CN113736728A - 一种小鼠体细胞核移植胚胎培养液及胚胎培养方法 - Google Patents
一种小鼠体细胞核移植胚胎培养液及胚胎培养方法 Download PDFInfo
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- CN113736728A CN113736728A CN202110962301.2A CN202110962301A CN113736728A CN 113736728 A CN113736728 A CN 113736728A CN 202110962301 A CN202110962301 A CN 202110962301A CN 113736728 A CN113736728 A CN 113736728A
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Abstract
本发明公开了一种小鼠体细胞核移植胚胎培养液及胚胎培养方法,属于生物技术领域,小鼠体细胞核移植胚胎培养液包括以下成分:NaB、乙二胺四乙酸二钠、氯化钠、氯化钾、磷酸二氢钾、七水硫酸镁、葡萄糖、碳酸氢钠、谷氨酰胺、丙酮酸钠、青霉素、链霉素、氯化钙、牛血清白蛋白、乳酸钠、必需氨基酸、非必需氨基酸、酚红和胚胎水。利用本发明的小鼠体细胞核移植胚胎培养液及培养方法,可以将小鼠胎儿成纤维细胞重构胚的卵裂率56.05%提高到68.28%(P<0.001)、囊胚率自11.07%提高到49.38%(P<0.001),体细胞核移植小鼠的出生率自0.23%提高到1.24%(P<0.01),在现有技术的基础上,显著改善了小鼠的胚胎2‑细胞阻滞情况,提高了小鼠体细胞核移植的效率。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及一种小鼠体细胞核移植胚胎培养液及胚胎培养方法。
背景技术
体细胞核移植是指通过显微操作技术将供体细胞的细胞核注入到去核的卵母细胞中,形成重构胚,最终发育成新个体的技术。自从1997年,体细胞核移植技术在绵羊上取得巨大的成功,在随后的几年时间,先后有十几种核移植动物成功出生。核移植技术的成功说明动物终末分化的体细胞基因组是有全能性的。在这个过程中,供体细胞基因组在受体细胞的转录因子和酶的作用下进行了重编程,去除分化细胞特异性表达的转录本和转录因子,重新建立胚胎细胞特异的DNA甲基化修饰和组蛋白修饰,并表达胚胎特异的转录因子,激活维持胚胎正常发育的基因的表达。探究体细胞核移植的机制有利于我们提高核移植效率,并进一步为医学应用做好准备。
体细胞核移植产生的胚胎可以正常发育并产生具有繁殖能力的个体,但是大部分动物的体细胞核移植动物的效率仍然很低,已经明确小鼠的核移植植入前胚胎会发生2-细胞阻滞,出生率极低。除此之外,很多体细胞克隆动物表现为发育异常,如胎盘巨大,出生缺陷,体质肥胖,过早死亡等。体细胞核移植的效率低下及发育异常是其后续应用的一个最主要的障碍。近些年来,大量研究人员通过各种方法试图提高核移植的效率,包括改进重构胚激活的方法,调整激活时间,缩短体外培养的时间等等,但仍然没有突破性的进展。
因此,提供一种提高体细胞核移植效率的产品或方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种小鼠体细胞核移植胚胎培养液,显著提高了小鼠胎儿成纤维细胞重构胚的卵裂率、囊胚率以及体细胞核移植小鼠的出生率。
发明思路:
由于体细胞高度分化,恢复全能性困难,体细胞核移植是唯一能够使已分化的细胞基因组具有全能性或多能性的再生生物学技术。体细胞核移植作为动物细胞工程技术的常用技术手段,其开发和利用对再生医学有重要意义。然而,体细胞核移植效率极低限制了此技术的实际应用,并在植入前胚胎发育过程中表现出ZGA阶段阻滞的现象。重构胚的体外培养作为核移植技术中不可缺少的一步,培养液的成分及环境对于重构胚的卵裂率、囊胚率及囊胚的质量至关重要,最终影响核移植动物的出生率。近些年来,大量学者对重构胚的体外培养条件进行改善,以期提高核移植重构胚的发育率。但目前为止,不同文献中报道的改进胚胎体外培养条件的方法不同,仍没有很好的培养体系更好地适用于小鼠胎儿成纤维重构胚的体外培养。因此,筛选出一套高效的体外胚胎培养系统十分必要。
为了实现上述目的,本发明采用如下技术方案:
一种小鼠体细胞核移植胚胎培养液,包括以下成分:丁酸钠(即NaB)、乙二胺四乙酸二钠、氯化钠、氯化钾、磷酸二氢钾、七水硫酸镁、葡萄糖、碳酸氢钠、谷氨酰胺、丙酮酸钠、青霉素、链霉素、氯化钙、牛血清白蛋白、乳酸钠、必需氨基酸、非必需氨基酸、酚红和胚胎水。
作为本发明优选的技术方案,所述小鼠体细胞核移植胚胎培养液每百毫升包括以下成分:0.01-0.03mmol丁酸钠、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸、0.1mL1%酚红和余量胚胎水。
更优选的,每百毫升小鼠体细胞核移植胚胎培养液含NaB 0.02mmol。
本发明通过试验证实,本发明的小鼠体细胞核移植胚胎培养液可以将小鼠胎儿成纤维细胞重构胚的卵裂率56.05%提高到68.28%(P<0.001)、囊胚率自11.07%提高到49.38%(P<0.001),体细胞核移植小鼠的出生率自0.23%提高到1.24%(P<0.01)。
本发明的另一目的是,提供上述小鼠体细胞核移植胚胎培养液的制备方法,先用胚胎水将除NaB以外的其他成分溶解,通过0.22μm的滤器除菌,再添加NaB,溶解均匀后,分装,-80℃保存。
本发明的再一目的是,提供一种小鼠体细胞核移植胚胎培养方法,体细胞核移植过程中,将重构胚的体外培养在上述的小鼠体细胞核移植胚胎培养液中进行。
作为本发明优选的技术方案,小鼠体细胞核移植的胚胎培养方法,包括以下具体步骤:
S1:供体细胞和受体细胞的制备;
S2:体细胞核移植操作;
S3:胚胎移植
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种小鼠体细胞核移植胚胎培养液及胚胎培养方法。利用本发明的小鼠体细胞核移植胚胎培养液及培养方法,可以将小鼠胎儿成纤维细胞重构胚的卵裂率56.05%提高到68.28%(P<0.001)、囊胚率自11.07%提高到49.38%(P<0.001),体细胞核移植小鼠的出生率自0.23%提高到1.24%(P<0.01),在现有技术的基础上,显著改善了小鼠的胚胎2-细胞阻滞情况,提高了小鼠体细胞核移植的效率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例2不同胚胎培养液中小鼠重构胚卵裂率结果示意图,其中横坐标0代表对比例1,0.1代表对比例2,0.2代表实施例1,0.3代表对比例3,0.4代表对比例4,*代表P<0.05差异显著,**代表P<0.01差异极显著;
图2附图为本发明实施例3不同胚胎培养液中体细胞核移植小鼠出生率结果示意图,其中横坐标0代表对比例1,0.2代表实施例1,**代表P<0.01差异极显著。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例公开了一种小鼠体细胞核移植胚胎培养液及小鼠体细胞核移植方法。所涉及试剂均为市售可得,例如必需氨基酸购自Gibco 11130-051、非必需氨基酸购自Gibco 11140-050,胚胎水购自Sigma W1503;所涉及试验方法如无特别提及,均为常规方法。
实施例1
小鼠体细胞核移植胚胎培养液的制备
准确称量取0.02mmol NaB、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解,1M盐酸或1M氢氧化钠调节pH至7.2-7.4,用胚胎水定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。标记为0.2mM NaB-KSOM-AA胚胎培养液。
对比例1
准确称量取0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解,1M盐酸或1M氢氧化钠调节pH至7.2-7.4,用胚胎水定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。标记为KSOM-AA胚胎培养液。
对比例2
准确称量取0.01mmol NaB、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解,1M盐酸或1M氢氧化钠调节pH至7.2-7.4,用胚胎水定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。标记为0.1mM NaB-KSOM-AA胚胎培养液。
对比例3
准确称量取0.03mmol NaB、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解,1M盐酸或1M氢氧化钠调节pH至7.2-7.4,用胚胎水定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。标记为0.3mM NaB-KSOM-AA胚胎培养液。
对比例4
准确称量取0.04mmol NaB、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解,1M盐酸或1M氢氧化钠调节pH至7.2-7.4,用胚胎水定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。标记为0.4mM NaB-KSOM-AA胚胎培养液。
实施例2
(考量实施例1及对比例1-4胚胎培养液中小鼠体细胞核移植重构胚的卵裂率)
1、试验材料
试验动物:
试验选择B6D2F1雌鼠提供稳定的卵母细胞;选择C57雌鼠和DBA雄鼠的后代B6D2F1胎儿提供成纤维细胞,以上小鼠均购自内蒙古大学实验动物研究中心SPF级实验动物繁育室。所进行的动物实验完全符合内蒙古大学实验动物研究中心《实验动物管理与使用操作规范》。鼠房温度22-24℃,湿度50-60%,日光灯进行光照控制,每天光照12h,光照时间为8:00-20:00。
试验试剂:
孕马血清促性腺激素(PMSG):用生理盐水(0.9%氯化钠溶液)溶解PMSG干粉至终浓度为100IU/mL,分装成1mL每管储存于-20℃,备用。
人绒毛膜促性腺激素(HCG):用生理盐水(0.9%氯化钠溶液)溶解HCG干粉至终浓度为100IU/mL,分装成1mL每管储存于-20℃,备用。
氯化锶(20×)浓储液:由2.666g六水氯化锶和50mL胚胎水配制而成,储存于-20℃,备用;
细胞松弛素B(100×)浓储液:由细胞松弛素B以1mg/mL的配比溶于二甲基亚砜溶液中配制而成,分装成2μL每管储存于-20℃,备用;
透明质酸酶(10×)浓储液:将100mg透明质酸酶溶解于20mL M2胚胎操作液中配制而成,分装成0.1mL每管储存于-20℃,备用。
M2胚胎操作液(pH=7.2-7.4):用胚胎水将0.545g氯化钠、0.036g氯化钾、0.016g磷酸二氢钾、0.029g七水硫酸镁、0.035g碳酸氢钠、0.004g丙酮酸钠、0.1g葡萄糖、0.497g4-羟乙基哌嗪乙磺酸、0.025g二水氯化钙、0.006g青霉素、0.005g链霉素、0.15mL1%酚红、0.4g牛血清白蛋白和0.35mL乳酸钠溶解并定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到15mL的离心管中,-20℃保存。
聚乙烯吡咯烷酮溶液(PVP):由1g PVP和10mL M2胚胎操作液配制成10%PVP溶液。由0.3g PVP和10mL M2胚胎操作液配制成3%PVP溶液。
无钙KSOM-AA胚胎培养液(pH=7.2-7.4):准确称量取0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸和0.1mL1%酚红,用胚胎水溶解并定容至100mL。配制完的液体用0.22μm的滤器过滤,分装到1.5mL的离心管中,-80℃保存。
试验器材:
Corning塑料培养皿、充油型注射仪、带有Nomarski或Hoffman光学系统的倒置显微镜、拉针仪、锻针仪、显微操作用针、压电陶瓷系统、水银。
2、小鼠体细胞核移植胚胎培养方法
S1:供体细胞和受体细胞的制备
①供体细胞的制备:
胎儿成纤维细胞的分离:将C57雌鼠进行超数排卵处理(皮下注射0.1mL(10IU)的PMSG,48h后腹腔注射0.1mL(10IU)的HCG)后与DBA雄鼠合笼,次日查阴道栓,见栓的记为0.5d。13.5d时解剖怀孕雌鼠,取出胎儿。剥离胎盘后,将胎儿浸泡在75%酒精中消毒30s,再用加有2%Pen Strep(Gibio,15140-122;下同)的PBS清洗3-5遍。用剪刀将胎儿的头部,尾巴、四肢和内脏去除,用加有2%Pen Strep的PBS清洗3-5遍,将剩余组织剪碎,转移到15mL离心管中,加入1mL0.05%胰酶,放于培养箱中消化。30min后,加入2mL的DMEM+10%FBS培养液终止消化,1500rpm离心5min,弃上清后,加入DMEM+10%FBS培养液吹匀,将消化出来的细胞转移到培养皿中,于37℃,5%CO2培养箱中培养。
胎儿成纤维细胞的冻存:次日观察分离的胎儿成纤维细胞,并于分离后的2d对细胞进行换液处理,换成新鲜的DMEM+10%FBS培养液继续培养,5d后待细胞长满培养皿后,对细胞进行冻存,冻存液为DMEM:FBS:DMSO=7:2:1。
胎儿成纤维细胞的准备:于核移植前两天解冻冻存的小鼠胎儿成纤维细胞,并于核移植前一晚换成无血清培养液,使成纤维细胞处于细胞间期。次日,消化细胞,用质量分数为3%的PVP溶液重悬细胞沉淀,放置于冰上保存,备用。
②受体细胞的制备:
取8-12周的B6D2F1雌鼠,皮下注射0.1mL(10IU)的PMSG,48h后腹腔注射0.1mL(10IU)的HCG。14-16h后脱颈处死小鼠,酒精消毒后用已灭菌的剪刀剪开腹部的皮肤,用眼科镊夹住子宫,剪刀剪下输卵管,放于干净的M2操作液滴中。在体视显微镜下找到输卵管上膨大的壶腹部,用注射器针头将其挑破,涌出的团状物质即为卵丘卵母细胞复合体。将卵丘卵母细胞复合体转移到加有5μL透明质酸酶的50μLM2胚胎操作液中,37℃消化5min左右,直至卵母细胞周围包裹的卵丘细胞消化完全,用口吸管迅速将去除卵丘的卵母细胞转移至干净的M2胚胎操作液液滴中,清洗3-5遍,将卵母细胞转移至平衡好的KSOM-AA胚胎培养液滴中,清洗3-5遍,放入37℃,5%CO2培养箱,备用。
S2:体细胞核移植操作
①准备显微操作用的操作滴:
准备100mm的培养皿的皿盖,将1μL细胞松弛素B(100×)浓储液加入到99μL的M2胚胎操作液中,混匀,作为去核和注核的操作滴。做质量分数为3%的PVP溶液滴,后期用于放供体细胞,做质量分数为10%的PVP溶液滴作为洗针液滴。上层覆盖石蜡油,放于37℃预热,备用。
②MII期卵母细胞的去核:
挑选已排出第一极体且形态规则、胞质均匀、透明带界限清晰、没有分裂碎片的MII期卵母细胞,用口吸管放到操作滴中,每组30-40枚。用持卵针将卵母细胞固定好,用去核针来回波动卵母细胞找到MⅡ期纺锤体,将纺锤体转到3点钟方向的位置,用去核针穿过透明带,吸出纺锤体,去核后的卵母细胞放于操作滴中静置10min后,移回KSOM-AA胚胎培养液中,放入37℃,5%CO2培养箱至少恢复30min,备用。
③供体核的注射:将去核的卵母细胞放入操作滴中,每组30-40枚。将前面准备好的胎儿成纤维细胞转移到3%的PVP液滴中,挑选生长状况和形态良好的供体细胞(胎儿成纤维细胞),用注射针吸取细胞,并不断地在针管内吹吸,使供体细胞的细胞质膜破裂,细胞核暴露出来。用持卵针将卵母细胞固定,注射针将供体细胞的细胞核注入到去核的卵母细胞中,形成重构胚,将重构胚放置在操作滴中缓冲10-15min后,移回KSOM-AA胚胎培养液液滴中,37℃,5%CO2培养箱中恢复培养至少30min。
④重构胚的激活:
取188μL的无钙KSOM-AA胚胎培养液,加入2μL的细胞松弛素B(100×)浓储液和10μL的氯化锶(20×)浓储液,混匀后,在35mm的培养皿中做滴,上层覆盖石蜡油,放入37℃,5%CO2培养箱中平衡至少1h,得激活液滴,备用。将恢复好的重构胚转移至激活液滴中,在激活液滴中清洗3-5遍后,放入37℃,5%CO2培养箱激活培养6h。
⑤重构胚的体外培养:
将重构胚转移至实施例1及对比例1-4的胚胎培养液中,放入37℃,5%CO2培养箱中培养15h,观察并计算各处理组重构胚的2-细胞比例,即卵裂率,
结果统计:
采用SPSS10.0统计学软件分析处理,结果如附图1所示,其中*代表P<0.05差异显著,**代表P<0.01差异极显著。由附图1数据可知,与对比例1不添加添加NaB的KSOM-AA胚胎培养液组的卵裂率54.21%相比,实施例1的0.2mM NaB-KSOM-AA胚胎培养液组卵裂率67.96%(差异极显著),对比例2的0.1mM NaB-KSOM-AA胚胎培养液组卵裂率59.32%(差异显著),对比例3的0.3mM NaB-KSOM-AA胚胎培养液组卵裂率60.59%(差异显著),对比例4的0.4mM NaB-KSOM-AA胚胎培养液组卵裂率53.35%(差异不显著)。
实施例3
(考量实施例1及对比例1胚胎培养液中小鼠体细胞核移植重构胚的卵裂率、囊胚率和小鼠出生率)
1、试验材料(同实施例2)
2、小鼠体细胞核移植胚胎培养方法
S1:供体细胞和受体细胞的制备(同实施例2)
S2:体细胞核移植操作(同实施例2)
S3:胚胎移植
A)结扎雄鼠的准备:
选取8周龄身体状况良好的CD1公鼠,腹腔注射麻药。用75%酒精对小鼠腹部进行消毒处理,向上轻推睾丸。在约与腿部平齐的地方,先用剪刀纵向剪开一个小口,再用剪刀缓慢撑开一个大口。用镊子夹住腹部皮肤,用钝头镊子伸向腹腔左侧夹出脂肪垫,找到睾丸下方的输精管,用眼科镊小心将其挑出,切勿碰到一旁的血管。用另一个镊子在酒精灯火焰上烧红后,迅速烧断输精管。然后小心将睾丸、脂肪等组织放回原位。同样,再将右侧的输精管烧断。两侧手术完毕后,用缝合针线分别将腹腔内外伤口缝合,并在伤口处洒一些青霉素粉末。将手术后的小鼠放于37℃热板上,待其麻药效力减弱至完全苏醒。将结扎后的雄鼠放回鼠房饲养,缓冲一周以上,备用。
B)假孕母鼠的准备:
选取6-8周龄处于发情期的CD1雌鼠,与结扎雄鼠1:1比例合笼,次日查栓,将见栓母鼠挑出,记为0.5d。
C)胚胎移植手术:
将胚胎采子宫移植的方式移植到假孕母鼠体内。具体操作方法如下:挑取见栓3.5d的母鼠,腹腔注射麻醉剂。将小鼠放于体式显微镜下,用75%酒精消毒小鼠背部皮肤,选取与腿平齐处背部皮肤,用灭菌剪刀在皮肤上纵向剪开一个小口,再用剪刀剪开肌肉层。用钝头镊子夹出腹腔左侧脂肪,并拖出输卵管和卵巢,用夹子固定脂肪组织。用注射器针头在子宫上扎一个眼。按照石蜡油-空气-KSOM-AA培养液-空气-胚胎的顺序依次装入移植针。将装好的移植针缓慢插入到子宫内,轻轻吹入胚胎。拔出移植针,小心将脂肪组织、卵巢、输卵管和子宫放回腹腔内。重复上述步骤,移植另一侧子宫。手术完成后,先缝合内部肌肉层,再缝合外部皮肤层。并在外部伤口处洒一些青霉素粉末。将小鼠放于37℃热板上,待其麻药效力减弱至完全苏醒后,放回鼠房。
结果统计:
该实施例重构胚在不同胚胎培养液中的卵裂率、囊胚率,以及小鼠出生率,采用SPSS10.0统计学软件对数据进行分析处理,其中重构胚在不同胚胎培养液中的卵裂率、囊胚率见表1,重构胚小鼠出生率见表2,P<0.05为差异显著,P<0.01为差异极显著,P<0.001为差异极其显著。
表1
表2
由表1结果可知,和对比例1 KSOM-AA胚胎培养液组的卵裂率56.05%相比较,胎儿成纤维重构胚在实施例10.2mM NaB-KSOM-AA胚胎培养液组的卵裂率显著提高,为68.28%(P<0.001);和对比例1 KSOM-AA胚胎培养液组的囊胚率11.07%相比较,胎儿成纤维重构胚在在实施例10.2mM NaB-KSOM-AA胚胎培养液组的囊胚率得到了显著地提升,为49.38%(P<0.001)。由表2结果可知,对比例1 KSOM-AA胚胎培养液组的重构胚小鼠出生率为0.23%,实施例10.2mM NaB-KSOM-AA胚胎培养液组的重构胚小鼠出生率得到了显著提高,为1.24%(P<0.01)。因此,本发明0.2mM NaB-KSOM-AA胚胎培养液作为一种小鼠体细胞核移植胚胎培养液和培养方法显著提高了小鼠胎儿成纤维细胞核移植重构胚的卵裂率、囊胚率和小鼠出生率。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.一种小鼠体细胞核移植胚胎培养液,其特征在于,包括以下成分:丁酸钠、乙二胺四乙酸二钠、氯化钠、氯化钾、磷酸二氢钾、七水硫酸镁、葡萄糖、碳酸氢钠、谷氨酰胺、丙酮酸钠、青霉素、链霉素、氯化钙、牛血清白蛋白、乳酸钠、必需氨基酸、非必需氨基酸、酚红和胚胎水。
2.根据权利要求1所述的小鼠体细胞核移植胚胎培养液,其特征在于,所述小鼠体细胞核移植胚胎培养液每百毫升包括以下成分:0.01-0.03mmol丁酸钠、0.38mg乙二胺四乙酸二钠、559.5mg氯化钠、18.5mg氯化钾、4.75mg磷酸二氢钾、4.95mg七水硫酸镁、3.6mg葡萄糖、210mg碳酸氢钠、14.5mg谷氨酰胺、2.2mg丙酮酸钠、6.3mg青霉素、5mg链霉素、25mg氯化钙、100mg牛血清白蛋白、0.174mL乳酸钠、1.0mL必需氨基酸、0.5mL非必需氨基酸、0.1mL 1%酚红和余量胚胎水。
3.根据权利要求1所述的小鼠体细胞核移植胚胎培养液,其特征在于,每百毫升小鼠体细胞核移植胚胎培养液含丁酸钠0.02mmol。
4.权利要求1-3任一所述小鼠体细胞核移植胚胎培养液的制备方法,其特征在于,先用胚胎水将除丁酸钠以外的其他成分溶解,通过0.22μm的滤器除菌,再添加丁酸钠,溶解均匀后,分装,-80℃保存。
5.一种小鼠体细胞核移植的胚胎培养方法,其特征在于,重构胚的体外培养在权利要求1-3任一所述的小鼠体细胞核移植胚胎培养液中进行。
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