本文選題：細(xì)胞外基質(zhì) + 真皮支架　； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：大面積燒傷、創(chuàng)傷患者救治的關(guān)鍵是如何及時(shí)有效的封閉創(chuàng)面。真皮成分的缺乏成為皮膚移植術(shù)后患者植皮區(qū)愈后外觀及功能的主要影響因素，因此真皮移植和/或真皮替代物的研究成為改善燒傷、創(chuàng)傷和整形患者的預(yù)后，提高其生活質(zhì)量的關(guān)鍵之一。由細(xì)胞外基質(zhì)(ECM)組成的生物支架材料已經(jīng)應(yīng)用于各種外科重建并作為一種再生醫(yī)學(xué)策略廣泛應(yīng)用于組織和器官替代。細(xì)胞外基質(zhì)成分生物支架材料的來源有多種動(dòng)物，由于細(xì)胞外基質(zhì)成分生物支架有特殊的機(jī)械性能、保留了生物活性可促進(jìn)組織再生，所以這些蛋白為基礎(chǔ)的材料與人工合成材料在外科和再生醫(yī)學(xué)的應(yīng)用中有許多天然的優(yōu)勢(shì)。由于異體真皮的來源及其有限且價(jià)格昂貴，因此有必要選擇一種與人真皮相對(duì)接近的異種真皮來源。在本課題組前期工作的基礎(chǔ)上，我們選擇了新生小牛皮膚，探討與人皮膚組織結(jié)構(gòu)、主要組成成分及其比例上的差異及生物力學(xué)性能特點(diǎn)，為臨床應(yīng)用牛真皮基質(zhì)提供實(shí)驗(yàn)依據(jù)。并且對(duì)制備新生小牛脫細(xì)胞真皮基質(zhì)的方法進(jìn)行了探索，利用滲透沖擊聯(lián)合超聲和去污劑處理的方法制備出了較為理想的異種脫細(xì)胞真皮基質(zhì)，對(duì)所制備的脫細(xì)胞真皮基質(zhì)的組織學(xué)和生物活性成分與天然真皮基質(zhì)進(jìn)行了對(duì)比。 目的 1.通過新生小牛與成人皮膚組織在形態(tài)學(xué)和生物力學(xué)性能方面的對(duì)比研究，尋找二者之間的異同點(diǎn)，為臨床應(yīng)用牛真皮基質(zhì)提供實(shí)驗(yàn)依據(jù)。 2.探索新生小牛脫細(xì)胞真皮基質(zhì)的制備方法，觀察處理前后真皮基質(zhì)的組織學(xué)和生物活性成分的變化。 方法 1.收集新生小牛和成人背部全層皮膚標(biāo)本，，采用大體觀察、HE染色、Masson三色染色、天狼猩紅染色、Gomori醛復(fù)紅染色、掃描電鏡和透射電鏡的方法，顯微照相后進(jìn)行形態(tài)學(xué)觀察和圖像分析軟件的測(cè)量并制備新生小牛和成人皮膚生物力學(xué)試件，采用力學(xué)生物材料試驗(yàn)機(jī)檢測(cè)皮膚的力學(xué)性能。 2.收集并制備新生小牛網(wǎng)狀層真皮，分別采用酶學(xué)法、滲透沖擊聯(lián)合去污劑法及滲透沖擊聯(lián)合超聲和去污劑法探索制備脫細(xì)胞真皮基質(zhì)，對(duì)制備出的脫細(xì)胞真皮基質(zhì)采用大體觀察和HE染色確定較為理想的制備方法； 3.對(duì)制備出的脫細(xì)胞真皮基質(zhì)進(jìn)行HE染色、DAPI染色和核酸電泳檢測(cè)細(xì)胞成分殘留情況；掃描電鏡(SEM)觀察膠原束結(jié)構(gòu)和細(xì)胞成分；PicoGreen法、DMMB法和BCA法對(duì)脫細(xì)胞前后真皮基質(zhì)的DNA、sGAG和蛋白質(zhì)含量進(jìn)行定量分析；ELISA法測(cè)定脫細(xì)胞前后真皮基質(zhì)內(nèi)TGF-β1、EGF、 bFGF和KGF含量。 結(jié)果 1.新生小牛與人的皮膚比較，真皮和表皮的厚度均明顯變薄，兩者的比值顯著減小；真皮內(nèi)Ⅰ型和Ⅲ型膠原纖維分別較人真皮增多和減少，但真皮膠原纖維束的間隙率[(41.72±1.81) vs (40.66±1.40)，P=0.467]和膠原纖維束粗細(xì)[(11.28±0.18) vs(10.88±0.66)， P=0.368]均沒有統(tǒng)計(jì)學(xué)差異； 2.掃描電鏡觀察可見新生牛真皮膠原纖維束與人真皮膠原纖維束均較纖細(xì)，排列疏松，且有一定的孔隙分布；透射電鏡顯示新生牛真皮膠原原纖維橫紋周期長(zhǎng)度較人真皮長(zhǎng)，但兩者的膠原原纖維直徑無顯著差異。 3.生物力學(xué)檢測(cè)示新生牛皮膚最大應(yīng)力(21.08±0.91) MPa和楊氏模量(82.12±1.23) MPa均要明顯高于人皮膚的最大應(yīng)力(12.76±1.60) MPa (P=0.001)和楊氏模量(48.63±5.50)MPa (P=0.001)，而最大應(yīng)變卻明顯低于人真皮[(0.51±0.002) mm vs (0.75±0.028) mm (P=0.001)]。 4.酶學(xué)法可以制備出脫細(xì)胞真皮，但對(duì)網(wǎng)狀層真皮要求較高，要求網(wǎng)狀層真皮厚薄均勻，并且對(duì)制作網(wǎng)狀層真皮的部位有一定要求；滲透沖擊聯(lián)合去污劑法所制備的脫細(xì)胞真皮HE染色觀察細(xì)胞核去除較徹底，但所使用去污劑對(duì)膠原有一定程度破壞，變現(xiàn)為膠原纖維束松散，部分呈團(tuán)塊狀；滲透沖擊聯(lián)合超聲去污劑處理制備的脫細(xì)胞真皮HE染色觀察細(xì)胞核去除較為徹底，膠原纖維束排列疏松，未見膠原纖維束結(jié)構(gòu)明顯破壞，DAPI染色未見真皮內(nèi)有DNA著色，掃描電鏡結(jié)果顯示膠原纖維束排列疏松，未發(fā)現(xiàn)細(xì)胞存留。 5.成分定量分析證實(shí)DNA含量[(2516.1±324.2) ng/mg vs (249.5±53.8) ng/mg,P=0.000)，下降了90.1%，sGAG含量[(5.92±0.50) μg/mg vs (2.48±0.24) μg/mg,P=0.000]，含量保留約41.9%。與脫細(xì)胞前比較，含水量有所下降[(72.83+1.43)%vs(69.68+1.78)%，P0.05]。細(xì)胞因子檢測(cè)TGF-β1含量[(478.8±196.1) pg/g vs (180.3±111.0) pg/g, P=0.009]和EGF含量[(10.52±2.78) pg/g vs未能檢測(cè)到EGF含量]均明顯減少；而bFGF含量[(788.6±333.8) pg/g vs (364.8±294.8) pg/g, P=0.424]下降了53.7%，KGF含量[(0.033±0.00019) pg/g vs (0.033±0.00022) pg/g, P=0.433]變化不顯著。 結(jié)論 1.新生牛皮膚雖然在表皮與真皮厚度、真皮膠原類型比例以及生物力學(xué)上與人皮膚有差異，但真皮的膠原纖維束三維結(jié)構(gòu)兩者之間存在較大相似性。 2.反復(fù)滲透沖擊聯(lián)合超聲和去污劑處理對(duì)新生牛真皮基質(zhì)明顯地清除了細(xì)胞及細(xì)胞碎片，較好地保留了真皮細(xì)胞外基質(zhì)的三維結(jié)構(gòu)和主要生物活性成分，為進(jìn)一步改進(jìn)脫細(xì)胞真皮的制備方法提供了重要實(shí)驗(yàn)依據(jù)。
[Abstract]:The key to the treatment of large area burns is how to close the wound in time and effectively. The deficiency of the dermal component is the main factor affecting the appearance and function of the skin graft after skin transplantation. Therefore, the study of dermis transplantation and / or dermis substitutes can improve the prognosis of burn, trauma and plastic patients and improve their life. One of the key points of quality. The scaffold material composed of extracellular matrix (ECM) has been applied to various surgical reconstruction and is widely used as a regenerative medicine strategy for tissue and organ substitution. The source of the extracellular matrix component biomaterial has a variety of animals, and the biological scaffold of the extracellular matrix has special mechanical properties. Well, the preservation of biological activity can promote tissue regeneration, so these protein based materials and synthetic materials have many natural advantages in the application of surgery and regenerative medicine. Because of the origin of the allogenic dermis and its limited and expensive, it is necessary to choose a heterologous dermal source which is relatively close to human dermal. On the basis of the earlier work of the group, we selected the newborn calf skin, explored the structure of the skin, the main components and its proportion and the biomechanical properties, and provided the experimental basis for the clinical application of the bovine dermal matrix. The ideal heterologous dermis matrix was prepared by the method of osmotic impact combined with ultrasonic and decontamination treatment. The histology and bioactive components of the prepared acellular dermal matrix were compared with the natural dermal matrix.
objective
1. compare the morphological and biomechanical properties of the newborn calves and adult skin tissues in terms of morphology and biomechanics, find the similarities and differences between the two, and provide the experimental basis for the clinical application of the bovine dermal matrix.
2. explore the preparation method of neonatal calf acellular dermal matrix, and observe the changes of histological and bioactive components of dermal matrix before and after treatment.
Method
1. the whole skin specimens of the back of newborn calves and adults were collected, using gross observation, HE staining, Masson tricolor staining, Sirius scarlet staining, Gomori aldehyde red staining, scanning electron microscopy and transmission electron microscopy, morphologic observation and image analysis software after microphotography and preparation of newborn calf and adult skin biomechanical specimens. A mechanical biomaterial testing machine was used to detect the mechanical properties of the skin.
2. the reticulated dermis of calf was collected and prepared, and the acellular dermal matrix was prepared by enzyme method, osmotic impact combined with decontamination agent and osmotic shock combined with ultrasonic and decontamination agent. The preparation method of the prepared acellular dermal matrix was determined by gross observation and HE staining.
3. HE staining, DAPI staining and nucleic acid electrophoresis were used to detect the residual cell composition of the acellular dermal matrix. The structure of collagen bundles and cell components were observed by scanning electron microscopy (SEM); PicoGreen, DMMB and BCA methods were used to quantitative analysis of the content of DNA, sGAG and egg white in the dermis matrix before and after the dehydration; the ELISA method was used to determine the cell dehydration. The contents of TGF- beta 1, EGF, bFGF and KGF in the dermal matrix before and after.
Result
1. newborn calves compared with human skin, the thickness of dermis and epidermis were significantly thinner, the ratio of the two was significantly reduced, and the number of type I and type III collagen fibers in the dermis increased and decreased respectively, but the clearance rate of the collagen fiber bundles was [(41.72 + 1.81) vs (40.66 + 1.40), P=0.467] and collagen fiber bundle thickness [(11.28 + 0.18) vs (10.88) (10.88). There was no statistical difference between P=0.368] and P=0.368].
2. the scanning electron microscope showed that the collagen fiber bundles of the newborn bovine dermis and the human dermal collagen fiber bundle were thin, loose and porous, and the transmission electron microscopy showed that the length of the collagen fibrils in the newborn bovine dermis was longer than that of the human dermis, but there was no significant difference in the diameter of the collagen fibrils between the two.
3. the biomechanical test showed that the maximum stress (21.08 + 0.91) MPa and Young's modulus (82.12 + 1.23) MPa of newborn cattle were significantly higher than the maximum stress (12.76 + 1.60) MPa (P=0.001) and young modulus (48.63 + 5.50) MPa (P=0.001) of the human skin, while the maximum strain was significantly lower than that of human dermis [(0.51 + 0.002) mm vs (0.75 + 0.028) mm (P=0.001)].
The 4. enzyme method can be used to prepare the dermis of the cells, but the dermis of the reticular layer is higher, and the reticular layer of dermis is uniform, and the site of the reticular skin is made. The removal of the cell nucleus by the acellular dermal HE staining prepared by the osmotic impact combined with the decontamination agent method is more thorough, but the decontamination agent is used for the collagen. There was a definite degree of destruction, which turned into a collagen fibrous bundle loose and partial mass. The removal of cellular dermis by HE staining with osmotic impact combined with ultrasonic decontamination was more thorough, the collagenous fiber bundles were loosely arranged, no collagen fiber bundles were destroyed, and the DNA coloring was not found in the dermis by DAPI staining, and the scanning electron microscope results were not found. The collagen bundles were loose and no cell retention was observed.
5. component quantitative analysis confirmed that the content of DNA was [(2516.1 + 324.2) ng/mg vs (249.5 + 53.8) ng/mg, P=0.000), decreased 90.1%, sGAG content [(5.92 + 0.50) mu g/mg vs (2.48 + 0.24) mu g/mg, P=0.000], and the content of the retention was compared with that before deactivation [(72.83+1.43)%]. The amount [(478.8 + 196.1) pg/g vs (180.3 + 111) pg/g, P=0.009] and EGF content [(10.52 + 2.78) pg/g vs failed to detect EGF content] decreased obviously, while bFGF content [(788.6 + 333.8) pg/g vs (364.8 + 294.8) pg/g, decreased 53.7%.
conclusion
Although the thickness of epidermis and dermis, the proportion of dermal collagen type and the biomechanics are different from those of human skin, there is a great similarity between the skin and the skin of 1. newborn cattle.
2. repeated osmotic shock combined with ultrasound and decontamination agent treatment of newborn bovine dermal matrix clearly cleared the cell and cell debris, preferably retained the three-dimensional structure of the extracellular matrix and the main bioactive components, and provided an important experimental basis for further improvement of the preparation of acellular dermal matrix.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.0

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