【摘要】：胚胎干細(xì)胞是由內(nèi)細(xì)胞團(tuán)分離培養(yǎng)得到的,在適當(dāng)?shù)臈l件下能分化成所有類型的細(xì)胞。為篩選出能夠維持綿羊類胚胎干細(xì)胞多能性的因子,以實(shí)驗(yàn)室建系的oESC-like為材料,培養(yǎng)細(xì)胞8d,從形態(tài)、增殖、AKP以及多能性候選基因Oct4、Sox2、Klf4、c-Myc、Lin28mRNA表達(dá)量的變化,分析綿羊類胚胎干細(xì)胞的多能性,確定維持綿羊類胚胎干細(xì)胞多能性的因子。主要研究結(jié)果： 在N2B27/CH中,添加PD、PD/hLIF、PD/BPM4、PDhLIF/BMP4培養(yǎng)8d,通過與本實(shí)驗(yàn)室原培養(yǎng)體系N2B27/CH/bFGF相比,結(jié)果顯示,添加PD、PD/hLIF、PD/BPM4、PD/hLIF/BMP4oESC-like細(xì)胞都表達(dá)AKP與Sox2、Oct4免疫熒光蛋白；添加PD、PD/hLIF oESC-like細(xì)胞之間的結(jié)合致密,oESC-like細(xì)胞的生長速度減慢；而添加PD/BPM4、PD/hLIF/BMP4oESC-like細(xì)胞之間的結(jié)合疏松,oESC-like細(xì)胞的生長速度增快。添加PD和PD/hLIF使多能性候選基因Lin28與c-Myc表達(dá)量升高差異極顯著(P0.01),Nestin的表達(dá)量降低差異極顯著(P0.01),加入PD/hLIF使Oct4表達(dá)量升高差異極顯著(P0.01),添加PD/BPM4、PDhLIF/BMP4使多能性候選基因Oct4、Sox2、c-Myc、Klf4的表達(dá)量降低差異極顯著(P0.01),Nestin與Lin28的表達(dá)量降低差異極顯著(P0.01)。 在培養(yǎng)體系N2B27/CH/bFGF中,分別加入PD、PD/hLIF、PD/BPM4、PDhLIF/BMP4培養(yǎng)細(xì)胞8d,與N2B27/CH/bFGF相比,結(jié)果顯示：AKP染色及Sox2與Oct4免疫熒光蛋白染色結(jié)果表明,添加不同因子后oESC-like顯示胚胎干細(xì)胞多能性,添加PD多能性候選基因Oct4與Klf4mRNA的表達(dá)量升高差異顯著(P0.01),Nestin與Lin28的表達(dá)量降低差異極顯著(P0.01),Sox2與c-Myc升高差異不顯著。添加hLIF/PD使多能性候選基因Oct4的表達(dá)量表達(dá)量升高差異極顯著(P0.01),c-Myc、Klf4、Lin28表達(dá)量升高差異不顯著,Nestin、Sox2表達(dá)量降低差異不顯著。添加PD/BPM4、 PD/hLIF/BMP4使Oct4、Sox2、Nestin表達(dá)量降低差異顯著(P0.01),使Lin28、c-Myc、Klf4表達(dá)量降低差異不顯著。 分別將選出的N2B27/CH/PD、N2B27/CH/PD/hLIF、N2B27/CH/bFGF/PD、N2B27/CH/bFGF/PD/hLIF進(jìn)行培養(yǎng),結(jié)果顯示,添加PD、PD/hLIF與bFGF/PD、bFGF/PD/hLIF相比,oESC-like細(xì)胞表達(dá)堿性磷酸酶、生長速度快、細(xì)胞之間的結(jié)合更加致密,且多能性候選基因Sox2與Oct4表達(dá)免疫熒光蛋白。添加PD與bFGF/PD相比,oESC-like細(xì)胞多能性候選基因Oct4、Klf4與c-Myc的表達(dá)量升高差異極顯著(P0.01),Sox2的表達(dá)量升高差異不顯著,c-Myc與Nestin的表達(dá)量降低差異極顯著(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).
【學(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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