模拟干细胞Niche同步实现表皮干细胞扩增与皮肤替代物构建的实验研究

发布时间：2018-05-14 21:11

本文选题：微载体 + 羊膜　；参考：《第二军医大学》2012年博士论文

【摘要】：研究背景表皮干细胞主要位于皮肤基底层和毛囊隆突部，由于细胞数量相对较少且增殖慢，因此如何快速扩增表皮干细胞一直是皮肤组织工程研究中的热点和难点。目前主要利用表皮干细胞对基底膜的显著黏附特性进行分离、纯化，采用二维平面的培养方式进行培养，同时为了保证干细胞的单一及清除分化增殖能力不强的细胞，传代扩增时需要首先将基底膜的主要成份之一Ⅳ型胶原平铺于培养皿，但仍然难以避免多次传代培养后细胞克隆形成率明显下降、细胞逐渐分化、增殖活性降低等缺点，而且由于表皮干细胞固有的慢增殖特性，传统的二维培养方式难以在较短时间内扩增足量的细胞。微载体培养是一种大规模扩增贴壁依赖型细胞的技术，提供了细胞单层培养时不具备的三维立体空间，模拟了细胞在体内的生长环境，不仅可以大量扩增细胞，而且有利于维持细胞表型、防止分化，现已广泛地应用于生物制药领域。最近，有研究者采用微载体培养技术扩增干细胞取得良好效果，如以Cultispher-S、Cytodex、Solohill、Matrigel-coated cellulose等扩增间充质干细胞及胚胎干细胞，不仅在短时间内可大量扩增，而且维持其多向分化潜能。因此我们设想，采用微载体技术为表皮干细胞提供更适于其生长、增殖的三维立体空间。然而，目前研制的微载体多以生物材料人工合成，尽管有研究者采用表面改良或修饰等仿生技术处理，但缺乏基底膜结构，仍难以模拟表皮干细胞生长的体内niche微环境，不利于表皮干细胞的粘附、增殖。研究表明人羊膜是理想的细胞培养扩增载体，具有天然的人体最厚的基底膜，其基本结构与皮肤、角膜基底膜相似，此外基质中含有丰富的生长因子，如NGF、HGF、KGF、bFGF、TGF-β1、EGF等，可以在体外模拟干细胞生长的niche微环境，因此羊膜被用于角膜缘干细胞、间充质干细胞的扩增和移植。本研究利用羊膜固有的生物学特性，制备具有完整基底膜结构和生物学活性的三维立体微粒羊膜，以其作为一种天然的新型微载体，结合旋转培养系统进行表皮干细胞的体外培养与扩增，评价其培养、扩增表皮干细胞的效果及维持干细胞特性的作用。在此基础上构建含表皮干细胞的皮肤替代物，并评估其作为皮肤替代物修复全层皮肤缺损的可行性。研究方法 1．羊膜微载体的制备及特征检测 1.1羊膜获取及微载体制备经医院伦理委员会批准，产妇知情同意，选择肝炎病毒抗体、梅毒抗体及HIV均为阴性的剖腹产妇的胎盘组织，钝性分离绒毛膜，剥离羊膜。采用反复冻融结合DNA酶消化法去除羊膜细胞，片状羊膜经冷冻裂解技术均质化制备微粒，然后在真空密闭条件下冷冻干燥，通过金属筛网筛选过滤，获得300-600μm的微粒羊膜。 1.2羊膜微载体物理特征及组织结构检测采用扫描电镜观察mAM的形状及大小，石蜡包埋切片HE染色及表面HE染色观察羊膜细胞的去除情况及组织结构，免疫组化染色和透射电镜观察mAM保留的基底膜结构。 1.3生长因子及DNA含量分析 Western blotting、Elisa测定mAM基质中的生长因子含量：EGF、bFGF、HGF、TGF-β1、KGF，NGF。DNA试剂盒测定DNA含量，Hoechst染色观察mAM表面残存DNA情况。 1.4mAM组织相容性检测通过mAM浸提液实验观察mAM基质对细胞的毒性，大鼠皮下移植实验评价mAM的组织相容性，Masson三色染色检测羊膜胶原基质降解过程。 2．mAM培养、扩增表皮干细胞并构建皮肤替代物 2.1mAM培养、扩增表皮干细胞分离培养表皮干细胞，以mAM作为微载体，结合旋转培养系统培养扩增表皮干细胞，定期取材标本以hoechst33342荧光标记，荧光显微镜下观察表皮干细胞黏附增殖情况。 2.2表皮干细胞增殖活性及特性检测以常规二维培养作为对照，采用CCK-8测定微载体培养、扩增表皮干细胞的增殖活性，，免疫组化检测表皮干细胞的表面特征标记：β1整合素、CK19、P63及CK10的表达情况。 2.3构建并扩增含表皮干细胞的皮肤替代物以mAM作为真皮支架，在三维旋转培养体系下，种植表皮干细胞，构建含表皮干细胞的皮肤替代物，借助“球碰球”传接技术，通过加入新的羊膜微载体扩增皮肤替代物。行组织切片HE染色及电镜扫描观察含表皮干细胞皮肤替代物的形态特征。 2.4皮肤替代物移植全层皮肤缺损创面以裸鼠全层皮肤缺损创面为动物移植模型，将构建好的含表皮干细胞的皮肤替代物移植创面，观察其修复全层皮肤缺损创面的效果，定期测定创面愈合率，并行组织学检测。研究结果 1.mAM物理特性及DNA含量通过物理反复冻融3次，辅助DNase酶消化可完全去除羊膜中的细胞及核酸，利用冷冻裂解技术可制备mAM，其外观白色、半透明状，呈六面体，形状不规则，大小在300-600μm之间，厚度为80-250μm。mAM基质中核酸基本去除干净，去除率达85±4.15%。 2.mAM组织结构及生长因子检测 mAM基底膜结构保存完整，羊膜上皮细胞、成纤维细胞等去除完全，胶原组织连续、排列规则，可见层粘连蛋白、Ⅳ型胶原蛋白连续分布，贯穿于整个基底膜，同时基质中保留了多种生长因子：NGF、HGF、KGF、bFGF、TGF-β1、EGF。 3.mAM浸提液毒性及组织相容性评价 mAM浸提液细胞培养发现不含任何毒性，而且以其培养细胞增殖速度明显高于对照组。大鼠皮下移植发现mAM组织相容性良好，移植后未见明显急性炎症或排斥反应，14d时显示许多微血管形成，30d时mAM开始降解，90d时基本降解完全，与周围组织融合，胶原纤维排列整齐。 4.mAM培养、扩增表皮干细胞表皮干细胞种植后半小时即可见粘附于mAM表面，呈立体三维生长。培养第3天开始mAM培养扩增细胞相对增殖活性即明显高于培养皿，培养7、14天时，相对增殖活性分别为326±28%、535±47%，远高于常规的培养皿培养方法（232±21%，307±32%，P＜0.05）。同时干细胞特征检测可见表皮干细胞高表达β1整合素、K19及P63。 5.构建并扩增含表皮干细胞的皮肤替代物以mAM为真皮支架，表皮干细胞为种子细胞可成功构建含表皮种子细胞的活性皮肤替代物，培养14d表皮干细胞已形成2-3层。利用“球碰球”传接技术，通过加入新的mAM即可实现快速扩增含表皮干细胞的皮肤替代物。 6.皮肤替代物移植修复全层皮肤缺损创面移植后2周，可见ESC-mAM组新生表皮形成，而对照组可见残存创面，创面收缩明显；移植后4周可见ESC-mAM、mAM组愈合表皮下微粒充填真皮基质，ESC-mAM组伴有表皮乳突样结构形成，类似正常皮肤，而对照组表皮细胞基底处平整未见乳突样结构形成。研究结论 1、反复冻融辅助DNA酶消化法可有效去除羊膜细胞成份，结合冷冻裂解可以制备微粒羊膜，其不仅具备一般微载体的特性，而且具有完整的基底膜结构，同时基质中保存了多种生物活性物质。 2、微粒羊膜作为一种新型的微载体，可以为表皮干细胞的体外培养、扩增提供近似体内生理的Niche微环境，可在迅速扩增表皮干细胞的同时，维持干细胞增殖活性，防止其分化。 3、微粒羊膜具备良好的组织相容性，可以作为真皮支架快速构建含表皮干细胞的皮肤替代物，并修复全层皮肤缺损创面，改善创面愈合质量。即mAM可以同步完成表皮干细胞的大量扩增与皮肤替代物的快速构建。 4、通过加入新的羊膜微载体的方式即可以实现含表皮干细胞皮肤替代物的快速扩增，缩短了体外培养时间，改善了皮肤替代物的传统构建模式，提高了皮肤替代物从实验室构建到临床应用的效率，为促进皮肤替代物向临床转化与应用提供新方法和新思路。
[Abstract]:Research background
Epidermal stem cells are mainly located in the basal layer of the skin and the protuberance of the hair follicle. As the number of cells is relatively small and the proliferation is slow, the rapid expansion of epidermal stem cells has always been a hot and difficult point in the research of skin tissue engineering. At present, the main use of epidermal stem cells to separate, purify the apparent adhesion properties of the basement membrane, and use Er Weiping At the same time, in order to ensure the single cell and clear the cells with low differentiation and proliferation, it is necessary to spread the type IV collagen, one of the main components of the basement membrane, to the culture dish, but it is still difficult to avoid the decrease of the cell clone formation rate and the cell differentiation. Moreover, due to the slow growth characteristics of epidermal stem cells, the traditional two dimensional culture method is difficult to expand enough cells in a relatively short time.
Microcarrier culture is a technique for large-scale amplification of adherent cells. It provides a three-dimensional space that is not available in single cell culture. It simulates the growth environment of cells in the body. It can not only expand cells in large quantities but also maintain cell phenotype and prevent differentiation. It has been widely used in the field of biopharmaceuticals. Recently, Some researchers use microcarrier culture technology to amplify stem cells to achieve good results, such as Cultispher-S, Cytodex, Solohill, Matrigel-coated cellulose and other MSCs and embryonic stem cells, not only in a short time, but also to maintain their multidirectional differentiation potential. Therefore, we assume that microcarrier technology is used as the table. The skin stem cells provide a three-dimensional space which is more suitable for its growth and proliferation. However, the microcarriers currently developed are synthesized by biomaterials. Although the researchers have been treated with biomimetic techniques such as surface modification or modification, it is difficult to simulate the niche microenvironment in the body of epidermal stem cells, which is not conducive to the epidermal stem. Cell adhesion and proliferation.
The research shows that the human amniotic membrane is an ideal carrier for cell culture amplification. It has the thickest basement membrane of natural human body. Its basic structure is similar to the skin and corneal basement membrane. In addition, the matrix contains rich growth factors, such as NGF, HGF, KGF, bFGF, TGF- beta 1, EGF, etc., and can simulate the niche microenvironment of stem cell growth in vitro. So the amniotic membrane is used. The proliferation and transplantation of mesenchymal stem cells from limbal stem cells.
This study makes use of the inherent biological characteristics of amniotic membrane to prepare a three-dimensional microparticle amniotic membrane with complete basement membrane structure and biological activity. The amniotic membrane is used as a new type of natural microcarrier and combined with rotating culture system for the culture and expansion of epidermal stem cells in vitro, to evaluate its culture, to amplify the effect of epidermal stem cells and to maintain the dry fine. On the basis of this, we constructed a skin substitute containing epidermal stem cells and evaluated its feasibility as a skin substitute to repair full-thickness skin defects.
research method
Preparation and characterization of 1. amniotic membrane microcarriers
1.1 amniotic membrane acquisition and microcarrier preparation
With the approval of the hospital ethics committee, the maternal informed consent, the hepatitis virus antibody, the syphilis antibody and the HIV were all negative for the placental tissue of the cesarean section, the blunt separation of the chorionic membrane and the stripping of the amniotic membrane.
The amniotic cells were removed by repeated freezing and thawing combined with DNA enzyme digestion. The lamellar amniotic membrane was homogenized by freezing lysis technology to prepare microparticles. Then, the microparticles were frozen and dried under the vacuum closed condition. The 300-600 micron amniotic membrane was obtained by screening and filtration by metal screen.
1.2 detection of physical characteristics and tissue structure of amniotic microcarriers
The shape and size of mAM were observed by scanning electron microscopy. The removal and tissue structure of amniotic membrane cells were observed by HE staining and surface HE staining in paraffin embedded section. The structure of the basement membrane retained by mAM was observed by immunohistochemical staining and transmission electron microscopy.
Analysis of 1.3 growth factor and DNA content
Western blotting and Elisa were used to determine the growth factor content in mAM matrix: EGF, bFGF, HGF, TGF- beta 1, KGF and NGF.DNA kits for DNA content. Hoechst staining was used to observe the residual condition of the surface.
Detection of histocompatibility of 1.4mAM
The cytotoxicity of mAM matrix was observed by mAM extract test. The tissue compatibility of mAM was evaluated by subcutaneous transplantation and Masson trichromatic staining was used to detect the degradation process of amniotic membrane collagen matrix.
2.mAM cultured and expanded epidermal stem cells and constructed skin substitute.
2.1mAM culture and amplification of epidermal stem cells
Epidermal stem cells were isolated and cultured. MAM was used as a microcarrier, and the epidermal stem cells were cultured and expanded in combination with rotating culture system. The samples were regularly labeled with hoechst33342 fluorescence, and the adhesion and proliferation of epidermal stem cells were observed under the fluorescence microscope.
2.2 epidermal stem cell proliferation activity and characterization
Using conventional two-dimensional culture as control, CCK-8 microcarrier culture was used to determine the proliferation activity of epidermal stem cells, and the surface characteristics of epidermal stem cells were detected by immunohistochemistry: the expression of beta 1 integrin, CK19, P63 and CK10.
2.3 construction and expansion of skin substitute containing epidermal stem cells
Using mAM as a dermal scaffold, the epidermal stem cells were planted in the three-dimensional rotation culture system, the skin substitutes containing epidermal stem cells were constructed, and the skin substitutes were amplified by the "ball touch" transfer technique. The skin substitutes were amplified by adding a new amniotic membrane microcarrier. The morphological characteristics of the skin substitutes containing epidermal stem cells were observed by tissue section HE staining and electron microscopy. Sign.
2.4 skin substitutes for full-thickness skin defect transplantation
The whole layer skin defect wound of nude mice was used as the animal transplantation model. The skin substitutes containing epidermal stem cells were constructed and the effect of the repair of the full layer skin defect was observed. The healing rate of the wound was measured regularly and the histological examination was carried out.
Research results
The physical properties of 1.mAM and the content of DNA
The cells and nucleic acids in the amniotic membrane can be completely removed by DNase enzyme digestion and DNase enzyme digestion. MAM can be prepared by freezing lysis technology. Its appearance is white, translucent, hexahedron, irregular shape, between 300-600 and m, and the nucleic acid in the thickness of 80-250 mu m.mAM is basically removed and the removal rate is 85 + 4.15%.
Detection of 2.mAM tissue structure and growth factor
The structure of mAM basement membrane is intact. The amniotic epithelial cells and fibroblasts are completely removed, the collagen tissue is continuously arranged and arranged regularly. The laminin and type IV collagen are continuously distributed throughout the basement membrane, and many growth factors are retained in the matrix: NGF, HGF, KGF, bFGF, TGF- beta 1, EGF.
Evaluation of toxicity and histocompatibility of 3.mAM extract
The cell culture of mAM extract showed no toxicity, and the proliferation rate of the cultured cells was significantly higher than that of the control group. Subcutaneous transplantation found that mAM had good histocompatibility, no obvious acute inflammation or rejection after transplantation. Many microvessels were formed at 14d, mAM began to degrade at 30d, and the basic degradation of 90d was complete, with the surrounding group. The collagenous fibers are arranged in neat arrangement.
4.mAM culture and amplification of epidermal stem cells
The epidermal stem cells were adhered to the mAM surface half an hour after planting. The relative proliferation activity of mAM culture amplification cells at third days was obviously higher than that of culture dish. The relative proliferation activity was 326 + 28% and 535 + 47% respectively when cultured for 7,14 days, which was much higher than that of the conventional culture dish culture method (232 + 21%, 307 + 32%, P < 0.05). The stem cells showed high expression of beta 1 integrin, K19 and P63. in stem cells.
5. construction and expansion of skin substitute containing epidermal stem cells
Using mAM as a dermal scaffold, epidermal stem cells can be used as seed cells to successfully construct active skin substitutes with epidermal seed cells, and 2-3 layers of 14d epidermal stem cells have been formed. Using the "ball touch" transfer technique, the skin substitutes with epidermal stem cells can be rapidly amplified by adding a new mAM.
6. skin substitute transplantation to repair full-thickness skin defects
2 weeks after transplantation, the formation of the newborn epidermis in the ESC-mAM group was seen, while the residual wound was found in the control group, and the wound contracted obviously. ESC-mAM was seen in the 4 weeks after the transplantation. The subsurface particles of the mAM group were filled with the dermal matrix, and the ESC-mAM group was accompanied by the epidermoid structure of the epidermis, similar to the normal skin, while the basal part of the epidermis of the control group had no papilloid structure. Form.
research conclusion
1, repeated freezing and thawing assisted DNA digestion method can effectively remove the amniotic membrane cells and prepare the amniotic membrane by freezing lysis, which not only possesses the characteristics of general microcarriers, but also has a complete basement membrane structure, and many bioactive substances are preserved in the matrix.
2, as a new type of microcarrier, the particle amniotic membrane can be used to culture the epidermal stem cells in vitro, and provide an Niche microenvironment that approximates the body's physiology. It can expand the epidermal stem cells quickly and maintain the proliferation of stem cells and prevent their differentiation.
3, the microgranular amniotic membrane has good histocompatibility. It can be used as dermal scaffold to quickly construct skin substitutes containing epidermal stem cells, repair the full layer skin defect wound and improve the quality of wound healing. That is, mAM can synchronously complete the large expansion of epidermal stem cells and the rapid construction of skin substitutes.
4, the rapid expansion of skin substitutes containing epidermal stem cells can be achieved by adding new amniotic membrane microcarriers, shortening the culture time in vitro, improving the traditional construction pattern of skin substitutes, improving the efficiency of skin substitutes from laboratory construction to clinical application, and promoting the transformation and application of skin substitutes to clinical applications. Provide new methods and new ideas.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【参考文献】