【作者】 朱晓芳；

【导师】 卜平；

【作者基本信息】 扬州大学 ， 中西医结合临床， 2013， 博士

【摘要】 研究背景：银屑病(Psoriasis)是一种常见的慢性炎症性皮肤病,病情顽固,且发病率在逐年增高。该病组织病理改变主要为表皮角质形成细胞(keratinocyte, KC)过度增殖和分化异常,真皮乳头毛细血管增生、扩张,以及真表皮内炎症细胞浸润。近年来,银屑病发病的免疫机制备受关注,对银屑病患者皮损处T细胞的特性及其产生细胞因子功能的研究发现,一种新的调节性T细胞即Th17细胞及其活化后产生的细胞因子Interleukin-17A(IL-17A)、 IL-17F和IL-22在银屑病的发病中具有重要作用。一个重要发现是在银屑病患者的皮损及血浆中IL-22表达均显著增加。研究表明,IL-22阻碍KC的终末分化,诱导KC的促炎基因表达和KC的迁移。然而目前并不十分明确IL-22介导的KC效应的细胞分子机制。Grb2-相关结合蛋白1/2(Grb2-associated binder1/2, Gab1/2)是一类脚手架／接头蛋白,主要参与膜表面受体酪氨酸激酶和非受体酪氨酸激酶的信号转导。磷酸化的Gab1通过与含有2个Src同源结构域(SH2)的蛋白酪氨酸磷酸酶(Src homology2domain-containing tyrosine phosphatase,Shp-2)相互作用,具有调节表皮细胞生长因子诱导的表皮生长和分化,但尚不清楚Gab1以及同样高表达于KC细胞上的Gab2在IL-22诱导的KC增殖、迁移和分化中是否发挥作用?银屑病属于中医“白疙”、“蛇虱”、“松皮癣”等范畴。清热解毒,凉血活血是中医治疗本病的重要治则。紫草味甘、咸,性寒,有凉血、活血、清热、解毒透疹等功效。紫草素(Shikonin)为紫草中的主要有效成分之一,具有多种药用价值,包括抗肿瘤、抗菌、抗病毒、抗炎、镇静、清热解毒、免疫调节以及促进伤口愈合等。在对紫草素诱导肿瘤细胞凋亡机制的研究中发现,紫草素可调节促分裂原活化蛋白激酶激酶(mitogen-ativated protein kinase kinase, MAPK)通路的活性,降低Erk, Jnk, Stat3磷酸化水平,从而抑制肿瘤细胞生长、增殖、存活。临床发现紫草素治疗银屑病具有较好的疗效,但其机制尚有待探讨。我们提出一假设,即紫草素通过拮抗IL-22刺激皮肤角质形成细胞引起的Gab1/2的磷酸化,进一步抑制其下游Erk/MAPK信号通路的活化,从而抑制KC增殖,影响KC分化及迁移等。本文的研究目的：第一部分：研究紫草素对IL-22刺激HaCaT细胞生物学行为影响1.观察紫草素对IL-22刺激HaCaT细胞增殖、迁移的影响;2.观察紫草素对IL-22刺激HaCaT细胞分泌S100A7, S100A8、S100A9的影响。第二部分：紫草素对IL-22刺激HaCaT细胞生物学行为改变的机制研究1.研究IL-22刺激HaCaT细胞中Jak1/Tyk2/Stat, Erk/MAPK信号通路中相关蛋白表达；2.研究IL-22刺激Gab1和Gab2突变体(Shp2结合缺陷型)感染的HaCaT细胞中Erk/MAPK信号通路中相关蛋白表达；3.研究IL-22刺激Gab1和Gab2基因沉默的HaCaT细胞中Erk/MAPK信号通路中相关蛋白表达；4.探讨Gab1和Gab2是否共同参与IL-22诱导的细胞增殖和迁移以及对细胞分化的影响；5.研究IL-22刺激HaCaT细胞后Gab1、Gab2以及Erkl/2磷酸化水平及紫草素干预后的变化。研究方法：1.WST-1法检测细胞增殖；2. Transwell法检测细胞迁移；3. Real-TimePCR法检测基因mRNA表达；4. Western blot法检测相关蛋白的表达；5.免疫沉淀技术检测特定蛋白间的相互作用：6.构建Shp2结合缺陷型Gabl和／或Gab2以及Gabl和Gab2siRNA干扰的重组腺病毒；7.统计学分析：采用SPSS17.0统计软件进行数据分析,P<0.05时差异有统计学意义。研究结果：1.紫草素抑制HaCaT细胞的生长不同浓度的紫草素对HaCaT细胞的生长有一定的抑制作用,但低浓度时(浓度为0.1和0.25μg/ml),紫草素本身不影响细胞活性,对HaCaT细胞无损伤及毒性作用。2.紫草素抑制IL-22介导的HaCaT细胞增殖和迁移实验分组：未处理(未加紫草素和IL-22)组、IL-22刺激24h组、IL-22+紫草素24h组, IL-22刺激48h组、IL-22+紫草素48h组。紫草素作用浓度选择为0.25μg/ml, IL-22推荐工作浓度为100ng/ml, IL-22刺激HaCaT细胞24h和48h后可促进细胞增殖和迁移,与未处理组相比,P均<0.05；加入紫草素组,IL-22诱导的HaCaT细胞增殖和迁移作用被抑制,P均<0.05。3.紫草素可下调IL-22诱导的促炎症因子S100A7、S100A8mRNA表达Real-Time PCR检测紫草素对IL-22刺激HaCaT细胞分泌促炎症因子S100A7、 S100A8、S100A9mRNA影响,根据扩增曲线由公式得出2-△Ct值(即实验组目的基因的表达相对于对照组基因的变化倍数),IL-22刺激S100A7S100A8mRNA表达与未处理组相比其差别倍数(2-△△Ct)均大于2,而加入紫草素组与IL-22组相比其差别倍数均小于1,表明IL-22可明显刺激HaCaT细胞分泌S100A7, S100A8,而紫草素可下调IL-22诱导的S100A7S100A8mRNA表达,但对S100A9mRNA的影响不明显。4.IL-22诱导HaCaT细胞Jak1/Tyk2/Stat通路、MAPK通路的活化HaCaT细胞饥饿处理16-18h后,加入IL-22(100ng/ml)刺激10min、20min、30min、60min以及120min,提取总蛋白,Western blot检测到IL-22刺激10min后即可见Jakl/Tyk2/Stat通路中Stat3Tyr705位酪氨酸磷酸化、Stat3Ser727位丝氨酸磷酸化、Jakl以及Tyk2磷酸化；MAPK通路中Erk1/2和p38磷酸化。Mek抑制剂PD98059(10μM)预处理HaCaT细胞1h,然后再用IL-22刺激,则Erk1/2活化被抑制,同时PD98059预处理后IL-22诱导的HaCaT细胞的增殖及迁移亦被抑制(P分别<0.01和<0.05),而KC细胞分化标志性蛋白Loricrin在IL-22刺激72h后仍未检测到表达,但PD98059预处理后再用IL-22刺激则Loricrin表达明显增加。5.IL-22刺激HaCaT细胞引起Gab1和Gab2磷酸化,并进一步活化Erk1/2IL-22刺激饥饿处理后的HaCaT细胞,10min后裂解细胞,采用免疫共沉淀技术及免疫印迹技术检测IL-22刺激后,Gab1和Gab2磷酸化水平明显升高,Shp2结合增强。以表达Shp2结合缺陷型Gab1和Gab2重组腺病毒(AdGab1FF、AdGab2F)以及野生型Gab1和Gab重组腺病毒(AdGab1WT、AdGab2WT)感染HaCaT,检测IL-22刺激引起的Erkl/2以及Stat3Tyr795蛋白活化水平,结果发现,Erkl/2磷酸化水平在(AdGab1FF)HaCaT、(AdGab2F)HaCaT组明显降低,Stat3Tyr795磷酸化水平在缺陷型组及野生型组基本没有变化；IL-22刺激siRNA沉默Gab1和／或Gab2腺病毒感染的HaCaT细胞(AdGab1siRNA) HaCaT、(AdGab2siRNA) HaCaT、(AdGab1siRNA+AdGab2siRNA) HaCaT,检测Erkl/2以及Stat3Tyr795蛋白活化水平,结果发现,Erkl/2磷酸化水平在基因沉默组中明显降低,磷酸化Stat3Tyr795水平依然没有变化。6.Gab1和Gab2影响IL-22诱导的HaCaT细胞增殖、细胞迁移及分化与Shp2相关IL-22刺激(AdGab1WT)HaCaT、(AdGab2WT)HaCaT、(AdGab1FF)HaCaT、(AdGab2F)HaCaT,24h和48h后分别检测细胞增殖率及细胞迁移,结果(AdGab1FF)HaCaT、(AdGab2F)HaCaT组细胞增殖率减低(P均<0.01),迁移细胞数减少(P均<0.01)；IL-22刺激(AdGab1siRNA) HaCaT、(AdGab2siRNA) HaCaT、(AdGab1siRNA+AdGab2siRNA) HaCaT,24h和48h后检测细胞增殖率和迁移,与感染空载体HaCaT细胞相比,细胞增殖率减低(P<0.05,P<0.01),细胞迁移数减少(P均<0.05)。IL-22刺激重组腺病毒感染的HaCaT的细胞72h后,检测Loricin蛋白的表达,结果发现,与野生型组相比,基因突变和沉默组细胞Loricin的表达量均明显增加。7.紫草素拮抗IL-22刺激皮肤角质细胞引起的Gab1, Gab2以及Erkl/2的磷酸化IL-22刺激经紫草素(0.25μg/ml)预处理HaCaT细胞,检测Gab1和Gab2以及Erkl/2磷酸化水平,与未经紫草素处理组相比,明显减低。研究结论：1.紫草素能够抑制IL-22诱导的HaCaT细胞的增殖和迁移,并下调IL-22诱导的HaCaT细胞S100A7, S100A8mRNA表达。2.IL-22诱导Jak1/Tyk2/Stat及MAPK通路在HaCaT细胞中的活化,介导细胞增殖、迁移和分化。3.Gab1和Gab2共同参与IL-22诱导的HaCaT细胞中的Erk/MAPK信号通路的活化,并共同参与IL-22介导的细胞增殖、迁移和分化。Shp2与Gab1和Gab2结合是IL-22介导的效应所必需的。4.紫草素抑制IL-22诱导的Gabl,Gab2以及Erkl/2磷酸化,从而推论紫草素治疗银屑病机制可能与抑制IL-22引起的Gab1、Gab2磷酸化,进一步抑制Erk1/2-MAPK通路活化有关。更多还原

【Abstract】 Background Psoriasis is a kind of common chronic inflammatory skin disease with increasing incidence year by year. The histopathological changes of psoriasis include hyperplasia of the epidermis (acanthosis), abnomal differentiation of keratinocyte (KC), dilation and growth of dermal papilla capillaries, and infiltration of leukocytes into both the dermis and epidermis. Recent studies have shed light on the immunologic mechanism of psoriasis. Based on the studies of the characteristics of T cells in the skin lesions of psoriasis patients and their function of inducing cytokines, researchers found that a new regulatory T cell subset that is, Th17cells secrete cytokines of Interleukin-17A(IL-17A), IL-17F and IL-22, which play important roles in the pathogenesis of psoriasis. Of note, the expression of IL-22in skin lesions and plasma of psoriasis patients is increased significantly. IL-22is known to not only block the terminal differentiation of KC but also induce KC migration and the expression of proinflammatory genes. However, the cellular and molecular mechanism of KC effect mediated by IL-22is not well understood so far. Grb2-associated binder1/2(Gab1/2) are a kind of adaptor proteins which are involved in signal transduction of membrane surface receptor tyrosine kinase and non-receptor tyrosine kinase. Phosphorylated Gabl regulates the epidermal growth and differentiation induced by epidermal growth factor (EGF) through interaction with Src homology2domain-containing tyrosine phosphatase(Shp-2), but it is not known whether Gabl and Gab2, which are highly expressed in KC, paly roles in proliferation, migration and differention of KC induced by IL-22.Psoriasis belongs to categories of "baibi","snake lice","loose skin tinea" in Traditional Chinese Medicine (TCM).Clearing heat and detoxification, cooling blood and invigorating circulation are the important TCM therapeutic principles for the disease. Zicao tastes sweet and salty with cold properties, and has effects of cooling blood, invigorating circulation, detoxification, as well as promoting eruptions. Shikonin is one major effective components of Zicao with a wide range of bioactivities. It has high medicine values including anti-tumor, antibacterial, antiviral, sedation, heat-clearing and detoxifying, immunoregulation, healing wounds. Investigating the apoptosis mechanism of tumor cell induced by shikonin revealed that it inhibits the proliferation of tumor cells by regulating the activity of MAPK pathway and reducing the phosphorylation of Erk, Jnk, and Stat3. Though the effect of shikonin treatment for psoriasis is well demonstrated, the underlying mechanism remains largely unknown.We speculate that shikonin exerts it effect on the proliferation, differentiation and migration of KC by antagonizing IL-22-induced phosphorylation of Gab1/2on KC, which resulting in the down-regulation of the activation of Erkl/2/MAPK singnal pathway.ObjectivesPart one:To investigate the effect of shikonin on cell biological behaviour of HaCaT mediated by IL-221. Effect of shikonin on proliferation and migration of HaCaT cell stimulated by IL-22;2. Effect of shikonin on expression of proinflammatory genes S100A7, S100A8, S100A9mRNA stimulated by IL-22.Part two:The mechanisms study of the effect of shikonin on IL-22-mediated HaCaT biological behaviour.1. Detect the expression of proteins in Erkl/2/MAPK pathway in HaCaT stimulated by IL-22;2. Explore the expression of proteins in Erkl/2/MAPK pathway in HaCaT infected with adenovirus expressing Gab1and Gab2mutants (Shp2-binding defective);3. Explore the expression of proteins in Erk1/2/MAPK pathway in HaCaT with adeno virus expressing siRNA targeting Gab1and Gab2;4. Study on whether both Gabl and Gab2play roles in cell proliferation, migration and differentiation of HaCaT induced by IL-22;5. Study on the phosphorylation of Gabl, Gab2and Erkl/2stimulated by IL-22and the intervention of shikonin.Methods1. Detection of cell proliferation by WST-1assay2. Detection of cell migration with Transwell assay3. Detection of the expression of mRN A by Real-Time PCR4. Detection of the expression of proteins by Western blot assay5. Deterction of the interactions between specific proteins by immunoprecipitation6. Construction of recombinant adenovirus (Shp2-binding defective Gabl and Gabl, siRNA knockouted Gab1and Gab2, siRNA knockouted Gab1+Gab2)7. Statistical analysis with SPSS17.0software (difference has statistical significance when P<0.05)Results1. Shikonin inhibited the growth of HaCaT cells in a dose-dependent manner. It should be noted that low concentration of shikonin (0.1and0.25ug/ml) didn’t affect the cell activity and had no damage and toxicity to HaCaT cells.2. Shikonin treatment decreased HaCaT cells proliferation and migration mediated by IL-22.. The experiment about the proliferation and migration of HaCaT inhibited by shikonin is performed as following groups:blank control (without shikonin and IL-22), incubate with IL-22for24h, incubate with IL-22and shikonin for24h, incubate with IL-22for48h, and incubate with IL-22and shikonin for48h. The concentration of shikonin and IL-22were0.25ug/ml and100ng/ml, respectively. After adding IL-22for24h and48h, the proliferation and migration of HaCaT were all increased (P<0.05), however these effects were inhibited by adding the shikonin (P<0.05). 3. Shikonin down-regulated the expression of S100A7, S100A8and S100A9induced by IL-22. We detected the effect of shikonin on the expression of S100A7, S100A8and S100A9mRNA induced by IL-22by Real-Time PCR. The results showed that2-△△Ct values of S100A7and S100A8induced by IL-22were all more than2, while adding shikonin, the2-△△Ct values were less than1, that indicates shikonin can down-regulate the mRNA expression of S100A7, S100A8induced by IL-22in HaCaT, but has no influence on the expression of S100A9.4. IL-22induced the activation of Jakl/Tyk2/Stat and MAPK pathway. HaCaT cells were serum starved for16-18h, and then stimuilated with or without IL-22(100ng/ml) for10min,20min,30min,60min and120min.and total cells lysates were analyzed by Western blot, The results of Western blot showed that Stat3Tyr705, Stat Ser727, Jakl and Tyk2in Jakl/Tyk2/Stat pathway and Erkl/2and p38in MAPK pathway were phosphorylated after10min stimulation by IL-22. The activation of Erkl/2, the proliferation and migration of HaCa T cells induced by IL-22were suppressed when the HaCaT cells were pretreated with Mek inhibitor PD98059(10μM) for1h. However, Loricrin, the marker protein of the differentiation of KC, was still not detected at72h after the stimulation of IL-22, while Loricrin expression of HaCaT was increased significantly when PD98059was added before IL-22stimulation.5. IL-22induced the phosphorylation of Gabl and Gab2, and then activated Erkl/2. After the stimulation of serum-starved HaCaT cells with IL-22for10min, co-immunoprecipitation and Western blot assays showed that the phosphorylation of Gabl and Gab2increased markedly and the binding with Shp2was also enhanced. The activation of Erkl/2and Stat3Tyr795were tested after the HaCaT cells were infected by recombinant adenovirus AdGab1EF, AdGab2F, AdGab1WT, AdGab2WT. The results showed that there was a marked decrease in the phosphorylation of Erkl/2in groups of (AdGablEF) HaCaT and (AdGab2F) HaCaT, while the phosphorylation of Stat3Tyr795in groups of (AdGab2F) HaCaT,(AdGab1WT) HaCaT and (AdGab2WT) HaCaT had no change. Besides, the activations of Erkl/2and Stat3Tyr795in (AdGablsiRNA) HaCaT,(AdGab2siRNA) HaCaT and (AdGablsiRNA+AdGab2siRNA) HaCaT were also tested. The results showed that the phosphorylation of Erkl/2all decreased in the three groups, but that of Stat3Tyr795still had no markedly changes.6. The influence of Gab1and Gab2on the proliferation, migration and differentiation of HaCaT induced by IL-22is related with Shp2. The cell proliferation rate and migration were detected after the stimulation of(AdGab1WT) HaCaT,(AdGab2WT) HaCaT,(AdGab1FF) HaCaT and (AdGab2F) HaCaT by IL-22for24h and48h. The results showed that the proliferation rate and migration cells of (AdGablFF) HaCaT and (AdGab2F) HaCaT all reduced (P<0.01). Similar results were obtained in (AdGab1siRNA) HaCaT、(AdGab2siRNA) HaCaT and (AdGab1siRNA+AdGab2siRNA) HaCaT. Compaired with the (AdU6Vector) HaCaT, cells proliferation rate decreased in the experimental groups within24h and48h(P<0.05and P<0.01, respectively);migration cells also decreaed(both P<0.05);Stimulating adenovirus-infected HaCaT with IL-22for72h, we observed that the expression of Loricin in mutant and silent taget gene groups were higher than that in wild type groups or the control virus.7. Shikonin inhibited IL-22-induced phosphorylation of Gab1, Gab2and Erkl/2on KC. The levels of phosphorylation of Gabl, Gab2and Erkl/2in shikonin-treated HaCaT cells upon IL-22stimulation were lower than in the groups without shikonin.Conclusions1. Shikonin inhibited the proliferation and migration of HaCaT cells and reduced the expression of S100A7and S100A8mRNA in HaCaT cells induced by IL-22.2. IL-22induced the activation of Jakl/Tyk2/Stat and MAPK pathways in HaCaT cells and mediated the cell proliferation, migration and differentiation.3. Gabl and Gab2participated jointly in IL-22mediated keratinocyte proliferation, migration and differentiation mainly via activation of Erk/MAPK pathway.4. Shikonin inhibited the phosphorylation of Gabl, Gab2and Erkl/2induced by IL-22, suggesting that the mechanism of shikonin treatment for psoriasis may be related with the inhibition of activation of Gabl, Gab2and futhur down-regulated the activation of Erkl/2/MAPK pathway.更多还原