【作者】 张艳；

【导师】 徐宏伟； 韩彦弢；

【作者基本信息】 青岛大学 ， 微生物与生化药学， 2016， 硕士

【摘要】 目的建立模拟日光紫外线B(180m J/cm2)长期照射昆明种无毛小鼠皮肤癌的模型,从AQP3,AQP9,EGFR,AKT的分子角度,研究扇贝多肽(PCF)和狭鳕鱼皮胶原蛋白肽(CPWPS)对UVB辐射诱导致皮肤癌的预防作用。方法CCK-8法检测不同浓度(50,100,200,300,500,1000,2000mg·L-1)的CPWPS对Ha Ca T细胞的毒性,筛选其对Haca T细胞的无毒性作用剂量,将PCF和CPWPS无毒性剂量作用于表皮鳞癌细胞A431,同法检测细胞存活率。动物实验分组:正常对照组、模型组、5%VC组,5%PCF组,5%CPWPS组,20%CPWPS组。除正常对照组外其他各组均进行180m J/cm2 UVB诱导,每个周照射3次共照射25周,建立皮肤癌的模型;肉眼观察,HE染色法探究5,10,15,20,25周的昆明种无毛小鼠的皮肤病理改变。采用免疫组化的方法检测25周时模型组和正常组PCNA的表达量及各组AQP3,AQP9,EGFR,P-AKT的蛋白表达量。结果细胞水平:CPWPS浓度高于300mg·L-1时对Ha Ca T细胞有毒性作用(P<0.05)。选取50,100,200mg·L-1的CPWPS和1.42,2.48,5.68mmol·L-1的PCF作用于表皮鳞癌A431细胞时,并未抑制细胞增殖(P>0.05),无统计学意义。动物水平:肉眼观察到随着持续的UVB照射,模型组第5周无毛鼠的皮肤出现红斑,长鳞屑,第10周,脱屑,掉毛,第15周开始出现帽针尖大小丘疹,第20周丘疹进一步增多,第25周形成肿瘤;同时期5%PCF组,20%CPWPS组较模型组均有不同程度的症状减轻。海洋抗氧化肽3组出现肿瘤的时间均比阳性对照组晚,且25周时平均每只老鼠背部肿瘤个数较VC组少,尤其是20%CPWPS组平均每只无毛鼠背部仅出现1.80±0.45个肿瘤(P<0.05)。HE的光镜结果显示,正常组昆明种无毛小鼠皮肤组织表皮层较有毛小鼠薄,毛囊及汗腺较有毛小鼠少,其他均无不同。UVB照射5周后,模型组无毛小鼠皮肤表皮层较非UVB照射组变厚;照射10周后,表皮角化过度,表皮突不规则轻度向下延伸;照射15周后,角化过度更加明显,开始出现癌巢;照射20周后,角化过度相对15周进一步明显,癌巢增多,黑色素轻微沉积,核浆比增大,突破基底层浸润,细胞排列较乱,异型性;照射25周后,高度的角化过度,弥漫的异型性细胞,癌巢进一步增多且明显,部分伴有鳞癌角珠。免疫组化结果显示模型组的PCNA蛋白表达量明显高于正常组(P<0.05),综合以上结果显示无毛鼠皮肤癌模型构建成功。而同时期各给药组较模型组均有不同程度的症状减轻。与正常组相比,模型组AQP3,AQP9,EGFR,P-AKT蛋白表达水平明显升高(P<0.05);与模型组相比,5%PCF组,5%CPWPS组,20%CPWPS组,5%VC各给药组中AQP3,AQP9,EGFR,P-AKT蛋白表达水平均降低(P<0.05),且20%CPWPS组各蛋白表达水平均降低的最为显著(P<0.05)。结论新发现AQP9与EGFR,AKT,AQP3共同参与了UVB诱导的皮肤癌的发生发展,外用海洋抗氧化肽(PCF和CPWPS)能预防180m J/cm2UVB辐射诱导所致的皮肤癌,且20%CPWPS组预防作用最为明显,其机制可能与抑制EGFR,AKT,AQP3,AQP9各蛋白的表达有关。更多还原

【Abstract】 Aim: The aim of this study was to establish chronic day-light mimicking UVB(180m J/cm2) induced skin carcinogenesis model in Kunming hairless mice and to investigate the preventive effects of marine antioxidant peptides: polypeptides from chlamys farreri(PCF) and collagen peptides from walleye pollock skin(CPWPS)against UVB induced skin carcinogenesis, focusing on AQP3, AQP9, EGFR and AKT as potential mechanisms. Methods: CPWPS cytotoxicity was screened with CCK-8method in Ha Ca T cells(50, 100, 200, 300, 500, 1000, 2000 mg·L-1), non-toxic doses were selected for subsequent experiments, in which the inhibitatory effect of PCF and CPWPS on skin squamous-cell carcinoma cell line A431 were assessed with CCK-8assay. Additionally, in vivo work randomly assigned Kunming hairless mice into different groups: control group, UVB model group, 5% vitamin C(VC) group, 5%PCF group, 5% CPWPS group and 20% CPWPS group. All the animals except for those in control group received 180 m J/cm2 UVB exposure three times a week for a total of 25 weeks to induce skin carcinogenesis. Pathological changes in skin after 5,10, 15, 20 or 25 week UVB exposure were assessed with visual observation and Hematoxylin and Eosin(HE) staining. Immunohistochemistry was utilized to assess the expression levels of PCNA in control and UVB model group, as well as the expression levels of AQP3, AQP9, EGFR and p-AKT in all the groups after 25 week treatment. Results: In vitro work revealed significant cytotoxicity of CPWPS only at concentrations higher than 300 mg·L-1(P<0.05). However, 1.42, 2.48, 5.68 mmol.L-1PCF and 50, 100 or 200 mg·L-1 CPWPS treatment in A431 cell did not remarkably inhibit cell proliferation(P>0.05). On the other hand, our in vivo work demonstrated directly observable rythema and scale on the hairless mice skin following five week chronic UVB exposure; 10 week exposure led to scale and linting;pin-point sized papula started to emerge from week 15; more papula were observed by week 20, and tumor formation was confirmed by week 25, these symptoms were alleviated to varied degrees in mice treated with marine antioxidant peptides at the same time. The tumor formation in all the three groups of mice treated with marine antioxidant peptides were delayed relative to model group mice, with less tumorformation on the back per mice. Notably, only 1.80±0.45 tumor on average were observed on the back per mice treated with 20% CPWPS by week 25(P<0.05). Under optic microscope, HE stained sections indicated no difference between Kunming hairless mice and normal Kunming mice, except for thinner epidermis, less follicles and sudoriferous. After five week UVB exposure, thicker epidermis was observed in the UVB model group hairless mice samples comparing to control; 10 week UVB exposure led to hyperkeratosis of epidermis and irregular trochanterellus that slightly stretched downward; more prominent hyperkeratosis was present after 15 week UVB exposure and nest of cancer started to emerge. After 20 week UVB exposure,hyperkeratosis became more obvious, more nest of cancer were present with slight deposit of melanin and increased nucleus/cytoplasm ratio. Infiltration and breaching of basal layer were observed. Irregular cell alignment and dysplasia were also noticeable. Finally, 25 week UVB exposure resulted in extensive hyperkeratosis,diffuse dysplasia and more prominent nests of cancer along with horn pearls.Immunohistochemistry revealed higher PCNA protein expression in UVB model group relative to control group(P<0.05). These data indicated successful establishment of skin cancer model in Kunming hairless mice. Meanwhile, these symptoms were alleviated to varied degrees in mice treated with marine antioxidant peptides at the same time. Moreover, immunohistochemistry demonstrated remarkably higher expression levels of AQP3, AQP9, EGFR and p-AKT in UVB model group comparing to control group(P<0.05), while 5% PCF, 5% CPWPS, 20%CPWPS and 5% VC treatment all resulted in significantly lower expression levels of AQP3, AQP9, EGR and p-AKT(P<0.05). Among different treatments, 20% CPWPS exhibited the most prominent effect reducing the expression levels(P<0.05).Conclusions: For the first time, involvement of EGFR, AKT, AQP3 and AQP9 in UVB induced skin carcinogenesis and tumor progression was revealed. Topical application of marine antioxidant peptides(PCF and CPWPS) exerted preventive effects against 180 m J/cm2 UVB induced skin carcinogenesis, in which 20% CPWPS had most remarkable effect. The underlying mechanism of these peptides is associated with inhibitory effects on the expression of EGFR, AKT, AQP3 and AQP9.更多还原