本文選題：羊膜 + 脫細胞��； 參考：《第二軍醫(yī)大學》2013年博士論文

【摘要】：研究背景：真皮替代物的研究一直是皮膚組織工程的重點和難點，也是復合型皮膚替代物發(fā)展的基礎。真皮替代物作為創(chuàng)面修復過程中的支架結構，不僅能促進表皮細胞的增殖、遷移和分化，調節(jié)基底膜的形成，而且能引導成纖維細胞和血管內皮細胞的浸潤增殖，沉積新的膠原和形成新的血管，從而實現(xiàn)真皮結構重建。迄今為止一系列的真皮替代物已經被成功研制出來，包括自然來源的材料，如異體或異種脫細胞真皮、膠原和透明質酸等，以及人工合成材料，，包括一些聚合體生物材料和其他納米材料等。另外的一些新型真皮替代物材料也正在被研制之中。在這些真皮替代物中，Integra和Tegaderm等已經被商品化并成功運用于臨床治療。大部分的真皮替代物都能很好地模擬正常真皮的組織結構特點，但是除了脫細胞真皮外，普遍都缺乏基底膜結構成分。而且即使是脫細胞真皮，為了去除表皮細胞層和真皮中的成纖維細胞和血管內皮細胞，一般需要很強的細胞清除劑處理，而后者會對真皮的基底膜成分產生嚴重的破壞。 基底膜成分是正常皮膚中重要的成分，位于是表皮和真皮連接處，對于維持皮膚的正常功能起到重要的作用。正常皮膚中的表皮干細胞主要位于基底層，通過半橋粒結構緊密連接在基底膜表面；后者可以調節(jié)表皮干細胞的增殖，遷移和分化。體外分離培養(yǎng)的表皮干細胞由于失去了基底膜的支撐和調節(jié)作用，逐漸喪失了增殖的能力，分化成角質形成細胞。大量的研究表明：在人工合成的真皮支架表面添加自然來源或者人工合成的基底膜成分如IV型膠原和纖維彈性蛋白等，可以有效地促進表皮細胞的增殖，改善所形成的表皮結構的形態(tài)和功能。 人羊膜基質(amniotic membrane, AM)可能是一種良好的真皮替代物材料。它來源于胎盤的最內層，可在分娩時獲得，主要由三部分結構組成：單層立方狀表皮細胞，富含細胞生長因子的厚基底膜，以及成纖維細胞散在其中的疏松網狀纖維基質。羊膜基質含有I型膠原、IV型膠原、VII型膠原、彈性蛋白和糖胺聚糖等成分，類似于人體的真皮。羊膜的基底膜主要成分為層粘連蛋白、IV型和VII型膠原，與皮膚和角膜的基底膜成分相似，是人體內最厚的基底膜。羊膜還具有促進上皮化、抑制瘢痕增生、抗炎癥、抗血管生成、抗菌和抗病毒等生物特性，且免疫原性極低，因而被廣泛用作外科手術材料和創(chuàng)面覆蓋物，尤其是用于眼科角膜重建治療。近年來更有研究者更是將羊膜作為角膜干細胞、間充質干細胞、以及其他體細胞擴增和移植的載體，用于修復各種組織缺損。羊膜使用時既可以保留失活的表皮細胞，也可以完全去除表皮細胞。完整羊膜含有大量的細胞生長因子，有利于細胞培育過程中干細胞特性的維持；而脫細胞羊膜(accelullar amniotic membrane, AAM)借助于良好的基質和基底膜成分，能促進培育細胞的增殖、遷移和分化；部分體細胞在脫細胞羊膜上的擴增速度要明顯快于完整羊膜。 在本課題中，我們利用反復凍融和DNase消化的方法消化處理人新鮮羊膜，獲得保留完整基底膜結構的羊膜基質；隨后又利用水溶性碳二亞胺對脫細胞羊膜進行適當?shù)慕宦?lián)處理，以改善脫細胞羊膜的機械強度和生物穩(wěn)定性。得到的交聯(lián)脫細胞羊膜具有良好的可操作性和抗酶降解能力，作為培養(yǎng)基質可以促進表皮細胞的體外增殖速度，而且保持了良好的生物組織相容性。將表皮細胞培養(yǎng)在交聯(lián)脫細胞羊膜表面形成復合型表皮-真皮替代物，并將其用于移植修復裸鼠全層皮膚缺損，結果發(fā)現(xiàn)這種復合皮膚替代物可以明顯促進創(chuàng)面愈合速度，改善新生表皮的厚度和功能，促進真皮結構重建，減輕創(chuàng)面收縮。 實驗方法： 一、將新鮮羊膜根據(jù)脫細胞方法的不同分為以下幾組：Dispase II消化+細胞刷處理組、Freeze-thaw+DNase消化組和完整羊膜對照組。通過下面的幾種檢測方法觀察不同組的脫細胞方法效果。 （一）HE表面和切片染色； （二）Hoechst細胞核染色； （三）免疫組織化學抗IV型膠原、VI型膠原、VII型膠原、層粘連蛋白（Laminin）、主要組織相容性抗原-I（MHC-I）、MHC-II和波形蛋白（Vimentin）染色； （四）掃描電鏡和透射電鏡檢查。 二、利用水溶性碳二亞胺（EDC,0.05mmol/mgAAM）分別交聯(lián)脫細胞羊膜5min，30min和6h，得到不同交聯(lián)程度的脫細胞羊膜；然后通過人表皮細胞經浸提液培養(yǎng)和直接接觸培養(yǎng)的方法觀察交聯(lián)脫細胞羊膜的細胞毒性效應；最后將交聯(lián)脫細胞羊膜皮下埋置于具有正常免疫功能的大鼠以觀察其體內組織相容性。通過以下的方法檢測交聯(lián)脫細胞羊膜的機械強度和生物穩(wěn)定性，以及細胞毒性效應和體內外生物組織相容性。 （一）茚三酮法測定脫細胞羊膜的交聯(lián)程度--交聯(lián)指數(shù)； （二）單軸張力計測定脫細胞羊膜交聯(lián)前后的最大張力和最大拉伸長度； （三）交聯(lián)前后脫細胞羊膜的體外膠原酶降解速度測定； （四）CCK-8法和Live/Dead染色觀察表皮細胞的增殖活性； （五）HE染色，Masson三色三膠原染色觀察交聯(lián)脫細胞羊膜體內埋置后的免疫炎癥反應和支架降解情況； （六）免疫組織化學抗CD31, CD11b, CD4, CD8, CD68和Vimenin染色觀察支架埋置后浸潤細胞的類型，評估炎癥反應。 三、以交聯(lián)脫細胞羊膜為載體體外擴增人表皮細胞，并與常規(guī)細胞培養(yǎng)皿相比較；構建表皮細胞-交聯(lián)脫細胞羊膜的復合型皮膚替代物，并將其用于移植修復裸鼠全層皮膚缺損創(chuàng)面，單純表皮膜片移植組和空白組作為對照。通過以下的方法檢測表皮細胞的體外擴增速度和復合皮膚替代物移植后的創(chuàng)面愈合及真皮重建情況。 （一）CCK-8法和Hoechst細胞核染色觀察兩組表皮細胞的擴增速度； （二）免疫組織化學抗P63染色觀察表皮細胞的增殖活性； （三）HE染色觀察復合皮膚替代物的形態(tài)； （四）觀察移植創(chuàng)面愈合過程的大體形態(tài)； （五）HE染色和免疫組織化學抗laminin染色觀察愈合創(chuàng)面的組織結構。 實驗結果： 一、反復液氮凍融+DNase消化處理可以完全清除羊膜的上皮細胞層和間質細胞，而Dispase II消化+細胞刷法處理后的脫細胞羊膜仍有少量上皮細胞殘留，間質細胞則基本不能清除。兩種方法處理后的脫細胞羊膜DNA總量均顯著降低，組間無明顯差異；但反復液氮凍融+DNase消化處理的脫細胞羊膜保留的總蛋白量明顯高于DispaseII消化+細胞刷處理的脫細胞羊膜（77.2±4.72%VS48.5±4.16%，p＜0.05）。反復液氮凍融+DNase消化處理的脫細胞羊膜的基底膜成分（層粘連蛋白、IV型膠原、VI型膠原和VII型膠原）基本保留，且對基質纖維結構無明顯影響；而Dispase II消化+細胞刷法處理的脫細胞羊膜基底膜被嚴重破壞，蛋白成分丟失明顯，且基質膠原纖維變得疏松，排列紊亂。正常羊膜中的表皮細胞和間質細胞均表達MHC-I抗原，不表達MHC-II抗原，其中間質細胞還表達Vimentin，而在反復液氮凍融+DNase消化處理的脫細胞羊膜中幾乎未檢測到MHC-I抗原和Vimentin的存在。 二、脫細胞羊膜的形態(tài)極其柔軟和光滑，經過EDC交聯(lián)5min后依然平整，并具有一定的硬度，而交聯(lián)30min和6h后逐漸變得卷曲和僵硬。隨交聯(lián)時間的增加，脫細胞羊膜的機械強度顯著增強，體外膠原酶完全降解時間也明顯延長，并與交聯(lián)程度存在正相關關系。不同交聯(lián)程度的脫細胞羊膜的浸提液培養(yǎng)7天對人表皮細胞的增殖活性無不良影響。交聯(lián)5min的脫細胞羊膜直接負載人表皮細胞培養(yǎng)7天后細胞增殖活性良好，與未交聯(lián)脫細胞羊膜組無明顯統(tǒng)計學差異（p＞0.05）；而交聯(lián)30min和6h的脫細胞羊膜組的細胞增殖活性明顯受損，兩組細胞在培養(yǎng)7天后的凋亡或死亡細胞比例分別為1.27±0.30%和10.02±1.43%，明顯高于未交聯(lián)和交聯(lián)5min脫細胞羊膜組的0.42±0.14%和0.44±0.18%（p均＜0.05）。皮下埋置后的組織相容性檢測結果顯示交聯(lián)5min的脫細胞羊膜在體內的完全降解時間約為4個月，降解后形成一層厚的皮下組織，膠原沉積和血管化良好，并無明顯的急慢性炎癥反應發(fā)生。 三、表皮細胞在交聯(lián)5min的脫細胞羊膜表面種植培養(yǎng)7和14天后的細胞相對增殖率分別為367±33%和631±43%，顯著高于同一時間點的常規(guī)培養(yǎng)皿組（294±30%和503±41%, p均0.05)。培養(yǎng)14天后表皮細胞在交聯(lián)脫細胞羊膜表面形成一個2-3層的復層表皮結構。免疫組化染色結果顯示交聯(lián)脫細胞羊膜上P63陽性表皮細胞的比例明顯高于對照的常規(guī)培養(yǎng)皿組（54.32±4.27%VS33.32±3.18%, p0.05）。將培養(yǎng)形成的表皮細胞-交聯(lián)脫細胞羊膜復合皮膚替代物移植于裸鼠全層皮膚缺損創(chuàng)面后，表皮細胞成活良好并完全封閉創(chuàng)面，形成類似正常皮膚的表皮。復合皮膚替代物移植組的創(chuàng)面修復效果要明顯優(yōu)于單純表皮膜片組和空白對照組，創(chuàng)面收縮明顯改善。創(chuàng)面組織學觀察結果提示復合皮膚替代物移植后創(chuàng)面的真皮結構重建良好，新生基底膜厚而完整。 實驗結論： 一、反復液氮凍融+DNase消化的脫細胞方法能有效去除羊膜的上皮細胞和間質細胞，優(yōu)于傳統(tǒng)的Dispase II消化+細胞刷處理方法；更重要的是該方法能有效保留羊膜的基質成分，尤其是基底膜結構，且脫細胞后羊膜的免疫原性極低。 二、EDC（0.05mmol/mg AAM）交聯(lián)5min的脫細胞羊膜，不僅具備改善了的機械強度和抗酶降解能力，而且無明顯細胞毒性效應，能有效負載表皮細胞的粘附和增殖。皮下埋置實驗結果顯示該交聯(lián)脫細胞羊膜具有良好的體內生物組織相容性和降解特性。 三、交聯(lián)脫細胞羊膜作為真皮替代物在體外可以負載并促進表皮細胞的快速擴增，有利于維持細胞的增殖能力。用交聯(lián)脫細胞羊膜構建復合型皮膚替代物并移植可以促進全層皮膚缺損創(chuàng)面的真皮結構和基底膜重建，改善創(chuàng)面愈合質量，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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R318.08

【參考文獻】

相關期刊論文 前1條

1 朱希山;曾麗芬;趙春華;;組織工程化人工皮膚研究的新進展[J];中國組織工程研究與臨床康復;2007年06期

本文編號：1873774