核心蛋白聚糖对皮肤创伤再生和瘢痕愈合中胶原纤维装配的影响

发布时间：2018-05-12 01:20

本文选题：核心蛋白聚糖 + 创面愈合　；参考：《兰州大学》2015年硕士论文

【摘要】：目的：正常真皮组织中胶原纤维是有序的结构,由胶原蛋白分子高度有序的组装构成。如果胶原蛋白排列、组装和相互连接异常,则形成结构异常的胶原纤维支架,在创伤愈合后则表现为瘢痕形成。核心蛋白聚糖(DCN)对真皮创伤再生和瘢痕愈合中胶原纤维装配影响的研究国内很少。本研究通过建立兔耳皮肤创伤再生修复和瘢痕愈合一体化模型,于创面愈合过程在创周分别注射生理盐水(NS),核心蛋白聚糖(DCN)及核心蛋白聚糖抗体(DCN抗体)。1.动态观察并比较创面愈合过程中不同分组及不同深浅的组织形态学差异。2.观察创面愈合过程中,Ⅰ型和Ⅲ型胶原蛋白在浅层和深层创面的表达差异。3.探讨核心蛋白聚糖对创面愈合过程中Ⅰ、Ⅲ型胶原蛋白表达的影响。4.分析胶原纤维在浅层创面和深层创面愈合过程中的排列的差异,以及核心蛋白聚糖对其结构的影响。为进一步研究皮肤再生修复和瘢痕愈合提供形态学依据。方法：1.50只日本大耳白兔,按性别、年龄、体重相近的大白兔分配出25对,然后将每一对分别分配到不同的两组中,分别为DCN组和DCN抗体组,每组25只,组内设空白对照,视为正常对照(NS)组。于耳腹做大小为10.0mmx10.0mm由浅至深的斜行创面。伤后当日起,左耳创周以30G针头注射浓度为5.0μg/ml的DCN(DCN组)或浓度为10.0μg/ml的DCN抗体(DCN抗体组),每次0.1ml,隔日一次,直至创面愈合；右耳注射等量生理盐水作为对照(NS组),方法同上。所有创面均以无菌辅料包扎并卷耳固定。分别于伤后0天、4天、8天、12天及30天大体观察和用标尺测量创面大小变化,并拍照存档以便后期统计数据。比较各组平均愈合时间和观察创面形态学改变。2.应用HE染色、免疫组化和计算机图像处理法检测日本大耳白兔耳腹侧皮肤创伤模型外用DCN和DCN抗体前后,深浅创面愈合过程中的组织形态结构变化和Ⅰ、Ⅲ型胶原蛋白的表达差异。3.利用扫描电镜技术观测胶原纤维在深浅创面愈合过程中的变化规律,以及DCN对其结构的影响。结果：1.兔耳浅侧创面(距浅侧创面边缘0-4.0mm内为浅侧创面)术后第8天,创面大部分结痂已脱离,再生修复。术后第30天,浅侧色泽与正常皮肤无明显差异,且界限不清,为再生愈合。深侧创面(距浅侧创缘内约6-10mm为深侧创面)直至术后第12天,创面仍见部分结痂存在,未完全上皮化,术后第30天,创面中央现小丘样凸起,明显高于皮面质硬,色泽较周围组织深,为瘢痕愈合。2.同一创面浅侧或深侧,DCN组创面较NS组创面愈合更快：创面DCN组创面愈合率第4天为(41.2±7.6)%、8天为(79.5±6.5)%、12天为(93.6±4.2)%,分别高于NS组的(36.6±4.3)%、(60.5±3.9)%和(85.2±4.3)%(P0.05)。应用DCN抗体后创面愈合率第4天(22.8±4.5)%、8天为(47.5±5.3)%、12天为(73.9±2.6)%,低于NS组(P0.05)；同时,DCN组愈合后组织色泽更接近正常组织,瘢痕组织更软更薄,而DCN抗体组创面愈合后色泽深,瘢痕厚且硬,NS组介于两者之间。3.创面干预后第30天时,DCN组再生面积(0.86±0.13)cm2高于NS组的(0.71±0.11)cm2(P0.05),而DCN抗体组再生面积(0.46±0.08)cm2低于NS组(P0.05)；DCN组瘢痕面积(0.14±0.07)cm2低于NS组的(0.29±0.09)cm2(P0.05),而DCN抗体组瘢痕面积(0.54±0.10)cm2高于NS组(P0.05)。4.创面Ⅰ、Ⅲ型胶原蛋白的表达：各个时间点Ⅰ、Ⅲ型胶原蛋白在浅侧创面的表达均低于深侧(P0.05)；各层次创面中Ⅰ胶原蛋白在各个时间点DCN组表达最低,DCN抗体组表达最高(P0.05),而Ⅲ型胶原蛋白在各组的表达无差异(P0.05)；各层次创面中Ⅲ/Ⅰ型胶原蛋白比例在各个时间点DCN组最高,DCN抗体组表达最低,差异明显(P0.05)。5.创面胶原纤维的排列：各组浅侧创面胶原纤维的排列在各个时间点均比深侧创面更规律,增生不明显,网状结构更典型,纤维更细。同深浅侧创面中,胶原纤维于各个时间点在DCN组排列更规律,纤维更细且增生不明显,网状结构更典型,而在DCN抗体组排列最紊乱,增生明显,未见网状结构且纤维最粗。结论：1.外源性DCN能调节Ⅲ/Ⅰ型胶原蛋白比例的作用,同时调节了胶原纤维的装配,起到了预防瘢痕形成甚至实现再生修复的作用。2.本实验成功建立了兔耳皮肤创伤再生和瘢痕愈合一体化模型,浅侧创面实现了再生愈合,而深侧创面实现了瘢痕愈合。
[Abstract]:Objective: the collagen fibers in normal dermal tissue are ordered structure, which are composed of collagen molecules highly ordered. If collagen is arranged, assembled, and interconnected, the collagen fibrous scaffold is formed with abnormal structure. After wound healing, it appears as scar formation. DCN There are few studies on the effect of collagen fiber assembly in scar healing. In this study, the integrated model of rabbit ear skin wound regeneration and scar healing was established, and the wound healing process was injected with physiological saline (NS), core proteoglycan (DCN) and core proteoglycan antibody (DCN antibody).1. in the wound healing process, and the wound healing was compared. Differences in histomorphological differences between different groups and different depths of the process.2. observation of the expression difference between type I and type III collagen in superficial and deep wounds during the wound healing process.3. probe into the effect of core proteoglycan on the expression of type I and III collagen in the wound healing process.4. analysis of the healing of collagen fibers in superficial and deep wounds The difference in the arrangement and the effect of the core proteoglycan on its structure. To provide morphological basis for further study of skin regeneration and scar healing. Methods: 1.50 Japanese white rabbits were assigned to 25 pairs by sex, age, and similar body weight, and each pair was assigned to two different groups, respectively. Group DCN and DCN antibody group, each group of 25, set blank control, considered as the normal control (NS) group. On the ear abdomen, the size of 10.0mmx10.0mm from shallow to deep of the oblique wound. From the day after injury, the left ear wound week with the 30G needle injection concentration of 5 mu DCN (DCN group) or the concentration of 10 micron DCN antibody (DCN antibody group), each time 0.1ml, straight every other day, straight To the wound healing, the right ear was injected with equal amount of saline as the control (group NS) and the method was same. All the wounds were wrapped with aseptic auxiliary materials and fixed with the ear. The size of the wound was measured at 0 days, 4 days, 8 days, 12 days and 30 days respectively. The morphological changes of the wound surface were observed by.2., HE staining, immunohistochemistry and computer image processing were used to detect the changes of tissue morphology and structure in the healing process of the deep and shallow wounds of the ears of Japanese big ear rabbits before and after the external use of DCN and DCN antibodies, and the difference of collagen type III with.3. was observed by scanning electron microscopy. The changes in the healing process of deep and shallow wounds and the effect of DCN on its structure. Results: 1. on the eighth day after the operation, most of the superficial wound of the rabbit ear (the edge of the superficial wound surface is the superficial wound in 0-4.0mm), most of the scab of the wound has been separated and regenerated. There is no obvious difference between the superficial color and the normal skin on the thirtieth day after the operation, and the boundary is not clear, for the regeneration of the skin. Healing. The deep side wound (about 6-10mm in the margin of the shallow margin) until twelfth days after the operation, the wound still found some scab, not completely epithelialization. On the thirtieth day after the operation, the center of the wound was a small hillock convex, obviously higher than the skin surface, and the color and lustre was deeper than the surrounding tissue. The wounds of the DCN group were wound in the superficial or deep side of the same wound, and the wound in group DCN was more than that of the NS group. The healing rate of the surface was faster: the wound healing rate in the wound DCN group was fourth days (41.2 + 7.6)%, 8 days (79.5 + 6.5)% and 12 days (93.6 + 4.2)%, respectively (36.6 + 4.3)%, (60.5 + 3.9)% and (93.6%)% (P0.05). The wound healing rate after the application of DCN antibody was (+ +)%, and was lower than that of the NS group (P0.05). At the same time, at the same time, After the healing of the DCN group, the tissue color was closer to the normal tissue, and the scar tissue was softer and thinner, while the wound healing was deep and the scar was thick and hard in the DCN antibody group. In the group NS, the regenerated area of the DCN group (0.86 + 0.13) cm2 was higher than that of the NS group (0.71 + 0.11) cm2 (P0.05), while the regeneration area of the DCN antibody group was (0.46 + 0.08) cm2. In group NS (P0.05), the scar area of group DCN (0.14 + 0.07) was lower than that of group NS (0.29 + 0.09) cm2 (P0.05), and the scar area of DCN antibody group (0.54 + 0.10) cm2 higher than that of NS group (P0.05).4. wound I, type III collagen expression: the expression of type III collagen egg white in the superficial wound was lower than that in the deep side. The expression of collagen in DCN group was the lowest at all time points, and the expression of DCN antibody group was the highest (P0.05), but there was no difference in the expression of type III collagen in each group (P0.05). The proportion of type III / I collagen in all layers was the highest in DCN group at every time point, and the expression of DCN antibody group was the lowest, and the difference was obvious (P0.05).5. wound collagen fiber arrangement: each group was in each group. The arrangement of collagen fibers in the superficial wound surface was more regular at each time point than the deep side wound. The proliferation was not obvious, the reticular structure was more typical and the fiber was finer. In the deep and shallow wounds, the collagen fibers were arranged more regularly in the DCN group at various time points, the fiber was finer and the proliferation was not obvious, the net structure was more typical, and the most disorder in the DCN antibody group was increased. Conclusion: 1. exogenous DCN can regulate the proportion of collagen type III / type I, adjust the assembly of collagen fiber and play the role of preventing scar formation and even realizing regenerative repair..2. experiment has successfully established the integrated model of wound regeneration and scar healing in the skin of rabbit ear skin. Wound healing was achieved and scar healing was achieved on deep side wounds.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622

【参考文献】