添加不同的因子对绵羊类胚胎干细胞多能性的影响
发布时间：2018-09-12 10:03
【摘要】：胚胎干细胞是由内细胞团分离培养得到的,在适当的条件下能分化成所有类型的细胞。为筛选出能够维持绵羊类胚胎干细胞多能性的因子,以实验室建系的oESC-like为材料,培养细胞8d,从形态、增殖、AKP以及多能性候选基因Oct4、Sox2、Klf4、c-Myc、Lin28mRNA表达量的变化,分析绵羊类胚胎干细胞的多能性,确定维持绵羊类胚胎干细胞多能性的因子。主要研究结果： 在N2B27/CH中,添加PD、PD/hLIF、PD/BPM4、PDhLIF/BMP4培养8d,通过与本实验室原培养体系N2B27/CH/bFGF相比,结果显示,添加PD、PD/hLIF、PD/BPM4、PD/hLIF/BMP4oESC-like细胞都表达AKP与Sox2、Oct4免疫荧光蛋白；添加PD、PD/hLIF oESC-like细胞之间的结合致密,oESC-like细胞的生长速度减慢；而添加PD/BPM4、PD/hLIF/BMP4oESC-like细胞之间的结合疏松,oESC-like细胞的生长速度增快。添加PD和PD/hLIF使多能性候选基因Lin28与c-Myc表达量升高差异极显著(P0.01),Nestin的表达量降低差异极显著(P0.01),加入PD/hLIF使Oct4表达量升高差异极显著(P0.01),添加PD/BPM4、PDhLIF/BMP4使多能性候选基因Oct4、Sox2、c-Myc、Klf4的表达量降低差异极显著(P0.01),Nestin与Lin28的表达量降低差异极显著(P0.01)。 在培养体系N2B27/CH/bFGF中,分别加入PD、PD/hLIF、PD/BPM4、PDhLIF/BMP4培养细胞8d,与N2B27/CH/bFGF相比,结果显示：AKP染色及Sox2与Oct4免疫荧光蛋白染色结果表明,添加不同因子后oESC-like显示胚胎干细胞多能性,添加PD多能性候选基因Oct4与Klf4mRNA的表达量升高差异显著(P0.01),Nestin与Lin28的表达量降低差异极显著(P0.01),Sox2与c-Myc升高差异不显著。添加hLIF/PD使多能性候选基因Oct4的表达量表达量升高差异极显著(P0.01),c-Myc、Klf4、Lin28表达量升高差异不显著,Nestin、Sox2表达量降低差异不显著。添加PD/BPM4、 PD/hLIF/BMP4使Oct4、Sox2、Nestin表达量降低差异显著(P0.01),使Lin28、c-Myc、Klf4表达量降低差异不显著。 分别将选出的N2B27/CH/PD、N2B27/CH/PD/hLIF、N2B27/CH/bFGF/PD、N2B27/CH/bFGF/PD/hLIF进行培养,结果显示,添加PD、PD/hLIF与bFGF/PD、bFGF/PD/hLIF相比,oESC-like细胞表达碱性磷酸酶、生长速度快、细胞之间的结合更加致密,且多能性候选基因Sox2与Oct4表达免疫荧光蛋白。添加PD与bFGF/PD相比,oESC-like细胞多能性候选基因Oct4、Klf4与c-Myc的表达量升高差异极显著(P0.01),Sox2的表达量升高差异不显著,c-Myc与Nestin的表达量降低差异极显著(P0.01)。
[Abstract]:Embryonic stem cells are isolated from inner cell clusters and can differentiate into all types of cells under suitable conditions. In order to screen out the factors that can maintain the pluripotency of sheep embryonic stem cells (ESCs), the cells were cultured for 8 days with oESC-like, and the changes of Oct4,Sox2,Klf4,c-Myc,Lin28mRNA expression in morphology, proliferation and pluripotent candidate genes were studied. To analyze the pluripotency of sheep embryonic stem cells and determine the factors that maintain the pluripotency of sheep embryonic stem cells. Main results: in N2B27/CH, adding PD,PD/hLIF,PD/BPM4,PDhLIF/BMP4 for 8 days, compared with the original culture system N2B27/CH/bFGF, the results showed that both AKP and Sox2,Oct4 immunofluorescence protein were expressed in PD,PD/hLIF,PD/BPM4,PD/hLIF/BMP4oESC-like cells. The growth rate of compact PD,PD/hLIF oESC-like cells was slower than that of PD/BPM4,PD/hLIF/BMP4oESC-like cells, while the growth rate of loose oESC-like cells increased rapidly. The addition of PD and PD/hLIF increased the expression of Lin28 and c-Myc, significantly decreased the expression of nestin (P0.01), increased the expression of Oct4 (P0.01) by adding PD/hLIF, and increased the expression of Oct4,Sox2,c-Myc,Klf4 (P0.01) by adding PD/BPM4,PDhLIF/BMP4. The quantity of nestin and Lin28 decreased significantly (P0.01). In the culture system of N2B27/CH/bFGF, the cells were cultured with PD,PD/hLIF,PD/BPM4,PDhLIF/BMP4 for 8 days. Compared with N2B27/CH/bFGF, the results showed that oESC-like showed the pluripotency of embryonic stem cells after adding different factors, compared with N2B27/CH/bFGF and Sox2 and Oct4 immunofluorescence protein staining. The expression of Oct4 and Klf4mRNA increased significantly (P0.01). The expression of nestin and Lin28 decreased significantly (P0.01). There was no significant difference between Sox2 and c-Myc. Addition of hLIF/PD increased the expression of multipotent candidate gene Oct4 significantly (P0.01). There was no significant difference in the expression level of c-Mycf4lf4hLin28. There was no significant difference in the expression of Nestinine hLIF/PD and Sox2. The addition of PD/BPM4, PD/hLIF/BMP4 reduced the expression of Oct4,Sox2,Nestin significantly (P0.01), but did not decrease the expression of Lin28,c-Myc,Klf4 (P0.01). The selected N2B27 / CHP / N2B27 / HLIFN2BFGF- / PDN2BFGF- / N2B27 / PD-hLIF was cultured respectively. The results showed that the addition of PD,PD/hLIF expressed alkaline phosphatase (ALP), grew faster, and expressed immunofluorescence protein (Oct4) than that of bFGF/PD,bFGF/PD/hLIF, and the candidate gene Sox2 and Oct4 expressed immunofluorescence protein. There was no significant difference in the expression of Oct4,Klf4 and c-Myc between PD and bFGF/PD (P0.01). The expression of c-Myc and Nestin decreased significantly (P0.01).
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Embryonic stem cells are isolated from inner cell clusters and can differentiate into all types of cells under suitable conditions. In order to screen out the factors that can maintain the pluripotency of sheep embryonic stem cells (ESCs), the cells were cultured for 8 days with oESC-like, and the changes of Oct4,Sox2,Klf4,c-Myc,Lin28mRNA expression in morphology, proliferation and pluripotent candidate genes were studied. To analyze the pluripotency of sheep embryonic stem cells and determine the factors that maintain the pluripotency of sheep embryonic stem cells. Main results: in N2B27/CH, adding PD,PD/hLIF,PD/BPM4,PDhLIF/BMP4 for 8 days, compared with the original culture system N2B27/CH/bFGF, the results showed that both AKP and Sox2,Oct4 immunofluorescence protein were expressed in PD,PD/hLIF,PD/BPM4,PD/hLIF/BMP4oESC-like cells. The growth rate of compact PD,PD/hLIF oESC-like cells was slower than that of PD/BPM4,PD/hLIF/BMP4oESC-like cells, while the growth rate of loose oESC-like cells increased rapidly. The addition of PD and PD/hLIF increased the expression of Lin28 and c-Myc, significantly decreased the expression of nestin (P0.01), increased the expression of Oct4 (P0.01) by adding PD/hLIF, and increased the expression of Oct4,Sox2,c-Myc,Klf4 (P0.01) by adding PD/BPM4,PDhLIF/BMP4. The quantity of nestin and Lin28 decreased significantly (P0.01). In the culture system of N2B27/CH/bFGF, the cells were cultured with PD,PD/hLIF,PD/BPM4,PDhLIF/BMP4 for 8 days. Compared with N2B27/CH/bFGF, the results showed that oESC-like showed the pluripotency of embryonic stem cells after adding different factors, compared with N2B27/CH/bFGF and Sox2 and Oct4 immunofluorescence protein staining. The expression of Oct4 and Klf4mRNA increased significantly (P0.01). The expression of nestin and Lin28 decreased significantly (P0.01). There was no significant difference between Sox2 and c-Myc. Addition of hLIF/PD increased the expression of multipotent candidate gene Oct4 significantly (P0.01). There was no significant difference in the expression level of c-Mycf4lf4hLin28. There was no significant difference in the expression of Nestinine hLIF/PD and Sox2. The addition of PD/BPM4, PD/hLIF/BMP4 reduced the expression of Oct4,Sox2,Nestin significantly (P0.01), but did not decrease the expression of Lin28,c-Myc,Klf4 (P0.01). The selected N2B27 / CHP / N2B27 / HLIFN2BFGF- / PDN2BFGF- / N2B27 / PD-hLIF was cultured respectively. The results showed that the addition of PD,PD/hLIF expressed alkaline phosphatase (ALP), grew faster, and expressed immunofluorescence protein (Oct4) than that of bFGF/PD,bFGF/PD/hLIF, and the candidate gene Sox2 and Oct4 expressed immunofluorescence protein. There was no significant difference in the expression of Oct4,Klf4 and c-Myc between PD and bFGF/PD (P0.01). The expression of c-Myc and Nestin decreased significantly (P0.01).
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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