Zebularine对绵羊卵丘细胞及其核移植胚胎发育效果的影响研究

发布时间：2018-09-07 12:41

【摘要】：体细胞核移植技术在生物学、医学等多个研究领域具有越来越重要的意义,但核移植效率低限制了其发展。目前研究人员普遍认为,核移植效率低主要是由于供体细胞核在卵母细胞中的重编程不完全造成的。而供体细胞DNA甲基化重编程异常是主要原因之一。本研究以绵羊卵丘细胞为供体细胞,拟通过添加DNA甲基转移酶抑制剂Zebularine,探讨其对绵羊卵丘细胞生物学特性、核移植重构胚发育及对胚胎甲基化水平的影响,尝试建立一种既不影响体细胞生物学特性,又可降低供体细胞细胞核的甲基化状态方法,从而提供一种提高体细胞核移植效率的有效途径。试验中使用不同浓度(0、2.5、5、10 nmol/L)的Zebularine处理绵羊卵丘细胞12 h,研究不同处理对绵羊卵丘细胞的存活率、染色体倍性、细胞周期、DNA甲基化水平、DNMT1基因表达水平、核移植胚胎发育能力的影响。同时,使用间接免疫荧光染色法检测了5nmol/L Zebularine处理绵羊卵丘细胞12 h后对核移植胚胎不同发育时期DNA甲基化水平的影响。结果显示:(1)经甲基转移酶抑制剂Zebularine(2.5-10 nmol/L)处理后的卵丘细胞存活率与未经过处理的对照组相比没有显著差异(P0.05);(2)采用常规染色体核型分析发现,不同浓度Zebularine处理组与对照组的细胞染色体整二倍体比例(96.11%、96.53%、95.13%、96.77%)均没有显著差异(P0.05);(3)通过流式细胞仪检测细胞周期,结果显示:绵羊卵丘细胞经不同浓度Zebularine处理12 h后,G0/G1的细胞比例呈逐渐上升趋势,S期细胞比例均呈逐渐下降的趋势,但与对照组之间均没有显著差异(P0.05);(4)使用间接免疫荧光染色法检测DNA甲基化水平,卵丘细胞DNA甲基化水平随着Zebularine处理浓度的升高而降低,其中5、10 nmol/L处理组DNA甲基化水平显著显著低于对照组(P0.05),但两处理组间没有显著差异(P0.05);(5)通过实时荧光定量PCR检测发现:卵丘中Dnmt1基因表达水平随着Zebularine处理浓度的升高而降低,其中5、10 nmol/L处理组Dnmt1基因表达水平显著低于对照组(P0.05),且10 nmol/L处理组显著低于5nmol/L处理组(P0.05);(6)绵羊卵丘细胞经不同浓度Zebularine处理12h后进行核移植,结果显示:Zebularine各处理组与对照组(0 nmol/L)间核移植胚胎的卵裂率差异均不显著(P0.05);5 nmol/L Zebularine处理组桑椹胚率显著高于对照组(P0.05),与其他各组间差异不显著(P0.05)。5 nmol/L Zebularine处理组囊胚率显著高于对照组及其他Zebularine处理组(P0.05)。(7)使用间接免疫荧光染色法检测5 nmol/L Zebularine处理绵羊卵丘细胞12 h后对核移植胚胎DNA甲基化水平的影响,结果显示:SCNT-Zebularine组胚胎DNA甲基化水平变化趋势与体外受精组、核移植对照组胚胎类似,并且不同发育时期胚胎DNA甲基化水平SCNT-Zebularine组均低于核移植对照组胚胎,其中2-细胞与8-细胞期显著降低(P0.05),但SCNT-Zebularine组胚胎DNA甲基化水平仍高于相应时期的体外受精胚胎(P0.05)。综上所述,绵羊卵丘细胞经过2.5、5、10 nmol/L Zebularine处理12h后,对存活率、染色体倍性、细胞周期均没有显著影响;5、10 nmol/L Zebularine处理12 h后可显著降低其DNA甲基化水平和Dnmt1基因的表达水平,且5 nmol/L Zebularine组可以显著提高核移植胚胎的发育效率,降低核移植胚胎的DNA甲基化水平。
[Abstract]:Somatic cell nuclear transfer (SCNT) is becoming more and more important in many fields, such as biology and medicine. However, the low efficiency of SCNT limits its development. Abnormality is one of the main causes. In this study, sheep cumulus cells were used as donor cells to investigate the biological characteristics of sheep cumulus cells, the effects of Zebularine on the development of reconstructed embryos and the level of embryo methylation after nuclear transfer, and try to establish a new method that does not affect the biological characteristics of somatic cells, but also reduces the level of embryo methylation. Low donor cell nucleus methylation provides an effective way to improve the efficiency of somatic cell nuclear transfer. In this experiment, sheep cumulus cells were treated with Zebularine at different concentrations (0,2.5,5,10 nmol/L) for 12 hours to study the survival rate, chromosome ploidy, cell cycle and DNA methylation water of sheep cumulus cells under different treatments. Meanwhile, the effects of 5 nmol/L Zebularine on DNA methylation at different developmental stages of nuclear transfer embryos were examined by indirect immunofluorescence staining. There was no significant difference in the survival rate of cumulus cells between the treated and untreated control groups (P 0.05); (2) There was no significant difference in the percentage of chromosome aneuploidy (96.11%, 96.53%, 95.13%, 96.77%) between Zebularine treated and untreated control groups (P 0.05); (3) There was no significant difference in the percentage of chromosome aneuploidy (96.11%, 96.53%, 95.13%, 96.77%) between Zebularine treated and untreated control groups (P 0. The results showed that after 12 hours of Zebularine treatment, the proportion of G0/G1 cells increased gradually and that of S-phase cells decreased gradually, but there was no significant difference between the control group and the control group (P 0.05); (4) DNA methylation level was detected by indirect immunofluorescence staining. Methylation level decreased with the increase of Zebularine treatment concentration, and DNA methylation level in 5,10 nmol/L treatment group was significantly lower than that in control group (P 0.05), but there was no significant difference between the two treatment groups (P 0.05); (5) Real-time fluorescence quantitative PCR detection showed that Dnmt1 gene expression level in cumulus increased with the increase of Zebularine treatment concentration. The expression level of Dnmt1 gene in 5,10 nmol/L treatment group was significantly lower than that in control group (P 0.05), and 10 nmol/L treatment group was significantly lower than that in 5 nmol/L treatment group (P 0.05); (6) Sheep cumulus cells were treated with different concentrations of Zebularine for 12 hours after nuclear transfer, the results showed that: Zebularine treatment group and control group (0 nmol/L) between the nuclear transfer embryos. The blastocyst rate of 5 nmol/L Zebularine treatment group was significantly higher than that of control group (P 0.05). The blastocyst rate of 5 nmol/L Zebularine treatment group was significantly higher than that of control group and other Zebularine treatment groups (P 0.05). The results showed that the changes of DNA methylation level in SCNT-Zebularine group were similar to those in IVF group and nuclear transfer control group, and the levels of DNA methylation in SCNT-Zebularine group were lower than those in nuclear transfer control group at different developmental stages. The DNA methylation level of SCNT-Zebularine group embryos was still higher than that of in vitro fertilized embryos (P 0.05). In conclusion, the cumulus cells treated with 2.5, 5, 10 nmol/L Zebularine for 12 hours had no significant effect on survival rate, chromosome ploidy and cell cycle. DNA methylation level and Dnmt1 gene expression level were significantly decreased after 12 hours of NE treatment, and the 5 nmol/L Zebularine group could significantly improve the development efficiency of nuclear transfer embryos and reduce the DNA methylation level of nuclear transfer embryos.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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