组织工程化羊膜促进表皮细胞扩增和真皮重建的实验研究

发布时间：2018-05-11 11:32

本文选题：羊膜 + 脱细胞　；参考：《第二军医大学》2013年博士论文

【摘要】：研究背景：真皮替代物的研究一直是皮肤组织工程的重点和难点，也是复合型皮肤替代物发展的基础。真皮替代物作为创面修复过程中的支架结构，不仅能促进表皮细胞的增殖、迁移和分化，调节基底膜的形成，而且能引导成纤维细胞和血管内皮细胞的浸润增殖，沉积新的胶原和形成新的血管，从而实现真皮结构重建。迄今为止一系列的真皮替代物已经被成功研制出来，包括自然来源的材料，如异体或异种脱细胞真皮、胶原和透明质酸等，以及人工合成材料，，包括一些聚合体生物材料和其他纳米材料等。另外的一些新型真皮替代物材料也正在被研制之中。在这些真皮替代物中，Integra和Tegaderm等已经被商品化并成功运用于临床治疗。大部分的真皮替代物都能很好地模拟正常真皮的组织结构特点，但是除了脱细胞真皮外，普遍都缺乏基底膜结构成分。而且即使是脱细胞真皮，为了去除表皮细胞层和真皮中的成纤维细胞和血管内皮细胞，一般需要很强的细胞清除剂处理，而后者会对真皮的基底膜成分产生严重的破坏。基底膜成分是正常皮肤中重要的成分，位于是表皮和真皮连接处，对于维持皮肤的正常功能起到重要的作用。正常皮肤中的表皮干细胞主要位于基底层，通过半桥粒结构紧密连接在基底膜表面；后者可以调节表皮干细胞的增殖，迁移和分化。体外分离培养的表皮干细胞由于失去了基底膜的支撑和调节作用，逐渐丧失了增殖的能力，分化成角质形成细胞。大量的研究表明：在人工合成的真皮支架表面添加自然来源或者人工合成的基底膜成分如IV型胶原和纤维弹性蛋白等，可以有效地促进表皮细胞的增殖，改善所形成的表皮结构的形态和功能。人羊膜基质(amniotic membrane, AM)可能是一种良好的真皮替代物材料。它来源于胎盘的最内层，可在分娩时获得，主要由三部分结构组成：单层立方状表皮细胞，富含细胞生长因子的厚基底膜，以及成纤维细胞散在其中的疏松网状纤维基质。羊膜基质含有I型胶原、IV型胶原、VII型胶原、弹性蛋白和糖胺聚糖等成分，类似于人体的真皮。羊膜的基底膜主要成分为层粘连蛋白、IV型和VII型胶原，与皮肤和角膜的基底膜成分相似，是人体内最厚的基底膜。羊膜还具有促进上皮化、抑制瘢痕增生、抗炎症、抗血管生成、抗菌和抗病毒等生物特性，且免疫原性极低，因而被广泛用作外科手术材料和创面覆盖物，尤其是用于眼科角膜重建治疗。近年来更有研究者更是将羊膜作为角膜干细胞、间充质干细胞、以及其他体细胞扩增和移植的载体，用于修复各种组织缺损。羊膜使用时既可以保留失活的表皮细胞，也可以完全去除表皮细胞。完整羊膜含有大量的细胞生长因子，有利于细胞培育过程中干细胞特性的维持；而脱细胞羊膜(accelullar amniotic membrane, AAM)借助于良好的基质和基底膜成分，能促进培育细胞的增殖、迁移和分化；部分体细胞在脱细胞羊膜上的扩增速度要明显快于完整羊膜。在本课题中，我们利用反复冻融和DNase消化的方法消化处理人新鲜羊膜，获得保留完整基底膜结构的羊膜基质；随后又利用水溶性碳二亚胺对脱细胞羊膜进行适当的交联处理，以改善脱细胞羊膜的机械强度和生物稳定性。得到的交联脱细胞羊膜具有良好的可操作性和抗酶降解能力，作为培养基质可以促进表皮细胞的体外增殖速度，而且保持了良好的生物组织相容性。将表皮细胞培养在交联脱细胞羊膜表面形成复合型表皮-真皮替代物，并将其用于移植修复裸鼠全层皮肤缺损，结果发现这种复合皮肤替代物可以明显促进创面愈合速度，改善新生表皮的厚度和功能，促进真皮结构重建，减轻创面收缩。实验方法：一、将新鲜羊膜根据脱细胞方法的不同分为以下几组：Dispase II消化+细胞刷处理组、Freeze-thaw+DNase消化组和完整羊膜对照组。通过下面的几种检测方法观察不同组的脱细胞方法效果。（一）HE表面和切片染色；（二）Hoechst细胞核染色；（三）免疫组织化学抗IV型胶原、VI型胶原、VII型胶原、层粘连蛋白（Laminin）、主要组织相容性抗原-I（MHC-I）、MHC-II和波形蛋白（Vimentin）染色；（四）扫描电镜和透射电镜检查。二、利用水溶性碳二亚胺（EDC,0.05mmol/mgAAM）分别交联脱细胞羊膜5min，30min和6h，得到不同交联程度的脱细胞羊膜；然后通过人表皮细胞经浸提液培养和直接接触培养的方法观察交联脱细胞羊膜的细胞毒性效应；最后将交联脱细胞羊膜皮下埋置于具有正常免疫功能的大鼠以观察其体内组织相容性。通过以下的方法检测交联脱细胞羊膜的机械强度和生物稳定性，以及细胞毒性效应和体内外生物组织相容性。（一）茚三酮法测定脱细胞羊膜的交联程度--交联指数；（二）单轴张力计测定脱细胞羊膜交联前后的最大张力和最大拉伸长度；（三）交联前后脱细胞羊膜的体外胶原酶降解速度测定；（四）CCK-8法和Live/Dead染色观察表皮细胞的增殖活性；（五）HE染色，Masson三色三胶原染色观察交联脱细胞羊膜体内埋置后的免疫炎症反应和支架降解情况；（六）免疫组织化学抗CD31, CD11b, CD4, CD8, CD68和Vimenin染色观察支架埋置后浸润细胞的类型，评估炎症反应。三、以交联脱细胞羊膜为载体体外扩增人表皮细胞，并与常规细胞培养皿相比较；构建表皮细胞-交联脱细胞羊膜的复合型皮肤替代物，并将其用于移植修复裸鼠全层皮肤缺损创面，单纯表皮膜片移植组和空白组作为对照。通过以下的方法检测表皮细胞的体外扩增速度和复合皮肤替代物移植后的创面愈合及真皮重建情况。（一）CCK-8法和Hoechst细胞核染色观察两组表皮细胞的扩增速度；（二）免疫组织化学抗P63染色观察表皮细胞的增殖活性；（三）HE染色观察复合皮肤替代物的形态；（四）观察移植创面愈合过程的大体形态；（五）HE染色和免疫组织化学抗laminin染色观察愈合创面的组织结构。实验结果：一、反复液氮冻融+DNase消化处理可以完全清除羊膜的上皮细胞层和间质细胞，而Dispase II消化+细胞刷法处理后的脱细胞羊膜仍有少量上皮细胞残留，间质细胞则基本不能清除。两种方法处理后的脱细胞羊膜DNA总量均显著降低，组间无明显差异；但反复液氮冻融+DNase消化处理的脱细胞羊膜保留的总蛋白量明显高于DispaseII消化+细胞刷处理的脱细胞羊膜（77.2±4.72%VS48.5±4.16%，p＜0.05）。反复液氮冻融+DNase消化处理的脱细胞羊膜的基底膜成分（层粘连蛋白、IV型胶原、VI型胶原和VII型胶原）基本保留，且对基质纤维结构无明显影响；而Dispase II消化+细胞刷法处理的脱细胞羊膜基底膜被严重破坏，蛋白成分丢失明显，且基质胶原纤维变得疏松，排列紊乱。正常羊膜中的表皮细胞和间质细胞均表达MHC-I抗原，不表达MHC-II抗原，其中间质细胞还表达Vimentin，而在反复液氮冻融+DNase消化处理的脱细胞羊膜中几乎未检测到MHC-I抗原和Vimentin的存在。二、脱细胞羊膜的形态极其柔软和光滑，经过EDC交联5min后依然平整，并具有一定的硬度，而交联30min和6h后逐渐变得卷曲和僵硬。随交联时间的增加，脱细胞羊膜的机械强度显著增强，体外胶原酶完全降解时间也明显延长，并与交联程度存在正相关关系。不同交联程度的脱细胞羊膜的浸提液培养7天对人表皮细胞的增殖活性无不良影响。交联5min的脱细胞羊膜直接负载人表皮细胞培养7天后细胞增殖活性良好，与未交联脱细胞羊膜组无明显统计学差异（p＞0.05）；而交联30min和6h的脱细胞羊膜组的细胞增殖活性明显受损，两组细胞在培养7天后的凋亡或死亡细胞比例分别为1.27±0.30%和10.02±1.43%，明显高于未交联和交联5min脱细胞羊膜组的0.42±0.14%和0.44±0.18%（p均＜0.05）。皮下埋置后的组织相容性检测结果显示交联5min的脱细胞羊膜在体内的完全降解时间约为4个月，降解后形成一层厚的皮下组织，胶原沉积和血管化良好，并无明显的急慢性炎症反应发生。三、表皮细胞在交联5min的脱细胞羊膜表面种植培养7和14天后的细胞相对增殖率分别为367±33%和631±43%，显著高于同一时间点的常规培养皿组（294±30%和503±41%, p均0.05)。培养14天后表皮细胞在交联脱细胞羊膜表面形成一个2-3层的复层表皮结构。免疫组化染色结果显示交联脱细胞羊膜上P63阳性表皮细胞的比例明显高于对照的常规培养皿组（54.32±4.27%VS33.32±3.18%, p0.05）。将培养形成的表皮细胞-交联脱细胞羊膜复合皮肤替代物移植于裸鼠全层皮肤缺损创面后，表皮细胞成活良好并完全封闭创面，形成类似正常皮肤的表皮。复合皮肤替代物移植组的创面修复效果要明显优于单纯表皮膜片组和空白对照组，创面收缩明显改善。创面组织学观察结果提示复合皮肤替代物移植后创面的真皮结构重建良好，新生基底膜厚而完整。实验结论：一、反复液氮冻融+DNase消化的脱细胞方法能有效去除羊膜的上皮细胞和间质细胞，优于传统的Dispase II消化+细胞刷处理方法；更重要的是该方法能有效保留羊膜的基质成分，尤其是基底膜结构，且脱细胞后羊膜的免疫原性极低。二、EDC（0.05mmol/mg AAM）交联5min的脱细胞羊膜，不仅具备改善了的机械强度和抗酶降解能力，而且无明显细胞毒性效应，能有效负载表皮细胞的粘附和增殖。皮下埋置实验结果显示该交联脱细胞羊膜具有良好的体内生物组织相容性和降解特性。三、交联脱细胞羊膜作为真皮替代物在体外可以负载并促进表皮细胞的快速扩增，有利于维持细胞的增殖能力。用交联脱细胞羊膜构建复合型皮肤替代物并移植可以促进全层皮肤缺损创面的真皮结构和基底膜重建，改善创面愈合质量，因而是理想的真皮支架材料。
[Abstract]:Background: the study of dermis substitutes is always the key and difficult point of skin tissue engineering. It is also the basis for the development of compound skin substitutes. As a scaffold structure in the process of wound repair, dermis substitutes can not only promote the proliferation, migration and differentiation of epidermal cells, but also regulate the formation of the basal membrane, and can guide the fibroblasts. A series of dermal substitutes have been successfully developed to date, including natural sources such as allogenic or xenogenic dermis, collagen and hyaluronic acid, as well as artificial synthetic materials, including some synthetic materials, including some synthetic materials, including some artificial synthetic materials. Other new dermal substitute materials are also being developed. In these dermis substitutes, Integra and Tegaderm have been commercialized and successfully applied to clinical treatment. Most dermis substitutes can well simulate the structural characteristics of normal dermis. In addition to acellular true skin, there is a common lack of basement membrane structure. And even if it is acellular dermal, in order to remove the fibroblasts and vascular endothelial cells in the epidermis and dermis, a strong cell scavenger is needed, and the latter will cause serious damage to the basal membrane components of the dermis.
The basilar membrane is an important component of the normal skin, located at the junction of the epidermis and the dermis, which plays an important role in maintaining the normal function of the skin. The epidermal stem cells in the normal skin are mainly located in the basal layer and are closely connected to the surface of the basement membrane through the half bridge structure; the latter can regulate the proliferation, migration and migration of epidermal stem cells. Differentiation. The epidermal stem cells isolated and cultured in vitro have lost their ability to proliferate and differentiate into keratinocytes due to the loss of the support and regulation of the basement membrane. A large number of studies have shown that natural or synthetic basement membrane components such as IV collagen and fibrous eggs are added to the surface of artificial dermal scaffolds. Bai et al can effectively promote the proliferation of epidermal cells and improve the morphology and function of epidermal structures.
Human amniotic matrix (amniotic membrane, AM) may be a good substitute material for dermis. It is derived from the most inner layer of the placenta and can be obtained during delivery. It consists mainly of three parts: a single cuboid epidermal cell, a thick basement membrane rich in cell growth factor, and a loose reticular fiber matrix scattered by fibroblasts. Amniotic membrane matrix contains type I collagen, type IV collagen, type VII collagen, elastin and glycosaminoglycan, similar to human dermal. The basement membrane of amniotic membrane is mainly composed of laminin, IV and VII collagen, similar to the basal membrane of the skin and cornea, the thickest basement membrane in the human body. The amniotic membrane also promotes epithelialization and inhibits Cicatricial hyperplasia, anti inflammation, anti angiogenesis, antibacterial and antiviral properties, and extremely low immunogenicity, are widely used as surgical materials and wound cover, especially in eye corneal reconstruction. In recent years, more researchers have used amniotic membrane as angular membrane stem cells, mesenchymal stem cells, and other somatic cells. An amniotic membrane can be used to repair a variety of tissue defects. The amniotic membrane can not only retain the inactivated epidermal cells but also completely remove the epidermal cells. The complete amniotic membrane contains a large number of cell growth factors, which are beneficial to the maintenance of stem cell characteristics in the process of cell cultivation; and the acellular amniotic membrane (accelullar amniotic membrane, AAM) is borrowed. The good matrix and basement membrane components can promote the proliferation, migration and differentiation of cells, and the rate of amplification in the amniotic membrane of partial somatic cells is faster than that of the complete amniotic membrane.
In this study, we use the method of repeated freezing and DNase digestion to digest the fresh amniotic membrane, obtain the amniotic membrane with the intact basement membrane structure, and then use the water-soluble carbon two imide to treat the acellular amniotic membrane properly, in order to improve the mechanical strength and biological stability of the acellular amniotic membrane. The acellular amniotic membrane has good maneuverability and anti enzyme degradation ability. As culture matrix, it can promote the proliferation of epidermal cells in vitro, and maintain good biocompatibility. The epidermal cells are cultured on the surface of the crosslinked amniotic membrane to form a compound epidermic skin substitute and use it to repair nude mice. It is found that the compound skin substitutes can obviously promote the healing speed of the wound, improve the thickness and function of the newborn epidermis, promote the reconstruction of the dermal structure, and reduce the wound contraction.
Experimental methods:
First, the fresh amniotic membrane was divided into the following groups according to the different methods of cell removal: Dispase II digestion + cell brush treatment group, Freeze-thaw+DNase digestion group and complete amniotic membrane control group. The effect of cell removal methods in different groups was observed by the following methods.
(1) HE surface and slice staining;
(two) Hoechst cell nuclear staining;
(three) immuno histochemical anti IV collagen, type VI collagen, type VII collagen, laminin (Laminin), major histocompatibility antigen -I (MHC-I), MHC-II and vimentin (Vimentin) staining;
(four) scanning electron microscope and transmission electron microscope examination.
Two, using water soluble carbon two imide (EDC, 0.05mmol/mgAAM) to cross cross the acellular amniotic membrane 5min, 30min and 6h to obtain the acellular amniotic membrane with different crosslinking degree, and then observe the cytotoxic effect of the cross linked acellular amniotic membrane through the culture and direct contact culture of human epidermal cells. Finally, the cross-linked acellular amniotic membrane will be cross linked. The following methods were used to detect the mechanical strength and biological stability of the crosslinked amniotic membrane, as well as the cytotoxicity and the biocompatibility of the biological tissue in vivo and in vivo.
(1) the crosslinking degree of acellular amniotic membrane was determined by the method of three - ketone.
(two) the maximum tension and maximum tensile length of acellular amniotic membrane before and after crosslinking were measured by uniaxial tension meter.
(three) the degradation rate of collagenase from acellular amniotic membrane before and after crosslinking was measured.
(four) CCK-8 and Live/Dead staining were used to observe the proliferative activity of epidermal cells.
(five) HE staining and Masson trichrome three collagen staining were used to observe the immune inflammatory reaction and stent degradation in the cross linked acellular amniotic membrane.
(six) immunohistochemical staining of CD31, CD11b, CD4, CD8, CD68 and Vimenin staining was used to observe the types of infiltrating cells after stent implantation, and to evaluate the inflammatory response.
Three, the cross linked acellular amniotic membrane was used as the carrier to amplify human epidermal cells in vitro, and compared with the conventional cell culture dish; the compound skin substitutes for the epidermal cells - cross-linked acellular amniotic membrane were constructed and used for transplantation to repair the full layer skin defect of nude mice. The simple epidermis graft group and the blank group were used as the control. Methods the in vitro expansion rate of epidermal cells and wound healing after composite skin substitute and dermal reconstruction were detected.
(1) CCK-8 and Hoechst nuclear staining were used to observe the amplification rate of epidermal cells in the two groups.
(two) immunohistochemistry was used to observe the proliferative activity of epidermal cells against P63 staining.
(three) HE staining was used to observe the morphology of compound skin substitute.
(four) to observe the gross morphology of the wound healing process.
(five) HE staining and immunohistochemical staining for laminin staining were used to observe the tissue structure of wound healing.
Experimental results:
First, repeated liquid nitrogen freezing and thawing +DNase digestion can completely remove the epithelial cells and interstitial cells of the amniotic membrane, while the Dispase II digestion + cell brush treatment still has a small amount of epithelial cells remaining in the acellular amniotic membrane, and the interstitial cells can not be removed. The total amount of DNA in the acellular amniotic membrane after treatment with the two methods is significantly reduced, and there is no clear between the groups. The total protein content of the acellular amniotic membrane retained by repeated liquid nitrogen freeze-thaw +DNase digestion was significantly higher than that of DispaseII digestible + cell brush treated acellular amniotic membrane (77.2 + 4.72%VS48.5 + 4.16%, P < 0.05). The base membrane components of the acellular amniotic membrane (laminin, IV collagen, VI collagen) treated by repeated liquid nitrogen freeze-thaw +DNase digestion And the type VII collagen was basically retained and had no obvious effect on the structure of the matrix fiber, while the Dispase II digestion + cell brush treatment of the acellular amniotic membrane was seriously damaged, the protein composition was lost, and the matrix collagen fiber became loose and disorganized. The epidermal cells and interstitial cells in the normal amniotic membrane all expressed MHC-I antigen, and did not express MHC-II antigen, in which stromal cells also expressed Vimentin, had almost no detection of MHC-I antigen and Vimentin in the acellular amniotic membrane treated with repeated liquid nitrogen freezing thawing +DNase.
Two, the morphology of the acellular amniotic membrane is very soft and smooth. After EDC cross-linked 5min, it is still smooth and has a certain hardness, and the crosslinking of 30min and 6h gradually becomes curly and rigid. With the increase of crosslinking time, the mechanical strength of the acellular amniotic membrane is significantly enhanced, and the total degradation time of collagenase in vitro is also prolonged, and the degree of cross-linking is stored with the degree of cross-linking. There was no adverse effect on the proliferation activity of human epidermal cells for 7 days in the positive correlation. The proliferation activity of human epidermal cells was not adversely affected by the culture of the extract of the acellular amniotic membrane with different crosslinking degrees. The proliferation activity of the cross linked 5min amniotic membrane was good for 7 days after the culture of human epidermal cells, and there was no significant difference between the uncross linked amniotic membrane group and the uncross linked amniotic membrane group (P > 0.05); and the cross-linked 30mi was cross linked with the amniotic membrane. The cell proliferation activity of the acellular amniotic membrane group of N and 6h was significantly damaged. The percentage of apoptotic or dead cells in the two groups was 1.27 + 0.30% and 10.02 + 1.43% respectively after 7 days of culture, which was significantly higher than 0.42 + 0.14% and 0.44 + 0.18% (P < 0.05) of the non crosslinked and cross-linked 5min decellular amniotic membrane group (P all < 0.05). The degrading time of the acellular amniotic membrane with cross-linked 5min was about 4 months in the body. After degradation, a thick layer of subcutaneous tissue was formed, and the collagen deposition and vascularization were good. There was no obvious acute and chronic inflammatory reaction.
Three, the relative proliferation rates of epidermal cells were 367 + 33% and 631 + 43% after 7 and 14 days after cultivation on the surface of the cross linked 5min amniotic membrane, which were significantly higher than those of the same time point (294 + 30% and 503 + 41%, P 0.05). The epidermal cells formed a layer of stratified epidermis on the surface of the cross-linked acellular amniotic membrane. Structure. The results of immunohistochemical staining showed that the proportion of P63 positive epidermal cells on the cross linked amniotic membrane was significantly higher than that of the conventional culture dish group (54.32 + 4.27%VS33.32 + 3.18%, P0.05). The wound healing was similar to that of the normal skin. The wound healing effect of the compound skin replacement group was obviously better than that of the simple skin patch group and the blank control group, and the wound contraction was obviously improved. The histological observation of the wound surface suggests that the dermal structure of the wound surface after the compound skin replacement is well rebuilt. The basement membrane is thick and complete.
Experimental conclusions:
First, the acellular method of repeated liquid nitrogen freeze-thaw +DNase digestion can effectively remove the epithelial cells and interstitial cells of amniotic membrane, which is better than the traditional Dispase II digestion + cell brush treatment method. More importantly, this method can effectively retain the matrix composition of amniotic membrane, especially the basement membrane structure, and the immunogenicity of amniotic membrane is extremely low.
Two, EDC (0.05mmol/mg AAM) cross linked 5min acellular amniotic membrane not only improved the mechanical strength and anti enzyme degradation ability, but also had no obvious cytotoxic effect, and could effectively load the adhesion and proliferation of epidermal cells. The results of subcutaneous embedding experiment showed that the cross linked acellular sheep membrane had good biocompatibility and degradation in vivo. Sex.
Three, cross linked acellular amniotic membrane as a dermal substitute can load and promote the rapid expansion of epidermal cells in vitro.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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