小鼠皮肤切创愈合过程中c-jun表达及其与损伤时间相关性的研究

发布时间：2018-04-19 18:12

本文选题：法医病理学 + 皮肤损伤　；参考：《中国医科大学》2009年硕士论文

【摘要】： 目的皮肤损伤愈合是一个复杂而连续的过程,包括血液成分凝集、炎症、肉芽组织形成、新生上皮覆盖、结缔组织收缩和纤维化等过程。原癌基因c-jun是即刻早期基因jun家族成员之一,属于碱性亮氨酸拉链家族核内转录因子成员,可以结合在许多基因的启动子上参与基因转录的调控,其表达产物在多种基因表达、细胞凋亡、增殖、分化等过程中起重要作用。本研究应用免疫组织化学染色和Westernblot技术,检测小鼠皮肤损伤区在损伤后不同时间段c-jun表达的变化情况,探讨c-jun在皮肤损伤愈合中的作用及其在损伤时间推断上应用的可能性。实验材料与方法健康成年(8周龄)清洁级昆明系小鼠55只,雌雄不限,体质量35～40g,随机分为11组。其中皮肤切创分为10个时间段组,另一组为正常对照组,每组均为5只小鼠。乙醚气体吸入麻醉,背部剪毛处理后,常规手术消毒,在背部中央做一纵行长1.5cm皮肤全层切口,深达筋膜。分别于伤后0h、3h、6h、12h、1d、3d、5d、7d、10d和14d将小鼠脱颈椎处死,取伤口处1.5cm×1.0cm皮肤组织。对照组小鼠麻醉后背部剪毛,不做切创,观察至14d,脱颈椎处死,取相同部位同等大小皮肤,以备HE染色、免疫组织化学染色和Western blot检测应用。应用免疫组织化学染色和Western blot技术检测55例小鼠皮肤切创后各时间段c-jun变化情况,以非切创小鼠皮肤组织作对照,显微镜×400倍下,在损伤区及损伤周边区随机选取10个视野,计数细胞总数、阳性细胞数及阳性细胞率。应用FluochemV2.0软件检测各时间段的平均光密度值,测得数据用均数±标准差((?)±s)表示。用SPSS13.0 for Windows进行单因素方差分析,以P＜0.05为差异有统计学意义。结果正常对照组小鼠皮肤中c-jun主要表达在表皮、毛囊和皮脂腺等处。伤后3h即可见少数多核粒细胞有c-jun表达。伤后6～24h,大量浸润的多核粒细胞和部分单个核细胞c-jun呈阳性。伤后1～3d,c-jun阳性细胞主要以单个核细胞和成纤维细胞为主。伤后5～14d,c-jun主要在成纤维细胞中表达。伤后0h时c-jun阳性细胞率比较低,随着伤后时间的延长,c-jun阳性细胞率逐渐增加,12h达到高峰。伤后1～5d一直呈高表达状态。伤后7d时c-jun阳性细胞率达到第二峰,随后逐渐下降。除伤后1d与3d阳性细胞率差异无统计学意义外(P＞0.05),其它各组与相邻上组的细胞总数及阳性细胞率差异均有统计学意义(P＜0.05)。Western blot结果显示正常对照皮肤内有c-jun阳性条带,皮肤切创伤后各时间段组c-jun表达强度有一定规律。伤后0～12h,表达逐渐增强;12h达高峰。1～5d稍有下降;伤后7d,c-jun表达稍有上升,随后表达渐降低。但12h组中c-jun表达相对较多,到1 4d时,c-jun基本与对照组表达情况一致。除0h与对照组间平均光密度值差异无统计学意义外(P＞0.05),其余各组间平均光密度值差异均具有统计学意义(P＜0.05)。结论 1、正常对照组小鼠皮肤的表皮、毛囊及皮脂腺中表达c-jun。 2、小鼠皮肤切创愈合过程中多核粒细胞、单个核细胞及成纤维细胞均表达c-jun。 3、小鼠皮肤切创愈合过程中c-jun对诱导多核粒细胞、单个核细胞及成纤维细胞的凋亡可能起着重要作用。 4、小鼠皮肤切创愈合过程中c-jun表达的规律性变化可用于皮肤切创损伤时间的推断。
[Abstract]:objective
Skin injury healing is a complex and continuous process, including blood component agglutination, inflammation, granulation tissue formation, newborn epithelial cover, connective tissue contraction and fibrosis. Proto oncogene c-jun is one of the immediate early gene jun family members, members of the alkaline leucine zipper family of transcription factors, can be combined in Xu. The multiple gene promoter participates in the regulation of gene transcription, and its expression products play an important role in the process of multiple gene expression, cell apoptosis, proliferation and differentiation. In this study, immunohistochemical staining and Westernblot technique were used to detect the changes of the expression of c-jun in the injured area of mice after injury in the same time period, and the c-Jun was discussed. The role of skin wound healing and the possibility of its application in the estimation of injury time.
Experimental materials and methods
55 healthy adult (8 weeks old) Kunming mice were divided into 11 groups, male and female, with body mass from 35 to 40g, and randomly divided into 11 groups. The skin incision was divided into 10 time groups, the other group was the normal control group, and the other groups were 5 mice. After the ether gas inhalation anesthesia, the back shearing treatment, the routine operation disinfection and the longitudinal length 1.5cm skin in the center of the back. After the injury, 0h, 3h, 6h, 12h, 1D, 3D, 5D, 7d, 10d and 14d respectively removed the mice from the cervical vertebra, and took the 1.5cm x 1.0cm skin tissue in the wound. The application of ern blot detection. The changes of c-jun in 55 mice were detected by immunohistochemistry and Western blot. The non cut mice skin tissue was used as the control, and the microscope x 400 times, the 10 fields were randomly selected in the injured area and the surrounding area. The number of cells, the number of positive cells and the positive cells were counted. The average optical density of each time period was detected by FluochemV2.0 software, and the measured data were measured with mean mean deviation ((?) + s). The single factor variance analysis was carried out with SPSS13.0 for Windows, and the difference was statistically significant with the difference of P < 0.05.
Result
C-Jun was mainly expressed in the skin, hair follicle and sebaceous gland in the skin of normal control mice. A few multinucleated granulocytes were expressed as c-Jun after injury. After injury, 6 to 24h, a large number of infiltrating multinucleated granulocytes and some mononuclear cells were c-Jun positive. 1 to 3D after injury, and the main c-Jun positive cells were mainly mononuclear cells and fibroblasts. 5 ~ 14d after injury, c-jun was mainly expressed in fibroblasts. The rate of c-jun positive cells was lower at 0h after injury. With the prolongation of the injury time, the rate of c-jun positive cells increased gradually and the 12h reached the peak. The rate of 1 to 5D after injury was highly expressed. The rate of c-Jun positive cells reached second peaks at 7d after injury, and then gradually decreased. 1D and 3D positive after injury. The difference of cell rate was not statistically significant (P > 0.05). The difference of cell number and positive cell rate between the other groups and adjacent groups were statistically significant (P < 0.05).Western blot results showed that there were c-Jun positive bands in the normal control skin, and the expression intensity of c-jun in each time group after the skin incisal trauma had a certain regularity. 0 to 12h after injury, expression by 0 to 12h after injury. Gradually, the peak of 12h reached a slight decrease in.1 to 5D, and the expression of 7D, c-jun increased slightly after the injury, and then the expression gradually decreased. But the expression of c-jun in 12h group was relatively more, and at 1 4D, the expression of c-jun was basically the same as that of the control group. The mean light density difference between 0h and control group was not statistically significant (P > 0.05), and the mean light density difference between the other groups was poor. The difference was statistically significant (P < 0.05).
conclusion
1, the epidermis, hair follicle and sebaceous glands of normal control mice were expressed in c-Jun..
2, multinucleated granulocytes, mononuclear cells and fibroblasts were expressed in c-Jun. during wound healing in mice.
3, c-jun may play an important role in inducing apoptosis of multinucleated granulocytes, mononuclear cells and fibroblasts during wound healing in mice.
4, the regular change of c-jun expression during wound healing in mice can be used to deduce the time of wound healing.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

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