本文關(guān)鍵詞： 色素上皮衍生因子 HaCaT細(xì)胞 銀屑病 表皮附屬器 外根鞘細(xì)胞　出處：《浙江大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：[研究背景]色素上皮衍生因子是一分子量為50-KDa的內(nèi)源性可溶性分泌蛋白,屬于絲氨酸蛋白酶抑制劑家族。近期的研究發(fā)現(xiàn),PEDF在人體廣泛分布,見于人角膜上皮、腎臟、胰腺、前列腺、骨組織、牙齒、成纖維細(xì)胞、心肌細(xì)胞以及外周血等組織。在體外培養(yǎng)的細(xì)胞中,PEDF表達(dá)于肺、皮膚和子宮內(nèi)膜間質(zhì)的成纖維細(xì)胞、血管平滑肌細(xì)胞、心肌細(xì)胞、視網(wǎng)膜色素上皮細(xì)胞和T淋巴細(xì)胞等。目前已證實(shí)PEDF是一個(gè)作用強(qiáng)效和廣泛的神經(jīng)營(yíng)養(yǎng)因子,具有誘導(dǎo)神經(jīng)元細(xì)胞分化和減少腦脊髓病變傷害作用。體外實(shí)驗(yàn)還證實(shí),PEDF能夠劑量依賴性抑制內(nèi)皮細(xì)胞遷移,強(qiáng)于其他血管新生抑制劑如內(nèi)皮抑素和凝血栓蛋白-1。在大部分的實(shí)體腫瘤和腫瘤細(xì)胞系中,PEDF表達(dá)降低或丟失。研究顯示PEDF的降低和多種腫瘤侵襲增加和預(yù)后不良相關(guān),如前列腺癌、胰腺癌、骨肉瘤、乳腺癌、神經(jīng)母細(xì)胞瘤、黑色素瘤和神經(jīng)膠質(zhì)瘤。近來(lái)一些實(shí)驗(yàn)證實(shí)PEDF還具有免疫調(diào)節(jié)和對(duì)抗氧化應(yīng)激等功能。在有關(guān)皮膚PEDF的研究中,最早有文獻(xiàn)報(bào)道發(fā)現(xiàn)PEDF主要分布于真皮層,而表皮層僅表達(dá)少量的PEDF。然而最近有Abe研究小組有不同的報(bào)道,他們發(fā)現(xiàn)PEDF在正常皮膚表皮和真皮層均表達(dá),且表皮表達(dá)明顯高于真皮層。因此,需要對(duì)皮膚中PEDF表達(dá)及功能深入進(jìn)行研究。 第一部分色素上皮衍生因子在HaCaT細(xì)胞上的表達(dá)和功能研究 [目的]明確PEDF在HaCaT細(xì)胞的表達(dá)情況以及對(duì)其增殖遷移和粘附等細(xì)胞功能的影響。 [方法]采用RT-PCR和Western-blot檢測(cè)HaCaT細(xì)胞PEDF以及PEDF受體mRNA和蛋白的表達(dá)；間接免疫熒光檢測(cè)PEDF的細(xì)胞定位情況；MTT方法檢測(cè)PEDF對(duì)HaCaT細(xì)胞增殖的影響；細(xì)胞劃痕實(shí)驗(yàn)和Transwell遷移實(shí)驗(yàn)方法檢測(cè)PEDF對(duì)HaCaT細(xì)胞遷移的影響；結(jié)晶紫染色法檢測(cè)PEDF對(duì)HaCaT細(xì)胞粘附功能的影響；Western-blot方法檢測(cè)PEDF對(duì)HaCaT細(xì)胞ERK1/2、p38、JNK和AKT信號(hào)通路的影響。 [結(jié)果]HaCaT細(xì)胞在mRNA和蛋白水平均可檢測(cè)到PEDF和PEDF受體的表達(dá)。PEDF熒光信號(hào)主要位于胞漿內(nèi)。此外,50ng/ml的VEGF165可以降低HaCaT細(xì)胞PEDF蛋白表達(dá)。50ng/ml的PEDF可以抑制HaCaT細(xì)胞的增殖和遷移,增加其粘附。PEDF可以降低ERK1/2的磷酸化,而對(duì)p38、JNK和AKT信號(hào)通路無(wú)影響。 [結(jié)論]PEDF表達(dá)于HaCaT細(xì)胞,PEDF可能通過(guò)ERK1/2通路抑制HaCaT細(xì)胞增殖和遷移。 第二部分正常和銀屑病皮膚PEDF表達(dá)的研究 [目的]明確PEDF在正常人和銀屑病皮膚組織、表皮角質(zhì)形成細(xì)胞和真皮成纖維細(xì)胞表達(dá)情況。 [方法]采用免疫組化方法檢測(cè)正常和銀屑病皮膚組織PEDF表達(dá)情況。分離正常及銀屑病患者表皮角質(zhì)形成細(xì)胞和真皮成纖維細(xì)胞。使用RT-PCR和Western-blot方法檢測(cè)正常健康人和銀屑病患者表皮角質(zhì)形成細(xì)胞和真皮成纖維細(xì)胞PEDF mRNA和蛋白的表達(dá)。 [結(jié)果]免疫組化提示在正常健康成人表皮中PEDF主要表達(dá)于基底層和鄰近基底層的棘細(xì)胞層,和正常皮膚、銀屑病非皮損區(qū)、鄰近皮損區(qū)皮膚相比,銀屑病皮損區(qū)PEDF表達(dá)較高。和正常皮膚來(lái)源的角質(zhì)形成細(xì)胞相比,銀屑病皮損區(qū)、鄰近皮損區(qū)和銀屑病非皮損區(qū)角質(zhì)形成細(xì)胞PEDF mRNA表達(dá)較高。而正常皮膚、銀屑病非皮損區(qū)、鄰近皮損區(qū)和銀屑病皮損區(qū)成纖維細(xì)胞PEDF mRNA表達(dá)無(wú)明顯差異。銀屑病來(lái)源的角質(zhì)形成細(xì)胞和成纖維細(xì)胞PEDF的蛋白表達(dá)水平類似mRNA表達(dá)。 [結(jié)論]PEDF在銀屑病皮損表皮表達(dá)明顯增加,可能參與了銀屑病的發(fā)病。 第三部分色素上皮衍生因子在皮膚附屬器表達(dá) [目的]明確PEDF在皮膚附屬器的表達(dá)情況以及相關(guān)功能。 [方法]采用免疫組化方法檢測(cè)正常皮膚附屬器的PEDF表達(dá)情況。使用RT-PCR、Western-blot方法和間接免疫熒光方法檢測(cè)外根鞘細(xì)胞PEDF mRNA和蛋白的表達(dá)。采用細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)PEDF對(duì)外根鞘細(xì)胞遷移的影響。 [結(jié)果]PEDF主要表達(dá)于毛囊的表皮基質(zhì)、內(nèi)毛根鞘、外毛根鞘和纖維鞘部位,在皮脂腺和汗腺等也有表達(dá)。體外培養(yǎng)的外根鞘細(xì)胞表達(dá)PEDF mRNA和蛋白,PEDF在其胞漿和胞核均有表達(dá)。25ng/ml的IL-4和IL-17尤其是IL-4可以上調(diào)外根鞘細(xì)胞PEDF的表達(dá)。此外,50ng/ml的PEDF可以抑制外根鞘細(xì)胞的遷移。 [結(jié)論]PEDF表達(dá)于毛囊、皮脂腺和汗腺以及體外培養(yǎng)的外根鞘細(xì)胞,可能對(duì)外根鞘細(xì)胞功能具有調(diào)節(jié)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R751

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本文編號(hào)：1517612