in vivo adipose tissue formation.[Conclusion]Adipose derived stem cells can be successfully obtained from the human liposuction extacat with enzymatic digestion. While attached to both solid collagen I scaffold and injectable fibrin glue scaffold, and then transplanted under the skin of nude mouse, new formed adipose tissue is found at a fairly satisfactory volume. Injectable fibrin glue scaffold represents inspring advantages like scarless, minimal invasive and plastic, which shows more value in vivo adipose tissue formation.更多还原