新生小牛与成人皮肤组织学及生物力学性能的比较和牛脱细胞真皮基质的制备

发布时间：2018-04-19 18:57

本文选题：细胞外基质 + 真皮支架　；参考：《第三军医大学》2012年硕士论文

【摘要】：大面积烧伤、创伤患者救治的关键是如何及时有效的封闭创面。真皮成分的缺乏成为皮肤移植术后患者植皮区愈后外观及功能的主要影响因素，因此真皮移植和/或真皮替代物的研究成为改善烧伤、创伤和整形患者的预后，提高其生活质量的关键之一。由细胞外基质(ECM)组成的生物支架材料已经应用于各种外科重建并作为一种再生医学策略广泛应用于组织和器官替代。细胞外基质成分生物支架材料的来源有多种动物，由于细胞外基质成分生物支架有特殊的机械性能、保留了生物活性可促进组织再生，所以这些蛋白为基础的材料与人工合成材料在外科和再生医学的应用中有许多天然的优势。由于异体真皮的来源及其有限且价格昂贵，因此有必要选择一种与人真皮相对接近的异种真皮来源。在本课题组前期工作的基础上，我们选择了新生小牛皮肤，探讨与人皮肤组织结构、主要组成成分及其比例上的差异及生物力学性能特点，为临床应用牛真皮基质提供实验依据。并且对制备新生小牛脱细胞真皮基质的方法进行了探索，利用渗透冲击联合超声和去污剂处理的方法制备出了较为理想的异种脱细胞真皮基质，对所制备的脱细胞真皮基质的组织学和生物活性成分与天然真皮基质进行了对比。目的 1.通过新生小牛与成人皮肤组织在形态学和生物力学性能方面的对比研究，寻找二者之间的异同点，为临床应用牛真皮基质提供实验依据。 2.探索新生小牛脱细胞真皮基质的制备方法，观察处理前后真皮基质的组织学和生物活性成分的变化。方法 1.收集新生小牛和成人背部全层皮肤标本，，采用大体观察、HE染色、Masson三色染色、天狼猩红染色、Gomori醛复红染色、扫描电镜和透射电镜的方法，显微照相后进行形态学观察和图像分析软件的测量并制备新生小牛和成人皮肤生物力学试件，采用力学生物材料试验机检测皮肤的力学性能。 2.收集并制备新生小牛网状层真皮，分别采用酶学法、渗透冲击联合去污剂法及渗透冲击联合超声和去污剂法探索制备脱细胞真皮基质，对制备出的脱细胞真皮基质采用大体观察和HE染色确定较为理想的制备方法； 3.对制备出的脱细胞真皮基质进行HE染色、DAPI染色和核酸电泳检测细胞成分残留情况；扫描电镜(SEM)观察胶原束结构和细胞成分；PicoGreen法、DMMB法和BCA法对脱细胞前后真皮基质的DNA、sGAG和蛋白质含量进行定量分析；ELISA法测定脱细胞前后真皮基质内TGF-β1、EGF、 bFGF和KGF含量。结果 1.新生小牛与人的皮肤比较，真皮和表皮的厚度均明显变薄，两者的比值显著减小；真皮内Ⅰ型和Ⅲ型胶原纤维分别较人真皮增多和减少，但真皮胶原纤维束的间隙率[(41.72±1.81) vs (40.66±1.40)，P=0.467]和胶原纤维束粗细[(11.28±0.18) vs(10.88±0.66)， P=0.368]均没有统计学差异； 2.扫描电镜观察可见新生牛真皮胶原纤维束与人真皮胶原纤维束均较纤细，排列疏松，且有一定的孔隙分布；透射电镜显示新生牛真皮胶原原纤维横纹周期长度较人真皮长，但两者的胶原原纤维直径无显著差异。 3.生物力学检测示新生牛皮肤最大应力(21.08±0.91) MPa和杨氏模量(82.12±1.23) MPa均要明显高于人皮肤的最大应力(12.76±1.60) MPa (P=0.001)和杨氏模量(48.63±5.50)MPa (P=0.001)，而最大应变却明显低于人真皮[(0.51±0.002) mm vs (0.75±0.028) mm (P=0.001)]。 4.酶学法可以制备出脱细胞真皮，但对网状层真皮要求较高，要求网状层真皮厚薄均匀，并且对制作网状层真皮的部位有一定要求；渗透冲击联合去污剂法所制备的脱细胞真皮HE染色观察细胞核去除较彻底，但所使用去污剂对胶原有一定程度破坏，变现为胶原纤维束松散，部分呈团块状；渗透冲击联合超声去污剂处理制备的脱细胞真皮HE染色观察细胞核去除较为彻底，胶原纤维束排列疏松，未见胶原纤维束结构明显破坏，DAPI染色未见真皮内有DNA着色，扫描电镜结果显示胶原纤维束排列疏松，未发现细胞存留。 5.成分定量分析证实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]，含量保留约41.9%。与脱细胞前比较，含水量有所下降[(72.83+1.43)%vs(69.68+1.78)%，P0.05]。细胞因子检测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未能检测到EGF含量]均明显减少；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]变化不显著。结论 1.新生牛皮肤虽然在表皮与真皮厚度、真皮胶原类型比例以及生物力学上与人皮肤有差异，但真皮的胶原纤维束三维结构两者之间存在较大相似性。 2.反复渗透冲击联合超声和去污剂处理对新生牛真皮基质明显地清除了细胞及细胞碎片，较好地保留了真皮细胞外基质的三维结构和主要生物活性成分，为进一步改进脱细胞真皮的制备方法提供了重要实验依据。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.0

【参考文献】