小鼠皮肤切创愈合过程中巨噬细胞炎性蛋白-1α的表达及其与损伤时间的关系

发布时间：2018-05-11 03:29

本文选题：MIP-1α + 趋化因子　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的:创伤愈合是一个包括损伤组织炎症反应,增生增殖和成熟阶段的一个复杂生物学反应。许多不同种类的生物物质与创伤愈合的各个阶段比如促进炎症反应,肉芽组织的生成以及血管反应等密切相关。在法医病理学中,这些生物物质在皮肤损伤中的表达可以用于损伤时间的推测。趋化性细胞因子又称趋化因子,它们参与白细胞迁移、激活和趋化,在炎症反应中起核心作用。MIP-1α属趋化因子CC亚族,分子量为8KD。MIP-1α可在不同因素刺激下被细菌,病毒及IL-1、TNF-α等炎性细胞因子诱导产生,据报道细胞来源的MIP-1α可由单核/巨噬细胞、中性粒细胞、成纤维细胞、内皮细胞、及某些肿瘤细胞等多种细胞产生。MIP-1α的靶细胞有单核细胞、中性粒细胞、酸性粒细胞、NK细胞、树突状细胞等,其通过受体介导与靶细胞结合发挥生物学活性.MIP-1α与其受体结合后,有趋化免疫细胞、抑制造血干细胞生长及抑制HIV感染的作用。目前国内外对MIP-1α研究多集中在临床领域,而在法医学领域的研究较少,尤其是国内对MIP-1α在皮肤机械性损伤修复时间的推断方面几乎未见报道。本研究通过建立小鼠皮肤切创损伤模型,运用免疫组化技术及其阳性细胞计数法对损伤组织进行MIP-1α的检测与分析,探讨在皮肤创伤愈合过程中其表达的时序性变化规律,以便为法医病理学损伤时间的推断提供一个新的理论依据。方法:采用完全随机分组方法,分为实验组和对照组。实验组又分为生前造创组和死后造创组,生前造创组根据造创后处死时间不同将实验小鼠随机分为伤后1h、3h、6h、12h、1d、3d、6d、10d、14d共9组,死后造创组分为0.5 h,1 h,3 h,6h共4组;另设一组为正常对照组,即未造创组。生前造创组在小鼠背部制做全层切创,在预定时间分别断颈处死后即刻取材,死后造创组于处死小鼠后在预定时间造创取材,应用免疫组织化学技术及阳性细胞计数法观察伤后小鼠切创组织中不同时程的生前伤和死后伤皮肤创伤局部MIP-1α的表达,并和无切创的小鼠作为对照,探讨MIP-1α在小鼠皮肤切创损伤愈合过程中的表达分布变化规律及其与损伤时间的关系. 结果:1、HE染色观察:伤后1d内,可见大量炎性细胞游出,主要以中性粒细胞浸润为主;伤后3～6d,肉芽组织逐渐形成并大量增生,炎性细胞以单核-巨噬细胞为主;伤后10～14d,肉芽组织逐渐成熟,炎性细胞消失,创口基本愈合。死后造创组及正常对照组镜下无上述改变。2、免疫组化SABC法染色观察:伤后1d内,MIP-1α在大量浸润的中性粒细胞中呈阳性染色及在单核细胞中呈散在阳性表达;伤后3d,MIP-1α在逐渐形成并大量增生的肉芽组织中呈阳性染色,其主要在大量的单核细胞内表达并在开始增生的成纤维细胞内表达;其后到14d MIP-1α阳性表达在部分成纤维细胞内,但染色范围和强度明显减弱。死后造创组(0.5h,1h组)及正常对照组在部分表皮细胞及毛囊等表达弱阳性,死后造创3h,6h组表达阴性。3、阳性细胞计数法结果:阳性细胞率(PR值)在伤后6h到伤后3d逐渐上升,伤后3d达到高峰,伤后10～14d,PR值逐渐下降。4、统计学处理结果:方差分析结果表明生前创伤各相邻两组之间MIP-1αPR值相比均具有显著差异(P0.01)。其中生前创伤3d组与其他各时间组相比均有显著差异(P0.01)。结论: 1、生前造创组MIP-1α在炎症细胞中的表达量的变化具有规律性,呈现出升高期-高峰期-下降期的时序性变化曲线,其中造创后3d达到表达最高峰。2、MIP-1α表达阳性细胞的变化具有规律性,呈现出早期以中性粒细胞为主,高峰期以单核-巨噬细胞为主,后期以部分成纤维细胞表达为主的动态变化趋势,死后造创组(0.5h,1h组)及正常对照组在部分表皮细胞及毛囊等表达。3、MIP-1α在小鼠皮肤切创愈合过程中呈现一定时序性变化,有望作为法医损伤时间推断的一个有用的参考指标,以及对于死后伤如1h以内的早期损伤也有着重要的法医学意义。
[Abstract]:Objective: wound healing is a complex biological response to the inflammatory response, proliferation and maturation of the injured tissue. Many different kinds of biological substances are closely related to the various stages of wound healing, such as the promotion of inflammation, the formation of granulation tissue, and the reaction of blood vessels. In forensic pathology, these biomarkers The expression of substance in skin damage can be used to speculate on the time of damage. Chemokines are also known as chemokines, which participate in leukocyte migration, activation and chemotaxis, and play a core role in the inflammatory response of.MIP-1 a CC subgroup of chemokine, and the molecular weight of 8KD.MIP-1 alpha can be affected by bacteria, viruses and IL-1, TNF- alpha under the same factors. Induced by sex cytokine, it is reported that the cell source MIP-1 alpha can be produced by mononuclear cells, neutrophils, acidic granulocytes, acid granulocytes, NK cells, dendritic cells and so on, which are mediated by the receptor and targeted by the target cells, which can be produced by a variety of cells, such as mononuclear / macrophage, neutrophils, fibroblasts, endothelial cells, and some tumor cells. Combining with biologically active.MIP-1 alpha and its receptor, cellular binding can chemotaxis immune cells, inhibit the growth of hematopoietic stem cells and inhibit the effect of HIV infection. At present, the study of MIP-1 alpha mainly focuses on the clinical field, but there are few studies in the field of forensic medicine, especially the time for the repair of MIP-1 alpha in the repair time of the mechanical damage of the skin. In this study, a mouse skin incision injury model was established, and the immunohistopathological technique and the positive cell count method were used to detect and analyze the MIP-1 alpha of the injured tissue, and the temporal variation of the expression during the healing process of skin wound was discussed in order to provide the inference for the time of forensic pathology injury. A new theoretical basis.
Methods: the experimental group and the control group were divided into the experimental group and the control group. The experimental group was divided into the pre birth and the postmortem group. The experimental mice were randomly divided into 9 groups, including 1H, 3h, 6h, 12h, 1D, 3D, 6D, 10d, 14d after the injury. The group was divided into 0.5 h, 1 h, 3 h, and 4 groups. In the normal control group, that is, the non creation group. The group was made in the back of the mice in the whole layer to cut the whole layer on the back of the mice, and immediately took the material after the death of the neck at the predetermined time. After death, the group was sacrificed at the predetermined time after the death of the mice. The immuno histochemical technique and the positive cell count were used to observe the different time injuries of the mice after the injury. The expression of local MIP-1 alpha in skin wounds after injury and injury was compared with that of noninvasive mice. The change of expression distribution of MIP-1 alpha in the healing process of skin incision injury in mice and its relationship with the time of injury were discussed.
Results: 1, HE staining observation: in 1D after injury, a large number of inflammatory cells were seen, mainly infiltration of inflammatory cells, mainly neutrophil infiltration, 3 to 6D after injury, granulation tissue gradually formed and proliferated, inflammatory cells were mainly mononuclear macrophages; 10 to 14d after injury, granulation tissue gradually mature, inflammatory cells disappeared, the wound was basically healed. Postmortem group and positive In the control group, there was no.2 and immunohistochemical SABC staining. In 1D, MIP-1 alpha was positive in a large number of infiltrating neutrophils and scattered in mononuclear cells, and 3D, MIP-1 alpha was positive after the injury, and it was mainly in a large number of mononuclear cells. Expression and expression in the fibroblasts which began to proliferate; then 14d MIP-1 alpha was positive in some fibroblasts, but the range and intensity of dyeing decreased obviously. After death, the expression of 0.5h, 1H group and normal control group were weakly positive in some epidermal cells and hair follicles. After death, 3h, 6h negative.3, positive cell count method were used. Results: the positive cell rate (PR) increased gradually from 6h to 3D after injury, and the 3D reached its peak after injury, 10 to 14d after injury, and the PR value gradually decreased by.4. The results of statistical analysis showed that the MIP-1 alpha PR values between the adjacent two groups were significantly different (P0.01). The preoperative trauma 3D group was compared with the other time groups. There were significant differences (P0.01).
Conclusion: 1, the changes in the expression of MIP-1 alpha in the inflammatory cells in the pre creation group are regular, showing a time series change curve of the peak period and the decline period, in which the 3D reached the peak of.2, and the change of the MIP-1 alpha expression positive cells is regular, and the early stage is mainly neutrophils, and the peak period is mononuclear at the peak period. - macrophage was the main trend of the expression of partial fibroblasts in the later period..3 was expressed in some epidermal cells and hair follicles in the postmortem group (0.5h, 1H group) and the normal control group. MIP-1 alpha showed certain temporal changes during the healing process of the skin incision in mice. It is expected to be a useful reference for forensic injury time. There is also an important forensic significance for the index and early injury of postmortem injury, such as 1H.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919

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