PEDF活性透皮短肽筛选及局部应用皮肤鳞癌的实验研究

Cutaneous squamous cell carcinoma is a common malignant cutaneous tumor and its incidence rates appear to be increasing over years. Pigment epithelium-derived factor (PEDF) is a potent endogenous anti-tumor protein, in recent years study showed that low molecular weight PEDF peptides can inhibit angiogenesis after percutaneous absorption.Our previous study found that PEDF was widely expressed in normal skin and cutaneous appendages, PEDF was down-regulated in tissues of human cutaneous squamous cell carcinoma and PEDF can inhibit the proliferation of A431 cells of cutaneous squamous cell carcinoma. These results suggested that PEDF may play an important role in the anti-tumor of cutaneous squamous cell carcinoma.This subject intends to explore the effect of PEDF on cell function and MAKP signaling pathway of cutaneous squamous cell carcinoma cells through stimulation in vitro and over-expression of PEDF at first. Further, through protein purification and synthesis of short peptide and other methods, we will screen 6-7 amino acid active peptide and observe their ability of skin permeability. At last,we will establish animal models of cutaneous squamous cell carcinoma and topically apply of PEDF active peptide to observe the anti-tumor effect, mechanism and target of PEDF short peptides.This study aims to explore the effect of PEDF on cutaneous squamous cell carcinoma cell function and screen active PEDF peptide of anti-tumor of cutaneous squamous cell carcinoma.These will provide theoretical support and experimental basis for carrying out an efficient, non-invasive local treatment method on cutaneous squamous cell carcinoma.

皮肤鳞状细胞癌是皮肤常见恶性肿瘤，发病率逐年上升。色素上皮衍生因子（PEDF）是一种强效的内源性抗肿瘤蛋白，近年来发现PEDF低分子短肽经皮吸收后可以抑制血管新生。我们前期研究发现：PEDF广泛表达于正常皮肤及附属器，在人皮肤鳞癌组织中表达降低且抑制皮肤鳞癌A431细胞增殖，故提示PEDF可能在抗皮肤鳞癌中发挥重要作用。本课题拟通过体外刺激以及皮肤鳞癌细胞过表达PEDF，探讨PEDF对皮肤鳞癌细胞功能及MAPK通路的影响；进一步采用蛋白表达纯化、合成短肽等方法，筛选出6-7氨基酸的活性短肽并检测其皮肤渗透性；最后建立皮肤鳞癌动物模型，局部应用PEDF活性短肽观察其抑瘤效果，探讨PEDF短肽的作用机制以及靶点。该研究旨在明确 PEDF对皮肤鳞癌细胞功能学影响,筛选出活性的PEDF抗皮肤鳞癌短肽，为开展皮肤鳞癌的高效、非损伤性局部治疗方法提供理论支持和实验依据。

皮肤鳞状细胞癌是皮肤常见恶性肿瘤。色素上皮衍生因子是一种强效的内源性抗肿瘤蛋白，且近年发现外涂小分子PEDF短肽可以改善小鼠银屑病模型血管新生和皮肤增厚情况。我们的课题研究目的就是筛选出抗皮肤鳞癌的PEDF活性短肽，经皮肤吸收后可以抑制皮肤鳞癌生长。我们发现皮肤鳞癌A431细胞表达PEDF mRNA和蛋白，PEDF抑制A431细胞增殖和迁移。为了观察皮肤鳞癌细胞过表达PEDF对其功能影响，我们构建了PEDF-pEGFP-N1表达载体，HeLa细胞瞬时转染该质粒后能表达PEDF融合蛋白，然而皮肤鳞癌细胞株A431细胞和SCL-1细胞转染该质粒后均未能稳定表达PEDF融合蛋白。此外，PEDF在正常生长期毛囊及体外培养的毛囊外毛根鞘细胞均有表达。PEDF在皮脂腺组织和体外培养的皮脂腺细胞均有表达。我们把PEDF分成PEDF1、PEDF2和PEDF3 3个长肽，通过构建原核表达载体，合成PEDF1、PEDF2和PEDF3融合蛋白并分离纯化。通过CCK-8增殖实验，我们发现PEDF3抑制A431细胞和SCL-1细胞增殖。我们把PEDF3分成3个更小的18-20氨基酸，人工合成短肽，发现短肽分子P12673和P12674可以抑制A431细胞和SCL-1细胞增殖。我们把P12673和P12674分成6个更小的6-7氨基酸，人工合成短肽，发现P13445和P13446 能抑制A431细胞和SCL-1增殖。我们采用生物素标记 P13445、P13446短肽分子，溶解后外涂于裸鼠的局部皮肤，2 小时后通过免疫荧光发现血管内和血管壁检测到P13445、P13446短肽表达。成功构建小鼠皮肤鳞癌模型，给予小鼠外涂P13445、P13446短肽分子，发现P13446短肽分子抑肿瘤生长。通过Western-blot检测发现，P13445组PCNA 和MMP-9蛋白表达降低，而P13446组更低。通过以上这些体内外的研究，我们发现P13346短肽分子能透过皮肤表皮，体内体外均证实对皮肤鳞癌有抑制作用，有希望作为皮肤鳞癌的高效、非损伤性局部治疗方法。