CN109679895A - 含输卵管源外泌体的胚胎培养液及胚胎培养方法 - Google Patents
含输卵管源外泌体的胚胎培养液及胚胎培养方法 Download PDFInfo
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Abstract
本发明公开了一种含输卵管源外泌体的胚胎培养液及胚胎培养方法:从输卵管液中提取输卵管源外泌体,将输卵管源外泌体添加到胚胎基础培养液中,得到含有输卵管源外泌体的胚胎培养液,所述胚胎培养液中输卵管源外泌体浓度为6.0~7.0×108个/mL;将动物(例如,兔子)胚胎移入含有输卵管源外泌体的胚胎培养液中进行培养。本发明通过添加输卵管源外泌体,使得胚胎在体外培养环境中获得了体内输卵管调控因子的作用,有利于胚胎在体外环境中的发育,提高胚胎的体外培养效率。
Description
技术领域
本发明涉及体外胚胎培养,具体涉及含有输卵管源外泌体的胚胎培养液及胚胎培养方法。
背景技术
兔作为重要的实验动物,广泛地用于人类疾病的研究。尤其在心血管疾病、脂代谢以及胚胎发育的研究中,以兔为模型发挥了重要的研究价值。此外,兔作为经济动物或宠物在人民的生活中占有重要的地位。随着基因编辑技术的发展,多种基因工程兔陆续诞生。然而关于兔的辅助生殖技术相比于其他物种并不成熟,截止目前,国内外并没有商品化的兔胚胎培养液。兔胚胎的体外培养处于相对较低的水平,这极大的限制了基因工程兔的生产效率和兔的人工繁育。因此兔胚胎培养液的开发具有重要意义。
哺乳动物的胚胎培养液成分较为相似,主要包含水、无机盐、碳水化合物、氨基酸、维生素、核酸前体、螯合剂、抗氧化剂、抗生素、大分子物质、激素和生长因子等。尽管胚胎培养液的组成不断优化,但体外培养的胚胎在形态、基因表达和发育能力方面表现出相对较差的状态,特别是对于兔胚胎培养,存在发育率低、细胞凋亡率高等实际问题。胚胎培养液成分的改良无疑是迫切和必要的。
目前,尚未见到将输卵管源外泌体应用于胚胎培养液的报道。
发明内容
本发明的目的在于提供一种含输卵管源外泌体的胚胎培养液及胚胎培养方法,可以提高胚胎的体外培养效率。
为达到上述目的,本发明采用了以下技术方案:
一种胚胎培养液,该胚胎培养液包括胚胎基础培养液以及输卵管源外泌体,所述胚胎培养液中输卵管源外泌体的浓度为6.0~7.0×108个/mL。
优选的,所述胚胎基础培养液包括用于模拟动物(例如,兔子)输卵管液的理化特征的无机盐组分、有机盐组分、氨基酸组分、葡萄糖及血清白蛋白(例如,牛血清白蛋白)。
优选的,所述胚胎基础培养液所含无机盐组分具体包括1.780~1.790mM的CaCl2·2H2O、1.510~1.520mM的MgSO4·7H2O、7.160~7.170mM的KCl、1.190~1.200mM的KH2PO4、25.000~25.010mM的NaHCO3以及107.590~107.690mM的NaCl。
优选的,所述胚胎基础培养液所含有机盐组分具体包括0.393~0.403mM的丙酮酸钠、4.053~4.063mM的乳酸钠以及0.340~0.350mM的柠檬酸钠。
优选的,所述胚胎基础培养液所含氨基酸组分具体包括0.080~0.090mM的丙氨酸、0.190~0.199mM的精氨酸、0.120~0.129mM的天冬酰胺、0.100~0.112mM的天冬氨酸、0.120~0.132mM的胱氨酸、0.090~0.100mM的谷氨酸、0.680~0.689mM的谷氨酰胺、3.000~3.035mM的甘氨酸、0.050~0.055mM的组氨酸、0.020~0.031mM的羟脯氨酸、0.280~0.291mM的异亮氨酸、0.440~0.451mM的亮氨酸、0.260~0.273mM的赖氨酸、0.110~0.121mM的蛋氨酸、0.130~0.139mM的苯丙氨酸、0.100~0.112mM的脯氨酸、0.150~0.156mM的丝氨酸、0.260~0.272mM的苏氨酸、0.050~0.059mM的色氨酸、0.130~0.139mM的酪氨酸以及0.260~0.270mM的缬氨酸。
优选的,所述胚胎基础培养液所含葡萄糖的浓度为2.770~2.780mM,胚胎基础培养液所含血清白蛋白的浓度为60~80g/L。
优选的,所述输卵管源外泌体是从动物(例如,兔子)输卵管液中提取得到的。
一种胚胎培养方法,包括以下步骤:
1)收集动物(例如,兔子)输卵管液,从所述输卵管液中提取输卵管源外泌体,将输卵管源外泌体添加到上述胚胎基础培养液中,得到含有输卵管源外泌体的胚胎培养液,所述含有输卵管源外泌体的胚胎培养液中输卵管源外泌体的浓度为6.0~7.0×108个/mL;
2)将动物(例如,兔子)胚胎移入所述含有输卵管源外泌体的胚胎培养液中,从而在模拟体内胚胎发育的仿生体液微环境中完成培养。
优选的,所述动物输卵管液的收集采用平衡盐溶液冲出的方式,具体包括以下步骤:对雌性动物供体进行超数排卵处理后与雄性动物供体交配,或者直接将雌性动物供体与雄性动物供体交配;交配20~24h后,麻醉雌性动物供体(例如,受孕兔子),从雌性动物供体子宫输卵管结合处注入平衡盐溶液,交配形成的受精卵随之冲出(自输卵管伞部冲出),收集冲出液,将冲出液静置后吸取上清(下部沉淀中含有受精卵),得动物输卵管液-平衡盐溶液的混合液。
优选的,所述平衡盐溶液(例如,DPBS)的用量为2~5mL(每侧输卵管)。
优选的,所述输卵管源外泌体的提取采用超速离心法,具体包括以下步骤:将上述动物输卵管液-平衡盐溶液的混合液在2~4℃、300~400g条件下离心10~15min,以去除大的杂物,得到上清液A;将离心所得上清液A在2~4℃、2000~2100g条件下离心20~25min,以去除死细胞,得到上清液B;将离心所得上清液B在2~4℃、10000~11000g条件下离心30~35min,以去除细胞碎片等杂物,得到上清液C;将离心所得上清液C在2~4℃、100000~110000g条件下超速离心2~2.5小时,超速离心所得沉淀即为提取得到的输卵管源外泌体。
优选的,所述步骤2)具体包括以下步骤:将含有输卵管源外泌体的胚胎培养液制作微滴并覆盖石蜡油,然后置于38.5℃、CO2体积分数为5%及完全相对湿度的培养箱中进行平衡以用于胚胎培养,将动物胚胎移入经过平衡后的含有输卵管源外泌体的胚胎培养液的微滴中,并继续置于所述培养箱中进行培养。
优选的,所述步骤2)中,动物胚胎选自动物(例如,兔子)的受精胚(例如,受精卵至囊胚期之前的任意时期的胚胎)。
本发明具有以下有益的技术效果:
本发明通过添加输卵管源外泌体,使得胚胎在体外培养环境中获得了体内输卵管调控因子的作用,发现其有利于胚胎在体外环境中的发育,提高胚胎的体外培养效率。
进一步的,所述胚胎基础培养液从模拟动物输卵管液的理化特征角度出发进行组分优化,从而确定无机盐组分、有机盐组分、氨基酸组分等的组成,结合添加的输卵管源外泌体,模拟受精胚体内发育微环境,更好的促进胚胎的体外发育。同时,解决了动物自身输卵管液有限、不适宜大量制取培养液(具体指基础培养液)的不足,从而使本发明更加适应工业化体外胚胎培养需求。
附图说明
图1为输卵管源外泌体(图中箭头所指为外泌体)透射电子显微镜图。
具体实施方式
下面结合附图和实施例对本发明做详细说明,所述实施例仅用于解释本发明,而不是对本发明保护范围的限制。
输卵管液中存在大量外泌体,输卵管源外泌体内含有RNA、蛋白质、脂类以及其它物质,这些物质使得输卵管源外泌体在胚胎和母体环境信号通讯中发挥作用。本发明通过提取输卵管源外泌体并利用含有该外泌体的胚胎培养液进行胚胎培养,提高了胚胎体外培养效率。
1.试剂的来源和制备
注射用垂体促卵泡素(PMSG)及注射用绒促性素(HCG)均购自宁波市三生药业有限公司。灭菌注射用水购自芮城县维尔富兽药有限公司。戊巴比妥钠购自Merck公司。DPBS购自Hyclone公司。
胚胎培养液(不含外泌体,记为A液)的配方,由以下(1)、(2)、(3)及(4)中的组分构成,采用纯化水配制,作为基础培养液:
(1)CaCl2·2H2O 1.780mM、MgSO4·7H2O 1.510mM、KCl 7.160mM、KH2PO4 1.190mM、NaHCO3 25.000mM,以及NaCl 107.590mM;
(2)丙酮酸钠(C3H3NaO3)0.393mM、乳酸钠(C3H5NaO3)4.053mM,以及柠檬酸钠(C6H5Na3O7·2H2O)0.340mM;
(3)丙氨酸0.080mM、精氨酸0.190mM、天冬酰胺0.120mM、天冬氨酸0.100mM、胱氨酸0.120mM、谷氨酸0.090mM、谷氨酰胺0.680mM、甘氨酸3.000mM、组氨酸0.050mM、羟脯氨酸0.020mM、异亮氨酸0.280mM、亮氨酸0.440mM、赖氨酸0.260mM、蛋氨酸0.110mM、苯丙氨酸0.130mM、脯氨酸0.100mM、丝氨酸0.150mM、苏氨酸0.260mM、色氨酸0.050mM、酪氨酸0.130mM,以及缬氨酸0.260mM;
(4)葡萄糖2.770mM,以及牛血清白蛋白60g/L。
2.用含有输卵管源外泌体的胚胎培养液培养兔胚胎
2.1超排程序
供体兔的制备:选用新西兰雌兔(西安交通大学实验动物中心)12只,6月龄,单笼饲养,自然采光,自由饮水采食。颈部皮下注射PMSG100IU/只。第四天中午将HCG用灭菌注射用水配制成500U/mL,当天12点半给前三天注射过PMSG的8只供体雌兔耳缘静脉注射HCG,0.2mL/只(100U/只)。选用6~8月龄生殖性能良好的雄兔(西安交通大学实验动物中心)8~10只,8只供体雌兔注射HCG后,按照1:1的比例与雄兔合笼交配。待第五天采集受精卵。
2.2兔胚胎的收集
取受精卵前用2mL注射器两支各吸取约2.5mL DPBS,置于37℃培养箱内预热,待冲卵用。在35mm*10mm的细胞培养皿内做体积200μL的胚胎培养液(A液)微滴,然后用胚胎级的矿物油覆盖微滴。供体雌兔交配20~24h后用3%戊巴比妥钠静脉注射过量麻醉后固定在解剖台上,腹面朝上,用75%乙醇喷洒兔子腹部尽量减少毛发的污染。用高压灭菌器械解剖切开腹腔,移开盘绕的肠子,找到子宫、输卵管和卵巢。用镊子提起输卵管、卵巢的脂肪垫,用小剪刀剥离使输卵管和卵巢与体腔壁分离,并剪开输卵管回折部分脂肪,使伞端暴露,伞端部分应尽量少带脂肪组织。将输卵管伞部及部分输卵管插入一次性无菌15mL离心管内接取冲出液。用预热过的DPBS从子宫输卵管结合处将胚胎依次从双侧输卵管伞部冲出。将收集冲卵液的离心管静置1分钟使受精卵沉淀至底部。用1mL移液枪缓缓吸出冲卵液的上清液至剩余约0.5mL液体,移液枪吹打使受精卵脱去卵丘颗粒细胞,重新悬浮后吸出,以液滴形式转移至无菌的培养皿内,在体视显微镜下,挑选出受精卵,并在DPBS液滴中清洗3~4次,使受精卵不含其它组织块或卵丘细胞。将挑选的420枚受精卵移入胚胎培养液(A液,提前平衡2小时)中进行短暂保藏(3小时,期间提取外泌体),培养环境为38.5℃、体积分数为5%的CO2、完全相对湿度的培养箱。
2.3输卵管源外泌体的提取
将吸取的冲卵液的上清液收集于一干燥无菌的离心管,在4℃条件下,300g离心10min以去除大的杂物,上清液转移至另一干燥无菌的离心管;而后在4℃条件下,2000g离心20min以去除死细胞,上清液转移至另一干燥无菌的离心管;而后在4℃条件下,10000g离心30min以去除细胞碎片等杂物,上清液转移至一干燥无菌的超速离心管;而后在4℃条件下,100000g超速离心2小时,小心取出超速离心管并标记沉淀所在位置,移除上清,取20μL胚胎培养液(A液)吹打重悬管壁沉淀,得悬液,置于4℃保存。
2.4输卵管源外泌体的鉴定
取1μL步骤2.3所得的悬液,滴于2mm的含碳载样铜网上,室温静置5min,用滤纸将多余液体轻轻吸去,用3%(质量浓度)磷钨酸钠溶液负染5min,室温晾干,透射电镜观察并拍照。结果见图1,外泌体的形态为杯状囊泡结构,直径大小为50~200nm。
2.5胚胎培养
经过鉴定,将输卵管源外泌体悬液(即剩余的19μL步骤2.3所得的悬液)加入胚胎培养液(A液,160μL),得到含有输卵管源外泌体的胚胎培养液(记为B液,输卵管源外泌体浓度为6.0~7.0×108个/mL),用相应胚胎培养液各制作4个40μL培养微滴并覆盖石蜡油,置于培养箱中(平衡2小时),用于胚胎培养。将获取的420枚受精卵,分为3组进行培养试验,培养环境为38.5℃、体积分数为5%的CO2、完全相对湿度的培养箱。空白对照组(胚胎培养液为A液)141枚,添加对照组(胚胎培养液为A液+上述超速离心后移除的上清液)138枚,添加外泌体组(胚胎培养液为B液)141枚。每组又分为4个小组,空白对照组4个培养微滴中培养胚胎数目为34枚、34枚、35枚、38枚,添加对照组4个培养微滴中培养胚胎数目为34枚、34枚、35枚、35枚。添加外泌体组的4个培养微滴中培养胚胎数目为34枚、34枚、35枚、38枚。
2.6结果记录与分析
体外培养一天后,统计卵裂率;第4天统计囊胚发育率,用卡方检验进行统计学分析。将囊胚用免疫染色液固定,并用凋亡试剂盒进行凋亡细胞染色,然后在荧光显微镜下观察,统计细胞凋亡数,统计囊胚细胞总数,采用单因素方差分析进行统计学分析。参见表1,3组之间的卵裂率并无显著差异,空白对照组和添加对照组的囊胚率、囊胚细胞凋亡率和囊胚细胞总数并无显著差异。添加外泌体组的囊胚率、囊胚细胞凋亡率和囊胚细胞总数显著高于空白对照组和添加对照组。结果表明,添加输卵管源外泌体能够显著提高胚胎体外囊胚发育率。
表1.添加输卵管液外泌体对胚胎发育率的影响
*代表差异显著(P<0.05)。
3.结果讨论
相比于体内环境,体外胚胎培养液缺失输卵管液是导致胚胎体外发育异常的重要原因,根据胚胎体外发育率、囊胚细胞凋亡率和囊胚总细胞数,表明外泌体中含有调控胚胎发育的重要调控因子,输卵管源外泌体作为胚胎体外培养液的添加物具有非常重要的作用。本发明对兔以及其他物种辅助生殖成功率的提高具有重要的借鉴作用,可促进动物(特别是兔)繁殖育种生产效率,也为开展基因修饰和克隆动物的研究提供了一定的参考。
Claims (10)
1.一种胚胎培养液,其特征在于:该胚胎培养液包括胚胎基础培养液以及输卵管源外泌体,所述胚胎培养液中输卵管源外泌体的浓度为6.0~7.0×108个/mL。
2.根据权利要求1所述一种胚胎培养液,其特征在于:所述胚胎基础培养液包括用于模拟动物输卵管液的理化特征的组分。
3.根据权利要求1或2所述一种胚胎培养液,其特征在于:所述胚胎培养液包括以下用于构成所述胚胎基础培养液的组分:无机盐组分、有机盐组分、氨基酸组分、葡萄糖及血清白蛋白。
4.根据权利要求3所述一种胚胎培养液,其特征在于:所述无机盐组分具体包括1.780~1.790mM的CaCl2·2H2O、1.510~1.520mM的MgSO4·7H2O、7.160~7.170mM的KCl、1.190~1.200mM的KH2PO4、25.000~25.010mM的NaHCO3以及107.590~107.690mM的NaCl。
5.根据权利要求3所述一种胚胎培养液,其特征在于:所述有机盐组分具体包括0.393~0.403mM的丙酮酸钠、4.053~4.063mM的乳酸钠以及0.340~0.350mM的柠檬酸钠。
6.根据权利要求3所述一种胚胎培养液,其特征在于:所述氨基酸组分具体包括0.080~0.090mM的丙氨酸、0.190~0.199mM的精氨酸、0.120~0.129mM的天冬酰胺、0.100~0.112mM的天冬氨酸、0.120~0.132mM的胱氨酸、0.090~0.100mM的谷氨酸、0.680~0.689mM的谷氨酰胺、3.000~3.035mM的甘氨酸、0.050~0.055mM的组氨酸、0.020~0.031mM的羟脯氨酸、0.280~0.291mM的异亮氨酸、0.440~0.451mM的亮氨酸、0.260~0.273mM的赖氨酸、0.110~0.121mM的蛋氨酸、0.130~0.139mM的苯丙氨酸、0.100~0.112mM的脯氨酸、0.150~0.156mM的丝氨酸、0.260~0.272mM的苏氨酸、0.050~0.059mM的色氨酸、0.130~0.139mM的酪氨酸以及0.260~0.270mM的缬氨酸。
7.根据权利要求3所述一种胚胎培养液,其特征在于:所述葡萄糖的浓度为2.770~2.780mM,血清白蛋白的浓度为60~80g/L。
8.根据权利要求1所述一种胚胎培养液,其特征在于:所述输卵管源外泌体是从动物输卵管液中提取得到的。
9.一种胚胎培养方法,其特征在于:包括以下步骤:
1)收集动物输卵管液,从所述输卵管液中提取输卵管源外泌体,将输卵管源外泌体添加到胚胎基础培养液中,得到含有输卵管源外泌体的胚胎培养液,所述胚胎培养液中输卵管源外泌体的浓度为6.0~7.0×108个/mL;
2)将动物胚胎移入含有输卵管源外泌体的胚胎培养液中进行培养。
10.根据权利要求9所述一种胚胎培养方法,其特征在于:所述步骤1)中,动物输卵管液的收集采用平衡盐溶液冲出的方式,所述输卵管源外泌体采用超速离心法提取;所述步骤2)中,动物胚胎选自动物的受精胚。
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