本文選題：微載體 + 羊膜　； 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究背景 表皮干細(xì)胞主要位于皮膚基底層和毛囊隆突部，由于細(xì)胞數(shù)量相對(duì)較少且增殖慢，因此如何快速擴(kuò)增表皮干細(xì)胞一直是皮膚組織工程研究中的熱點(diǎn)和難點(diǎn)。目前主要利用表皮干細(xì)胞對(duì)基底膜的顯著黏附特性進(jìn)行分離、純化，采用二維平面的培養(yǎng)方式進(jìn)行培養(yǎng)，同時(shí)為了保證干細(xì)胞的單一及清除分化增殖能力不強(qiáng)的細(xì)胞，傳代擴(kuò)增時(shí)需要首先將基底膜的主要成份之一Ⅳ型膠原平鋪于培養(yǎng)皿，但仍然難以避免多次傳代培養(yǎng)后細(xì)胞克隆形成率明顯下降、細(xì)胞逐漸分化、增殖活性降低等缺點(diǎn)，而且由于表皮干細(xì)胞固有的慢增殖特性，傳統(tǒng)的二維培養(yǎng)方式難以在較短時(shí)間內(nèi)擴(kuò)增足量的細(xì)胞。 微載體培養(yǎng)是一種大規(guī)模擴(kuò)增貼壁依賴型細(xì)胞的技術(shù)，提供了細(xì)胞單層培養(yǎng)時(shí)不具備的三維立體空間，模擬了細(xì)胞在體內(nèi)的生長(zhǎng)環(huán)境，不僅可以大量擴(kuò)增細(xì)胞，而且有利于維持細(xì)胞表型、防止分化，現(xiàn)已廣泛地應(yīng)用于生物制藥領(lǐng)域。最近，有研究者采用微載體培養(yǎng)技術(shù)擴(kuò)增干細(xì)胞取得良好效果，如以Cultispher-S、Cytodex、Solohill、Matrigel-coated cellulose等擴(kuò)增間充質(zhì)干細(xì)胞及胚胎干細(xì)胞，不僅在短時(shí)間內(nèi)可大量擴(kuò)增，而且維持其多向分化潛能。因此我們?cè)O(shè)想，采用微載體技術(shù)為表皮干細(xì)胞提供更適于其生長(zhǎng)、增殖的三維立體空間。然而，目前研制的微載體多以生物材料人工合成，盡管有研究者采用表面改良或修飾等仿生技術(shù)處理，但缺乏基底膜結(jié)構(gòu)，仍難以模擬表皮干細(xì)胞生長(zhǎng)的體內(nèi)niche微環(huán)境，不利于表皮干細(xì)胞的粘附、增殖。 研究表明人羊膜是理想的細(xì)胞培養(yǎng)擴(kuò)增載體，具有天然的人體最厚的基底膜，其基本結(jié)構(gòu)與皮膚、角膜基底膜相似，此外基質(zhì)中含有豐富的生長(zhǎng)因子，如NGF、HGF、KGF、bFGF、TGF-β1、EGF等，可以在體外模擬干細(xì)胞生長(zhǎng)的niche微環(huán)境，因此羊膜被用于角膜緣干細(xì)胞、間充質(zhì)干細(xì)胞的擴(kuò)增和移植。 本研究利用羊膜固有的生物學(xué)特性，制備具有完整基底膜結(jié)構(gòu)和生物學(xué)活性的三維立體微粒羊膜，以其作為一種天然的新型微載體，結(jié)合旋轉(zhuǎn)培養(yǎng)系統(tǒng)進(jìn)行表皮干細(xì)胞的體外培養(yǎng)與擴(kuò)增，評(píng)價(jià)其培養(yǎng)、擴(kuò)增表皮干細(xì)胞的效果及維持干細(xì)胞特性的作用。在此基礎(chǔ)上構(gòu)建含表皮干細(xì)胞的皮膚替代物，并評(píng)估其作為皮膚替代物修復(fù)全層皮膚缺損的可行性。 研究方法 1．羊膜微載體的制備及特征檢測(cè) 1.1羊膜獲取及微載體制備 經(jīng)醫(yī)院倫理委員會(huì)批準(zhǔn)，產(chǎn)婦知情同意，選擇肝炎病毒抗體、梅毒抗體及HIV均為陰性的剖腹產(chǎn)婦的胎盤組織，鈍性分離絨毛膜，剝離羊膜。 采用反復(fù)凍融結(jié)合DNA酶消化法去除羊膜細(xì)胞，片狀羊膜經(jīng)冷凍裂解技術(shù)均質(zhì)化制備微粒，然后在真空密閉條件下冷凍干燥，通過(guò)金屬篩網(wǎng)篩選過(guò)濾，獲得300-600μm的微粒羊膜。 1.2羊膜微載體物理特征及組織結(jié)構(gòu)檢測(cè) 采用掃描電鏡觀察mAM的形狀及大小，石蠟包埋切片HE染色及表面HE染色觀察羊膜細(xì)胞的去除情況及組織結(jié)構(gòu)，免疫組化染色和透射電鏡觀察mAM保留的基底膜結(jié)構(gòu)。 1.3生長(zhǎng)因子及DNA含量分析 Western blotting、Elisa測(cè)定mAM基質(zhì)中的生長(zhǎng)因子含量：EGF、bFGF、HGF、TGF-β1、KGF，NGF。DNA試劑盒測(cè)定DNA含量，Hoechst染色觀察mAM表面殘存DNA情況。 1.4mAM組織相容性檢測(cè) 通過(guò)mAM浸提液實(shí)驗(yàn)觀察mAM基質(zhì)對(duì)細(xì)胞的毒性，大鼠皮下移植實(shí)驗(yàn)評(píng)價(jià)mAM的組織相容性，Masson三色染色檢測(cè)羊膜膠原基質(zhì)降解過(guò)程。 2．mAM培養(yǎng)、擴(kuò)增表皮干細(xì)胞并構(gòu)建皮膚替代物 2.1mAM培養(yǎng)、擴(kuò)增表皮干細(xì)胞 分離培養(yǎng)表皮干細(xì)胞，以mAM作為微載體，結(jié)合旋轉(zhuǎn)培養(yǎng)系統(tǒng)培養(yǎng)擴(kuò)增表皮干細(xì)胞，定期取材標(biāo)本以hoechst33342熒光標(biāo)記，熒光顯微鏡下觀察表皮干細(xì)胞黏附增殖情況。 2.2表皮干細(xì)胞增殖活性及特性檢測(cè) 以常規(guī)二維培養(yǎng)作為對(duì)照，采用CCK-8測(cè)定微載體培養(yǎng)、擴(kuò)增表皮干細(xì)胞的增殖活性，，免疫組化檢測(cè)表皮干細(xì)胞的表面特征標(biāo)記：β1整合素、CK19、P63及CK10的表達(dá)情況。 2.3構(gòu)建并擴(kuò)增含表皮干細(xì)胞的皮膚替代物 以mAM作為真皮支架，在三維旋轉(zhuǎn)培養(yǎng)體系下，種植表皮干細(xì)胞，構(gòu)建含表皮干細(xì)胞的皮膚替代物，借助“球碰球”傳接技術(shù)，通過(guò)加入新的羊膜微載體擴(kuò)增皮膚替代物。行組織切片HE染色及電鏡掃描觀察含表皮干細(xì)胞皮膚替代物的形態(tài)特征。 2.4皮膚替代物移植全層皮膚缺損創(chuàng)面 以裸鼠全層皮膚缺損創(chuàng)面為動(dòng)物移植模型，將構(gòu)建好的含表皮干細(xì)胞的皮膚替代物移植創(chuàng)面，觀察其修復(fù)全層皮膚缺損創(chuàng)面的效果，定期測(cè)定創(chuàng)面愈合率，并行組織學(xué)檢測(cè)。 研究結(jié)果 1.mAM物理特性及DNA含量 通過(guò)物理反復(fù)凍融3次，輔助DNase酶消化可完全去除羊膜中的細(xì)胞及核酸，利用冷凍裂解技術(shù)可制備mAM，其外觀白色、半透明狀，呈六面體，形狀不規(guī)則，大小在300-600μm之間，厚度為80-250μm。mAM基質(zhì)中核酸基本去除干凈，去除率達(dá)85±4.15%。 2.mAM組織結(jié)構(gòu)及生長(zhǎng)因子檢測(cè) mAM基底膜結(jié)構(gòu)保存完整，羊膜上皮細(xì)胞、成纖維細(xì)胞等去除完全，膠原組織連續(xù)、排列規(guī)則，可見(jiàn)層粘連蛋白、Ⅳ型膠原蛋白連續(xù)分布，貫穿于整個(gè)基底膜，同時(shí)基質(zhì)中保留了多種生長(zhǎng)因子：NGF、HGF、KGF、bFGF、TGF-β1、EGF。 3.mAM浸提液毒性及組織相容性評(píng)價(jià) mAM浸提液細(xì)胞培養(yǎng)發(fā)現(xiàn)不含任何毒性，而且以其培養(yǎng)細(xì)胞增殖速度明顯高于對(duì)照組。大鼠皮下移植發(fā)現(xiàn)mAM組織相容性良好，移植后未見(jiàn)明顯急性炎癥或排斥反應(yīng)，14d時(shí)顯示許多微血管形成，30d時(shí)mAM開(kāi)始降解，90d時(shí)基本降解完全，與周圍組織融合，膠原纖維排列整齊。 4.mAM培養(yǎng)、擴(kuò)增表皮干細(xì)胞 表皮干細(xì)胞種植后半小時(shí)即可見(jiàn)粘附于mAM表面，呈立體三維生長(zhǎng)。培養(yǎng)第3天開(kāi)始mAM培養(yǎng)擴(kuò)增細(xì)胞相對(duì)增殖活性即明顯高于培養(yǎng)皿，培養(yǎng)7、14天時(shí)，相對(duì)增殖活性分別為326±28%、535±47%，遠(yuǎn)高于常規(guī)的培養(yǎng)皿培養(yǎng)方法（232±21%，307±32%，P＜0.05）。同時(shí)干細(xì)胞特征檢測(cè)可見(jiàn)表皮干細(xì)胞高表達(dá)β1整合素、K19及P63。 5.構(gòu)建并擴(kuò)增含表皮干細(xì)胞的皮膚替代物 以mAM為真皮支架，表皮干細(xì)胞為種子細(xì)胞可成功構(gòu)建含表皮種子細(xì)胞的活性皮膚替代物，培養(yǎng)14d表皮干細(xì)胞已形成2-3層。利用“球碰球”傳接技術(shù)，通過(guò)加入新的mAM即可實(shí)現(xiàn)快速擴(kuò)增含表皮干細(xì)胞的皮膚替代物。 6.皮膚替代物移植修復(fù)全層皮膚缺損創(chuàng)面 移植后2周，可見(jiàn)ESC-mAM組新生表皮形成，而對(duì)照組可見(jiàn)殘存創(chuàng)面，創(chuàng)面收縮明顯；移植后4周可見(jiàn)ESC-mAM、mAM組愈合表皮下微粒充填真皮基質(zhì)，ESC-mAM組伴有表皮乳突樣結(jié)構(gòu)形成，類似正常皮膚，而對(duì)照組表皮細(xì)胞基底處平整未見(jiàn)乳突樣結(jié)構(gòu)形成。 研究結(jié)論 1、反復(fù)凍融輔助DNA酶消化法可有效去除羊膜細(xì)胞成份，結(jié)合冷凍裂解可以制備微粒羊膜，其不僅具備一般微載體的特性，而且具有完整的基底膜結(jié)構(gòu)，同時(shí)基質(zhì)中保存了多種生物活性物質(zhì)。 2、微粒羊膜作為一種新型的微載體，可以為表皮干細(xì)胞的體外培養(yǎng)、擴(kuò)增提供近似體內(nèi)生理的Niche微環(huán)境，可在迅速擴(kuò)增表皮干細(xì)胞的同時(shí)，維持干細(xì)胞增殖活性，防止其分化。 3、微粒羊膜具備良好的組織相容性，可以作為真皮支架快速構(gòu)建含表皮干細(xì)胞的皮膚替代物，并修復(fù)全層皮膚缺損創(chuàng)面，改善創(chuàng)面愈合質(zhì)量。即mAM可以同步完成表皮干細(xì)胞的大量擴(kuò)增與皮膚替代物的快速構(gòu)建。 4、通過(guò)加入新的羊膜微載體的方式即可以實(shí)現(xiàn)含表皮干細(xì)胞皮膚替代物的快速擴(kuò)增，縮短了體外培養(yǎng)時(shí)間，改善了皮膚替代物的傳統(tǒng)構(gòu)建模式，提高了皮膚替代物從實(shí)驗(yàn)室構(gòu)建到臨床應(yīng)用的效率，為促進(jìn)皮膚替代物向臨床轉(zhuǎn)化與應(yīng)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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