TSA促進梅花鹿和牛種間克隆胚胎發(fā)育的研究
發(fā)布時間：2018-10-25 20:25
【摘要】：自從1997年第一只克隆羊多莉出生以后,至今已有19種哺乳動物通過體細胞核移植獲得克隆后代,在世界掀起了一陣“克隆”熱潮。雖然同屬鹿科的赤鹿已經(jīng)成功被克隆了后代,但是梅花鹿被成功克隆還未報道。梅花鹿是國家一級保護動物,除了可以為人類提供多種名貴的醫(yī)療保健藥材外,它還具有很高的觀賞性和科學研究價值。但是有關(guān)梅花鹿克隆的研究處于停滯不前的境地,主要是由于梅花鹿本身數(shù)量的減少和屠殺數(shù)量的減少,這樣就不能提供豐富的卵巢來源,所以極大地限制了梅花鹿克隆胚胎方面的研究進展。所以尋找代替梅花鹿卵母細胞的替代物是梅花鹿種間克隆研究中一個值得關(guān)注的關(guān)鍵性問題。本試驗探討了梅花鹿-牛種間克隆胚胎體外培養(yǎng)的條件,試驗數(shù)據(jù)顯示梅花鹿胎兒成纖維細胞作為核供體細胞所構(gòu)建的種間重構(gòu)胚胎的卵裂率(71.7±4.0%)及囊胚率(13.1±1.7%)顯著高于梅花鹿耳成纖維細胞作為核供體細胞的卵裂率(53.8±0.1%)和囊胚率(7.1±0.2%);以280V、脈沖時間10μs、2次直流脈沖條件下進行重構(gòu)胚胎的細胞融合所獲得的梅花鹿-牛種間克隆胚胎的卵裂率(60.9±0.5%)顯著高于230V-300V融合電壓的其他試驗組;牛胚胎培養(yǎng)液SOFAA和梅花鹿胚胎培養(yǎng)液SDSOF對梅花鹿-牛種間重構(gòu)胚胎的卵裂率沒有顯著差異,分別是60.7±1.9%及61.7±1.7%,但SDSOF培養(yǎng)基中所獲得的種間重構(gòu)胚胎囊胚率(10.7%±1.2%)顯著高于SOFAA培養(yǎng)基中獲得的囊胚率(5.6±1.2%)。同時探討TSA對梅花鹿和牛種間克隆胚胎發(fā)育作用。為了更加直觀的觀察種間胚胎的發(fā)育情況,首先建立了梅花鹿pEGFP-N1成纖維細胞系作為核供體細胞。以不處理組為對照組,選擇不同濃度的TSA處理核供體細胞24h和種間重構(gòu)克隆胚胎10h為試驗組。結(jié)果表明:最適合梅花鹿-牛種間克隆重構(gòu)胚胎發(fā)育的TSA濃度是50nM,篩選的pEGFP-N1成纖維細胞系2及3作為核供體細胞獲得的囊胚率顯著高于克隆1(9.2%和10.7%vs.4.3%,P0.05)。綜上所述,牛卵母細胞可以支持梅花鹿體細胞的重編程及去分化。
[Abstract]:Since the birth of Dolly, the first cloned sheep in 1997, 19 mammals have obtained cloned offspring through somatic cell nuclear transfer, which has caused a wave of "cloning" in the world. Although red deer belonging to the same family have been successfully cloned, the successful cloning of sika deer has not been reported. Sika deer is one of the first class protected animals in the country. In addition to providing a variety of valuable medical and health care materials for human beings, sika deer also has high ornamental and scientific research value. But the research on sika deer cloning is at a standstill, mainly due to the decline in the number of sika deer themselves and the reduction in the number of people slaughtered, which does not provide a rich source of ovaries. Therefore, the research progress in cloned embryos of sika deer is greatly restricted. Therefore, the search for alternative oocytes is a key issue in interspecific cloning of sika deer. In this experiment, the conditions for the culture of sika deer and bovine interspecific cloned embryos in vitro were studied. Experimental data showed that the cleavage rate (71.7 鹵4.0%) and blastocyst rate (13.1 鹵1.7%) of interspecific reconstructed embryos constructed by sika deer fetal fibroblasts as nuclear donor cells were significantly higher than those of sika deer ear fibroblasts. The cleavage rate and blastocyst rate of nuclear donor cells were 53.8 鹵0.1% and 7.1 鹵0.2%, respectively. The cleavage rate (60.9 鹵0.5%) of sika deer interspecific cloned embryos obtained by cell fusion of reconstructed embryos at 280V and 10 渭 s of pulse time was significantly higher than that of other experimental groups with 230V-300V fusion voltage. There was no significant difference between bovine embryo culture medium (SOFAA) and sika deer embryo culture medium (SDSOF) in cleavage rate of sika deer and bovine interspecific reconstructed embryos, which were 60.7 鹵1.9% and 61.7 鹵1.7%, respectively. But the rate of blastocysts obtained in SDSOF medium (10.7% 鹵1.2%) was significantly higher than that in SOFAA medium (5.6% 鹵1.2%). At the same time, the effects of TSA on the development of interspecific cloned embryos between sika deer and cattle were studied. In order to observe the development of interspecific embryos more intuitively, a sika deer pEGFP-N1 fibroblast cell line was established as a nuclear donor cell. The untreated group was used as the control group and the nuclear donor cells were treated with different concentrations of TSA for 24 h and the interspecific reconstructed cloned embryos for 10 h were selected as the experimental group. The results showed that the optimal concentration of TSA for embryo development of sika deer and bovine interspecific clone reconstruction was 50 nm. The blastocyst rate of pEGFP-N1 fibroblasts 2 and 3 as nuclear donor cells was significantly higher than that of clone 1 (9.2% vs 10.7vs.4.3p0.05). In conclusion, bovine oocytes can support the reprogramming and dedifferentiation of sika deer somatic cells.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S814
本文編號：2294746
[Abstract]:Since the birth of Dolly, the first cloned sheep in 1997, 19 mammals have obtained cloned offspring through somatic cell nuclear transfer, which has caused a wave of "cloning" in the world. Although red deer belonging to the same family have been successfully cloned, the successful cloning of sika deer has not been reported. Sika deer is one of the first class protected animals in the country. In addition to providing a variety of valuable medical and health care materials for human beings, sika deer also has high ornamental and scientific research value. But the research on sika deer cloning is at a standstill, mainly due to the decline in the number of sika deer themselves and the reduction in the number of people slaughtered, which does not provide a rich source of ovaries. Therefore, the research progress in cloned embryos of sika deer is greatly restricted. Therefore, the search for alternative oocytes is a key issue in interspecific cloning of sika deer. In this experiment, the conditions for the culture of sika deer and bovine interspecific cloned embryos in vitro were studied. Experimental data showed that the cleavage rate (71.7 鹵4.0%) and blastocyst rate (13.1 鹵1.7%) of interspecific reconstructed embryos constructed by sika deer fetal fibroblasts as nuclear donor cells were significantly higher than those of sika deer ear fibroblasts. The cleavage rate and blastocyst rate of nuclear donor cells were 53.8 鹵0.1% and 7.1 鹵0.2%, respectively. The cleavage rate (60.9 鹵0.5%) of sika deer interspecific cloned embryos obtained by cell fusion of reconstructed embryos at 280V and 10 渭 s of pulse time was significantly higher than that of other experimental groups with 230V-300V fusion voltage. There was no significant difference between bovine embryo culture medium (SOFAA) and sika deer embryo culture medium (SDSOF) in cleavage rate of sika deer and bovine interspecific reconstructed embryos, which were 60.7 鹵1.9% and 61.7 鹵1.7%, respectively. But the rate of blastocysts obtained in SDSOF medium (10.7% 鹵1.2%) was significantly higher than that in SOFAA medium (5.6% 鹵1.2%). At the same time, the effects of TSA on the development of interspecific cloned embryos between sika deer and cattle were studied. In order to observe the development of interspecific embryos more intuitively, a sika deer pEGFP-N1 fibroblast cell line was established as a nuclear donor cell. The untreated group was used as the control group and the nuclear donor cells were treated with different concentrations of TSA for 24 h and the interspecific reconstructed cloned embryos for 10 h were selected as the experimental group. The results showed that the optimal concentration of TSA for embryo development of sika deer and bovine interspecific clone reconstruction was 50 nm. The blastocyst rate of pEGFP-N1 fibroblasts 2 and 3 as nuclear donor cells was significantly higher than that of clone 1 (9.2% vs 10.7vs.4.3p0.05). In conclusion, bovine oocytes can support the reprogramming and dedifferentiation of sika deer somatic cells.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S814
【參考文獻】
中國期刊全文數(shù)據(jù)庫 前2條
1 ;Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleucd) embryos[J];Chinese Science Bulletin;2002年06期
2 殷宗俊;龍學仁;許瑞鋒;徐福樂;朱建兒;王霖;;受體牛胎次與黃體等級對胚胎移植妊娠率的影響[J];中國奶牛;2006年07期
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