【作者】 周玲；

【导师】 刘敏； 谢胜松； 徐德全；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2019， 硕士

【摘要】 诱导多能干细胞(induced Pluripotent Stem Cells,i PSC)是通过改变特定基因的表达量将已分化的体细胞重编程为具有自我更新与增殖分化能力的多能干细胞。与胚胎干细胞(Embryonic Stem Cells,ESC)相比,i PSC来源于体细胞不受限于伦理限制,能够自我更新和维持未分化状态,具有体内和体外分化成三胚层的能力,还可以减少免疫排斥和生物安全问题,这些特性为再生医学提供了新的希望,也为细胞重编程机制、人类疾病发病机制研究及治疗、保持物种多样性研究等方面提供了新的思路和方法。猪作为肉用大家畜,不仅在畜牧业中有着较重要地位,其生理状态和器官大小、解剖结构与人非常相似,更是理想的实验模型,因此研究猪i PSC(Porcine i PSC,Pi PSC)有着重要意义。但Pi PSC一直存在着诱导效率低的问题。本研究通过2种方式为提高Pi PSC诱导效率做准备:(1)借鉴i CD1诱导培养液可以高效、快速的将小鼠胎儿成纤维(Mouse Embryonic Fibroblasts,MEF)细胞重编程为i PSC的方法,尝试用i CD1培养液诱导Pi PSC,并和先前报道的猪的MXV诱导培养液比较诱导效率。(2)CRISPR/d Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/dead associated protein 9)可以作为调节基因表达的工具。本研究用CRISPR/d Cas9中的SAM(Synergistic Activation Mediator)激活系统分别筛选靶向猪OSKM和mi R-302/367高效激活的sg RNAs(Single-guide RNA),构建串联的sg RNA簇,实现同步激活猪内源性OSKM和mi R-302/367的转录,为激活猪胎儿成纤维(Porcine Embryonic Fibroblasts,PEF)细胞中重编程因子获得Pi PSC做准备。主要研究结果如下:1.i CD1诱导培养基可将PEF细胞诱导产生Pi PSC。外源过表达GV358-OSKM、FUGW-mi R-302/367质粒,并通过慢病毒导入到PEF细胞。在i CD1诱导培养液中经重编程产生ESC样克隆,传代后用碱性磷酸酶染色鉴定,克隆呈阳性表达。2.i CD1比MXV诱导培养液诱导产生Pi PSC的效率高且周期短。i CD1诱导培养液诱导i PSC的周期为10天,MXV诱导培养液诱导Pi PSC的周期为16天,且i CD1诱导产生的克隆是MXV诱导克隆的3倍。3.为了提高SAM激活系统的激活效率,构建PK-15-d Cas9-MS2稳定细胞系。通过慢病毒感染的方式将包含SAM激活系统元件的lenti d CAS-VP64＿Blast、lenti MS2-P65-HSF1＿Hygro质粒导入PK-15细胞中,用blasticidin和hygromycin药物筛选稳定表达lenti d CAS-VP64＿Blast、lenti MS2-P65-HSF1-Hygro的PK-15细胞,命名为PK-15-d Cas9-MS2。4.分别设计靶向猪OSKM和mi R-302/367的sg RNA,构建sg RNA质粒和双荧光素酶报告基因质粒。靶向猪OSKM基因和mi R-302/367的启动子区域分别设计了6-10条sg RNAs,将每条sg RNA连接在激活载体lenti sg RNA(MS2)＿zeo上,构建靶向Oct4和c-Myc基因的sg RNA质粒各7个,靶向Sox2和Klf4基因的sg RNA质粒各6个,靶向mi R-302/367的sg RNA质粒10个。预测OSKM和mi R-302/367的启动子区域,通过PCR扩增后连接到p GL3-basic载体上,成功构建了5个双荧光素酶报告基因质粒。5.利用PK-15-d Cas9-MS2稳定表达细胞系,通过双荧光素酶实验检测不同sg RNA靶向激活目标基因启动子的活性水平。靶向OSKM、mi R-302/367启动子sg RNA均有活性,靶向同一个基因启动子不同位置的sg RNA活性有差别,并分别筛选出OSKM、mi R-302/367活性排名靠前的两条sg RNA。6.通过双荧光素酶实验检测发现,单个sg RNA和同时表达2条sg RNA均能显著激活靶标基因启动子活性。但是,同时表达2条sg RNA激活启动子活性比单个sg RNA激活启动子活性高。进一步RT-q PCR定量检测同时表达2条sg RNA时对应的靶基因和mi RNA的表达量均有显著提高。7.为了测试采用SAM激活策略是否能同时促进OSKM和mi R-302/367的表达,构建分别靶向目标重编程因子启动子的串联sg RNA激活质粒。通过双荧光素酶实验结果显示,串联sg RNA的质粒可以在PK-15-d Cas9-MS细胞中激活OSKM、mi R-302/367的转录活性。综上所述,在GV358-OSKM和FUGW-mi R-302/367混合诱导Pi PSC中i CD1诱导培养液的诱导效率高于MXV诱导培养液。i CD1诱导培养液为猪的i PSC的诱导效率和速率提供了新的方法。SAM激活系统筛选靶向猪OSKM和mi R-302/367的启动子sg RNA,采用串联sg RNA策略同步激活猪OSKM、mi R-302/367转录,为提高诱导Pi PSC提供了新的思路。更多还原

【Abstract】 Somatic cells can be reprogrammed into induced Pluripotent Stem Cells(i PSC)with the overexpression of key reprogramming factors.IPSC have self-renewal capacity and can differentiate into three germ layers in vivo and vitro.IPSC offer great potential for regenerative medicine.Pig are valuable animal models in pre-clinical research due to their anatomical and similarity to human-beings.Therefore,it is important to study Pi PSC.But Pi PSC have a problem of low efficiency.In this study,we aimed to improve the Pi PSC efficiency:(1)MEF(Mouse Embryonic Fibroblasts)cells can be induced into i PSC by i CD1 medium efficiently and quickly.We try to induce Pi PSC by i CD1 medium and compared efficiency with the previously reported MXV medium.(2)CRISPR/d Cas9(Clustered Regular Interspaced Short Palindromic Repeats/dead associated protein9)can activate gene expression.We used the SAM(Synergistic activation mediator)activation system select efficient sg RNA(Single-guide RNA)for target activating porcine Oct4,Sox2,Klf4 and c-Myc(OSKM)and mi R-302/367.Obtaining combine five sg RNAs tandem vector to simultaneous activation of porcine endogenous OSKM and mi R-302/367 transcription,preparing for reprogram PEF cells to obtain Pi PSC.The main findings are as follows:1.PEF cells can be reprogrammed into Pi PSC with i CD1 Medium.PEF cells were incubated with the lentiviral of GV358-OSKM and FUGW-mi R-302/367,and then PEF cells were reprogramed with i CD1 Medium.Pi PSC clones were generated and passage,and which was positively stained against alkaline phosphatase.2.Pi PSC clones reprogramming are more efficient and shorter time in i CD1 medium than in the MXV medium.Pi PSC clones need to be cultured in the i CD1 Medium for 10 day,and the Pi PSC clones need 16 day in MXV medium.The number of Pi PSC clones in i CD1 medium were three times that of MXV medium.3.PK-15-d Cas9-MS2 cells were selected to increased activation efficiency of the SAM system.PK-15 cells were used to incubate lentiviral of lenti d CAS-VP64＿Blast,lenti MS2-P65-HSF1＿Hygro for 2 days,then these cells were treated with blasticidin(10μg/m L)and hygromycin(500 μg/m L)for 6 days,the PK-15 cell line was obtained with stable expression of lenti d CAS-VP64＿Blast and lenti MS2-P65-HSF1＿Hygro,named PK-15-d Cas9-MS2.4.Six or ten sg RNAs were designed to target porcine reprogramming factors OSKM and mi R-302/367 in their promoter region.Each sg RNA was ligated into lenti sg RNA(MS2)＿zeo backbone vector.Seven Sg RNAs plasmids were constructed to target Oct4 and c-Myc genes,six sg RNAs plasmids were constructed to target Sox2 and Klf4 genes,and ten sg RNAs plasmids were constructed to target mi R-302/367.The luciferase reporter plasmids of OSKM and mi R-302/367 were generated by PCR amplification and ligation with p GL3-basic plasmid.5.Dual luciferase assay was used to detect the efficiency of different sg RNA in PK-15-d Cas9-MS2 cell.Targeting OSKM and mi R-302/367 sg RNAs were tested actively,these sg RNAs targeting in different gene region had different editing efficiencies.We selected two highly active sg RNAs.6.Gene promoter can significantly be activated by single sg RNA or simultaneous expression two sg RNAs.However,promoter activity was higher by two sg RNAs than single sg RNA.Further RT-q PCR detection showed that the expression levels of target genes and mi RNA were significantly increased when two sg RNAs were simultaneous expressed.7.To test whether the SAM activation can simultaneous activate promote activity of OSKM and mi R-302/367.We construct a tandem sg RNAs vector.The dual luciferase assay showed that the plasmids of tandem sg RNA could activate the transcriptional activities of OSKM and mi R-302/367 in PK-d Cas9-MS cells.In summary,Pi PSC clones reprogramming are more efficient and faster in i CD1 medium than in the MXV medium.ICD1 medium provides a new method for Pi PSC.We selected efficient sg RNA(Single-guide RNA)for activating porcine OSKM and mi R-302/367 by SAM activation system and obtained the plasmids of tandem sg RNA in this study.The strategy of tandem sg RNA provides a new idea for reprogramming PEF cells to obtain Pi PSC.更多还原