【作者】 王敏；

【导师】 方杰；

【作者基本信息】 江苏大学 ， 妇产科学（专业学位）， 2021， 硕士

【摘要】 目的:生殖细胞肿瘤可以伴随精子发生障碍,是男性不育的重要原因。生殖干细胞的功能异常可导致生殖细胞肿瘤的发生。生殖干细胞的自我更新和分化潜能不仅受其内源性信号的影响,而且经历干细胞微环境的有序调控。模式动物果蝇的睾丸是研究生殖干细胞微环境调控非常理想的模型。核糖体小亚基蛋白RpS13被鉴定为调控果蝇睾丸生殖干细胞自我更新和分化的重要因子,然而,其在生殖干细胞微环境中的具体调控机制尚不明确。本研究旨在探索和讨论RpS13调控果蝇睾丸生殖干细胞自我更新和分化过程的作用机理。方法:(1)利用UAS/GAL4系统介导的RNAi干涉沉默方法,分别使果蝇睾丸的早期生殖细胞(Nos-Gal4)、包囊细胞(Tj-Gal4)和精原细胞(Bam-Gal4)中产生特异性的RpS13功能丧失(Nos>RpS13 RNAi、Tj>RpS13 RNAi和Bam>RpS13 RNAi)。通过雄性果蝇生育率测试观察RpS13是否影响雄性生育力。(2)应用Vasa、1B1、Eya、Zfh1、Fas III标记果蝇睾丸各细胞类型的抗体和PH3标记增殖细胞的抗体对WT野生型果蝇睾丸(对照组)和RpS13 RNAi果蝇睾丸(实验组)进行免疫荧光染色,观察RpS13对果蝇睾丸生殖细胞的自我更新和分化过程的影响和结局。(3)运用小干扰RNA(siRNA)在果蝇胚胎干细胞(S2细胞)中实现RpS13基因表达沉默,使用qRT-PCR检测阴性对照组(NC)和实验组(siRpS13-16和siRpS13-234)中RpS13 m RNA表达水平,验证干涉效率。利用PH3免疫荧光染色检测各组细胞增殖情况,TUNEL染色和流式细胞术检测各组细胞凋亡情况,观察和分析RpS13是否调节S2细胞的增殖和凋亡过程,以此探索RpS13在体外的功能。(4)利用果蝇UAS/GAL4系统和标记蛋白的免疫荧光染色,观察Rho1分别在生殖细胞、体细胞和精原细胞中表达缺失后的睾丸表型是否与RpS13RNAi相似(雄性果蝇生育能力和睾丸功能改变)。(5)利用qRT-PCR检测阴性对照组(NC)和实验组(siRho1-457和siRho1-374)S2细胞中Rho1 m RNA表达水平,验证干涉效率。再分别用PH3免疫荧光染色、TUNEL染色和流式细胞术检测各组细胞增殖与凋亡情况,观察Rho1是否调控S2细胞的生长,其体外功能是否与RpS13相似。(6)利用免疫荧光染色检测果蝇生殖细胞和体细胞中RpS13功能丧失后粘附蛋白Rho1、DE-cad和Arm的表达模式,观察RpS13缺失是否会引起干细胞之间的粘附功能障碍。同时利用免疫荧光染色检测果蝇Rho1功能丧失的情况下DE-cad和Arm的表达模式是否与RpS13 RNAi相似。(7)利用qRT-PCR分别在RpS13和Rho1表达沉默的S2细胞中检测核糖体大亚基(Rp L6、Rp L14、Rp L19、Rp L22、Rp L27和Rp L30)和核糖体小亚基(RpS2、RpS8、RpS9、RpS14a和RpS16)的m RNA表达水平。结果:(1)生育率测试结果显示,与WT果蝇相比,Nos>RpS13 RNAi、Tj>RpS13RNAi和Bam>RpS13 RNAi的雄性果蝇生育能力降低。(2)免疫荧光染色和激光共聚焦显微镜成像结果显示,一方面,果蝇生殖细胞中RpS13表达缺失导致生殖细胞完全丢失和体细胞异常增殖,睾丸形态发生改变;另一方面,体细胞中RpS13功能缺失则导致未分化的生殖细胞堆积和包囊细胞分化受阻,生殖细胞样肿瘤形成;此外,RpS13在精原细胞中的表达缺失也出现精原细胞分化障碍。(3)果蝇S2细胞中RpS13基因沉默导致细胞增殖和凋亡增多。(4)免疫荧光染色和激光共聚焦显微镜成像结果显示,Rho1 RNAi模拟了果蝇睾丸中RpS13 RNAi导致生殖细胞自我更新和分化障碍的表型。(5)果蝇S2细胞中Rho1表达沉默也导致细胞增殖和凋亡增多,完全模拟RpS13调控S2细胞生长的功能。(6)免疫荧光染色结果显示,果蝇睾丸中RpS13功能缺陷导致粘附蛋白Rho1、DE-cad和Arm的表达水平增加;同时,Nos-Gal4和Tj-Gal4驱动的Rho1RNAi果蝇睾丸中Rho1表达水平降低,DE-cad和Arm蛋白的表达出现异位累积。(7)qRT-PCR结果分析显示RpS13和Rho1能够竞争性地调节重要核糖体亚基的m RNA表达水平。结论:我们利用基因遗传操作的方法,研究了核糖体蛋白S13(RpS13)在睾丸和S2细胞中的作用。我们证明RpS13是GSCs自我更新和分化所必需的,并进一步影响了精原细胞的分化,同时,RpS13参与了果蝇S2细胞增殖和凋亡过程的调节。在机制上,我们发现RpS13调节核糖体亚基的表达水平,以及Rho1、DEcad和Arm蛋白的表达模式。此外,果蝇体内和体外实验都证明Rho1模拟了RpS13的表型。这些结果提示我们,RpS13与Rho1相互竞争,并通过募集粘附蛋白DE-cad和Arm来破坏干细胞微环境中的细胞粘附信号,从而导致果蝇睾丸生殖系微环境缺陷。这些发现揭示了RpS13在果蝇睾丸干细胞生态位中介导Rho1信号的新机制,可能为阐明干细胞微环境的调控网络和睾丸生殖细胞肿瘤的发病机制提供新的见解。更多还原

【Abstract】 Objective:Germ cell tumors can be accompanied by spermatogenesis disorder,which is an important cause of male infertility.Abnormal function of germline stem cells(GSCs)can lead to germ cell tumors.The potential self-renewal and differentiation of GSCs are regulated by their intrinsic signals and stem cell microenvironment.For researchers,Drosophila testis is an ideal model to study GSC.Ribosomal small subunit protein RpS13 has been identified as an important factor in regulating GSCs self-renewal and differentiation in Drosophila testes.However,its specific regulatory mechanism in germline stem cell niche is not well understood.The purpose of this study was to explore and discuss the mechanism of RpS13,which regulates the self-renewal and differentiation of germline stem cells in Drosophila testes.Methods:(1)Using RNAi interference silencing method mediated by UAS/Gal4 system,the specific loss of RpS13 function(Nos>RpS13 RNAi,Tj>RpS13 RNAi and Bam >RpS13 RNAi)was induced in early germ cells(Nos-Gal4),cyst cells(Tj-Gal4)and spermatogonia(Bam-Gal4)of Drosophila testes.The fertility of male Drosophila melanogaster was tested to observe whether RpS13 affected male fertility.(2)Immunofluorescence staining was performed with antibodies Vasa,1B1,Eya,Zfh1 and Fas III,which labeled different cell types in testis,and PhosphoHistone H3(PH3)labeled proliferating cells in WT wild-type Drosophila testes(control group)and RpS13 RNAi Drosophila testes(experimental group)to observe the effect of RpS13 on GSCs self-renewal and differentiation in Drosophila testes.(3)Small interfering RNA(siRNA)was utilized to silence RpS13 gene expression in Drosophila embryonic stem cells(S2 cells).Quantitative real-time PCR was used to detect the expression of RpS13 m RNA in negative control group(NC)and experimental group(siRpS13-16 and siRpS13-234)to verify the efficiency of interference.The cell proliferation was detected by PH3 immunofluorescence staining,and cell apoptosis was detected by TUNEL staining and flow cytometry.(4)The expression of Rho1 in germ cells,somatic cells and spermatogonia was detected by immunofluorescence staining of UAS/Gal4 system and marker protein in Drosophila melanogaster.The testicular phenotype was similar to that of RpS13 RNAi(changes of fertility and testicular function in male Drosophila).(5)QRT-PCR was used to detect the expression of Rho1 m RNA in S2 cells of negative control group(NC)and experimental group(siRho1-457 and siRho1-374)to verify the interference efficiency.The proliferation and apoptosis of S2 cells were respectively detected by PH3 immunofluorescence staining,TUNEL staining and flow cytometry to observe whether Rho1 regulates the growth of S2 cells and whether its function in vitro is similar to that of RpS13.(6)The expression patterns of adhesion proteins Rho1,DE-cad and Arm were detected by immunofluorescence staining in germline cells and somatic cells of RpS13 RNAi Drosophila.At the same time,immunofluorescence staining was used to detect whether the expression patterns of DE-cad and Arm in Rho1 RNAi Drosophila mimicked those of RpS13 RNAi in testis.(7)QRT-PCR was used to detect the m RNA expression levels of large ribosomal subunits(Rp L6,Rp L14,Rp L19,Rp L22,Rp L27 and Rp L30)and small ribosomal subunits(RpS2,RpS8,RpS9,RpS14 a and RpS16)in RpS13 and Rho1 silenced S2 cells.Results:(1)The results of fertility test showed that the fertility of male Drosophila with Nos>RpS13 RNAi,Tj>RpS13 RNAi and Bam>RpS13 RNAi was lower than that of WT Drosophila.(2)Immunofluorescence staining and laser confocal microscopy showed that on the one hand,knockdown of RpS13 in early germ cells resulted in complete loss of germ cells and abnormal proliferation of somatic cells,along with tiny testicular morphology;On the other hand,RpS13 deficiency in somatic cells leads to an accumulation of undifferentiated germ cells and the differentiation of cyst cells were blocked,and the formation of germ cell like tumor;What’s more,RpS13 deficiency in spermatogonia caused spermatogonia differentiation defects.(3)The deletion of RpS13 increased the proliferation and apoptosis of Drosophila S2 cells;(4)The results of immunofluorescence staining and confocal laser microscopy showed that Rho1 RNAi mimicked the phenotype of RpS13 RNAi in Drosophila testes leading to the dysfunction of GSCs self-renewal and differentiation.(5)Knocking down of Rho1 in Drosophila S2 cells also led to the increase of cell proliferation and apoptosis,which fully simulated the function of RpS13 in regulating the growth of S2 cells.(6)Immunofluorescence staining results showed that the functional defect of RpS13 in Drosophila testes led the expression level of adhesion proteins Rho1,DE-cad and Arm increased;at the same time,the expression level of Rho1 in Drosophila testes was decreased in Nos>Rho1 RNAi and Tj>Rho1 RNAi,and the expression levels of DE-cad and Arm were ectopic accumulation.(7)QRT-PCR analysis showed that RpS13 and Rho1 could competitively regulate the m RNA expression of important ribosomal subunits.Conclusions:Herein,using a genetic manipulation approach,we investigated the role of ribosomal protein S13(RpS13)in testes and S2 cells.We reported that RpS13 was required for GSCs self-renewal and differentiation,and further affected the differentiation of spermatogonia.We also demonstrated that RpS13 participated in the regulation of proliferation and apoptosis of Drosophila S2 cells.Mechanistically,we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression patterns of the Rho1,DE-cad and Arm proteins.Notably,Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells.These results suggest that RpS13 and Rho1 compete with each other and damage cell adhesions,which was mediated by the DE-cad and Arm proteins,to disorder cell adhesion signal,thus leading to the defects of stem cell niche in Drosophila testes.These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis,which may provide new insights into the regulatory network of stem cell niche and the pathogenesis of testicular germ cell tumors.更多还原