大鼠皮肤挫伤修复过程中caspase-6的表达及其时间规律性研究

发布时间：2018-02-09 00:49

本文关键词： 法医病理学皮肤挫伤 caspase-6 挫伤时间推断　出处：《中国医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的探讨皮肤挫伤修复过程中，caspase-6在挫伤区及挫伤周边区不同时间表达的变化规律。方法 1、动物模型的建立和分组健康成年雄性SD大鼠30只，体质量220-250g，随机分为10组，每组3只，其中9组为实验组，1组为正常对照组。用2％戊巴比妥钠(0.45mg／kg体重)腹腔注射麻醉后，参照Kami等的大鼠骨骼肌急性钝挫伤模型制作方法，用重500g的自制打击器，从54cm高处自由垂直落下打击动物右小腿皮肤。分别于伤后3h、6h、12h、1d、3d、5d、7d、10d、14d将大鼠脱颈椎处死，取做好标记的皮肤组织，对照组大鼠同样方法处理后不作打击，处死后取与损伤组大鼠相同部位，大小相等的皮肤组织作为对照，OCT包埋后，液氮速冻，5μm厚度切片。 2、免疫组织化学染色(SP法) (1)室温晾干；(2)3％H_2O_2／PBS，室温孵育20min；(3)0.3％Triton X-100中20min；(4)非免疫血清封闭，室温孵育20min；(5)一抗1∶100倍稀释，4℃孵育过夜；(6)生物素化兔抗山羊二抗，室温孵育20min；(7)SP试剂，室温孵育20min；(8)DAB显色，3min，苏木素核复染、透明、中性树胶封片。同时进行常规H．E染色。caspase-6抗体购自美国Santa Cruz公司、SP试剂盒购自北京中杉金桥生物试剂公司。 3、Caspase-6的Western blot检测 (1)蛋白定量；(2)电泳；(3)转印；(4)封闭抗原，采用含有0.1％Tween-20的5％脱脂奶粉封闭液；(5)山羊抗大鼠caspase-6抗体孵育；(6)兔抗山羊二抗孵育；(7)ECL显色。结果 1、皮肤挫伤的宏观所见伤后3h组出血、水肿明显，呈鲜红色；6h组出血、水肿程度减轻；1d组挫伤区仍有水肿，出血明显减轻；3d组水肿几乎全部吸收，挫伤处皮肤呈棕黄色；5d组挫伤区皮肤仅可见呈浅黄色改变；7d～14d组挫伤区皮肤与正常对照组皮肤相同。 2、组织学所见伤后3h组挫伤处表皮层与真皮层分离，真皮内毛细血管扩张充血，少量多型核细胞浸润；6h组挫伤周边区中性粒细胞浸润增多；12h组单个核细胞开始大量浸润，1d达高峰；3d组多型核细胞明显减少，成纤维细胞和单核细胞大量浸润；5d组胶原成分增多，细胞数目明显减少；7d～14d大量胶原纤维形成。 3、Caspase-6在皮肤损伤修复过程中损伤区及损伤周边区的表达对照组标本中，caspase-6只在表皮层、毛囊、皮脂腺处表达。挫伤组标本中，伤后3h组挫伤区真皮层内有成纤维细胞浸润，6h组成纤维细胞阳性染色进一步增多，12h、1d、3d组达到高峰，5d组成纤维细胞阳性染色减少，7d组、10d组成纤维细胞阳性染色继续下降，14d组降至最低。伤后3h组挫伤区皮下组织处开始有多型核细胞浸润，6h组、12h组挫伤区浸润的细胞数量逐渐增多，caspase-6阳性细胞主要为多型核细胞和单个核细胞，1d组多型核细胞和单个核细胞数目最多，3d组出现大量成纤维细胞阳性染色，5d～14d组caspase-6阳性细胞以成纤维细胞为主。免疫组织化学染色阴性对照组切片caspase-6无阳性染色。阳性细胞计数及统计学分析：伤后3h组，阳性细胞比率为(25.78％±1.38％)，6h组阳性细胞比率继续增高(36.21％±4.25％)，12h组阳性细胞比率增高明显(47.70％±5.14％)，1d组阳性细胞比率进一步增高(50.62％±5.42％)，3d组caspase-6阳性细胞率达到最高(54.58％±5.64％)。伤后5d组开始下降(28.39％±4.50％)，7d、10d组进一步下降，且阳性细胞率基本维持在一个相对稳定的水平(23.75％±3.12％，，20.43％±2.36％)，14d组阳性细胞率下降至(11.31％±2.19％)。经统计学方差分析，各时间段相邻两组之间caspase-6阳性细胞率相比均存在显著差异(P＜0.01)。 4、Western blot分析实验组中，1d、3d、5d组可见明显的10kD caspase-6活化片段。结论大鼠皮肤挫伤修复过程中，caspase-6在挫伤后的炎症反应中发挥重要作用；同时，caspase-6在挫伤区内多型核细胞、单个核细胞及成纤维细胞中的表达呈规律性变化，可对挫伤时间的推断提供参考。
[Abstract]:objective
To investigate the changes of caspase-6 expression in the contusion area and the peripheral zone of contusion during the repair of skin contusion.
Method
1, establishment and grouping of animal models
30 adult male SD rats, weighing 220-250g, were randomly divided into 10 groups, 3 rats in each group, the 9 groups as the experimental group, the 1 group was the normal control group. With 2% sodium pentobarbital (0.45mg / kg body weight) intraperitoneal injection of anesthesia, method of making acute blunt skeletal muscle contusion in rats model reference Kami, homemade blow 500g, free fall from a height of 54cm vertical strike animal right calf skin. After injury, 3h, 6h, 12h, 1D, 3D, 5D, 7d, 10d, 14d rats were sacrificed by cervical dislocation, and marked the skin tissue of rats in the control group the same method does not blow after sacrificed and injury rats in the same parts of equal size skin tissue as control, OCT embedding, quick-frozen in liquid nitrogen, 5 mu m thick slices.
2, immunohistochemical staining (SP)
Dry at room temperature (1); (2) 3%H_2O_2 / PBS, incubated at room temperature for 20min; (3) 20min 0.3%Triton X-100; (4) non immune serum blocking, and incubated at room temperature for 20min; (5) - 1: 100 times dilution, 4 C incubated overnight; (6) the biotinylated anti rabbit two anti goat, incubated at room temperature for 20min; (7) SP reagent, incubated at room temperature for 20min; (8) DAB color, 3min, hematoxylin nuclear staining, transparent, neutral balata. Routine H.E staining of.Caspase-6 antibody was purchased from American Santa Cruz company at the same time, SP Kit was purchased from Beijing golden bridge the biological reagent company.
3, Western blot detection of Caspase-6
(1) protein quantification; (2) electrophoresis; (3) transfer; (4) blocking antigen, using 5% skim milk powder containing 0.1%Tween-20, and (5) Goat anti rat caspase-6 antibody incubation; (6) Rabbit anti goat two anti incubation; (7) ECL color.
Result
1, the macroscopic view of the skin contusion
The group of 3H hemorrhage, edema, bright red; group 6h hemorrhage, edema decreased in 1D group; the contusion area still have edema, hemorrhage significantly reduced; 3D group edema is almost completely absorbed, the skin contusion was brown; 5D group of Skin Contusion area only visible pale yellow; 7d ~ 14d contusion group the skin and the normal control group the same skin.
2, histology
The group of 3H injured epidermis and dermis, dermis angiotelectasis hyperemia, a few polymorphonuclear cell infiltration; 6h group of surrounding area increased neutrophil infiltration; 12h group began a large number of mononuclear cells infiltration, reached the peak at 1D; 3D group of polymorphonuclear cells significantly decreased, into a large number of fibroblasts and infiltration mononuclear cells; group 5D collagen increased, the number of cells was significantly reduced; 7d ~ 14d a large number of collagen fibers.
3, Caspase-6 expression in the damaged area and the surrounding area during the repair of skin injury
Samples in the control group, caspase-6 only in the epidermis, hair follicles and sebaceous glands. The expression of contusion group specimens after injury, contusion group 3H in dermal fibroblast infiltration, 6h positive staining cells further increased, 12h, 1D, 3D group reached the peak, 5D fibroblasts positive staining decreased 7d group, 10d positive staining cells continued to decline, fell to the lowest in group 14d after injury in 3H group. The subcutaneous tissue contusion area starting with polymorphonuclear cell infiltration, 6h group, 12h group the number of cells infiltrating the contusion area gradually increased, caspase-6 positive cells were polymorphonuclear cells and mononuclear cells, 1D group polymorphonuclear cells and mononuclear cell number, a large number of fibroblasts positive staining of 3D group, 5D group 14d ~ caspase-6 positive cells with fibroblast. Immunohistochemical staining in negative control group were caspase-6 positive staining.
Positive cell counting and statistical analysis: after injury in 3H group, positive cells ratio was (25.78% + 1.38%), the ratio of positive cells in the 6h group continue to increase (36.21% + 4.25%) 12h group, the rate of positive cells increased significantly (47.70% + 5.14%), 1D group further increased the rate of positive cells (50.62% + 5.42%), 3D group caspase-6 the rate of positive cells reached the highest (54.58% + 5.64%). The group of 5D began to decline (28.39% + 4.50%), 7d group, 10d decreased further, and the positive cell rate remained at a relatively stable level (23.75% + 3.12%, 20.43% + 2.36%), 14d group (positive cell rate dropped to 11.31% + 2.19%).
The statistical analysis of variance showed that there were significant differences in the rate of caspase-6 positive cells between the two adjacent groups (P < 0.01).
4, Western blot analysis
In the experimental group, 1D, 3D, and 5D groups showed significant 10kD caspase-6 activation fragments.
conclusion
In the process of Skin Contusion repair, caspase-6 plays an important role in the inflammatory response after contusion. Meanwhile, the expression of caspase-6 changes regularly in various types of nuclear cells, mononuclear cells and fibroblasts in the contusion area, which can provide a reference for the inference of contusion time.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

【参考文献】