C/EBPα调控角质形成细胞增殖和分化在寻常型银屑病皮损形成中的作用
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摘要
银屑病(Psoriasis)又称“牛皮癣”,是临床常见的一种有遗传背景的与免疫反应异常有关的红斑鳞屑性皮肤病,国内发病率约0.123%-0.470%。银屑病最早于公元前三百多年由苏格拉底首先报道,是人类认识的最古老的疾病之一,其易复发、难治愈,严重影响患者的身心健康,影响程度与心肌梗死、高血压及2型糖尿病相当,甚至约15.9%的银屑病患者曾有过轻生的想法。然而,银屑病确切的病因和发病机制至今仍未完全阐明,是皮肤科领域亟待解决的难题之一。
国内外对银屑病的病因及发病机制均进行了较多的研究,虽然取得了一定进展,但至今尚未完全阐明。目前认为银屑病的发病机制与遗传因素、免疫因素、感染因素、内分泌因素、神经精神因素及生活习惯、药物和环境因素等密切相关,多种因素的共同作用最终导致角质形成细胞的增殖、分化、凋亡异常和真皮乳头层血管内皮细胞的异常新生,形成了临床可见的银屑病特征性皮损,表现为表面覆盖银白色鳞屑的红色斑块,具有薄膜现象、蜡滴现象和Auspitz征。
CCAAT/增强子结合蛋白α(C/EBPα)是转录因子C/EBP家族中的重要成员,该基因最早于小鼠的NIH-3T3细胞中被克隆,定位于7号染色体上。C/EBPα位于人类染色体19q13.1,eDNA全长2385bp,内含多个翻译起始位点。C/EBPα在脂肪细胞、骨髓细胞、肝细胞、角质形成细胞和肺泡细胞等正常组织的发育、细胞增殖与分化的调节、磷脂的合成与代谢中发挥重要作用。C/EBP α表达在表皮的基底层,角质形成细胞中C/EBP α的过表达可导致细胞周期停滞。C/EBP α在小鼠和人类表皮中高表达,表明其在复层鳞状上皮细胞的分化中起转录因子的作用。癌基因Ras的过表达导致永生化角质形成细胞中C/EBP α减少,从而使C/EBPα抑制细胞增殖的能力减弱。皮脂腺中C/EBP α与C/EBP β的迅速联合消融导致硬脂酰CoA去饱和酶(SCD3)和黑皮素5受体(FC5R)的表达减少,最终引起严重的形态缺陷和皮脂细胞分化受阻。由此可见,C/EBP α对于正常皮肤中表皮角质形成细胞的增殖、分化发挥重要的调控作用。那么,在以角质形成细胞增殖和分化异常为基础的寻常型银屑病皮损中,C/EBP α如何表达?是否参与了病理改变的过程呢?目前未见国内外文献报道。
为明确C/EBP α在银屑病发病中的作用,本研究从mRNA和蛋白水平检测寻常型银屑病皮损中C/EBP α的表达,并与角质形成细胞异常增殖及临床皮损面积和严重程度指数(PASI评分)之间进行相关性分析,旨在探讨其异常表达的临床意义。应用针对C/EBP α的siRNA沉默人原代角质形成细胞C/EBP α基因的表达,探究C/EBP α基因对角质形成细胞增殖和分化的影响。采用银屑病发病过程中Th17和Th22细胞分泌的关键细胞因子IL-22刺激角质形成细胞,检测MAPK信号通路中关键信号分子JNK、ERK、p38的磷酸化水平及C/EBP α的表达水平,探究IL-22是否能够通过MAPK信号通路调控C/EBP α的表达,参与寻常型银屑病皮损的形成过程。
第一部分
C/EBP α mRNA和蛋白在人健康皮肤及银屑病皮损中的表达及意义
目的
探究C/EBP α mRNA和蛋白在寻常型银屑病皮损中的表达及其与角质形成细胞异常增殖和银屑病皮损面积和严重程度指数(PASI)评分之间的相关性。
方法
1、标本收集：收集2011年1月—2011年12月山东大学齐鲁医院皮肤科病理室留存的寻常型银屑病蜡块标本共计30例。诊断标准根据《临床诊疗指南—皮肤病与性病分册》(中华医学会编著,人民卫生出版社)、《临床技术操作规范—皮肤病与性病分册》(中华医学会编著,人民军医出版社)、《中国银屑病治疗指南》(中华医学会皮肤科分会银屑病学组,2008年)。所有患者在本次发病以来均未经系统治疗,符合入组标准(详见正文)。另选取本院皮肤科留取的健康对照皮肤标本30例,在年龄、性别、取材部位上与寻常型银屑病组相匹配。
2、免疫组化法检测C/EBP α的表达：采用免疫组化二步法检测寻常型银屑病皮损和健康对照组织中C/EBP α的表达,以灰度值代表C/EBP α的表达水平,采用Image J2.1.4.7软件分析计算灰度值,两组之间的灰度值采用t检验。
3、C/EBPa的表达与角质形成细胞增殖指数及PASI评分之间的相关性分析：免疫组化法检测Ki-67的表达,根据Ki-67的阳性表达,计算细胞增殖指数(PI),采用Pearson相关性分析,统计分析C/EBP α的表达水平与PI及PASI评分之间的相关性。
4、RT-PCR和Western Blot法检测银屑病皮损和健康对照组织中C/EBPa mRNA和蛋白的表达。
结果
1、患者临床资料：30例寻常型银屑病患者中,年龄15--49岁,平均25.2岁,其中男28例、女12例,皮损发生于头面部10例,躯干10例,四肢10例。
2、C/EBPα主要在角质形成细胞胞浆中表达,寻常型银屑病皮损中C/EBP α的表达较健康对照皮肤明显下调,差异具有统计学意义(t=7.819,P<0.05)。
3、Ki-67主要在角质形成细胞胞核中表达,寻常型银屑病皮损中角质形成细胞的增殖指数明显高于健康对照皮肤,差异具有统计学意义(t=4.535,P<0.05)。
4、经Pearson相关分析,寻常型银屑病皮损中C/EBP α的灰度值与增殖指数呈正相关(r=0.654,P<0.05),与银屑病皮损面积和严重程度指数(PASI评分)呈正相关(r=0.693,P<0.05)。
5.RT-PCR及Western Blot检测结果发现,寻常型银屑病皮损中C/EBP α mRNA和蛋白的表达水平较健康对照皮肤中明显降低,差异具有统计学意义(tRT-PCR=2.981,tmestern=2.756,P<0.05)。
结论
C/EBPα mRNA和蛋白在寻常型银屑病皮损中表达明显下调,其蛋白的表达水平与角质形成细胞的增殖指数及银屑病皮损面积和严重程度指数(PASI评分)呈负相关。由此推测C/EBP α蛋白可能参与了寻常型银屑病皮损中角质形成细胞异常增殖的过程,可作为一种反映寻常型银屑病皮损严重程度的指标。
第二部分
C/EBP α基因在角质形成细胞中的表达及沉默C/EBP α基因对细胞增殖、分化的影响
目的
1、探究原代培养的人角质形成细胞中C/EBPα mRNA和蛋白的表达
2、探究siRNA介导的C/EBP α基因沉默对原代培养的角质形成细胞增殖、分化的影响。
方法
1、原代细胞培养：本研究采用原代培养的人皮肤表皮角质形成细胞。皮肤标本取自山东大学附属山东省立医院小儿外科(泌尿外科组)术中留取的幼儿包皮标本,在实验室中PBS冲洗血污,碘伏浸泡消毒,PBS彻底冲洗后,切成小块,0.25%胰酶4℃消化过夜,待表、真皮分离后取下表皮,0.25%胰酶-0.01%EDTS消化液中消化成单细胞,加入Epilife-HKGS培养液,37℃、5%CO2、饱和湿度条件下贴壁培养,隔天换液1次,待细胞融合至70%时传代,传代2—3次后黑素细胞、成纤维细胞即可全部死亡,得到纯净的角质形成细胞培养物,经鉴定后取第3代、第4代细胞用于后续研究工作。
2、C/EBPα siRNA的转染：C/EBPα siRNA由山东大学生命科学学院动物发育与基因调控实验室张燕君教授馈赠,已经过前期预实验验证转染效率、干扰效率及转染浓度等,由德国QIAGEN公司合成。将对数生长期的角质形成细胞以2×105个/well数目接种于6孔板中,当细胞融合率达到70%左右,用脂质体lipofectamine2000转染细胞,步骤严格按照说明书进行。
3、C/EBPα基因干扰效率的检测：转染后24h收集细胞,提取细胞总RNA,应用RT-PCR法检测细胞中C/EBP α基因的表达。转染后72h收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中C/EBP α蛋白的表达。
4、CCK-8法检测C/EBP α基因沉默对角质形成细胞增殖的影响：取对数生长期siRNA转染组、空载体组和空白对照组的细胞,消化计数后按5×103个/孔接种于96孔细胞培养板中,于第12、24、48、72h采用CCK-8比色法检测各组细胞增殖情况。
5、角质形成细胞分化标志蛋白CK10、Involucrin的表达：收集转染后72h的细胞,提取细胞总蛋白,应用Western Blot法检测CK10、Involucrin蛋白的表达水平。
结果
1、C/EBPα siRNA能够有效下调角质形成细胞C/EBP α基因的表达：siRNA转染后24h,转染组细胞中C/EBP α mRNA的表达均较对照组下降47%；转染后72h,转染组细胞C/EBP α蛋白表达亦显著下调(P<0.05)。
2、应用CCK-8法检测C/EBP α基因沉默后角质形成细胞增殖活性,结果发现种板后第12h siRNA转染组、空载体组、空白对照组三组角质形成细胞的增殖活性未见明显差异(P>0.05),第24h开始,转染组细胞的增殖活性与空白对照组相比出现降低,统计学上有显著性差异(第24、48、72h的P<0.05)。
3、应用Western Blot法检测CK10、Involucrin蛋白的表达,结果显示转染第72h后,角质形成细胞分化的标志蛋白CK10、Involucrin表达水平较空白对照组明显下调,具有显著统计学差异(P<0.05)。
结论
1、C/EBP α siRNA能够有效的抑制角质形成细胞中C/EBP α的表达,C/EBP α基因沉默后角质形成细胞的增殖率提高、分化水平降低。
2、C/EBPα基因是治疗角质形成细胞增殖、分化异常性疾病的潜在靶基因。
第三部分
IL-22刺激对角质形成细胞MAPK信号通路及C/EBP α表达的影响
目的
1、探究IL-22对角质形成细胞MAPK信号通路关键蛋白磷酸化水平的影响
2、探究IL-22对角质形成细胞C/EBP a基因表达的影响
3、探究IL-22对角质形成细胞增殖和分化的影响及其与C/EBP α基因表达水平的相关性
方法
1、原代细胞培养：同第二部分
2、IL-22刺激角质形成细胞：以原代培养的角质形成细胞为研究对象,待细胞融合至60%左右时,加入终浓度为30ng/mL、60ng/mL、90ng/mL的人IL-22融合蛋白,在加药刺激后不同时间点收集细胞进行相关实验。
3、Western Blot法检测MAPK信号通路中关键分子的磷酸化水平：IL-22刺激后60min收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中JNK、p-JNK、 p38、p-p38、ERK、p-ERK的表达水平。
4、CCK-8法检测IL-22刺激对角质形成细胞增殖的影响：取对数生长期角质形成细胞,消化计数后按5×103/孔接种于96孔细胞培养板中,加入终浓度为30ng/mL、60ng/mL、90ng/mL的人IL-22融合蛋白,于加药后第12、24、48、72h采用CCK-8比色法检测各组细胞增殖情况。
5、Western Blot法检测IL-22刺激对角质形成细胞分化标志蛋白CKI0、Involucrin表达的影响：IL-22刺激后48h收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中CK10、Involucrin蛋白的表达水平。
6、RT-PCR和Western Blot法检测C/EBPαmRNA和蛋白的表达水平：IL-22刺激后48h收集细胞,提取细胞总RNA和总蛋白,应用RT-PCR和Western Blot法检测细胞中C/EBP a mRNA和蛋白的表达水平。
结果
1、IL-22刺激可显著上调JNK、ERK、p38的磷酸化水平：IL-22刺激后60mmin,p-JNK、p-ERK、p-p38均较对照组表达上调,差异具有统计学意义(P<0.05)。
2、IL-22可显著促进角质形成细胞的增殖：应用CCK-8比色法检测发现,种板后第12h开始,细胞增殖率明显升高(P<0.05),呈明显的时间和剂量依赖性。
3、IL-22可显著抑制角质形成细胞的分化：Western Blot检测发现,60ng/mL的IL-22可抑制角质形成细胞分化标志蛋白CK10、Involucrin的表达水平(P<0.05),且呈明显的浓度依赖性。
4、IL-22可显著下调C/EBP a mRNA和蛋白的表达水平：RT-PCR和Western Blot检测发现,IL-22可抑制角质形成细胞C/EBPα mRNA和蛋白的表达水平(P<0.05),且呈明显的浓度依赖性。
结论
1、IL-22可以显著调控角质形成细胞的增殖和分化过程。
2、IL-22可显著调控角质形成细胞中MAPK信号通路及C/EBP α的表达水平。
3、IL-22可能通过MAPK信号通路及下游的C/EBP α的表达调控角质形成细胞的增殖和分化
Psoriasis, which is associated with a genetic background and abnormal immune response, is one of the most common clinical erythematous desquamative dermatosis in China with an incidence of0.123%-0.470%. Because of easy recurrence and difficult to cure, psoriasis seriously influences patients health and quality of life. However, until now, the exact etiology and pathogenesis of psoriasis have not yet been fully elucidated, which becomes one of the most urgent problems to be solved in dermatology field.
In recent years, many studies have been conducted on psoriasis in both China and abroad with some progresses, nevertheless, the cause and pathogenesis of the psoriasis have not been completely clarified so far. The fundamental pathophysiology of the psoriasis is supposed to be related to the genetic factors, immune factors, infection factors, endocrine factors, neuropsychiatric factors, habits of life, drugs and environmental factors. All of these above factors lead to the abnormality for the proliferation, differentiation and apoptosis of keratinocyte and the abnormal neogenesis of the vascular endothelial cell in the dermal papilla layer, which result in the clinically characteristic skin lesions of psoriasis such as red patches covered with silvery white scale trifles, the phenomenon of membrane, the phenomenon of candle dripping and the Auspitz sign.
CCAAT/enhancer binding protein a (C/EBPa), an important member of the transcription factor C/EBP family, plays a vital role in the synthesis and metabolism of phospholipids and the development, proliferation, differentiation of variable cell types, for example fat cells, bone marrow cells, liver cells, keratinocyte and alveolar cells. C/EBPa is mainly expressed in the keratinocytes from the basal layer of the epidermis and the overexpression of C/EBPa may cause cell cycle arrest. The high expression of C/EBPa in the epidermis of mice and human indicates that C/EBPa plays a transcription factor role in the differentiation of stratified squamous epithelium cells. The overexpression of oncogene Ras leads to the decrease of C/EBPa in immortalized keratinocytes. Consequently, the inhibition effect of cell proliferation by C/EBPa is reduced. Thus it can be seen that C/EBPa plays an important role in regulating the proliferation and differentiation of epidermal keratinocytes in normal skin. How dose C/EBPa express in the skin lesions of psoriasis vulgaris with abnormal proliferation and differentiation of keratinocyte? Whether does C/EBPa participate in the course of pathological changes of psoriasis vulgaris or not? Up to now, there is no literature reported on those relevant issues in China and abroad.
In order to investigate the role of C/EBPa in the pathogenesis of psoriasis, the mRNA and protein expressions of C/EBPa in the skin lesions of psoriasis were detected. To explore the clinical significance of abnormal C/EBPa expression, the relationship of the expression of C/EBPa between the psoriasis area and severity index (PASI), the proliferation and differentiation of keratinocytes were investigated. To explore the effect of C/EBPa gene on the proliferation of keratinocytes, the expression of C/EBPa gene in primary human keratinocyte was silenced by short interfering RNA (siRNA) for C/EBPa. We applied the key cytokines IL-22, which was secreted by Thl7cells and Th22cells in the pathogenesis of psoriasis, to stimulate the keratinocyte,and then detected the expression of C/EBPa and the phosphorylation levels of JNK, ERK and p38, the key signaling molecules in MAPK signaling pathways. Through the above investigations, we wanted to make clear that if the expression of C/EBPa could participate in the formation of the skin lesions in psoriasis vulgaris through MAPK signaling pathways.
Part I. The mRNA and protein expressions of C/EBPa in the lesions of psoriasis with the clinical significance
Objective:To detect the mRNA and protein expressions of C/EBPa in the lesions of psoriasis and investigate their relationships with the abnormal proliferations of keratinocytes and the PASI.
Methods:1. Specimens Collection:Between January2011and December2011,30cases of paraffin-embedded specimens of psoriasis vulgaris were collected as the experimental group in the Pathological Lab of the Department of Dermatology in Qilu Hospital of Shandong University. Meanwhile,30cases of health skin specimens matched for ages, gender and site of sampling with the experimental group were collected as the control group in the Department of Dermatology of our hospital.2. Immunohistochemical method to detect the expression of C/EBPa and Ki-67:The expressions of C/EBPa, Ki-67and CK10were detected by two-step immunohistochemical method in the control and experimental groups. Semi-quantitative scores were recorded according to the number of positive cells and the degree of dyeing. The difference of expression between two groups was analyzed by Chi-square test.
3. The correlations between the expression of C/EBPa and the proliferation index (PI) of keratinocytes and the PASI:The PI of keratinocytes was calculated according to the positive expression of Ki-67and the associations between the expression of C/EBPa and PI and PASI were analyzed by Pearson correlation analysis.
4. The mRNA and protein expressions of C/EBPa in lesions of psoriasis and health skin were detected by RT-PCR and western blot.
Results:1. Clinical data of patients:The30cases of psoriasis vulgaris patients with a mean age of25.2years (range15to49years) included18males and12females. For the lesions among the30cases of psoriasis vulgaris,10cases were on the head and face,10cases on the trunk and10cases the on extremities.2. The expression of C/EBPa was localized dominantly in the cytoplasm of keratinocytes and the expression of C/EBPa was significantly lower in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=7.819, P<0.05).
3. The expression of Ki-67was detected mainly in the nucleus of keratinocytes. The PI was significantly higher in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=4.535, P<0.05).
4. According to the results of Pearson correlation analysis, the gray values of C/EBPa were positively correlated with the PI and PASI in the lesions of psoriasis vulgaris (r=0.654, P<0.05and r=0.693, P<0.05, respectively).
5. According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa were significantly lower in the lesions of psoriasis vulgaris than in control group with a statistical difference (P<0.05).
Conclusions:The mRNA and protein expressions of C/EBPa were significantly reduced in the lesions of psoriasis vulgaris. The protein expression of C/EBPa was negatively correlated with the PI of keratinocytes and PASI of lesions of psoriasis vulgaris. It was speculated that C/EBPα, which could be used as an indicator reflecting the severity of psoriasis lesions, might play a role in the process of the abnormal proliferation of keratinocytes.
Part Ⅱ. The gene expression of C/EBP α in keratinocytes and the effects on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBP α gene silencing
Objective:1. To detect the mRNA and protein expressions of C/EBPa in primary human keratinocytes.
2. To investigate the effects of C/EBPα on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBPα gene silencing.
Methods:1. The culture of primary cells:The primary culture of human epidermal keratinocytes was used in the study. The skin specimens from the young children's foreskin were obtained by the Department of Paediatric Urology of Shandong Province Hospital affiliated to Shandong University. The specimens were digested with0.25%trypsin at4℃for overnight to dissect epidermis from dermis. Then, the epidermis specimens were digested with0.25%trypsin-0.01%EDTA mixture to obtain single cell suspension, which was added into Epilife-HKGS culture solution. The adherent cells were gained by cultured in saturated humidity conditions with5%CO2at37℃and the medium was changed every other day. Following3-4stable passages, the cells were obtained for further study.
2. The siRNA transfection for C/EBPα:The siRNA sequence for C/EBPaproduced by QIAGEN Company. The transfection efficiency, interference efficiency and transfection concentration for the siRNA had been verified in the pretest. The keratinocytes in the logarithmic phase were inoculated into6-well plates with2×105 cells/well. When the primary keratinocytes were fused to70%, siRNA was transfected into the cells by lipofectamine2000according to the manual strictly, as siRNA transfection group. Meanwhile, the empty vector transfection group and non-transfection group were also established.
3. The detection of the interferential efficiency for C/EBPa gene:Total RNA was extracted after24h of transfection. The mRNA expression of C/EBP a was detected by RT-PCR. Total protein was extracted after72h of transfection. The protein expression of C/EBPa was detected by western blot.
4. The effect of C/EBPa gene silencing on the proliferation of keratinocytes by CCK-8colorimetric analysis:KC-C and KC cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. The proliferation of cells was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.
5. The expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:Total protein was extracted after72h of transfection. The protein expressions of CK10and involucrin were detected by western blot.
Results:1. The expression of C/EBPa was inhibited effectively by siRNA:The mRNA expression of C/EBPa was inhibited by47%compared with those in control group72h after siRNA transfection and the expressions of C/EBP a protein were also down-regulated significantly(P<0.05).
2. CCK-8colorimetric analysis was used to detect the effect of C/EBPa gene silencing on the proliferation of keratinocytes. There was no significant difference in the proliferation of keratinocytes in the siRNA transfection group, empty vector transfection group and non-transfection group after12h inoculating. However, after inoculating for24h,48h and72h, the proliferation of keratinocytes in the siRNA transfection group was significantly decreased with a statistical difference compared to the empty vector transfection group and non-transfection group.(P<0.05at24h,48h and72h, respectively)
3. According to the results of western blot, the protein expressions of CK10and involucrin were significantly lower in the siRNA transfection group than in the empty vector transfection group and non-transfection group with a statistical difference (P<0.05).
Conelusions:1. The siRNA for C/EBPa could inhibit the expression of C/EBPa in keratinocytes. After C/EBPa gene silencing, the proliferation rate of keratinocytes was increased and the differentiation level of keratinocytes was decreased.
2. The C/EBPa gene was a potential gene to cure the diseases withgabnormal proliferation and differentiation in keratinocytes.
Part Ⅲ:The effect of IL-22stimulation on MAPK signaling pathway and the expression of C/EBPa in keratinocytes
Objective:1. To investigate the effect of IL-22on the phosphorylation of key proteins in MAPK signaling pathway in keratinocytes.
2. To investigate the effect of IL-22on the gene expression of C/EBPa in keratinocytes.
3. To investigate the effect of IL-22on the proliferation and differentiation of keratinocytes and its correlation with the gene expression of C/EBPa.
Methods:1. The culture of primary cells referred to Part II.
2. The stimulation of IL-22on keratinocytes:The primary cultured cells of keratinocytes were used as the research subjects. When the primary keratinocytes were fused to60%, human IL-22fusion protein was added into the cells with the final concentration of30ng/mL,60ng/mL,90ng/mL, respectively. And then the cells with treatment were collected at different time points for subsequent experiments.
3. The detection of the phosphorylation level of key protein in MAPK signaling pathway by western blot:Total protein was extracted after72h of transfection. The protein expressions of JNK, p-JNK, p38, p-p38, ERK and p-ERK were detected by western blot.
4. The effect of IL-22on the proliferation of keratinocytes by CCK-8colorimetric analysis:The keratinocytes cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. Then, human IL-22fusion protein was added into every well with the final concentration of30ng/mL,60ng/mL and90ng/mL, respectively. The proliferation of keratinocytes was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.
5. The effect of IL-22on the expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:After the stimulation of IL-22for48h, total protein was extracted. The protein expressions of CK10and involucrin were detected by western blot.
6. The detections of the mRNA and protein expressions of C/EBPa by RT-PCR and western blot:After the stimulation of IL-22for48h, total RNA and total protein were extracted, respectively. The mRNA and protein expressions of C/EBPa were detected by RT-PCR and western blot, respectively.
Results:1. The phosphorylation levels of JNK, ERK and p38were significantly increased by the stimulation of IL-22:After the stimulation of IL-22for60minutes, p-JNK, p-ERK and p-p38were all significantly increased compared with the control group with the statistical difference (P<0.05).
2. The proliferation of keratinocytes was significantly increased by IL-22:According to the results of CCK-8colorimetric analysis, the cell proliferation was significantly increased, with a significant time and concentration dependent manner, from12h after the cells were inoculated into the plate.
3. The differentiation of keratinocytes was significantly inhibited by IL-22:According to the results of western blot, the protein expressions of CK10and involucrin in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.
4. The mRNA and protein expressions of C/EBPa were significantly inhibited by IL-22:According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.
Conelusions:1. IL-22can significantly regulate the proliferation and differentiation of keratinocytes.
2. IL-22can significantly regulate the MAPK signaling pathway and the expression of C/EBPa in keratinocytes.
3. IL-22may regulate the proliferation and differentiation of keratinocytes through MAPK signaling pathway and the expression of downstream molecular, C/EBPa.
国内外对银屑病的病因及发病机制均进行了较多的研究,虽然取得了一定进展,但至今尚未完全阐明。目前认为银屑病的发病机制与遗传因素、免疫因素、感染因素、内分泌因素、神经精神因素及生活习惯、药物和环境因素等密切相关,多种因素的共同作用最终导致角质形成细胞的增殖、分化、凋亡异常和真皮乳头层血管内皮细胞的异常新生,形成了临床可见的银屑病特征性皮损,表现为表面覆盖银白色鳞屑的红色斑块,具有薄膜现象、蜡滴现象和Auspitz征。
CCAAT/增强子结合蛋白α(C/EBPα)是转录因子C/EBP家族中的重要成员,该基因最早于小鼠的NIH-3T3细胞中被克隆,定位于7号染色体上。C/EBPα位于人类染色体19q13.1,eDNA全长2385bp,内含多个翻译起始位点。C/EBPα在脂肪细胞、骨髓细胞、肝细胞、角质形成细胞和肺泡细胞等正常组织的发育、细胞增殖与分化的调节、磷脂的合成与代谢中发挥重要作用。C/EBP α表达在表皮的基底层,角质形成细胞中C/EBP α的过表达可导致细胞周期停滞。C/EBP α在小鼠和人类表皮中高表达,表明其在复层鳞状上皮细胞的分化中起转录因子的作用。癌基因Ras的过表达导致永生化角质形成细胞中C/EBP α减少,从而使C/EBPα抑制细胞增殖的能力减弱。皮脂腺中C/EBP α与C/EBP β的迅速联合消融导致硬脂酰CoA去饱和酶(SCD3)和黑皮素5受体(FC5R)的表达减少,最终引起严重的形态缺陷和皮脂细胞分化受阻。由此可见,C/EBP α对于正常皮肤中表皮角质形成细胞的增殖、分化发挥重要的调控作用。那么,在以角质形成细胞增殖和分化异常为基础的寻常型银屑病皮损中,C/EBP α如何表达?是否参与了病理改变的过程呢?目前未见国内外文献报道。
为明确C/EBP α在银屑病发病中的作用,本研究从mRNA和蛋白水平检测寻常型银屑病皮损中C/EBP α的表达,并与角质形成细胞异常增殖及临床皮损面积和严重程度指数(PASI评分)之间进行相关性分析,旨在探讨其异常表达的临床意义。应用针对C/EBP α的siRNA沉默人原代角质形成细胞C/EBP α基因的表达,探究C/EBP α基因对角质形成细胞增殖和分化的影响。采用银屑病发病过程中Th17和Th22细胞分泌的关键细胞因子IL-22刺激角质形成细胞,检测MAPK信号通路中关键信号分子JNK、ERK、p38的磷酸化水平及C/EBP α的表达水平,探究IL-22是否能够通过MAPK信号通路调控C/EBP α的表达,参与寻常型银屑病皮损的形成过程。
第一部分
C/EBP α mRNA和蛋白在人健康皮肤及银屑病皮损中的表达及意义
目的
探究C/EBP α mRNA和蛋白在寻常型银屑病皮损中的表达及其与角质形成细胞异常增殖和银屑病皮损面积和严重程度指数(PASI)评分之间的相关性。
方法
1、标本收集：收集2011年1月—2011年12月山东大学齐鲁医院皮肤科病理室留存的寻常型银屑病蜡块标本共计30例。诊断标准根据《临床诊疗指南—皮肤病与性病分册》(中华医学会编著,人民卫生出版社)、《临床技术操作规范—皮肤病与性病分册》(中华医学会编著,人民军医出版社)、《中国银屑病治疗指南》(中华医学会皮肤科分会银屑病学组,2008年)。所有患者在本次发病以来均未经系统治疗,符合入组标准(详见正文)。另选取本院皮肤科留取的健康对照皮肤标本30例,在年龄、性别、取材部位上与寻常型银屑病组相匹配。
2、免疫组化法检测C/EBP α的表达：采用免疫组化二步法检测寻常型银屑病皮损和健康对照组织中C/EBP α的表达,以灰度值代表C/EBP α的表达水平,采用Image J2.1.4.7软件分析计算灰度值,两组之间的灰度值采用t检验。
3、C/EBPa的表达与角质形成细胞增殖指数及PASI评分之间的相关性分析：免疫组化法检测Ki-67的表达,根据Ki-67的阳性表达,计算细胞增殖指数(PI),采用Pearson相关性分析,统计分析C/EBP α的表达水平与PI及PASI评分之间的相关性。
4、RT-PCR和Western Blot法检测银屑病皮损和健康对照组织中C/EBPa mRNA和蛋白的表达。
结果
1、患者临床资料：30例寻常型银屑病患者中,年龄15--49岁,平均25.2岁,其中男28例、女12例,皮损发生于头面部10例,躯干10例,四肢10例。
2、C/EBPα主要在角质形成细胞胞浆中表达,寻常型银屑病皮损中C/EBP α的表达较健康对照皮肤明显下调,差异具有统计学意义(t=7.819,P<0.05)。
3、Ki-67主要在角质形成细胞胞核中表达,寻常型银屑病皮损中角质形成细胞的增殖指数明显高于健康对照皮肤,差异具有统计学意义(t=4.535,P<0.05)。
4、经Pearson相关分析,寻常型银屑病皮损中C/EBP α的灰度值与增殖指数呈正相关(r=0.654,P<0.05),与银屑病皮损面积和严重程度指数(PASI评分)呈正相关(r=0.693,P<0.05)。
5.RT-PCR及Western Blot检测结果发现,寻常型银屑病皮损中C/EBP α mRNA和蛋白的表达水平较健康对照皮肤中明显降低,差异具有统计学意义(tRT-PCR=2.981,tmestern=2.756,P<0.05)。
结论
C/EBPα mRNA和蛋白在寻常型银屑病皮损中表达明显下调,其蛋白的表达水平与角质形成细胞的增殖指数及银屑病皮损面积和严重程度指数(PASI评分)呈负相关。由此推测C/EBP α蛋白可能参与了寻常型银屑病皮损中角质形成细胞异常增殖的过程,可作为一种反映寻常型银屑病皮损严重程度的指标。
第二部分
C/EBP α基因在角质形成细胞中的表达及沉默C/EBP α基因对细胞增殖、分化的影响
目的
1、探究原代培养的人角质形成细胞中C/EBPα mRNA和蛋白的表达
2、探究siRNA介导的C/EBP α基因沉默对原代培养的角质形成细胞增殖、分化的影响。
方法
1、原代细胞培养：本研究采用原代培养的人皮肤表皮角质形成细胞。皮肤标本取自山东大学附属山东省立医院小儿外科(泌尿外科组)术中留取的幼儿包皮标本,在实验室中PBS冲洗血污,碘伏浸泡消毒,PBS彻底冲洗后,切成小块,0.25%胰酶4℃消化过夜,待表、真皮分离后取下表皮,0.25%胰酶-0.01%EDTS消化液中消化成单细胞,加入Epilife-HKGS培养液,37℃、5%CO2、饱和湿度条件下贴壁培养,隔天换液1次,待细胞融合至70%时传代,传代2—3次后黑素细胞、成纤维细胞即可全部死亡,得到纯净的角质形成细胞培养物,经鉴定后取第3代、第4代细胞用于后续研究工作。
2、C/EBPα siRNA的转染：C/EBPα siRNA由山东大学生命科学学院动物发育与基因调控实验室张燕君教授馈赠,已经过前期预实验验证转染效率、干扰效率及转染浓度等,由德国QIAGEN公司合成。将对数生长期的角质形成细胞以2×105个/well数目接种于6孔板中,当细胞融合率达到70%左右,用脂质体lipofectamine2000转染细胞,步骤严格按照说明书进行。
3、C/EBPα基因干扰效率的检测：转染后24h收集细胞,提取细胞总RNA,应用RT-PCR法检测细胞中C/EBP α基因的表达。转染后72h收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中C/EBP α蛋白的表达。
4、CCK-8法检测C/EBP α基因沉默对角质形成细胞增殖的影响：取对数生长期siRNA转染组、空载体组和空白对照组的细胞,消化计数后按5×103个/孔接种于96孔细胞培养板中,于第12、24、48、72h采用CCK-8比色法检测各组细胞增殖情况。
5、角质形成细胞分化标志蛋白CK10、Involucrin的表达：收集转染后72h的细胞,提取细胞总蛋白,应用Western Blot法检测CK10、Involucrin蛋白的表达水平。
结果
1、C/EBPα siRNA能够有效下调角质形成细胞C/EBP α基因的表达：siRNA转染后24h,转染组细胞中C/EBP α mRNA的表达均较对照组下降47%；转染后72h,转染组细胞C/EBP α蛋白表达亦显著下调(P<0.05)。
2、应用CCK-8法检测C/EBP α基因沉默后角质形成细胞增殖活性,结果发现种板后第12h siRNA转染组、空载体组、空白对照组三组角质形成细胞的增殖活性未见明显差异(P>0.05),第24h开始,转染组细胞的增殖活性与空白对照组相比出现降低,统计学上有显著性差异(第24、48、72h的P<0.05)。
3、应用Western Blot法检测CK10、Involucrin蛋白的表达,结果显示转染第72h后,角质形成细胞分化的标志蛋白CK10、Involucrin表达水平较空白对照组明显下调,具有显著统计学差异(P<0.05)。
结论
1、C/EBP α siRNA能够有效的抑制角质形成细胞中C/EBP α的表达,C/EBP α基因沉默后角质形成细胞的增殖率提高、分化水平降低。
2、C/EBPα基因是治疗角质形成细胞增殖、分化异常性疾病的潜在靶基因。
第三部分
IL-22刺激对角质形成细胞MAPK信号通路及C/EBP α表达的影响
目的
1、探究IL-22对角质形成细胞MAPK信号通路关键蛋白磷酸化水平的影响
2、探究IL-22对角质形成细胞C/EBP a基因表达的影响
3、探究IL-22对角质形成细胞增殖和分化的影响及其与C/EBP α基因表达水平的相关性
方法
1、原代细胞培养：同第二部分
2、IL-22刺激角质形成细胞：以原代培养的角质形成细胞为研究对象,待细胞融合至60%左右时,加入终浓度为30ng/mL、60ng/mL、90ng/mL的人IL-22融合蛋白,在加药刺激后不同时间点收集细胞进行相关实验。
3、Western Blot法检测MAPK信号通路中关键分子的磷酸化水平：IL-22刺激后60min收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中JNK、p-JNK、 p38、p-p38、ERK、p-ERK的表达水平。
4、CCK-8法检测IL-22刺激对角质形成细胞增殖的影响：取对数生长期角质形成细胞,消化计数后按5×103/孔接种于96孔细胞培养板中,加入终浓度为30ng/mL、60ng/mL、90ng/mL的人IL-22融合蛋白,于加药后第12、24、48、72h采用CCK-8比色法检测各组细胞增殖情况。
5、Western Blot法检测IL-22刺激对角质形成细胞分化标志蛋白CKI0、Involucrin表达的影响：IL-22刺激后48h收集细胞,提取细胞总蛋白,应用Western Blot法检测细胞中CK10、Involucrin蛋白的表达水平。
6、RT-PCR和Western Blot法检测C/EBPαmRNA和蛋白的表达水平：IL-22刺激后48h收集细胞,提取细胞总RNA和总蛋白,应用RT-PCR和Western Blot法检测细胞中C/EBP a mRNA和蛋白的表达水平。
结果
1、IL-22刺激可显著上调JNK、ERK、p38的磷酸化水平：IL-22刺激后60mmin,p-JNK、p-ERK、p-p38均较对照组表达上调,差异具有统计学意义(P<0.05)。
2、IL-22可显著促进角质形成细胞的增殖：应用CCK-8比色法检测发现,种板后第12h开始,细胞增殖率明显升高(P<0.05),呈明显的时间和剂量依赖性。
3、IL-22可显著抑制角质形成细胞的分化：Western Blot检测发现,60ng/mL的IL-22可抑制角质形成细胞分化标志蛋白CK10、Involucrin的表达水平(P<0.05),且呈明显的浓度依赖性。
4、IL-22可显著下调C/EBP a mRNA和蛋白的表达水平：RT-PCR和Western Blot检测发现,IL-22可抑制角质形成细胞C/EBPα mRNA和蛋白的表达水平(P<0.05),且呈明显的浓度依赖性。
结论
1、IL-22可以显著调控角质形成细胞的增殖和分化过程。
2、IL-22可显著调控角质形成细胞中MAPK信号通路及C/EBP α的表达水平。
3、IL-22可能通过MAPK信号通路及下游的C/EBP α的表达调控角质形成细胞的增殖和分化
Psoriasis, which is associated with a genetic background and abnormal immune response, is one of the most common clinical erythematous desquamative dermatosis in China with an incidence of0.123%-0.470%. Because of easy recurrence and difficult to cure, psoriasis seriously influences patients health and quality of life. However, until now, the exact etiology and pathogenesis of psoriasis have not yet been fully elucidated, which becomes one of the most urgent problems to be solved in dermatology field.
In recent years, many studies have been conducted on psoriasis in both China and abroad with some progresses, nevertheless, the cause and pathogenesis of the psoriasis have not been completely clarified so far. The fundamental pathophysiology of the psoriasis is supposed to be related to the genetic factors, immune factors, infection factors, endocrine factors, neuropsychiatric factors, habits of life, drugs and environmental factors. All of these above factors lead to the abnormality for the proliferation, differentiation and apoptosis of keratinocyte and the abnormal neogenesis of the vascular endothelial cell in the dermal papilla layer, which result in the clinically characteristic skin lesions of psoriasis such as red patches covered with silvery white scale trifles, the phenomenon of membrane, the phenomenon of candle dripping and the Auspitz sign.
CCAAT/enhancer binding protein a (C/EBPa), an important member of the transcription factor C/EBP family, plays a vital role in the synthesis and metabolism of phospholipids and the development, proliferation, differentiation of variable cell types, for example fat cells, bone marrow cells, liver cells, keratinocyte and alveolar cells. C/EBPa is mainly expressed in the keratinocytes from the basal layer of the epidermis and the overexpression of C/EBPa may cause cell cycle arrest. The high expression of C/EBPa in the epidermis of mice and human indicates that C/EBPa plays a transcription factor role in the differentiation of stratified squamous epithelium cells. The overexpression of oncogene Ras leads to the decrease of C/EBPa in immortalized keratinocytes. Consequently, the inhibition effect of cell proliferation by C/EBPa is reduced. Thus it can be seen that C/EBPa plays an important role in regulating the proliferation and differentiation of epidermal keratinocytes in normal skin. How dose C/EBPa express in the skin lesions of psoriasis vulgaris with abnormal proliferation and differentiation of keratinocyte? Whether does C/EBPa participate in the course of pathological changes of psoriasis vulgaris or not? Up to now, there is no literature reported on those relevant issues in China and abroad.
In order to investigate the role of C/EBPa in the pathogenesis of psoriasis, the mRNA and protein expressions of C/EBPa in the skin lesions of psoriasis were detected. To explore the clinical significance of abnormal C/EBPa expression, the relationship of the expression of C/EBPa between the psoriasis area and severity index (PASI), the proliferation and differentiation of keratinocytes were investigated. To explore the effect of C/EBPa gene on the proliferation of keratinocytes, the expression of C/EBPa gene in primary human keratinocyte was silenced by short interfering RNA (siRNA) for C/EBPa. We applied the key cytokines IL-22, which was secreted by Thl7cells and Th22cells in the pathogenesis of psoriasis, to stimulate the keratinocyte,and then detected the expression of C/EBPa and the phosphorylation levels of JNK, ERK and p38, the key signaling molecules in MAPK signaling pathways. Through the above investigations, we wanted to make clear that if the expression of C/EBPa could participate in the formation of the skin lesions in psoriasis vulgaris through MAPK signaling pathways.
Part I. The mRNA and protein expressions of C/EBPa in the lesions of psoriasis with the clinical significance
Objective:To detect the mRNA and protein expressions of C/EBPa in the lesions of psoriasis and investigate their relationships with the abnormal proliferations of keratinocytes and the PASI.
Methods:1. Specimens Collection:Between January2011and December2011,30cases of paraffin-embedded specimens of psoriasis vulgaris were collected as the experimental group in the Pathological Lab of the Department of Dermatology in Qilu Hospital of Shandong University. Meanwhile,30cases of health skin specimens matched for ages, gender and site of sampling with the experimental group were collected as the control group in the Department of Dermatology of our hospital.2. Immunohistochemical method to detect the expression of C/EBPa and Ki-67:The expressions of C/EBPa, Ki-67and CK10were detected by two-step immunohistochemical method in the control and experimental groups. Semi-quantitative scores were recorded according to the number of positive cells and the degree of dyeing. The difference of expression between two groups was analyzed by Chi-square test.
3. The correlations between the expression of C/EBPa and the proliferation index (PI) of keratinocytes and the PASI:The PI of keratinocytes was calculated according to the positive expression of Ki-67and the associations between the expression of C/EBPa and PI and PASI were analyzed by Pearson correlation analysis.
4. The mRNA and protein expressions of C/EBPa in lesions of psoriasis and health skin were detected by RT-PCR and western blot.
Results:1. Clinical data of patients:The30cases of psoriasis vulgaris patients with a mean age of25.2years (range15to49years) included18males and12females. For the lesions among the30cases of psoriasis vulgaris,10cases were on the head and face,10cases on the trunk and10cases the on extremities.2. The expression of C/EBPa was localized dominantly in the cytoplasm of keratinocytes and the expression of C/EBPa was significantly lower in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=7.819, P<0.05).
3. The expression of Ki-67was detected mainly in the nucleus of keratinocytes. The PI was significantly higher in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=4.535, P<0.05).
4. According to the results of Pearson correlation analysis, the gray values of C/EBPa were positively correlated with the PI and PASI in the lesions of psoriasis vulgaris (r=0.654, P<0.05and r=0.693, P<0.05, respectively).
5. According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa were significantly lower in the lesions of psoriasis vulgaris than in control group with a statistical difference (P<0.05).
Conclusions:The mRNA and protein expressions of C/EBPa were significantly reduced in the lesions of psoriasis vulgaris. The protein expression of C/EBPa was negatively correlated with the PI of keratinocytes and PASI of lesions of psoriasis vulgaris. It was speculated that C/EBPα, which could be used as an indicator reflecting the severity of psoriasis lesions, might play a role in the process of the abnormal proliferation of keratinocytes.
Part Ⅱ. The gene expression of C/EBP α in keratinocytes and the effects on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBP α gene silencing
Objective:1. To detect the mRNA and protein expressions of C/EBPa in primary human keratinocytes.
2. To investigate the effects of C/EBPα on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBPα gene silencing.
Methods:1. The culture of primary cells:The primary culture of human epidermal keratinocytes was used in the study. The skin specimens from the young children's foreskin were obtained by the Department of Paediatric Urology of Shandong Province Hospital affiliated to Shandong University. The specimens were digested with0.25%trypsin at4℃for overnight to dissect epidermis from dermis. Then, the epidermis specimens were digested with0.25%trypsin-0.01%EDTA mixture to obtain single cell suspension, which was added into Epilife-HKGS culture solution. The adherent cells were gained by cultured in saturated humidity conditions with5%CO2at37℃and the medium was changed every other day. Following3-4stable passages, the cells were obtained for further study.
2. The siRNA transfection for C/EBPα:The siRNA sequence for C/EBPaproduced by QIAGEN Company. The transfection efficiency, interference efficiency and transfection concentration for the siRNA had been verified in the pretest. The keratinocytes in the logarithmic phase were inoculated into6-well plates with2×105 cells/well. When the primary keratinocytes were fused to70%, siRNA was transfected into the cells by lipofectamine2000according to the manual strictly, as siRNA transfection group. Meanwhile, the empty vector transfection group and non-transfection group were also established.
3. The detection of the interferential efficiency for C/EBPa gene:Total RNA was extracted after24h of transfection. The mRNA expression of C/EBP a was detected by RT-PCR. Total protein was extracted after72h of transfection. The protein expression of C/EBPa was detected by western blot.
4. The effect of C/EBPa gene silencing on the proliferation of keratinocytes by CCK-8colorimetric analysis:KC-C and KC cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. The proliferation of cells was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.
5. The expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:Total protein was extracted after72h of transfection. The protein expressions of CK10and involucrin were detected by western blot.
Results:1. The expression of C/EBPa was inhibited effectively by siRNA:The mRNA expression of C/EBPa was inhibited by47%compared with those in control group72h after siRNA transfection and the expressions of C/EBP a protein were also down-regulated significantly(P<0.05).
2. CCK-8colorimetric analysis was used to detect the effect of C/EBPa gene silencing on the proliferation of keratinocytes. There was no significant difference in the proliferation of keratinocytes in the siRNA transfection group, empty vector transfection group and non-transfection group after12h inoculating. However, after inoculating for24h,48h and72h, the proliferation of keratinocytes in the siRNA transfection group was significantly decreased with a statistical difference compared to the empty vector transfection group and non-transfection group.(P<0.05at24h,48h and72h, respectively)
3. According to the results of western blot, the protein expressions of CK10and involucrin were significantly lower in the siRNA transfection group than in the empty vector transfection group and non-transfection group with a statistical difference (P<0.05).
Conelusions:1. The siRNA for C/EBPa could inhibit the expression of C/EBPa in keratinocytes. After C/EBPa gene silencing, the proliferation rate of keratinocytes was increased and the differentiation level of keratinocytes was decreased.
2. The C/EBPa gene was a potential gene to cure the diseases withgabnormal proliferation and differentiation in keratinocytes.
Part Ⅲ:The effect of IL-22stimulation on MAPK signaling pathway and the expression of C/EBPa in keratinocytes
Objective:1. To investigate the effect of IL-22on the phosphorylation of key proteins in MAPK signaling pathway in keratinocytes.
2. To investigate the effect of IL-22on the gene expression of C/EBPa in keratinocytes.
3. To investigate the effect of IL-22on the proliferation and differentiation of keratinocytes and its correlation with the gene expression of C/EBPa.
Methods:1. The culture of primary cells referred to Part II.
2. The stimulation of IL-22on keratinocytes:The primary cultured cells of keratinocytes were used as the research subjects. When the primary keratinocytes were fused to60%, human IL-22fusion protein was added into the cells with the final concentration of30ng/mL,60ng/mL,90ng/mL, respectively. And then the cells with treatment were collected at different time points for subsequent experiments.
3. The detection of the phosphorylation level of key protein in MAPK signaling pathway by western blot:Total protein was extracted after72h of transfection. The protein expressions of JNK, p-JNK, p38, p-p38, ERK and p-ERK were detected by western blot.
4. The effect of IL-22on the proliferation of keratinocytes by CCK-8colorimetric analysis:The keratinocytes cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. Then, human IL-22fusion protein was added into every well with the final concentration of30ng/mL,60ng/mL and90ng/mL, respectively. The proliferation of keratinocytes was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.
5. The effect of IL-22on the expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:After the stimulation of IL-22for48h, total protein was extracted. The protein expressions of CK10and involucrin were detected by western blot.
6. The detections of the mRNA and protein expressions of C/EBPa by RT-PCR and western blot:After the stimulation of IL-22for48h, total RNA and total protein were extracted, respectively. The mRNA and protein expressions of C/EBPa were detected by RT-PCR and western blot, respectively.
Results:1. The phosphorylation levels of JNK, ERK and p38were significantly increased by the stimulation of IL-22:After the stimulation of IL-22for60minutes, p-JNK, p-ERK and p-p38were all significantly increased compared with the control group with the statistical difference (P<0.05).
2. The proliferation of keratinocytes was significantly increased by IL-22:According to the results of CCK-8colorimetric analysis, the cell proliferation was significantly increased, with a significant time and concentration dependent manner, from12h after the cells were inoculated into the plate.
3. The differentiation of keratinocytes was significantly inhibited by IL-22:According to the results of western blot, the protein expressions of CK10and involucrin in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.
4. The mRNA and protein expressions of C/EBPa were significantly inhibited by IL-22:According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.
Conelusions:1. IL-22can significantly regulate the proliferation and differentiation of keratinocytes.
2. IL-22can significantly regulate the MAPK signaling pathway and the expression of C/EBPa in keratinocytes.
3. IL-22may regulate the proliferation and differentiation of keratinocytes through MAPK signaling pathway and the expression of downstream molecular, C/EBPa.
引文
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