【作者】 刘岚；

【导师】 李奎；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 目前,胚胎干细胞的研究主要集中在分离纯化、扩增技术和定向诱导分化几个方面。本研究以胚胎干细胞作为研究对象,研究内容共分以下两部分:第一部分主要是猪胚胎生殖细胞(EG)的分离培养研究。本实验室采用STO细胞作饲养层,在培养基中添加分化抑制因子的方法,已成功分离培养获得猪的EG细胞。为进一步探索猪EG细胞更为理想的培养体系,并获得能够稳定传代的EG细胞系,第一部分以三个实验展开了对猪EG细胞体外培养条件的进一步研究。实验一研究了条件培养基对猪EG细胞分离培养的影响。在原代分离猪PGCs的过程中,可以同时分离获得猪胎儿成纤维细胞(PEF)和胎儿心脏成纤维细胞(EHF);经培养纯化后,提取这两种细胞的RNA并反转录;RT-PCR检测结果表明,PEF和EHF中均表达猪的LIF基因。因此,分别收集两种细胞的培养液制成条件培养基,用来培养猪的EG细胞。分别使用两种条件培养基或将条件培养基与STO饲养层细胞联合使用来分离培养猪的EG细胞,结果发现条件培养基必须与饲养层细胞联合使用,才能维持EG细胞的增殖未分化状态。实验二研究了Knockout培养基对猪EG细胞分离培养的影响。本实验共分三组,其中实验组I为Knockout DMEM与Knockout SR组成的Knockout培养基,实验组II为Knockout DMEM/F-12+15%FCS的低渗培养基,对照组为DMEM+15%FCS的普通培养基,三个实验组中均添加了细胞生长因子。结果表明实验组I与对照组都可以获得能够传代的EG细胞,实验组I培养效果好于对照组;而实验组II不能维持EG细胞稳定传代。因此,Knockout DMEM与Knockout SR组成的Knockout培养基可以用于分离培养猪的EG细胞。实验三将PGCs与同源胎儿成纤维细胞共培养分离培养猪的EG细胞。培养获得的细胞经AKP染色,SSEA-1免疫荧光染色,转录因子Oct-4免疫荧光表达,核型分析,体外分化实验及电镜观察等多种方法进行鉴定,证明其具有胚胎干细胞的特征。因此,PGCs与同源胎儿成纤维细胞共培养的方法可以用于分离培养猪EG细胞。第二部分以小鼠ES细胞作为研究对象,分三个实验对不同诱导剂在胚胎干细胞定向分化为胰岛素分泌细胞(IPCs)中的作用以及基因转染方法诱导ES细胞分化为IPCs进行了初步探讨。实验一研究了GLP-1在诱导小鼠ES细胞向IPCs分化中的作用。诱导一周后,经DTZ染色鉴定,阳性细胞很少;诱导两周后,提取细胞总RNA,经RT-PCR检测发现,GLP-1可以使与胰腺发育相关的重要基因Pdx1的表达量上调,但诱导两周仍未能获得大量IPCs。实验二研究了bFGF在诱导小鼠ES细胞向IPCs分化中的作用。诱导后提取细胞总RNA,通过RT-PCR未能检测到IPCs相关基因的表达。实验结果表明,在本实验条件下,向培养基中单一添加bFGF并不能有效地诱导小鼠ES细胞向IPCs分化。实验三采用基因转染的方法将Pdx1基因与Isl1基因导入小鼠ES细胞诱导其分化。转染24h后,通过RT-PCR的方法检测到Pdx1基因与Isl1基因在小鼠ES细胞中过表达,其中Pdx1表达量明显上调,但是仍未检测到其它IPCs相关基因的表达。这一结果表明,转染时间不够,转染24h后过表达的基因未能诱导小鼠ES细胞的分化。但是由于过表达效果明显,如果添加其他诱导剂在培养基中,继续传代培养,将会有比较好的诱导结果。更多还原

【Abstract】 Now,the work of Embryonic stem(ES)cells mainly focused on isolation and purification,passage and enlarge,inducement and differentiation. ES cells were research objective in this study,the research work included two parts:One part of the research was isolating and culturing porcine embryonic germ(EG)cells.We had got porcine EG cells when plating PGCs on STO feeder cells and adding differentiation inhibitory activities (DIA)in the medium. To find optimization culture system and get successive EG cells lines,this part was composed of three experiments to research the culture condition of porcine EG cells in.vitro.The first experiment discussed the effect of condition medium(CM)on isolating and culturing porcine EG cells.When isolating the PGCs,the porcine embryonic fibroblas(tPEF)and embryonic heart fibroblast(EHF)could be got. After culturing and purifying,we extracted RNA from two cells,then reverse transcripted them to cDNA. The results of RT-PCR showed that LIF gene expressed both in PEF and EHF. So, we collected the cell mediums and mixed them with DMEM to get CM. We cultured porcine EG cells using CM only or together with STO feeder cells. The results indicated porcine EG cells could be got only while using CM together with STO feeder cells.The second experiment discussed the effect of knockout medium on isolating and culturing porcine EG cells. The experiment group I was composed of Knockout DMEM and Knockout SR, the group II was Knockout DMEM/F-12+15%FCS,the control group was DMEM+15%FCS. The growth factors were supplemented in three groups. The results indicated that group I and control group could get the porcine EG cells,group I was better than control group;but group II could not maintain the EG cells. So group I could be used to isolate and culture porcine EG cells.The third experiment was isolating PGCs in co-culture with homologous fetal fibroblast to get porcine EG cells. The EG colonies were AKP-positive, reacted with SSEA-1 antibody and their immunity fluorescence staining of Oct-4 was positive. The EG cells had correct karyotype. The colonies placed in suspension culture in medium without growth factors differentiated into simple embryoid bodies(EBs)in vitro. The EG cells were adhesive by observing them using the scanning and transmission electron microscope. So we confirmed these cells have many characteristics of embryonic stem cells as described previously and they were EG cells. So porcine EG cells could be obtained by isolating PGCs in co-culture with homologous fetal fibroblast.Another part of the research included three experiments. It briefly discussed the effect of different inducers and different method on inducing mouse ES cells to insulin-producing cells(IPCs). The first experiment discussed the function of glucagons-like peptide-1(GLP-1)to inducing MES cells to IPCs. After one week,the DTZ-positive cells was little. Two weeks later,we extracted RNA from cells,the results of RT-PCR showed that GLP-1 improved the expression of Pdx1,but it could not induce MES to IPCs in two weeks.The second experiment discussed the function of bFGF to induce MES cells to IPCs. We extracted RNA from cells after inducing and checking the related genes expression of IPCs using RT-PCR. The results indicated,in this condition,MES could not be induced to IPCs when only adding bFGF into medium.The third experiment used a method of gene transfection to induce MES cells differentiation. Pdx1 gene and Isl1 gene were transfected to MES cells. About 24h later,Pdx1 gene and Isl1 genes overexpessed in MES cells,Pdx1 gene improved obviously,but other relational genes of IPCs could not be checked by RT-PCR.The results showed transfection time was not enough and the gene overexpressed for 24h in MES cells could not induce the cells differentiation. But the effect of gene overexpression was visibly. So the results maybe better,if we added the inducer into medium more and cultured the cells continuly.更多还原