【作者】 张智广；

【导师】 周瑞祥；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2023， 博士

【摘要】 褪黑素(Melatonin,MLT)是一种保守的小分子吲哚激素,可调节昼夜节律,在哺乳动物体内由松果体以及消化系统、生殖系统等多种组织合成,具有抗炎、抗肿瘤等作用。在结肠癌患者肿瘤组织中发现褪黑素合成酶表达降低,细胞和动物实验证实加入褪黑素具有抗结肠癌作用,提示褪黑素与结肠癌关系密切,但作用机制尚未明确。本研究旨在利用C57BL/6J小鼠探寻褪黑素在结肠癌发生发展过程中的免疫调节机制。第一部分 C57BL/6J小鼠褪黑素水平及其合成酶表达分析C57BL/6J作为实验室常用模式生物,建立了大量转基因小鼠,作为模式生物被广泛使用。C57BL/6J小鼠是否存在褪黑素合成,科学界存在争议。本部分实验首先采用LC-MSMS测定C57BL/6J小鼠松果体、结肠、脾脏、睾丸、血清等组织褪黑素含量,结果表明这些组织中均有一定浓度褪黑素,其中松果体褪黑素含量最高,呈现昼低夜高节律,结肠褪黑素含量次之。褪黑素在动物体内由五羟色胺经Aanat转乙酰基和Asmt转甲基合成。免疫印迹结果显示松果体、结肠等组织有Aanat和Asmt表达。C57BL/6J小鼠松果体组织经mRNA转录组测序,结果显示Aanat和Asmt mRNA在松果体中高表达,其丰度甚至接近内参基因。松果体组织4D非标定量质谱,结果显示Asmt有高表达。本部分研究表明C57BL/6J小鼠松果体、结肠高表达褪黑素合成酶Aanat和Asmt,能合成褪黑素,且松果体中褪黑素合成存在典型昼夜节律。第二部分 褪黑素合成酶Aanat和Asmt双基因敲除纯合子小鼠模型的建立褪黑素在C57BL/6J小鼠松果体、结肠等组织存在。松果体褪黑素含量呈昼夜节律性改变,并随着机体需求呈现动态调整,通过单纯补充褪黑素无法准确模拟其生理功能。本部分实验旨在建立无体内褪黑素合成的动物模型,探寻褪黑素功能。首先利用CRISPR-CAS9技术建立C57BL/6J Aanat和Asmt两个基因的单基因敲除小鼠,随后繁育出Aanat&Asmt双基因敲除纯合子小鼠(Double gene knockout homozygote,DHO)。经过基因组PCR测序鉴定,确定建立了Aanat第1外显子和第4外显子之间缺失3548bp和3676bp两个类型Aanat基因删除小鼠,和Asmt第二个外显子和第七个外显子之间缺失3434和3470个碱基3个系Asmt基因删除小鼠。随后取DHO小鼠松果体组织进行mRNA转录组测序、蛋白质4D质谱测序,结果与基因组测序结果一致。跟踪观察显示DHO小鼠生育和体重生长曲线,松果体、结肠形态结构等与WT小鼠无明显差异。对松果体、结肠、脾脏、血清进行褪黑素测定,发现DHO小鼠各组织均含有褪黑素,松果体和脾脏与WT小鼠无差异,血清褪黑素浓度时辰时间(Zeitgeber time,ZT)12显著下降(P<0.001),结肠褪黑素浓度ZT4和ZT12显著下降(P<0.01)。小鼠粪便褪黑素浓度远低于结肠。以上结果提示:成功建立了Aanat和Asmt双基因敲除纯合子小鼠(DHO)。DHO小鼠ZT12血清和ZT4、ZT12结肠褪黑素含量降低。提示小鼠体内存在褪黑素合成代偿途径。第三部分 褪黑素在AOM/DSS诱导性小鼠结肠癌模型中对Cd8~+T和B细胞的免疫调节作用C57BL/6J小鼠用AOM/DSS诱导结肠癌,发现DHO小鼠的体重,结肠重量,结肠长度,肿瘤体积,肿瘤侵犯肠壁等病变指标均严重于WT小鼠。DHO小鼠经傍晚补充20mg/kg褪黑素后(DHO-MLT)其病变症状明显缓解。提示在结肠癌发生过程中褪黑素具有抗肿瘤作用。为探寻褪黑素的免疫调节机制,在AOM/DSS诱导结肠癌发病的早期阶段,取结肠组织解离分选Cd45~+免疫细胞进行单细胞mRNA转录组测序,发现DHO组结肠Cd8~+T细胞、Cd19~+记忆性B细胞和中性粒细胞减少,Ig A~+B细胞增加。DHO-MLT组可部分回复以上表型。取诱导结肠癌发病的早期(75天)脾脏解离,流式细胞术检测,结果显示Cd8~+T细胞在DHO和WT组无差异。取同期结肠进行解离,流式细胞术检测,结果显示DHO组中总Cd8a~+T,传统Cd8a~+Cd8b~+T,Trm Cd8a~+Cd8b~+Cd69~+T,效应记忆T细胞EM等均显著下降。DHO-MLT组代表活化和组织驻留标记的Cd8~+T细胞(Cd8a~+Cd8b~+Cd69~+T)的下降被逆转。为验证在结肠癌组织中的Cd8~+T细胞,取AOM/DSS诱导结肠癌晚期(110天)肿瘤组织和癌旁结肠组织进行流式细胞分析。结果显示晚期结肠肿瘤组织DHO组Cd8a~+总T细胞,Cd8a~+Cd8b~+T细胞,Cd8a~+Cd8b~-T细胞,Cd8a~+Cd8b~+Cd69~+T细胞显著下降。晚期结肠组织固有层DHO组Cd8~+T效应细胞下降。DHO加MLT后结肠肿瘤组织中代表活化和组织驻留的Cd8~+T细胞(Cd8a~+Cd8b~+Cd69~+T)的下降被逆转。本研究结果提示C57BL/6J小鼠体内存在MLT合成,MLT在体内的合成存在Aanat和Asmt之外的代偿途径,具体路径尚待深入探讨。在C57BL/6J小鼠敲除Aanat&Asmt后傍晚时段(ZT12时)血清和结肠褪黑素含量下降,这种下降在AOM/DSS诱导结肠癌过程中其病变症状会更严重,补充褪黑素可逆转该现象。DHO小鼠中癌症症状的加重与Cd8~+T细胞特别是Cd8a~+Cd8b~+Cd69~+T细胞下降有关,傍晚补充褪黑素可缓解Cd8a~+Cd8b~+Cd69~+T细胞下降,改善症状。表明褪黑素在AOM/DSS诱导性结肠癌过程中可通过维持Cd8~+T细胞的活化和驻留发挥抗肿瘤作用。更多还原

【Abstract】 Melatonin(MLT)is a conserved small molecule indole hormone that regulates circadian rhythms,plays roles in anti-inflammatory and anti-tumor and is synthesized in mammals by the pineal gland,digestive system,and so on.Decreased expression of melatonin synthetase was found in tumor tissues of colon cancer patients.Cell and animal experiments confirmed that high concentration of melatonin has certain anti-tumor effects,suggesting that melatonin is closely related to tumor,but the mechanism of action is not yet clear.The aim of this study was to explore the immunomodulatory mechanism of melatonin in the occurrence and development of colon cancer in C57BL/6J mice.Part I: Analysis of Melatonin Level and Synthase Expression in C57BL/6J MiceC57BL/6J,as a model organism commonly used in laboratories,has established a large number of transgenic mice and been widely used as model organisms.The existence of melatonin synthesis in C57BL/6J mice is controversial in the scientific community.In this part of the experiment,LC-MSMS was used to determine the melatonin content in the pineal gland,colon,spleen,testis,serum and other tissues of C57BL/6J mice.The results showed that there was a certain concentration of melatonin in these tissues,among which the pineal gland had the highest melatonin content,showing a low day and high night rhythm,followed melatonin content by the colon.Melatonin is synthesized in animals from serotonin via Aanat to acetyl and Asmt to methyl.Western blotting showed Aanat and Asmt expression in pineal colon and other tissues.Transcriptome sequencing of pineal mRNA in C57BL/6J mice showed that Aanat and Asmt were highly expressed in pineal tissues,and their abundance was even close to that of reference genes.Pineal tissue 4D lable free quantitative mass spectrometry showed high expression of Asmt.This part of the study showed that C57BL/6J mice had melatonin synthesis in the pineal gland,colon and other tissues,melatonin synthesis in the pineal gland had a circadian rhythm,and melatonin synthetase Aanat and Asmt were highly expressed.Part Ⅱ: Establishment of homozygous mice with melatonin synthetase Aanat and Asmt double gene knockoutMelatonin was found in pineal gland,colon and other tissues of C57BL/6J mice.The melatonin content of pineal gland changes rhythmically,and the physiological function of pineal gland cannot be accurately simulated by simply supplementing melatonin.This part of the experiment aims to establish an animal model without in vivo melatonin synthesis and explore the function of melatonin.First,to establish C57BL/6J Aanat and Asmt single-gene knockout mice by CRISPR-CAS9 technique,and then to obtain Aanat&Asmt double-gene knockout homozygous mice(DHO)by two single-gene knockout mice.After genome PCR sequencing,two lines mice with 3548 bp and 3676 bp deletion between exon 1 and exon 4 of Asmt gene were established,and three lines mice with 3434 and 3470 bp deletion between exon 2 and exon 7 of Asmt gene were identified.Subsequently,the pineal tissues of DHO mice were sequenced by mRNA transcriptome and protein 4D mass spectrometry,and the results were consistent with the genome sequencing results.Growth and weight growth curves,pineal gland,colon morphology and structure of DHO mice were not significantly different from WT mice.Melatonin was detected in the pineal gland,colon,spleen and serum of DHO mice.There was no difference between the pineal gland and spleen of DHO mice and that of WT.The serum melatonin concentration was significantly decreased at ZT12(P<0.001),and the colon melatonin concentration was significantly decreased at ZT4 and ZT12(P<0.01).And the concentration of melatonin in mouse feces was much lower than that in colon.These results suggest that Aanat and Asmt double knockout homozygous mice(DHO)were successfully established.The melatonin content of ZT12 in serum and ZT4 or ZT12 in colon was decreased in DHO mice.There may be a compensatory pathway for melatonin synthesis in mice.Part Ⅲ: Immunomodulatory effects of melatonin on Cd8~+T and B cells in an AOM/DSS induced colon cancerIn AOM/DSS induced Colon cancer with C57BL/6J mice,it was found that the body weight,colon weight,colon length,tumor volume and tumor invasion of the intestinal wall of DHO group were more serious than those of WT group.DHO mice were treated with 20mg/kg melatonin supplementation in the evening(DHO-MLT).It is suggested that melatonin has antitumor effect in the process of colon cancer.In order to explore the immunomodulatory mechanism of melatonin,Cd45+ immune cells were dissociated from colon tissue at the early stage of colon cancer induced by AOM/DSS,and single-cell mRNA sequencing was performed.It was found that Cd8~+T cells,Cd19+ memory B cells and neutrophils decreased in DHO group,while Ig A+B cells increased.Partially recover the above phenotypes in DHO-MLT group.Flow cytometry showed that Cd8~+T cells in spleen were not different in DHO and WT groups at the early stage.And at the same stage,the total Cd8a~+T,conventional Cd8a+Cd8b~+T,Trm Cd8a+Cd8b+Cd69~+T and effector memory T cells EM in colon decreased significantly in DHO group.Declines in Cd8~+T cells(Cd8a+Cd8b+Cd69~+T)representing activation and tissue-resident markers were reversed in the DHO-MLT group.In order to verify Cd8~+T cells in colon cancer tissues,advanced colon tumor tissues and paracancellular colon tissues were selected for flow cytometry analysis on day 110 of AOM/DSS induction.The results showed that Cd8a+ total T cells,Cd8a+Cd8b~+T cells,Cd8a+Cd8b-T cells,Cd8a+Cd8b+Cd69~+T cells decreased significantly in DHO group of advanced colon tumor tissue.Cd8~+T effector cells in Lamina propria colica decreased in DHO group at the late stage.DHO plus MLT reversed the decline in Cd8~+T cells(Cd8a+Cd8b+Cd69~+T)representing activation and tissue retention in colon tumor tissue.The results of this study suggest that there is MLT synthesis in C57BL/6J mice,and there is a compensatory pathway for MLT synthesis in vivo besides Aanat and Asmt.After Aanat&Asmt knockout in C57BL/6J mice,serum and colon melatonin levels decreased in the evening(ZT12),which was more serious in the process of colon cancer induced by AOM/DSS,and melatonin supplementation alleviated the symptoms.The exacerbation of cancer symptoms in DHO mice is associated with the decline of Cd8a+Cd8b+Cd69~+T cells in Cd8~+T cells,and melatonin supplementation in the evening can alleviate the decline of Cd8a+ Cd8b+Cd69~+T cells and improve the symptoms.The results of this study suggest that melatonin plays an antitumor role in AOM/DSS induced colon cancer by maintaining the activation and retention of Cd8~+T cells.更多还原