小鼠皮肤切创愈合过程中Ⅱ型大麻素受体（CB2R）表达的研究
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摘要
目的
皮肤损伤愈合是一个复杂而连续的过程,其大致可以分为三个阶段：炎症期、增殖期和重建修复期,其各阶段是相互联系,相互影响的,有大量细胞、细胞因子和炎症介质的参与其间。Ⅱ型大麻素受体(cannabinoid receptor type 2, CB2R)为G蛋白偶联受体,能激活多种信号转导通路,主要分布于外周,如免疫系统和造血细胞。新近研究发现CB2R与多种不同疾病和组织损伤有关,发挥重要的抗炎和抗纤维化的作用。本研究应用免疫组织化学染色和Western blot技术,检测小鼠皮肤损伤区在损伤后不同时间CB2R表达的变化情况,探讨CB2R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
健康成年清洁级昆明系小鼠50只,体质量35g-40g,随机分为10组,每组5只,其中1组为正常对照组。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,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蛋白印迹技术应用。应用免疫组织化学和Western blot方法检测50例小鼠皮肤切创后不同时间CB2R变化情况,以非切创小鼠皮肤组织作对照,显微镜x400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软件测各片段的平均光密度值,测得数据用均数±标准差(x±s)表示。用SPSS13.0 for Windows进行单因素方差分析,以P<0.05为差异有显著性意义。
免疫组织化学结果显示,对照组CB2R表达于表皮、毛囊、皮脂腺、皮肌层、血管平滑肌、神经纤维外膜及神经纤维束束膜；伤后6h切创皮肤损伤区及周边区多核粒细胞少量表达CB2R,12h-24h以单个核细胞阳性表达为主,3d以单个核细胞和梭形成纤维细胞表达为主,5d-14d以梭形成纤维细胞为主。CB2R阳性细胞率于0h-3d逐渐升高,5d达最高峰,随后逐渐下降。经Western blotting检测显示,对照及各个损伤时间段都有CB2R阳性条带,其中5d为CB2R表达高峰。
结论
小鼠皮肤切创愈合过程中,CB2R于损伤区及周边区多核粒细胞、单个核细胞、成纤维细胞中表达,并具有规律性,提示CB2R可能参与皮肤损伤愈合,并可用于皮肤损伤时间的推断。
Objective
Skin wound healing is continuous and complex process, which has been simply divided into three phases:inflammation, proliferation, and remodeling. Within each phase, a myriad of orchestrated reactions and interactions between cells, cytokine and inflammatory mediators are put into action. cannabinoid receptor type 2 (CB2R), is a G-protein-coupled receptor that activates in a multitude of signal transductional pathways. CB2R is predominantly expressed in peripherally, such as immune system and hematopoietic cells. More recent studies have found that CB2R is involved in a wide range of disparate diseases and tissue injuries, and plays an important role of anti-inflammatory and anti-fibrosis. In this study, the level of CB2R in skin incised wound was detected by immunohistochemical technique and western blotting. The aim of this investigation is to explore the possible function of CB2R in skin wound healing and its applicability to the wound age estimation.
Materials and Methods
A total of 50 mice, each weighting 35-40g, were divided into ten groups randomly, one control group and nine experiment groups, A 1.5cm-long full-thickness incision deep to the fascia was made with a scalpel in the skin layer on the central dorsum of each mouse under sterile technique after the mouse was anesthetized by inhalating diethyl ether. After injury, each mouse was housed individually and fed with sterilized food and distilled water to prevent from infection. The 1.5cm×1.0cm specimens were taken from the wounded site after the mice were executed by cervical dislocation at Oh,6h,12h, 1d,3d,5d,7d, 10d and 14d postinjury to prepare for the subsequent procedures. The level of CB2R in mouse skin incised wound was detected by immunohistochemical staining and Western blot, and the non-incised mouse skin was used as control. The number and ratio of CB2R positive cells were counted and analyzed by microscope. The band AverGray was analyzed by software of Fluochem V2.0. The data were analyzed comparatively by the software of SPSS13.0 for Windows, and there was statistical significance comparing with the neighboring group when P<0.05.
Results
CB2R immuroreactivity was detected in epidemical layer, hair follicle, sebaceous gland, dermomuscular layer, vascular smooth muscle, nerve fiber adventitia and perimysium in the control skin; weak CB2R immunostaining was detected in polymorphonuclear cells (PMNs) in the wound and peripheral region in aged 6h, a large number of CB2R-positive mononuclear cells (MNCs) were identified from 12h to 24h, Afterwards, the CB2R-positive cells were mostly MNCs and spindle-shaped fibroblastic cells (FBCs) at 3d postinjury, and mainly in spindle-shaped fibroblastic cells from 5d to 14d postinjury. The ratio of the CB2R-positive cells increased gradually in the wound specimens from Oh to 3d, and maximized at 5d, and decreased from 7d to 14d postinjury. The bands of CB2R expression were observed throughout the wound healing stages by Western blotting, and maximized at 5d.
Conclusion
The results suggested that CB2R was expressed in PMNs, MNCs, FBCs during healing process of skin wound in mice, and may be used as a newmarker for the wound age determination.
皮肤损伤愈合是一个复杂而连续的过程,其大致可以分为三个阶段：炎症期、增殖期和重建修复期,其各阶段是相互联系,相互影响的,有大量细胞、细胞因子和炎症介质的参与其间。Ⅱ型大麻素受体(cannabinoid receptor type 2, CB2R)为G蛋白偶联受体,能激活多种信号转导通路,主要分布于外周,如免疫系统和造血细胞。新近研究发现CB2R与多种不同疾病和组织损伤有关,发挥重要的抗炎和抗纤维化的作用。本研究应用免疫组织化学染色和Western blot技术,检测小鼠皮肤损伤区在损伤后不同时间CB2R表达的变化情况,探讨CB2R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
健康成年清洁级昆明系小鼠50只,体质量35g-40g,随机分为10组,每组5只,其中1组为正常对照组。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,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蛋白印迹技术应用。应用免疫组织化学和Western blot方法检测50例小鼠皮肤切创后不同时间CB2R变化情况,以非切创小鼠皮肤组织作对照,显微镜x400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软件测各片段的平均光密度值,测得数据用均数±标准差(x±s)表示。用SPSS13.0 for Windows进行单因素方差分析,以P<0.05为差异有显著性意义。
免疫组织化学结果显示,对照组CB2R表达于表皮、毛囊、皮脂腺、皮肌层、血管平滑肌、神经纤维外膜及神经纤维束束膜；伤后6h切创皮肤损伤区及周边区多核粒细胞少量表达CB2R,12h-24h以单个核细胞阳性表达为主,3d以单个核细胞和梭形成纤维细胞表达为主,5d-14d以梭形成纤维细胞为主。CB2R阳性细胞率于0h-3d逐渐升高,5d达最高峰,随后逐渐下降。经Western blotting检测显示,对照及各个损伤时间段都有CB2R阳性条带,其中5d为CB2R表达高峰。
结论
小鼠皮肤切创愈合过程中,CB2R于损伤区及周边区多核粒细胞、单个核细胞、成纤维细胞中表达,并具有规律性,提示CB2R可能参与皮肤损伤愈合,并可用于皮肤损伤时间的推断。
Objective
Skin wound healing is continuous and complex process, which has been simply divided into three phases:inflammation, proliferation, and remodeling. Within each phase, a myriad of orchestrated reactions and interactions between cells, cytokine and inflammatory mediators are put into action. cannabinoid receptor type 2 (CB2R), is a G-protein-coupled receptor that activates in a multitude of signal transductional pathways. CB2R is predominantly expressed in peripherally, such as immune system and hematopoietic cells. More recent studies have found that CB2R is involved in a wide range of disparate diseases and tissue injuries, and plays an important role of anti-inflammatory and anti-fibrosis. In this study, the level of CB2R in skin incised wound was detected by immunohistochemical technique and western blotting. The aim of this investigation is to explore the possible function of CB2R in skin wound healing and its applicability to the wound age estimation.
Materials and Methods
A total of 50 mice, each weighting 35-40g, were divided into ten groups randomly, one control group and nine experiment groups, A 1.5cm-long full-thickness incision deep to the fascia was made with a scalpel in the skin layer on the central dorsum of each mouse under sterile technique after the mouse was anesthetized by inhalating diethyl ether. After injury, each mouse was housed individually and fed with sterilized food and distilled water to prevent from infection. The 1.5cm×1.0cm specimens were taken from the wounded site after the mice were executed by cervical dislocation at Oh,6h,12h, 1d,3d,5d,7d, 10d and 14d postinjury to prepare for the subsequent procedures. The level of CB2R in mouse skin incised wound was detected by immunohistochemical staining and Western blot, and the non-incised mouse skin was used as control. The number and ratio of CB2R positive cells were counted and analyzed by microscope. The band AverGray was analyzed by software of Fluochem V2.0. The data were analyzed comparatively by the software of SPSS13.0 for Windows, and there was statistical significance comparing with the neighboring group when P<0.05.
Results
CB2R immuroreactivity was detected in epidemical layer, hair follicle, sebaceous gland, dermomuscular layer, vascular smooth muscle, nerve fiber adventitia and perimysium in the control skin; weak CB2R immunostaining was detected in polymorphonuclear cells (PMNs) in the wound and peripheral region in aged 6h, a large number of CB2R-positive mononuclear cells (MNCs) were identified from 12h to 24h, Afterwards, the CB2R-positive cells were mostly MNCs and spindle-shaped fibroblastic cells (FBCs) at 3d postinjury, and mainly in spindle-shaped fibroblastic cells from 5d to 14d postinjury. The ratio of the CB2R-positive cells increased gradually in the wound specimens from Oh to 3d, and maximized at 5d, and decreased from 7d to 14d postinjury. The bands of CB2R expression were observed throughout the wound healing stages by Western blotting, and maximized at 5d.
Conclusion
The results suggested that CB2R was expressed in PMNs, MNCs, FBCs during healing process of skin wound in mice, and may be used as a newmarker for the wound age determination.
引文
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17 Eming SA, Krieg T, Davidson JM. Inflammation in wound repair:molecular and cellular mechanisms. J Invest Dermatol.2007; 127(3):514-525.
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2 L. Bellocchio, C. Cervino, R. Pasquali, et al. The Endocannabinoid System and Energy Metabolism. Journal of Neuroendocrinology.2008; 20:850-857.
3 Pacher P, Gao B. Endocannabinoids and liver disease. Ⅲ. Endocannabinoid effects on immune cells:implications for inflammatory liver diseases. Am J Physiol Gastrointest Liver Physiol.2008; 294(4):G850-854.
4 Caraceni P, Domenicali M, Giannone F, et al. The role of the endocannabinoid system in liver diseases. Best Pract Res Clin Endocrinol Metab.2009; 23(1):65-77.
5 Richardson D, Pearson RG, Kurian N, et al. Characterisation of the cannabinoid receptor system in synovial tissue and fluid in patients with osteoarthritis and rheumatoid arthritis. Arthritis Res Ther.2008; 10(2):R43.
6 Marquez L, Abanades S, Andreu M. et al. Endocannabinoid system and bowel inflammation. Med Clin (Barc).2008; 131(13):513-517.
7 P Pacher P, Ungvari Z. Pleiotropic effects of the CB2 cannabinoid receptor activation on human monocyte migration:implications for atherosclerosis and inflammatory diseases. Am J Physiol Heart Circ Physiol.2008; 294(3):H1133-1134.
8 Di Filippo C, Rossi F, Rossi S, et al. Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia-reperfusion injury:involvement of cytokine/chemokines and PMN. J Leukoc Biol.2004; 75(3):453-459.
9 Pacher P, Hasko G. Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning. Br J Pharmacol.2008; 153(2):252-262.
10 Sonja Stander a, Martin Schmelz b, Dieter Metze a, et al. Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve fibers and adnexal structures in human skin. J Dermatol Sci.2005; 38:177-188.
11 Akhmetshina A, Dees C, Busch N, et al. The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis. Arthritis Rheum.2009; 60(4):1129-1136.
12 Steffens S, Mach F. Cannabinoid receptors in atherosclerosis. Curr Opin Lipidol.2006; 17(5):519-526.
13 Garcia-Gonzalez E, Selvi E, Balistreri E, et al. Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts. Rheumatology (Oxford).2009; 48(9):1050-1056.
14 Rieder SA, Chauhan A, Singh U, et al. Cannabinoid-induced apoptosis in immune cells as a pathway to immunosuppression. Immunobiology. doi:10.1016/j. imbio.2009.04.001.
15 Teixeira-Clerc F, Julien B, et al.CB 1 cannabinoid receptor antagonism:a new strategy forthe treatment of liver fibrosis. Nat Med.2006; 12(6):671-676.
16 Miller MC, Nanchahal J. Advances in the modulation of cutaneous wound healing and scarring. BioDrugs.2005; 19(6):363-381.
17 Eming SA, Krieg T, Davidson JM. Inflammation in wound repair:molecular and cellular mechanisms. J Invest Dermatol.2007; 127(3):514-525.
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