色素上皮衍生因子在皮肤中的表达及功能研究

发布时间：2018-02-17 08:11

本文关键词： 色素上皮衍生因子 HaCaT细胞银屑病表皮附属器外根鞘细胞　出处：《浙江大学》2011年博士论文　论文类型：学位论文

【摘要】：[研究背景]色素上皮衍生因子是一分子量为50-KDa的内源性可溶性分泌蛋白,属于丝氨酸蛋白酶抑制剂家族。近期的研究发现,PEDF在人体广泛分布,见于人角膜上皮、肾脏、胰腺、前列腺、骨组织、牙齿、成纤维细胞、心肌细胞以及外周血等组织。在体外培养的细胞中,PEDF表达于肺、皮肤和子宫内膜间质的成纤维细胞、血管平滑肌细胞、心肌细胞、视网膜色素上皮细胞和T淋巴细胞等。目前已证实PEDF是一个作用强效和广泛的神经营养因子,具有诱导神经元细胞分化和减少脑脊髓病变伤害作用。体外实验还证实,PEDF能够剂量依赖性抑制内皮细胞迁移,强于其他血管新生抑制剂如内皮抑素和凝血栓蛋白-1。在大部分的实体肿瘤和肿瘤细胞系中,PEDF表达降低或丢失。研究显示PEDF的降低和多种肿瘤侵袭增加和预后不良相关,如前列腺癌、胰腺癌、骨肉瘤、乳腺癌、神经母细胞瘤、黑色素瘤和神经胶质瘤。近来一些实验证实PEDF还具有免疫调节和对抗氧化应激等功能。在有关皮肤PEDF的研究中,最早有文献报道发现PEDF主要分布于真皮层,而表皮层仅表达少量的PEDF。然而最近有Abe研究小组有不同的报道,他们发现PEDF在正常皮肤表皮和真皮层均表达,且表皮表达明显高于真皮层。因此,需要对皮肤中PEDF表达及功能深入进行研究。第一部分色素上皮衍生因子在HaCaT细胞上的表达和功能研究 [目的]明确PEDF在HaCaT细胞的表达情况以及对其增殖迁移和粘附等细胞功能的影响。 [方法]采用RT-PCR和Western-blot检测HaCaT细胞PEDF以及PEDF受体mRNA和蛋白的表达；间接免疫荧光检测PEDF的细胞定位情况；MTT方法检测PEDF对HaCaT细胞增殖的影响；细胞划痕实验和Transwell迁移实验方法检测PEDF对HaCaT细胞迁移的影响；结晶紫染色法检测PEDF对HaCaT细胞粘附功能的影响；Western-blot方法检测PEDF对HaCaT细胞ERK1/2、p38、JNK和AKT信号通路的影响。 [结果]HaCaT细胞在mRNA和蛋白水平均可检测到PEDF和PEDF受体的表达。PEDF荧光信号主要位于胞浆内。此外,50ng/ml的VEGF165可以降低HaCaT细胞PEDF蛋白表达。50ng/ml的PEDF可以抑制HaCaT细胞的增殖和迁移,增加其粘附。PEDF可以降低ERK1/2的磷酸化,而对p38、JNK和AKT信号通路无影响。 [结论]PEDF表达于HaCaT细胞,PEDF可能通过ERK1/2通路抑制HaCaT细胞增殖和迁移。第二部分正常和银屑病皮肤PEDF表达的研究 [目的]明确PEDF在正常人和银屑病皮肤组织、表皮角质形成细胞和真皮成纤维细胞表达情况。 [方法]采用免疫组化方法检测正常和银屑病皮肤组织PEDF表达情况。分离正常及银屑病患者表皮角质形成细胞和真皮成纤维细胞。使用RT-PCR和Western-blot方法检测正常健康人和银屑病患者表皮角质形成细胞和真皮成纤维细胞PEDF mRNA和蛋白的表达。 [结果]免疫组化提示在正常健康成人表皮中PEDF主要表达于基底层和邻近基底层的棘细胞层,和正常皮肤、银屑病非皮损区、邻近皮损区皮肤相比,银屑病皮损区PEDF表达较高。和正常皮肤来源的角质形成细胞相比,银屑病皮损区、邻近皮损区和银屑病非皮损区角质形成细胞PEDF mRNA表达较高。而正常皮肤、银屑病非皮损区、邻近皮损区和银屑病皮损区成纤维细胞PEDF mRNA表达无明显差异。银屑病来源的角质形成细胞和成纤维细胞PEDF的蛋白表达水平类似mRNA表达。 [结论]PEDF在银屑病皮损表皮表达明显增加,可能参与了银屑病的发病。第三部分色素上皮衍生因子在皮肤附属器表达 [目的]明确PEDF在皮肤附属器的表达情况以及相关功能。 [方法]采用免疫组化方法检测正常皮肤附属器的PEDF表达情况。使用RT-PCR、Western-blot方法和间接免疫荧光方法检测外根鞘细胞PEDF mRNA和蛋白的表达。采用细胞划痕实验检测PEDF对外根鞘细胞迁移的影响。 [结果]PEDF主要表达于毛囊的表皮基质、内毛根鞘、外毛根鞘和纤维鞘部位,在皮脂腺和汗腺等也有表达。体外培养的外根鞘细胞表达PEDF mRNA和蛋白,PEDF在其胞浆和胞核均有表达。25ng/ml的IL-4和IL-17尤其是IL-4可以上调外根鞘细胞PEDF的表达。此外,50ng/ml的PEDF可以抑制外根鞘细胞的迁移。 [结论]PEDF表达于毛囊、皮脂腺和汗腺以及体外培养的外根鞘细胞,可能对外根鞘细胞功能具有调节作用。
[Abstract]:[background] pigment epithelium derived factor is a molecular weight of endogenous soluble 50-KDa proteins belong to the serine protease inhibitor family. Recent studies have found that PEDF is widely distributed in the human body, found in human corneal epithelium, kidney, pancreas, prostate, bone, teeth, fibroblast cells, myocardial cells and peripheral blood. Tissue. In cultured cells, the expression of PEDF in lung, skin fibroblasts and endometrial stromal and vascular smooth muscle cells, myocardial cells, retinal pigment epithelial cells and T lymphocytes. The confirmed PEDF is a potent and widely neurotrophic factors have induced neuronal cell differentiation and spinal cord lesions reduce brain damage effect. In vitro experiments also confirmed that PEDF can dose dependently inhibit the migration of endothelial cells, is stronger than other angiogenesis inhibitors such as endostatin and coagulation of egg bolt White -1. in most solid tumors and tumor cell lines, PEDF expression decreased or lost. Research shows that the decrease of PEDF and increase the variety of tumor invasion and poor prognosis, such as prostate cancer, pancreatic cancer, osteosarcoma, breast cancer, neuroblastoma, melanoma and glioma. Some recent experiments PEDF also has immunomodulatory and anti oxidative stress function. In the study of the skin PEDF, the earliest reported that PEDF was mainly distributed in the epidermis and dermis, only a small amount of PEDF. expression but recently Abe research group have different reports, they found that PEDF was expressed in normal epidermis and dermis. And the skin was significantly higher than that of the dermis. Therefore, the need for the expression of PEDF in the skin and the function of in-depth study.
The first part of the expression and function of pigment epithelium derived factor in HaCaT cells
[Objective] to clarify the expression of PEDF in HaCaT cells and the effect on cell functions such as proliferation, migration and adhesion.
[method] using RT-PCR and Western-blot detection of HaCaT cell PEDF and PEDF receptor mRNA and protein expression; cellular localization of indirect immunofluorescence detection of PEDF; effect of MTT method for detection of PEDF on the proliferation of HaCaT cells; cell scratch assay and Transwell migration assay. PEDF on HaCaT cell migration; crystal violet staining method was used to detect the effect of PEDF on HaCaT cell adhesion function; Western-blot PEDF method for the detection of HaCaT cell ERK1/2, p38, effects of JNK and AKT signaling pathway.
The]HaCaT cells at the level of mRNA and protein expression was detected in.PEDF fluorescence signals of PEDF and PEDF receptor mainly located in the cytoplasm. In addition, the proliferation and migration of 50ng/ml VEGF165 HaCaT can reduce the expression of PEDF protein.50ng/ml PEDF could inhibit HaCaT cells, increase the adhesion of.PEDF can reduce the phosphorylation of ERK1/2, and the p38, no effect of JNK and AKT signaling pathways.
[conclusion]PEDF is expressed in HaCaT cells, and PEDF may inhibit the proliferation and migration of HaCaT cells through the ERK1/2 pathway.
The study of the expression of PEDF in the second part of normal and psoriasis skin
[Objective] to clarify the expression of PEDF in normal and psoriatic skin tissues, epidermal keratinocytes and dermal fibroblasts.
[Methods] expressions were investigated by immunohistochemistry in normal and psoriatic skin tissue PEDF. The epidermis was separated from patients with normal and psoriatic keratinocytes and dermal fibroblasts. Using RT-PCR and Western-blot methods to detect the normal and psoriatic epidermal keratinocytes and dermal fibroblasts expressing PEDF and mRNA proteins.
[results] immunohistochemistry showed the prickle cell layer in the epidermis in normal healthy adult PEDF is mainly expressed in the basal layer and adjacent to the basal layer, and normal skin and psoriatic nonlesional skin lesions, compared with adjacent, PEDF expression in the psoriatic lesions and normal skin. High angle matter source formation than cells in lesions of patients with psoriasis. Nearby, the lesions and non lesional skin of psoriatic keratinocytes and PEDF mRNA was higher than normal skin, non lesional skin of psoriasis, psoriatic lesion and adjacent area fibroblast PEDF mRNA expression had no significant difference. The expression of cell and fibroblast PEDF protein levels similar to mRNA expression in human keratinocytes from psoriasis.
[conclusion the expression of]PEDF in the epidermis of psoriatic lesions is significantly increased, which may be involved in the pathogenesis of psoriasis.
The third part expression of PEDF in skin appendages
[Objective] clear expression of PEDF in skin appendages and related functions.
[Methods] immunohistochemical method was used to detect the expression of PEDF in normal skin appendages. The use of RT-PCR, Western-blot expression method and indirect immunofluorescence method to detect the outer root sheath cell of PEDF mRNA and protein. The effect of cell scratch assay PEDF external root sheath cell migration.
The cuticular matrix]PEDF is mainly expressed in the hair follicle, inner root sheath, outer root sheath and sheath fiber parts in the sebaceous glands and sweat glands is expressed. The expression of PEDF mRNA protein and outer root sheath cells in vitro, PEDF in the cytoplasm and nucleus expressed.25ng/ml IL-4 and IL-17 in particular is the expression of IL-4 was up-regulated in outer root sheath cells of PEDF. In addition, 50ng/ml PEDF can inhibit the migration of outer root sheath cells.
[conclusion]PEDF is expressed in hair follicles, sebaceous glands and sweat glands, as well as external root sheath cells in vitro, which may regulate the function of external root sheath cells.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R751

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