小鼠皮肤切创愈合过程中磷酸化JNK表达及其与损伤时间相关性的研究

发布时间：2018-05-16 02:15

本文选题：法医病理 + 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。c-Jun氨基末端激酶(c-JunN-terminal kinase/stress-activated protein kinase,JNK/SAPK)为丝裂酶原活化蛋白激酶(mitogen-activated protein kinase,MAPK)家族的一个重要的亚族,可通过细胞性压力和炎症刺激发生磷酸化而激活,与重要的细胞反应有关,比如炎性反应和凋亡。本研究应用免疫组织化学和Western blot技术,检测小鼠皮肤损伤区切创后各时间段磷酸化JNK(phospho-JNK,p-JNK)的变化情况,探讨p-JNK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,耳义伤口处1.5cm×1.0cm皮肤组织。正常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。应用免疫组织化学和Western blot技术检测55例小鼠皮肤切创后各时间段p-JNK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Fluochem V2.0软件检测各时间段的平均灰度值,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P0.05为差异有统计学意义。结果正常对照组小鼠皮肤的表皮、毛囊及皮脂腺中表达p-JNK。皮肤切创伤后6h,可见少数多核粒细胞有p-JNK表达。伤后12～24h,大量浸润的多核粒细胞和部分单个核细胞p-JNK呈阳性。伤后1～3d随着时间的延长,p-JNK阳性细胞主要以单个核细胞为主。伤后5～14d p-JNK表达主要在成纤维细胞中。伤后0～3h,p-JNK阳性细胞率较低,随着伤后时间的延长,p-JNK阳性细胞率逐渐增加,1d达到高峰。伤后3～5d一直呈高表达状态。伤后7d p-JNK阳性细胞率达到第二峰,随后逐渐下降。除伤后3d与5d阳性细胞率无统计学差异外(P0.05),其他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(P0.05)。Westernblot结果显示正常对照皮肤内有p-JNK阳性条带。皮肤切创伤后各时间段组p-JNK表达强度有一定规律。伤后0～12h,阳性条带逐渐增强;1d,稍有下降;3d,p-JNK达到高峰。伤后5d、10d,较少;但伤后7d p-JNK相对较深。到伤后14d p-JNK阳性条带基本与正常对照组条带情况一致。除0h与正常对照组间平均灰度值差异无统计学意义外(P0.05),其余各组间平均灰度值差异均具有统计学意义(P0.05)。结论 1、正常对照组小鼠皮肤的表皮、毛囊及皮脂腺中表达p-JNK。 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表达p-JNK。 3、小鼠皮肤切创愈合过程中p-JNK对诱导多核粒细胞、单个核细胞及成纤维细胞的凋亡可能具有调节作用。 4、小鼠皮肤切创愈合过程中p-JNK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 process, including blood component agglutination, inflammation, granulation tissue formation, new epithelial cover, connective tissue contraction and fibrosis,.C-Jun amino terminal kinase (c-JunN-terminal kinase/stress-activated protein kinase, JNK/SAPK) is a cleft zymogen activated protein kinase (mitogen-activated protei). An important subfamily of the family of N kinase, MAPK, which can be activated by phosphorylation of cellular stress and inflammatory stimulation, related to important cellular responses, such as inflammatory reactions and apoptosis. This study used immunohistochemistry and Western blot to detect the phosphorylated JNK (phospho-JNK, p-J) at every time after the wound in the skin of mice. NK, to explore the role of p-JNK in 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. 0h, 3h, 6h, 12h, 12h, 1D, 3D, 5D, 7d, 10d and 14d respectively removed the mice from the cervical vertebra after the injury. The application of color and Western blot detection. The changes of p-JNK in 55 mice were detected by immunohistochemistry and Western blot. The non cut mouse skin tissue was used as the control, and the microscope x 400 times, the 10 fields were selected randomly in the injured area and the surrounding area. The number of cells, the number of positive cells and the positive size were counted. The average gray value of each time period was detected by Fluochem V2.0 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 of P0.05 was statistically significant.
Result
The skin epidermis, hair follicle and sebaceous gland of normal control mice expressed 6h after p-JNK. skin incision, and a few multinucleated granulocytes had p-JNK expression. 12 to 24h after injury, and p-JNK positive in large number of infiltrating multinucleated granulocytes and some mononuclear cells. After 1 to 3D, p-JNK positive cells mainly were mononuclear cells. The expression of 5 ~ 14d p-JNK after injury was mainly in fibroblasts. The rate of p-JNK positive cells was low after injury. With the prolongation of the injury time, the rate of p-JNK positive cells increased gradually and the 1D reached the peak. The rate of 3 to 5D after injury was highly expressed. The rate of 7D p-JNK positive cells reached second peak after injury, and then decreased gradually. 3D and 5D positive cells after injury. There was no statistical difference (P0.05). The difference of the number of cells and the positive cell rate between the other groups and the adjacent groups were statistically significant (P0.05).Westernblot results showed that there were p-JNK positive bands in the normal control skin. The expression intensity of p-JNK in each time group after the skin incision was 0 to 12h, and the positive bands gradually increased; 1D 3D, p-JNK reached the peak. 5D, 10d, less after injury, but 7d p-JNK was relatively deep after injury. The positive band of 14d p-JNK after injury was basically the same as that in normal control group. The mean gray value difference between 0h and normal control group was not statistically significant (P0.05), and the difference of average gray value between the remaining groups was statistically significant (P0.0). 5).
conclusion
1, the epidermis, hair follicle and sebaceous glands of normal control mice were expressed in p-JNK..
2, multinucleated granulocytes, mononuclear cells and fibroblasts express p-JNK. during wound healing in mice.
3, p-JNK may play a regulatory role in inducing apoptosis of multinucleated granulocytes, mononuclear cells and fibroblasts during wound healing in mice.
4, the regular change of p-JNK 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

【相似文献】