大鼠皮肤挫伤修复过程中NFκB P65及IκBα表达的免疫组织化学研究

发布时间：2018-05-13 23:41

本文选题：皮肤挫伤 + NFкB　；参考：《中国医科大学》2004年硕士论文

【摘要】： 前言皮肤挫伤是法医学鉴定中常见的损伤，因此，皮肤挫伤时间的推断成为多年来国内外法医学界研究的热点内容。最初根据挫伤皮肤颜色的变化推断挫伤时间，但由于人种肤色、年龄等个体差异，无法精确判断皮肤挫伤时间，只能粗略估计。后来通过光学显微镜观察皮肤挫伤后炎症反应中炎性细胞的数量来推断挫伤时间，但由于各种变化的时相互相重叠，给确切推断挫伤时间带来了困难，所以，人们一直在寻找可以相对准确地推断皮肤挫伤时间的方法。随着医学界对创伤后分子病理学认识的提高，创伤后生物物质的变化越来越受到人们的重视，也为法医工作者推断皮肤挫伤时间提供了新的方法。核转录因子NFκB(nuclear factor kappa B，NFκB)是一种重要的生物因子，调控多种细胞因子如生长因子、细胞因子、粘附分子等编码基因的表达，近来研究表明，NFκB与创伤关系密切。IκBα因其抑制NFκB活性，也越来越受到人们的重视，故本实验应用免疫组织化学方法观察NFκB家族的重要成员NFκB P65与其抑制因子IκBα在大鼠皮肤挫伤后不同时间在多核粒细胞、单核巨噬细胞及成纤维细胞中的表达变化，揭示其与皮肤挫伤时间的关系，为皮肤挫伤时间的判定提供新的理论依据。材料和方法 1．动物模型制作及分组健康成年Sprague-Dawley大鼠33只，雌雄不限，体重180±20g，随机分为11组，每组3只，其中10组作为实验组，1组作为对照组。用250g重锤在150cm高度自由落下，造成右后肢股四头肌处皮肤挫伤，分别于挫伤后0h，3h，6h，9h，12h，1d，3d，，5d，7d，10d将大鼠脱颈椎处死，于挫伤处取1.5cm×1.5cm皮肤组织。对照组大鼠取与挫伤大鼠相同部位同等大小皮肤组织作为对照。4％多聚甲醛／PBS固定后石蜡包埋，连续5μm厚度切片。 2．实验方法应用免疫组织化学SABC法，步骤如下: (1)切片脱蜡水化; (2)3%过氧化氢孵育20而n，微波抗原修复(PH6 .0的柠檬酸缓冲液中，微波炉加热至沸腾，间隔smin，连续三次); (3)正常山羊血清孵育，室温下20面n; (4)加人1:200倍稀释的兔抗鼠多克隆NFKB巧5抗体，IKBa抗体，4℃ 过夜; (5)滴加生物素标记的山羊抗兔I娇抗体，保湿盒内25℃温箱中孵育 20min; (6)滴加SABC试剂，保湿盒内25℃温箱中孵育20而n; (7)DAB显色5一10而n，水洗、苏木素复染、透明、中性树胶封片。以上抗体及SABC试剂盒均购自武汉博士德生物有限公司。 3.统计学分析采用SPSS for而ndows n .0软件包，应用方差分析进行统计学处理。实验结果 1.NFKB P65与IKBa在挫伤修复过程中的表达变化正常对照组皮肤中，NFKB P65与IKBa在表皮基底细胞层、棘细胞层、颗粒细胞层和真皮层皮脂腺、汗腺上皮细胞呈阳性表达。实验组标本中，伤后Oh皮肤染色与正常皮肤基本相同;伤后3h组，在浸润的少量多核粒细胞的胞质中NFKB P65与IKBa呈阳性表达;伤后6h一12h，表达增加，伤后ld，在浸润的几乎所有的单核细胞和多核粒细胞的胞质和胞核中NFKB巧5呈强阳性表达，IKBa也在大多数细胞中表达;伤后3d组，成纤维细胞大量增生，阳性细胞主要以成纤维细胞为主;5d一10d组阳性细胞数逐渐减少。 2.阳性细胞计数及统计学分析 2.1 NFKB P65阳性细胞计数:伤后3h，NFKB P65阳性细胞率为(3. 20%士0.58%);伤后6h，NFKB P65阳性细胞率开始升高(11.33%士1. 53%);gh一12h继续升高，ld达高峰(97.33%土1.53%);伤后3d，阳性细胞率开始下降(79.33%土3.05%);sd一7d继续下降，伤后10d，阳性细胞率下降至(17.67%士2.52%)。 2.2 IKBa阳性细胞计数:伤后3h，IKBa阳性细胞率为(3.33%*0. 58%)，伤后6h，IKBa阳性细胞率开始升高(1 1 .00%士1 .00%)，gh一12h继续升高，ld达高峰(84.67%土2.52%);伤后3d，阳性细胞率开始下降(73. 00%土2.00%)，sd一7d继续下降，伤后10d，阳性细胞率下降至(13.33% 土1 .53%)。 2 .3统计学方差分析:经统计学方差分析，相邻两组阳性细胞率均存在显著差异(P0.01)，且各组与1d组相比阳性细胞率均存在显著差异(P 0 .01)。讨论核转录因子NFKB(nuelear factor kappaB，NFKB)是1986年由Sen和 Baltimere首先从B淋巴细胞抽提物中检测到的一种能与免疫球蛋白k链基因增强子B位点结合，调控免疫球蛋白k链基因转录的核蛋白因子。细胞未受到刺激时，细胞中的NFKB处于未活化状态，不具有调节基因转录的功能，此时，NFKB成员通常以同源二聚体或异源二聚体形式与其抑制蛋白 IKB结合形成三聚体复合物，以非活性形式存在于细胞质中。当细胞受到病毒、放射线、氧自由基、细胞因子如TNFa、IL一1等细胞外因素刺激时， NFKB从NFKB一IKB复合物中解离出来，并迅速移位至细胞核，从而发挥转录调节功能。越来越多的研究表明，NFKB在炎症反应、免疫应答中起重要作用。 NFKB P65是NFKB家族中最为重要的成员，而IKBa是NFKB的主要抑制蛋白之一。本实验应用免疫组织化学技术观察到在皮肤挫伤修复过程中 NFKB P65与 IKBa在皮肤挫伤区及周边区?
[Abstract]:Preface
Skin contusion is a common injury in forensic identification. Therefore, the inference of the time of skin contusion has become a hot topic in the field of forensic medicine at home and abroad for many years.
The initial contusion time was deduced based on the changes in the skin color of the contusion, but the time of Skin Contusion could not be accurately judged by the individual difference of skin color and age. The amount of inflammatory cells in the inflammatory reaction after the skin contusion was observed by the optical microscope. It is difficult to accurately infer the time of contusion, so people have been looking for a relatively accurate method to infer the time of skin contusion. With the improvement of the posttraumatic molecular pathology in the medical field, the changes in the biological substances after the trauma are being paid more and more attention to, and for the forensic workers to infer the skin contusion. A new way is provided.
The nuclear transcription factor NF kappa B (nuclear factor kappa B, NF kappa B) is an important biological factor that regulates the expression of a variety of cytokines, such as growth factors, cytokines and adhesion molecules. Recent studies have shown that NF kappa B is closely related to the trauma of.I kappa B alpha because it inhibits the activity of nuclear kappa. Therefore, it has also been paid more and more attention. Therefore, this experiment is applied to this experiment. The expression of NF kappa B P65, an important member of the NF kappa B family, and its inhibitory factor I kappa B alpha in the rat skin contusion, and the changes in the expression of the mononuclear macrophages and fibroblasts at different time after the skin contusion in rats were observed by immunohistochemical method. The relationship between the time of Skin Contusion and the time of skin contusion was revealed, which provided a new theoretical basis for the determination of the time of skin contusion.
Materials and methods
1. animal model making and grouping
33 healthy adult Sprague-Dawley rats, male and female, 180 + 20g, were randomly divided into 11 groups, 3 in each group, of which 10 groups were used as experimental group and 1 group as control group. The 250g weight was used as the control group. The skin contusion of the four head of the right hind leg of the right hind limb was caused by 0h, 3h, 6h, 9h, 12h, 1D, 3D, 5D. 1.5cm x 1.5cm skin tissue was taken at the contusion site. The same size skin tissue of the control group was taken as the control.4% paraformaldehyde / PBS fixed paraffin embedded in the control group, and the thickness of the skin was 5 mu m continuously.
2. experimental method
The immunohistochemical SABC method was used as follows:
(1) the slices were dewaxing and hydrated;
(2) 3% H2O2 incubated with 20 and N, microwave antigen repair (PH6.0 citric acid buffer)
The microwave oven was heated to boiling, separated by smin for three consecutive times.
(3) the normal goat serum was incubated and 20 N at room temperature.
(4) adding 1:200 times diluted Rabbit anti mouse polyclonal NFKB Qiao 5 antibody, IKBa antibody, 4 degrees centigrade
Pass the night;
(5) the Goat anti rabbit I antibody labeled with biotin was incubated in the incubator at 25 degrees centigrade incubator.
20min;
(6) SABC reagent was added to the incubator and incubated with 20 and N in the 25 degree incubator.
(7) DAB chromogenic 5 1 10 and N, washed, hematoxylin dyed, transparent, neutral gum wrap.
Antibodies and SABC kit were purchased from Wuhan doctorate biological Co., Ltd.
3. statistical analysis
Using SPSS for and ndows n.0 software package, ANOVA was used for statistical analysis.
experimental result
Expression changes of 1.NFKB P65 and IKBa during contusion repair
In the skin of normal control group, NFKB P65 and IKBa were found in the basal cell layer and spinous cell layer of epidermis.
The granular cell layer and the sebaceous glands of the dermis were positive for sweat gland epithelial cells.
After Oh, the skin staining was basically the same as that of the normal skin. In the 3H group, a small number of multinucleated granulocytes were infiltrated.
The expression of NFKB P65 and IKBa in the cytoplasm was positive, and the expression of 6h 12h increased after injury, LD after injury.
In the cytoplasm and nucleus of almost all mononuclear cells and multinucleated granulocytes, NFKB was 5.
IKBa was also expressed in most of the cells. After 3D, there was a large increase in fibroblasts.
The positive cells were mainly fibroblasts, while the number of positive cells in 5D 10d group gradually decreased.
2. positive cells count and statistical analysis
2.1 NFKB P65 positive cell count: 3h after injury, NFKB P65 positive cell rate was (3.
20%, 0.58%). The rate of 6h, NFKB P65 positive cells began to increase after injury (11.33% 1.).
53%); GH 12h continued to rise, LD reached the peak (97.33% soil 1.53%); 3D after injury, positive.
The cell rate began to decrease (79.33% soil 3.05%); SD 1 7d continued to decline, and 10d positive cells after injury.
The rate dropped to (17.67%. 2.52%).
2.2 IKBa positive cell count: 3h after injury, IKBa positive cell rate was (3.33%*0.
58%) after injury, the positive rate of 6h and IKBa cells began to increase (11% 1%), and GH one 12h continued.
The LD reached the peak (84.67% soil 2.52%) and the percentage of positive cells began to decrease after 3D (73.
00% soil 2%), SD 7d continued to decline, 10d after injury, the percentage of positive cells decreased to (13.33%
Soil 1.53%).
2.3 statistical variance analysis: by statistical variance analysis, the positive rates of two adjacent groups all exist.
Significant difference (P0.01), and the rates of positive cells in each group were significantly different from those in group 1D (P
0.01).
discuss
The nuclear transcription factor NFKB (Nuelear factor kappaB, NFKB) was formed in 1986 by Sen and
Baltimere first detected a K chain from immunoglobulin in B lymphocyte extracts.
B enhancer locates the nuclear protein factor that regulates the transcription of immunoglobulin K chain gene.
When the cell is not stimulated, the NFKB in the cell is not activated and does not regulate gene transcription.
At this time, NFKB members usually form homologous two dimers or heterologous two dimers and their inhibitory proteins.
IKB forms a trimer complex and forms in the cytoplasm in inactive form.
When virus, radiation, oxygen free radicals, cytokines such as TNFa, IL 1 and other extracellular factors are stimulated,
NFKB dissociated from the NFKB IKB complex and rapidly shifted to the nucleus, thus making the switch.
More and more studies have shown that NFKB plays an important role in inflammatory response and immune response.
Effect.
NFKB P65 is the most important member of NFKB family, and IKBa is the main inhibition of NFKB.
In this experiment, immunohistochemical staining was used to observe the process of Skin Contusion repair.
NFKB P65 and IKBa in the skin contusion area and surrounding area?

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2004
【分类号】：D919

【参考文献】