大鼠皮肤切创愈合过程中细胞间粘附分子及P选择素表达的研究

发布时间：2018-05-12 03:46

本文选题：皮肤损伤 + 细胞间粘附分子-1　；参考：《中国医科大学》2003年硕士论文

【摘要】： 前言皮肤损伤愈合是一个极其复杂的生物学过程，通常其包括三个阶段：炎症期、增殖期及组织重构期。多种生物学介质如：生长因子、细胞因子及粘附分子等与皮肤损伤愈合过程密切相关，尤为值得提出的是，粘附分子在炎症的发生的起始过程中起着至关重要的作用，这些因子在细胞外基质的形成、血管的发生、肉芽组织的生成及上皮的再形成方面等均具有重要作用。对于法医病理工作者来说，对这些生物物质变化过程的研究，可以为判定损伤活性及时间提供很好的方法。粘附分子之所以称之为粘附分子，是因为其首要功能就是将淋巴细胞、单核细胞及粒细胞粘附在血管内皮细胞上，使其穿越血管内皮，到达炎症反应处。本研究应用免疫组织化学方法，观察大鼠皮肤切创后不同时间细胞间粘附分子(ICAM-1)及P选择素的表达变化，以期确定这两种物质表达与损伤时间变化的规律，为皮肤损伤时间判定提供有效的免疫组织化学指标。材料与方法 1．材料取雄性健康SD大鼠39只，随机分为13组，每组3只。于伤后5min-7d将大鼠断颈处死，于伤口边缘处取全层皮肤组织。取9只雄性健康SD大鼠，随机分为3组做为死后伤对照组，取3只雄性健康SD大鼠，取与致伤大鼠相同部位皮肤作为对照。石蜡包埋，进行常规石蜡切片，厚度为 5 4m。 2．方法应用免疫组织化学SABC法，步骤如下：＊石蜡切片常规脱蜡至水； 2．3％的过氧化氢一甲醇孵育10分钟，微波抗原修复； 3．3％正常山羊血清孵育，室温下 20分钟。 4．力人适当稀释的一抗（1＊M－1：1：20O＊选择素1。10o）， 4℃过夜； 5．滴加生物素标记的山羊抗兔IgG，室温下20分钟； 6．滴加试剂SABC，室温孵育20分钟； 7．DAB显色 5则分钟，水洗，苏木素复染、透明、中性树胶封片。以上抗体及SABC试剂盒均购自于武汉博士德生物工程有限公司c 3．统计学分析采用 SPSS for windowslo．0软件包，应用方差分析进行统计学处理。实验结果 ICAM－1在正常皮肤对照组的表皮层、毛囊、皮脂腺中呈淡黄色染色；死后伤组中毛囊及皮脂腺着色较淡或不表达。P选择素在正常皮肤对照组及死后伤组中均未见表达。在伤后lh，即可见大鼠损伤边缘处表皮ICAM一互呈黄褐色染色，伤后3h组， ICAM一豆在表皮层中呈深黄褐色，尤以基底细胞及棘细胞表达为明显，在毛囊皮脂腺的染色亦增强，上述变化一直持续至伤后＊，伤后3d组ICAM一二染色转淡，呈黄褐色染色，在伤后sd及7d 组，ICAM一表达基本恢复至正常状态。在伤后10分钟即可见P 选择素在皮肤组织中呈阳性表达，主要表达在创缘底部毛细血管 ·2· 内皮细胞及白细胞上，一直持续至伤后sh组，至伤后7h＊选择素已恢复至正常状态。除此之外，ICAM一二还在炎症细胞及纤维母细胞中表达，且随损伤时间变化而呈规律性变化（第1组：损伤时间sndn 一lh，第＊组：3h一7h，第Ill 组：gb一12h，第IV组：id，第 V组：3d，第VI组：sd－7d人J［CAM一且在第二组（平均值。标准误：0．41 LO．73％）中阳性细胞．率极低，第＊组”．79。3．74％）及第＊组K3．33 LI．10％）均呈显著性增高，至第IV组门0．58。 z 65％）时达高峰，其后逐渐减少。经统计学方差分析，相邻两组阳性细胞率均存在显著差异，且各组与第IV组比较阳性细胞率亦存在显著差异。h＜0．05或0．of人讨论 ICAM司属于粘附分子中的免疫球蛋白超家族，为LFA－1 的配体，ICAM一二与LFA一二的相互作用可介导上皮细胞与淋巴细胞间的粘附；介导单核巨噬细胞、中性粒细胞的粘附。为细胞粘附所必需。选择素P主要参与介导白细胞与内皮细胞的起始粘附。法医病理学上关于 ICAM·一的研究已有很多，主要集中在应用ICAMJ判定心肌缺血及脑损伤时间可行性方面。 ICAM人在正常皮肤的表皮层、毛囊、皮脂腺中呈淡黄色染色，由于皮肤经常接受各种外来刺激，需要相对较快的细胞更新，而在正常皮肤中出现ICAM－l的表达，说明其在维持皮肤生物学稳态方面有重要作用。本实验研究证明，皮肤损伤后表皮层 ICAM1表达增高，其表达呈现一定时间规律性，在损伤后 lh，损伤边缘表皮细胞即呈黄褐色染色，伤后 3 h组，ICAM一二在表皮层中呈深黄褐色，并一直持续至伤后 id，伤后 3 d组 ICAM－1染色转淡，呈黄褐色染色，在伤后sd及7d组，1＊＊M一二表达基本恢复至正常状态。这一方面可以说明角质细胞可表达ICAM－＊另一方 ·3· 面也说明了ICAM一二在角质细胞的再生过程中有着重要作用。有学者认为，特定粘附分子及其相应配体的表达水平和结合的亲合力是不同类型炎症发生过程中重要的分子基础，在炎症起始阶段，粘附分子在炎症因子的刺激作用下而活化，使白细胞粘附、穿越血管内皮细胞向炎症部位移行。我们应用免疫组化技术证明皮肤损伤区中?
[Abstract]:Foreword

The healing of skin lesions is a very complicated biological process , which usually consists of three stages : inflammation stage , proliferation period and tissue reconstruction period . It is especially worth mentioning that adhesion molecules play a crucial role in the process of inflammation . These factors play an important role in the formation of extracellular matrix , angiogenesis , formation of granulation tissue and the formation of epithelium .

The adhesion molecule is called an adhesion molecule , because its primary function is to adhere lymphocytes , monocytes , and neutrophils to vascular endothelial cells , through the vascular endothelium , to the inflammatory reaction .

In this study , the expression of intercellular adhesion molecule ( ICAM - 1 ) and P - selectin ( P - selectin ) at different time after skin - cutting were observed by immunohistochemical method .

Materials and Methods

1 . Materials

Thirty - nine male healthy SD rats were randomly divided into 13 groups . The rats were sacrificed at 5 min to 7 d after injury . All the skin tissues were taken at the edge of the wound . Nine male healthy SD rats were randomly divided into 3 groups .

Wax was embedded and paraffin sections were paraffin sections with a thickness of 5 m .

2 . Method

The immunohistochemistry SABC method was applied in the following steps :

removing paraffin sections from normal paraffin sections to water ;

2 . 3 % hydrogen peroxide and methanol are incubated for 10 minutes , and the microwave antigen is repaired ;

3.3 % normal goat serum was incubated at room temperature for 20 minutes .

4 . An anti - ( 1 * M - 1 : 1 : 20 O * selectin 1.10o ) diluted appropriately by a force person ,

overnight at 4 & deg ; C ;

5.dripping biotin - labeled goat anti - rabbit IgG at room temperature for 20 minutes ;

6 . dropwise adding reagent SABC and incubating at room temperature for 20 minutes ;

7 . DAB color development for 5 minutes , water washing , Su - lignin compound dyeing , transparent , neutral gum

The above antibody and SABC kit are purchased from Wuhan Docket Biological Engineering Co . , Ltd .

Limit company c

3 . Statistical Analysis

Statistical analysis was carried out by SPSS for windowsill . 0 software package .

Learn to deal with it .

experimental results

The expression of ICAM - 1 in the epidermis , hair follicle and fat gland of the normal skin control group was pale .

Yellow staining ;
The color of the hair follicle and the skin glands in the injured group were less or not expressed . P selection

In the normal skin control group and the injured group , the expression was not found in the control group and the injured group after injury .

The expression of ICAM - 1 in the epidermis of the injured rat was yellow - brown stain , 3h after injury ,

The expression of ICAM - 1 in the epidermal layer is dark brown , especially in basal cells and ratchet cells .

Obviously , the staining of the hair follicle epithelial glands is also enhanced , and the above - mentioned changes have been continued until after injury * ,

The expression of ICAM - 1 in the 3d group was pale yellow and brown , and sd and 7d after injury .

The expression of ICAM - 1 was restored to the normal state , and the expression of ICAM - 1 was observed in 10 minutes after injury .

The positive expression of selectin in skin tissue was mainly expressed at the bottom of the wound .

路 2 路

The endothelial cells and the white blood cells were continuously injured in the post - wound sh group , and then to 7 h after injury .

It has been restored to normal state . In addition , ICAM - 1 is also in inflammatory cells and fibers .

Expression in cells and changes in regularity with time of injury ( group 1 : injury )

intersndn - 1 h , group * : 3h - 7 h , group III : gb - 12 h , group IV : id , p .

Group V : 3d , Group VI : sd - 7d Human J &bra; CAM - 1 &ket; and in the second group ( mean .

False : 0.41 LO . 73 % ) positive cells . Very low , group * " . 79 . 3.74 % ) and

There was a significant increase in Group * ( K3 . 33 LI . 10 % ) to the group IV gate 0.58 .

Up to the peak at the time of z 65 % , followed by a gradual decrease . After statistical analysis , the two groups were adjacent .

The positive rate of positive cells was significantly different , and the rate of positive cells in each group was also higher than that in group IV .

There was a significant difference . h & lt ; 0.05 or 0 . of people

discussion

The ICAM - 1 belongs to the immunoglobulin superfamily of adhesion molecules .

The interaction between the ligand , the ICAM - 1 and the lA - 2 can mediate the epithelial and lymph nodes .

intercellular adhesion ;
mediate adhesion of mononuclear phagocytes and neutrophils .

The essential part of selectin P is involved in mediating the initial viscosity of leukocytes and endothelial cells .

There are many researches on ICAM - 1 in forensic pathology , which mainly focus on the study of ICAM - 1 .

ICAMJ was used to determine the temporal feasibility of myocardial ischemia and brain injury .

The ICAM was pale yellow in the epidermis , hair follicle , and the skin of the normal skin .

color , a relatively fast cell update is required because the skin is often subjected to a variety of external stimuli ,

The expression of ICAM - 1 in normal skin indicated that it was in the maintenance of skin biology .

There is an important role in the steady - state aspect of the study . This experimental study demonstrates that skin lesions after skin damage

The expression of ICAM1 increased , and the expression of ICAM1 showed a certain time regularity . After injury , the expression of ICAM1 increased .

The epidermal cells of the wound were yellow - brown stain , 3 h after injury , ICAM - 1 in the epidermal layer .

The medium was dark brown , and persisted to the post - wound id , and ICAM - 1 in the 3 d group after injury was stained .

The expression of 1 * * M was basically recovered in the sd and 7d groups after injury .

Normal status . This aspect may indicate that keratinocytes can express ICAM - *

路 3 路

The expression of ICAM - 2 plays an important role in the regeneration of keratinocytes .

Some scholars believe that the expression level and binding affinity of specific adhesion molecules and their corresponding ligands

The resultant force is an important molecular basis in the process of the occurrence of different types of inflammation , and in the initial stage of inflammation

The adhesion molecule is activated by the stimulation of an inflammatory factor to cause leukocyte adhesion , penetration ,

The more vascular endothelial cells migrate to the site of inflammation .

Skin damage 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】：2003
【分类号】：D919

【参考文献】