【作者】 李明；

【导师】 唐瞻贵；

【作者基本信息】 中南大学 ， 外科学， 2024， 博士

【摘要】 研究背景:口腔黏膜纤维性变(Oral submucous fibrosis,OSF)是以炎症表现为主的口腔黏膜结缔组织进行性纤维性变疾病,可伴随口腔上皮不典型增生或萎缩,其临床表现为口腔黏膜可触及片状或条索状纤维硬化带,面颊部内陷、张口受限以及口腔黏膜对辛辣刺激不耐受等症状。OSF的发生与咀嚼槟榔、营养不良、免疫异常等因素有关,其中咀嚼槟榔被认为是OSF发生的主要致病因素。目前尚无有效治疗OSF的药物,急切需要相关靶向药物的研制。槟榔果中的槟榔生物碱、多酚类物质、鞣酸类物质以及微量金属元素都具有一定程度的细胞毒性。此外,经过脱水加工的商品槟榔果壳表面坚韧,果壳内含致密的粗纤维,在咀嚼过程中常磨损口腔黏膜上皮角化层,加重了槟榔中有害物质及其代谢产物对口腔角质细胞的危害。口腔黏膜上皮层细胞做为口腔组织的第一道屏障,在OSF发生发展中有较大的研究价值。然而,目前的相关研究中关于槟榔对口腔角质细胞的生物学影响,以及在槟榔刺激下的口腔角质细胞对口腔成纤维细胞的生物学影响尚未深入研究。因此,明确槟榔对口腔角质细胞的生物学影响,以及槟榔刺激下的口腔角质细胞对口腔成纤维细胞的“信息传递”内容及机制对OSF的预防和治疗具有重要意义。研究方法:第一部分研究方法:采用高效液相色谱法测定槟榔提取物中槟榔生物碱的含量。采用Cell Counting Kit-8(CCK-8)实验分析了梯度浓度槟榔提取物(Areca nut extract,ANE)对人口腔角质细胞(Human oral keratinocytes,HOK)增殖活性的影响。采用第二代转录组测序分析实验组HOK细胞与对照组HOK细胞的差异表达基因(P值小于0.05,log2倍数大于1),并采用生物信息学分析两组间差异高表达基因涉及的细胞信号通路和分子功能。采用流式细胞术检测HOK细胞周期或细胞凋亡改变;采用免疫印迹分析HOK细胞中Fox O信号通路相关蛋白(FOXO1、FOXO3、p-FOXO1和p-FOXO3)、细胞自噬相关蛋白(LC3-Ⅰ/Ⅱ和p62)、上皮间充质转化(Epithelial mesenchymal transition,EMT)相关蛋白(SMAD2,p-SMAD2,E-cadherin和Vimentin)表达;采用免疫荧光实验检测HOK细胞中γH2AX蛋白表达;采用β-半乳糖苷酶染色试剂盒检测HOK细胞衰老;采用Real-time PCR检测HOK细胞中衰老相关表型基因(IL6、MMP2、TGFB1、CDKN2A和CDKN1A)表达;采用ELISA实验检测HOK细胞外分泌蛋白TGF-β1表达;光学显微镜下观察ANE干预后的HOK细胞形态。第二部分研究方法:筛选10 mg/mL ANE组、500μg/mL ANE组转录组测序结果中涉及MAPK信号通路的高表达配体基因,并采用Real-time PCR验证其表达;采用Real-time PCR和免疫印迹实验检测AREG敲低、过表达质粒对HOK细胞的转染效果;预先敲低AREG基因后采用500μg/mL ANE处理HOK细胞,随后采用CCK-8、划痕实验、Transwell实验检测实验组细胞较对照组HOK细胞增殖、迁移能力的改变;过表达AREG后,采用CCK-8、划痕实验、Transwell实验检测实验组HOK细胞较对照组HOK细胞增殖、迁移能力的改变;过表达、敲低HOK细胞中AREG基因后,采用免疫印迹检测HOK细胞中MAPK信号通路蛋白(MEK1、ERK1+2、p-MEK1和p-ERK1+2)和EMT相关蛋白(E-cadherin和Vimentin)表达。第三部分研究方法:采用免疫组化检测AREG蛋白在正常人口腔组织和OSF组织中的表达,并结合OSF患者临床病理信息,分析OSF组织中AREG蛋白表达与OSF患者临床病理因素的关系;采用免疫荧光检测OSF组织中AREG蛋白分布;采用CCK-8、流式细胞术、划痕实验、Transwell迁移实验、免疫印迹实验分析重组人AREG蛋白对人口腔成纤维细胞(Human oral fibroblasts,HOF)的增殖、凋亡、迁移和胶原合成相关蛋白(Collagen-1α、Collagen-3α和α-SMA)表达的影响;将HOF细胞与500μg/mL ANE预处理的HOK细胞共培养后,采用CCK-8、流式细胞术、划痕实验、Transwell迁移实验和免疫印迹实验分析HOF细胞的增殖、凋亡、迁移和胶原合成能力改变;采用EGFR受体阻滞剂Erlotinib处理HOF细胞后再加入rhAREG蛋白,采用CCK-8、流式细胞术、划痕实验、Transwell迁移实验、免疫印迹实验分析HOF细胞生物功能改变及其涉及的信号通路。构建槟榔相关OSF大鼠模型,采用Masson染色检测大鼠口腔组织纤维化改变,采用免疫组化体内验证AREG、α-SMA、Collagen-1α 和Collagen-3α蛋白表达情况,采用免疫荧光分析OSF大鼠模型体内AREG与α-SMA、Collagen-1α和Collagen-3α蛋白的表达的相关性。研究结果:第一部分研究结果:本研究提取的ANE中成功地检测到槟榔果特异性成分槟榔碱、去甲基槟榔碱、槟榔次碱和去甲基槟榔次碱。CCK-8结果显示500μg/mL的ANE可促进HOK细胞增殖;10 mg/mL及以上浓度的ANE抑制了HOK细胞增殖;其余浓度的ANE组HOK细胞与对照组HOK细胞的增殖活性差异无统计学意义。在500μg/mL ANE组、10 mg/mL ANE组与对照组HOK细胞的转录组测序结果中可见10 mg/mL ANE和500μg/mL ANE均可使HOK细胞中MAKP信号通路相关基因表达上调,10 mg/mL ANE可引起HOK细胞中细胞周期、P53、自噬、Fox O、细胞衰老等信号通路相关基因高表达。流式细胞术结果显示10 mg/mL ANE可诱导HOK细胞凋亡和G2/M期阻滞。与对照组相比,10 mg/mL ANE组HOK细胞中衰老细胞数量增加,衰老相关表型基因IL6、MMP2、TGFB1、CDKN2A和CDKN1A表达上调。10 mg/mL ANE可促使HOK细胞的双链DNA损伤标记蛋白γH2AX表达增加。在对Fox O信号通路的验证中发现10 mg/mL ANE上调了HOK细胞中p-FOXO1和p-FOXO3蛋白表达。在对细胞自噬信号通路的验证中发现10 mg/mL ANE可促使HOK细胞中LC3-Ⅱ蛋白表达上调,LC3-Ⅰ和P62蛋白表达下调。采用2 mg/mL ANE处理HOK细胞30天后,细胞外分泌TGF-β1蛋白表达升高,细胞体积变大,呈梭形和不规则条形改变,且细胞中p-SMAD2和Vimentin蛋白表达较增加,E-cadherin蛋白表达下降。第二部分研究结果:经10 mg/mL ANE或500μg/mL ANE处理HOK细胞后,Real-time PCR结果显示HOK细胞中AREG、EREG和VEGFA基因表达均升高,其中AREG基因表达上调倍率最高。CCK-8、划痕实验、Transwell迁移实验和免疫印迹实验结果显示,500μg/mL ANE促进了HOK细胞的增殖、迁移能力,并上调了p-MEK1、p-ERK1+2和Vimentin蛋白表达,抑制E-cadherin蛋白表达;而敲低AREG可抑制500μg/mL ANE对HOK促增殖、迁移影响,且抑制p-MEK1、p-ERK1+2和Vimentin蛋白表达,提高了E-cadherin蛋白表达;过表达AREG后,HOK细胞的增殖、迁移能力提升,且p-MEK1、p-ERK1+2和Vimentin蛋白表达增加,E-cadherin蛋白表达减少。第三部分研究结果:免疫组化结果显示,AREG在OSF组织中高表达,其与OSF患者临床分级、病理分期和咀嚼槟榔年限因素相关。免疫荧光结果显示OSF组织中的AREG蛋白主要来源于上皮细胞。在HOF细胞中加入rhAREG蛋白,或与500μg/mL ANE预处理的HOK细胞共培养后,HOF细胞增殖、迁移和抗凋亡能力增加,且胶原合成相关蛋白(α-SMA、Collagen-1α和Collagen-3α)及其编码基因表达增高。将HOF细胞中EGFR受体封闭后,rhAREG对HOF细胞的促增殖、迁移、抗凋亡和胶原生成能力被抑制。免疫印迹结果显示,HOF细胞中EGFR受体被封闭后,rhAREG蛋白对HOF细胞中MAPK和PI3k-Akt信号通路的激活能力被抑制。Masson染色实验结果显示,ANE成功诱导出OSF大鼠动物模型。免疫组化结果表明,OSF组大鼠口腔组织中AREG、α-SMA、Collagen-1α和Collagen-3α蛋白表达较对照组增加。免疫荧光结果表明,OSF组大鼠口腔组织中AREG蛋白与α-SMA、Collagen-1α和Collagen-3α蛋白表达均呈正相关。研究结论:1.本研究发现ANE对HOK细胞增殖活性具有双向调节作用,较低浓度ANE(500μg/mL)可促进HOK细胞增殖;较高浓度ANE(10 mg/mL及以上)可抑制HOK细胞增殖,并诱导细胞凋亡、G2/M期阻滞、衰老、DNA损伤、自噬和EMT,形成促癌细胞微环境。2.本研究发现ANE可诱导HOK细胞AREG基因高表达,较低浓度ANE(500μg/mL)通过诱导HOK细胞高表达AREG促进自身增殖、迁移,并激活HOK细胞MAPK信号通路和EMT进程。3.本研究发现OSF组织中AREG蛋白表达与患者临床分级、病理分期和咀嚼槟榔年限有关。ANE诱导的口腔角质细胞源性AREG蛋白通过EGFR及其下游PI3k-Akt和MAPK信号通路促进HOF细胞增殖、迁移、抗凋亡和胶原合成,其可能是OSF发生发展的关键因素。图190幅,表5个,参考文献173篇更多还原

【Abstract】 Research background: Oral submucous fibrosis(OSF)is a progressive fibrosis disease of the oral connective tissue mainly characterized by inflammation,accompanied by atypical hyperplasia or atrophy of the oral epithelial tissue.The clinical Symptoms of OSF includes lamellar or stringy fibrous sclerotic bands under oral mucosa,cheek invagination,restricted mouth opening and oral mucosa intolerance to spicy stimulation.The pathogenic factors of OSF may be related to chewing areca nut,malnutrition,immune abnormalities,etc.Chewing areca nut is considered to be the main cause of OSF.Areca alkaloids,polyphenols,tannic acids and trace metals in areca nut have cytotoxicity to some extent.In addition,the dehydrated commercial areca nut shell surface is tough,the shell contains dense coarse fiber,often wear the oral mucosal epithelial keratosis layer during chewing,promote the harmful substances in areca nut and its metabolites on the oral epithelial cells.As the first barrier of oral tissue,oral mucosal epithelial cells play an important role in the occurrence and development of OSF.However,the biological effects of areca nut on oral keratinocytes and the biological effects of oral keratinocytes stimulated by areca nut on oral fibroblasts have not been thoroughly studied.Therefore,it is of great significance for the prevention and treatment of OSF to clarify the biological effects of areca nut on oral keratinocytes and the content and mechanism of "information transmission" between oral keratinocytes stimulated by areca nut and fibroblasts.Research methods:The first part of research methods: The content of areca alkaloids in areca extract was determined by high performance liquid chromatography.Cell Counting Kit-8(CCK-8)assay was used to analyze the effect of gradient concentration of areca nut extract(ANE)on the proliferation activity of human oral keratinocytes(HOK).The differentially expressed genes of HOK cells in ANE group and control group were analyzed by second-generation transcriptome sequencing(P value less than 0.05,log2 multiple greater than 1),and the signaling pathways and molecular functions of differentially expressed genes between the two groups were analyzed by bioinformatics.The changes of HOK cell cycle or apoptosis were detected by flow cytometry.Immunostaining was used to analyze Fox O signaling pathway-related proteins(FOXO1,FOXO3,p-FOXO1 and p-FOXO3),autophagy related proteins(LC3-Ⅰ/Ⅱ and p62),Epithelial mesenchymal transition,epithelial mesenchymal transition,epithelial mesenchymal transition,and epithelial mesenchymal transition(EMT)-related proteins(SMAD2,p-SMAD2,E-cadherin and Vimentin);The expression of γH2AX proteins in HOK cells was detected by immunofluorescence assay.HOK cell senescence was detected byβ-galactosidase staining kit.The expression of age-related phenotype genes(IL6,MMP2,TGFB1,CDKN2 A in HOK cells was detected by Real-time PCR.The expression of exocrine protein TGF-β1 in HOK cells was detected by ELISA.The morphology of HOK cells was observed under optical microscope.The second part of research methods: The high-expression ligand genes involved in MAPK signaling pathway in the 10 mg/m L ANE group and 500 μg/m L ANE group were intermixed by transcriptome sequencing,and their expression was verified by Real-time PCR.The source cells of AREG protein in OSF tissue were detected by immunofluorescence.Real-time PCR and western blot were used to detect the effect of short hairpin RNA on HOK cells with low or overexpressed AREG plasmid.After AREG was knocked down,HOK cells were treated with 500 μg/m L ANE.CCK-8,wound healing and transwell assays were used to detect the changes in the proliferation and migration ability of HOK cells in the experimental group and the control group.After AREG overexpression,CCK-8,wound healing and transwell assays were used to detect the changes of HOK cell proliferation and migration ability in the experimental group and the control group.After overexpression or knockdown of AREG gene in HOK cells,the expression of MAPK signaling pathway proteins(MEK1,ERK1+2,p-MEK1 and p-ERK1+2) and EMT-related proteins(E-cadherin and Vimentin)in HOK cells was detected by western blot.The third part of research methods: The expression of AREG protein in normal oral tissues and OSF tissues was detected by immunohistochemistry,and the relationship between the expression of AREG protein in OSF tissues and the clinicopathological factors in OSF patients was analyzed according to the clinicopathological information of OSF patients.The AREG protein distribution in OSF tissues was detected by immunofluorescence.The effects of exogenous AREG protein on proliferation,apoptosis,migration and the expression of collagen-synthesis-associated proteins of human oral fibroblasts(HOF)were analyzed by CCK-8,flow cytometry,wound healing,transwell migration and western blot assays.After HOF cells were co-cultured with500 μ g/m L ANE pre-treated HOK cells,the proliferation,apoptosis,migration and collagen synthesis of HOF cells were analyzed by CCK-8,flow cytometry,wound healing,transwell migration and western blot assays.After HOF cells were treated with EGFR receptor blocker Erlotinib,rhAREG protein was added,then CCK-8,flow cytometry,wound healing,transwell migration and western blot assays were used to analyze the biological function changes of HOF cells and signaling pathways involved.The areca nut-related OSF rat model was established.Masson staining was used to detect the changes of fibrosis in oral tissue of OSF rat,and the expression of AREG,α-SMA,Collagen-1α and Collagen-3α were verified in vivo by immunohistochemistry.The correlation between the expression of AREG and α-SMA,Collagen-1αand Collagen-3α protein in OSF rat was analyzed by immunofluorescence.Research results:The first part of research results: Arecoline,guvacine,arecaidine,and guvacoline were detected successfully in the ANE extracted in this study.The results of CCK-8 showed that ANE at 500 μ g/m L could promote HOK cell proliferation.ANE at 10 mg/m L or above inhibited the proliferation of HOK cells.There was no difference in the proliferation of HOK cells between other ANE group and the control group.Transcriptome sequencing results of HOK cells in 500 μ g/m L ANE group,10 mg/m L ANE group and control group showed that 10 mg/m L ANE and 500 mg/m L ANE could cause high expression of genes related to MAKP signaling pathway in HOK cells.Moreover,10 mg/m L ANE could induce the high expression of genes related to cell cycle,P53,autophagy,Fox O and cell senescence signal pathway in HOK cells.Flow cytometry showed that 10 mg/m L ANE induced apoptosis and G2/M phase arrest of HOK cells.Compared with the control group,the number of senescent cells in HOK cells of 10 mg/m L ANE group increased,and the expression of senescence-related phenotype genes IL6,MMP2,TGFB1,CDKN2 A and CDKN1 A was up-regulated.10 mg/m L ANE can increase the expression of double-stranded DNA damage marker γH2AX protein in HOK cells.Western blot assay found that 10 mg/m L ANE up-regulated the expression of p-FOXO1 and p-FOXO3 proteins in HOK cells.In the verification of autophagy signaling pathway,it was found that 10 mg/m L ANE induced the up-regulation of expression of LC3-Ⅱprotein in HOK cells,and down-regulation of expression of LC3-Ⅰ and P62 protein.HOK cells were treated with 2 mg/m L ANE for one month,the expression of TGF-β1 protein in exocrine cell was increased,the volume of the cells was enlarged,and the expression of p-SMAD2 and Vimentin protein was increased,while the expression of E-cadherin protein was decreased.The second part of research results: After treatment with 10 mg/mL ANE or 500 μg/m L ANE,the Real-time PCR results showed that the expression of AREG,EREG and VEGFA genes in HOK cells was increased,and the up-regulation rate of AREG gene expression was the highest.CCK-8,wound healing,transwell migration and western blot assays showed that 500 μg/m L ANE promoted the proliferation and migration of HOK cells,up-regulated the expression of p-MEK1,p-ERK1+2 and Vimentin,and inhibited the expression of E-cadherin protein.Knockdown of AREG can inhibit the effect of 500 μg/m L ANE on proliferation and migration of HOK cells,inhibit the expression of p-MEK1,p-ERK1+2 and Vimentin,and increase the expression of E-cadherin protein.After AREG overexpression,the proliferation and migration ability of HOK cells were improved,and the expression of p-MEK1,p-ERK1+2 and Vimentin was increased,while the expression of E-cadherin protein was decreased.The third part of the research results: Immunohistochemical results showed that AREG was highly expressed in OSF tissues,which was related to clinical grade,pathological stage and chewing time of patients with OSF.Immunofluorescence results showed that AREG protein in OSF was mainly derived from epithelial cells.rhAREG protein was added to HOF cells or HOF cells co-cultured with HOK cells pretreated with 500 μg/m L ANE,the proliferation,migration and anti-apoptosis of HOF cells were increased,and the expression of collagen-synthesis-related proteins α-SMA,Collagen-1α and Collagen-3αand their coding genes were increased.After blocking EGFR receptor in HOF cells,the ability of promoting proliferation,migration,anti-apoptosis and collagen production induced by rhAREG was inhibited in HOF cells.The results of western blot showed that the activation ability of rhAREG protein to MAPK and PI3k-Akt signaling pathways was inhibited after EGFR receptor blockade in HOF cells.Masson staining showed that ANE successfully induced OSF rat model.The immunohistochemical results showed that the expressions of AREG,α-SMA,collagen-1α and collagen-3α in oral tissues of OSF rats were increased compared with those of control groups.The results of immunofluorescence showed that the expression of AREG protein was positively correlated with α-SMA,Collagen-1α and Collagen-3α in oral tissue of OSF rats.Research conclusions:1.In this study,it was found that ANE had a bidirectional regulation effect on the proliferation activity of HOK cells,and a lower concentration of ANE(500 μg/m L)promoted the proliferation of HOK cells.Relative high concentration(10 mg/m L or above)of ANE inhibited HOK cell proliferation and induced apoptosis,G2/M phase arrest,senescence,DNA damage,autophagy and EMT,forming a pro-cancer cellular microenvironment.2.In this study,it was found that ANE could induce high expression of AREG gene in HOK cells,and a lower concentration of ANE(500 μg/m L)could induce high expression of AREG in HOK cells to promote self-proliferation and migration,and activate MAPK signaling pathway and EMT process in HOK cells.3.In this study,it was found that AREG protein expression in OSF tissue was related to clinical grade,pathological stage and chewing areca nut years of patients.ANE induced oral keratinocyte derived AREG protein promotes HOF cell proliferation,migration,anti-apoptosis and collagen synthesis through EGFR and its downstream PI3k-Akt and MAPK signaling pathways,which may be a key factor in the development and development of OSF.更多还原