【作者】 刘峰；

【导师】 雷闽湘； 陈慧玲；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的体外诱导小鼠胚胎干细胞SF1-G定向诱导分化为胰岛样细胞,观察诱导分化培养过程中印记基因Kcnq1、Cdknlc印记变化,探讨胚胎干细胞体外诱导分化培养过程中表观遗传学的稳定性。方法1.从孕小鼠胚胎分离培养小鼠胚胎成纤维细胞,丝裂霉素C处理第3-5代小鼠胚胎成纤维细胞制备饲养层细胞,在饲养层细胞上扩增培养小鼠胚胎干细胞株SF1-G细胞。2.参照Shi等人的三阶段法,定向诱导小鼠胚胎干细胞SF1-G向胰岛样细胞分化；细胞免疫荧光染色和RT-PCR检测分化细胞中胰岛细胞特异性标志物的表达。3.在诱导分化培养的不同阶段收集细胞,逆转录-聚合酶链式反应／限制性内切酶片段长度多态性(RT-PCR/RFLP)检测印记基因Kcnq1、Cdknlc表达的亲本来源,分析其印记状态。结果1.SF1-G细胞能在饲养层细胞上保持未分化状态增殖培养。2.RT-PCR结果显示,在诱导分化的第三阶段,细胞逐渐出现胰岛细胞特异性基因的表达；细胞免疫荧光结果显示,分化终末细胞可表达胰岛细胞特异性激素蛋白,证实成功将胚胎干细胞诱导分化为胰岛样细胞。3.RT-PCR/RFLP分析结果显示,诱导分化后的细胞印记基因Kcnq1、Cdknlc从母源单等位基因表达转变成双等位基因表达,出现印记丢失(LOI),而持续传代培养的ES细胞中Kcnq1、Cdknlc仍表现为母源单等位基因表达,即印记保持(MOI)。结论1.参照Shi等人的方法,我们能将小鼠胚胎干细胞体外定向诱导培养分化为胰岛样细胞。2.是诱导分化培养作用,而不是单纯的细胞传代培养过程导致印记基因表达异常,出现印记丢失,即细胞诱导分化培养过程导致了表观遗传性状的不稳定。目的观察胚胎干细胞SF1-G在诱导分化前后印记基因Kcnq1、Cdknlc印记调控区(ICR)差异性DNA甲基化区KvDMR1的甲基化状态,以及分化各阶段细胞中DNA甲基转移酶水平变化,以探讨胚胎干细胞在体外诱导分化培养过程中印记基因Kcnq1、Cdknlc表达变化的机理。方法1.重亚硫酸盐PCR测序法检测分化前后Kcnq1、Cdknlc基因印记调控区KvDMR1的CpG位点甲基化状态：1)收集未分化的细胞及诱导分化终末的细胞,提取基因组DNA,重亚硫酸盐处理DNA,以特异性引物PCR扩增差异性DNA甲基化区KvDMR1;2)将PCR产物连接到氨苄抗性T载体质粒后转入感受态细菌,用不含氨苄的LB培养基摇菌后均匀涂在氨苄琼脂糖平板上,次日挑取阳性细菌克隆,用含氨苄的LB培养基筛选阳性细菌；3)PCR验证细菌中存在目的片段后,将菌液送送上海生工生物工程公司测序,测序结果通过软件进行分析,了解分化前后细胞的KvDMR1的甲基化状态。2. Western blot检测诱导分化各阶段细胞中DNA甲基转移酶Dmnt1和Dmnt3b的表达水平。结果1.测序检测了KvDMR1区23个CpG位点的甲基化状态,在分化前的胚胎干细胞中,9个测序结果显示,有4个表现为1-23个CpG位点全部甲基化,另外5个表现为所有CpG位点都未甲基化,即KvDMR1区的CpG位点表现为全甲基化或全未甲基化,符合印记调控区域差异性DNA甲基化区(DMR)的特点；而在分化后的细胞中,11个测序结果显示,其中5个表现为1-23个CpG位点全部甲基化,4个在第18-23个CpG位点发生甲基化,而其余CpG位点未甲基化,其余2个表现为所有CpG位点都未甲基化,提示原来未甲基化的KvDMR1发生了甲基化。2. Western blot结果显示,胚胎干细胞诱导分化后,从第二阶段开始DNA甲基转移酶Dmnt1水平显著升高；DNA甲基转移酶Dmnt3b在第三阶段也显著升高,而同期传代培养的未分化细胞DNA甲基转移酶水平没有明显变化。结论1.诱导分化过程中印记调控区域的差异性DNA甲基化区KvDMR1的第18-23个CpG位点甲基化状态的改变可能与印记基因Kcnq1、Cdknlc的印记丢失相关。2.诱导分化过程中DNA甲基转移酶水平升高,可能介导了KvDMR1区甲基化状态的改变,最终引起基因印记状态发生改变,导致印记丢失。更多还原

【Abstract】 Chapter one expression of imprinted genes Kcnql, Cdknlc in the course of mouse embryonic stem cells SF1-G induced to differentiate into islet-like cells in vitroObjectiveMouse embryonic stem cells SF1-G were induced to differentiate into islet-like cells in vitro. Epigenetic stability of cells at different stages was observed by testing the parental origin of imprinted gene Kcnq1 and Cdknlc.Methods 1. Mouse embryonic fibroblasts (MEFs) was isolated from pregnant mice embryos. Fibroblast feeder cells were prepared by treating 3-5th generations MEFs with Mitomycin C. Embryonic stem cell line SF1-G cells was expanded on feeder cells in vitro.2. Refering to a three-phase protocol from Shi, mouse embryonic stem cells were induced into islet-like cells directly. Immunof-luorescence staining and RT-PCR were used to detect the expression of islet cell-specific marker in differentiated cells.3.Cells were collected at various stages during differentiation process. Imprinting status of imprinted genes Kcnq1, Cdkn1c was tested by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR/RFLP)Results1. SF1-G cells can be cultured and proliferated maintaining the undifferentiated state on the feeder cells.2. RT-PCR results showed cells appeared islet cell-specific gene expression; immunofluorescence showed that islet cell-specific hormone protein can be measured at stage 3, confirmed that the embryonic stem cells can be successful inducted into islet-like cells in vitro.3. RT-PCR/RFLP analysis showed that imprinted genes Kcnq1, Cdknlc were biallelic expression in the differentiated cells, suggesting that they were loss of imprinting (LOI), while these genes were maternal monoallelic expression in the undifferentiated cells continued subculture, which marked the maintenance of imprinting (MOI).Conclusions1. Refering to Shi’protocol, mouse embryonic stem cells were induced into islet-like cells in vitro.2. Differentiation, not merely the process of cell culture can lead to abnormal expression of some imprinted genes, suggesting that epigenetic instability exist during the process of differentiation in vitro. ObjectiveTo explore the mechanism of expression of imprinted genes Kcnq1, Cdknlc in embryonic stem cells culture and differentiation process in vitro, we observed the differentially DNA-methylated region KvDMRl methylation status in SF1-G cells before and after differentiation, which is a part of the imprinting contol region(ICR) of imprinted genes Kcnql, Cdknlc, as well as the DNA methyltransferase levels in various stages of differentiation.Methods1. The methylation status of CpG sites of imprinting control region KvDMRl of imprinted genes kcnql, cdknlc in SF1-G cells before and after differentiation was detected with Bisulfite sequencing PCR.1) undifferentiated cells and inducing terminal differentiated cells were collected, extraction of genomic DNA, bisulfite treatment of DNA, with specific primers PCR amplification of the differentially DNA-methylated region KvDMR1,2) PCR product connected to the ampicillin resistance T Vector plasmid was transformed into Competent bacteria, the latter was evenly coated in the ampicillin resistance on agar plate culture. positive bacterial clones were picked the next day, the LB medium containing ampicillin screened positive bacteria,3) PCR was used to verify that the purpose fragment was existence in bacteria, positive bacteria was sent to Shanghai sangon biotechnology services company sequencing, the results were analyzed by software to show the methylation status in KvDMR1 before and after differentiation.2. Western blot was performed to detect methyltransferase Dmnt1 and Dmnt3b level at various stages of cells differentiation.Results1.We totally sequenced 23 CpG methylation sites of KvDMR1 region. Among 9 DNA sequencing results from embryonic stem cells before differentiation, there were 4 showed 1-23 CpG sites were methylation, the remaining 5 showed unmethylated, so KvDMR1 region showed all CpG methylation or no methylation in all, which is consistent with the characteristic of differentially DNA-methylated region(DMR). In differentiated cells,11 DNA sequencing results showed that 5 of them have methylation of all CpG sites,4 samples have methylation in the first 18-23 CpG sites, while the remaining CpG sites did not methylate, and the other 2 showed none of all CpG sites methylation, suggesting the methylation occurred in unmethylated KvDMR1.2. Western blot results showed that after the induction of cells differentiation from the second stage, DNA methyltransferase Dmnt1 level significantly increased, DNA methyltransferase Dmnt3b significantly higher in the third stage, while simultaneously subcultured undifferentiated cells have no significant change.Conclusions1. In the course of differentiation, the changes in the first 18-23CpG sites methylation status of differentially DNA-methylated region KvDMR1 in imprinting control region may relate to imprinted genes Kcnq1, Cdknlc loss of imprinting.2. In the course of differentiation, the increase of DNA methyltransferase level may mediate KvDMR1 methylation status changes, ultimately lead to changes in imprinted gene activity, as well as leading to loss of imprinting.更多还原