【作者】 赵欣；

【导师】 于海泉；

【作者基本信息】 内蒙古大学 ， 生物学， 2020， 硕士

【摘要】 原始生殖细胞（Primordial germ cells,PGCs）是精子和卵子的前体细胞。PGCs由特化部位向生殖嵴迁移的过程被精细调节,这一过程由其自身的细胞特性以及周围环境共同作用决定,同时伴随着细胞增殖和凋亡等现象,目前有关PGCs迁移的分子机制仍有待深入研究。本研究通过分析不同发育阶段mPGCs细胞的RNA-seq数据,获得差异表达基因,经KEGG pathway分析后筛选出候选基因,对其中的Diaph1基因进行表达和功能分析,通过制作敲除小鼠,研究了Diaph1对小鼠繁殖力和生长特性的影响,以及对mPGCs的数量、增殖、凋亡和粘附等的影响。本研究首先通过检测不同分子的表达,对mPGCs的定位和筛选标记进行研究。通过组织切片免疫荧光法证明Oct4可以作为mPGCs定位与筛选的标记。从Oct4标记小鼠的胚胎生殖嵴中利用流式细胞法分选mPGCs,9.5天胚龄（E9.5）分选效率约为0.20%,E12.5分选效率约为2.28%;而使用Ssea1抗体标记后E12.5中mPGCs分选效率约为2.91%,分选后细胞经碱性磷酸酶染色呈阳性蓝紫色。qPCR进一步检测表明mPGCs标记基因Fkbp6、Mov10l1、Spo11、4930432K21Riken等,多能性基因Nanog、Oct4等和生殖特异性基因Mvh、Dazl等mRNA表达在阳性细胞显著高于阴性细胞。因此,Oct4和Ssea1均可作为mPGCs鉴定和筛选的标记分子。本研究对实验室前期检测的胚胎E9.5天和E12.5天阶段mPGCs的RNA-seq数据进行分析,从中选择ErbB signaling pathway和Focal adhesion通路中的基因进行qPCR验证,表达变化趋势与测序结果一致。随后查阅文献分析筛选出与细胞迁移相关的透明基因相关成蛋白1（Diaphanous-related formins1,Diaph1）和Ypel1（yippee like 1）候选基因,二者mRNA均在mPGCs迁移过程中高表达,到达生殖嵴后表达降低,且具有显著性差异。为进一步验证Diaph1和Ypel1的作用,本研究利用CRISPR/Cas9技术分别制作Diaph1^+/-和Ypel1^+/-敲除小鼠,经繁殖后对个体、组织和mPGCs细胞特征变化及相关分子机制进行研究。结果表明两种敲除的小鼠繁殖力均减弱,尤其是Ypel1敲除小鼠繁殖数量无法进行下一步检测。繁殖实验发现,敲除Diaph1的小鼠后代基因型不符合孟德尔遗传定律,说明其敲除对繁殖力产生影响。与野生型小鼠比较,敲除小鼠的睾丸、卵巢重量均显著下降。敲除小鼠的mPGCs数量减少,且与增殖相关的Dicer1、Mcm9基因表达下调。TUNEL实验表明,敲除小鼠发生凋亡的mPGCs细胞增加,且维持细胞存活的Map2k5、Rest基因表达下调。与粘附相关的E-cad蛋白和Cdh1表达下调,与迁移相关的Cxcr4、Hmgcr、Dazl基因表达下调。综上所述,Diaph1与mPGCs迁移相关,在迁移的mPGCs中mRNA表达较高,迁移终止时表达下调。敲除Diaph1后小鼠繁殖力减弱,mPGCs增殖受到抑制,发生凋亡的细胞增加导致其数量减少,细胞粘附和迁移相关基因表达受到抑制。本研究为进一步认识mPGC迁移分子机制奠定了基础。更多还原

【Abstract】 Primordial germ cells(PGCs)are the precursor cells of sperm and ovum.The migration of PGCs from the specialization sites to the genital ridge is finely regulated,which is determined by its own cell characteristics and the cross-talking with surrounding environment and accompanied by cell proliferation and apoptosis.At present,the molecular mechanism of PGCs migration remains to be further studied.In this study,the differentially expressed genes were obtained by analyzed the RNA-seq data of mPGCs at different developmental stages,and candidate genes were selected after KEGG pathway analysis.The expression and function of Diaph1 gene were analyzed with Diaph1 knockout mouse strains,the effect of Diaph1 on mouse fertility and growth,as well as the on number,proliferation,apoptosis and adhesion of mPGCs were studied.In this study,the location of mPGCs and the selection markers were studied by detecting the expression of different molecules.By using immunofluorescence method with tissue secyion,the results showed that Oct4 can be used as a marker for mPGCs localization and sorting.The flow cytometry were used to sort mPGCs from the embryonic genital ridge of the Oct4-labeled mouse,the sorting efficiency was about 0.20% for E9.5 embryos,and about 2.28% for E12.5 embryos.When Ssea1 antibody was used as marker,the sorting efficiency of mPGCs in E12.5 were2.91%.After sorting,the cells were positive blue-purple after Alkaline Phosphatase staining.qPCR results showed that mRNA expression of the following gene were was significantly higher in positive cells than in negative cells,including,mPGCs marker genes Fkbp6,Mov101,Spo11 and 4930432K21 Riken,pluripotency genes Nanog and Oct4,reproductive-specific genes Mvh and Dazl,etc.Therefore,both Oct4 and Ssea1 can be used as markers for identification and sorting of mPGCs.This study analyzed our previous RNA-seq data of mPGCs at the E9.5 and E12.5,the differentially expressed genes were enriched into regulatory pathways.The genes of key nodes in ErbB signaling pathway and Focal adhesion pathway were selected for qPCR verification,and the expression change trend was consistent with the sequencing results.Subsequently,the literature analysis was performed to screen out the candidate genes,Diaphanous-related formins 1(Diaph1)and yippee like 1(Ypel1),associated with cell migration.The mRNAs expression of both gene wer increased in migrating mPGCs,while decreased after reached genital ridge,and had significant differences.To further verify the role of Diaph1 and Ypel1,knockout mouse were by CRISPR/Cas9 technology,respectively,and the effect on individual,tissue and mPGCs characteristics and the mechanisms were studied.The results showed that the fertility were reduced after knockout,especially in Ypel1 knockout mouse,could not support the further studies.The results from the reproduction experiments found that the genotypes of Diaph1 knockout mouse did not comply with Mendel’s genetic law,indicating that knockout had an impact on fertility.Compared to be wild-type mouse,in knockout mouse,the weight of testis and ovaries decreased significantly,the number of mPGCs decreased,and the expression of Dicer1 and Mcm9 genes which related to proliferation was down-regulated.TUNEL experiments showed that the number of mPGCs,which undergone apoptosis were increased in the knockout mouse,and the expression of Map2k5 and Rest genes which maintained cell survival were down-regulated.Theexpression of E-cad protein and Cdh1 which related to adhesion were down-regulated,and the expression of Cxcr4,Hmgcr and Dazl genes which related to migration were down-regulated.In conclusion,Diaph1 is association with mPGCs migration,mRNA expression is higher in migrating mPGCs,and down-regulated after migration.After knocking of Diaph1,the reproductive capacity of mouse declined,the proliferation of mPGCs was inhibited,the apoptotic cells increased and the number of mPGCs decreased,in addition,the expression of genes releated to cell adhesion and migration-related were inhibited.This study laid the foundation for further understanding the molecular mechanism of mPGC migration.更多还原