摘要
中药是我国特有的资源,中药中的有效成分在动物繁殖中治疗不孕症、流产、保胎及促进精子活力等方面作用突出,还具有抗过氧化效应、清除自由基、促进卵母细胞成熟和细胞增殖等作用。共培养是近10多年来在胚胎培养方面最引人注目的变革之一。共培养能改善胚胎的形态、促进卵裂并有较高的细胞数、提高囊胚的形成率及孵化率。目前,胚胎体外生产技术的累积效率还很低,卵母细胞的存活力与异常卵母细胞的产生也是制约胚胎体外生产的瓶颈之一。本研究旨在探讨一种稳定的适合小鼠子宫内膜上皮细胞培养的方法,筛选能促进体外受精及胚胎体外发育的中药添加剂,并探讨其合适的添加时间,从而构建一种理想的培养体系,以提高胚胎体外生产的累积效率。
本试验为了研究适宜的中药培养液对小鼠体外受精及胚胎发育的影响,进行了三个阶段试验研究:第一阶段是筛选了适合于小鼠子宫内膜上皮细胞的培养方法,获得了高纯度子宫内膜上皮细胞;第二阶段在小鼠胚胎不同培养阶段在培养液中分别加入三种中药添加剂,观察其对体外受精及胚胎体外发育的影响,筛选适宜的中药培养液并确定其合适的添加时间;第三阶段结合前两项试验结果,构建新的共培养体系,观察小鼠体外受精胚胎体外发育的效果。试验结果表明:
(1)采用酶消化法可以获得小鼠子宫内膜上皮细胞,但酶会影响细胞活力,致使细胞增殖缓慢,且酶消化法获得的上皮细胞纯度不高;采用子宫翻转组织块培养法培养小鼠子宫内膜上皮细胞,细胞活力高,增殖速度快,可以获得较好的原代培养效果。该方法为下一步研究胚胎共培养提供了可靠的体细胞来源。
(2)经筛选试验表明,0.5mg/L川芎嗪与4mg/L黄芩苷对小鼠体外受精及早期胚胎发育的作用并不十分显著(P>0.05),且会延缓2-细胞阶段以后胚胎体外发育的速度; 0.1mg/L小檗碱在体外受精过程中能显著提高受精卵存活率(49.5%对照组7.5%,P<0.05),但会严重阻碍受精卵后续阶段的胚胎培养(P<0.05)。
(3)共培养体系能显著降低2-细胞发育阻滞率(46.2%对照组34.2%,P<0.05)。
Traditional Chinese drug is our country’s endemic resource. Active component’s combination in traditional Chinese drug have special effect on treating barrenness, abortion, tocolysis and promoting motility of sperm in animal breeding. And it also have the effect of Anti-lipid peroxidation, cleaning up free radical, promoting oocyte maturation, cell proliferation. Co-culture is a new cultural method in vitro.It can prolong the culture time of embryo in vitro.Accum efficiency of embryo producting in votro is low.The neck of restricting is the longevity of oocyte and abnor oocyte.The purpose of this study is to elevating the accum efficiency through approaching a safe method which can culture mouse endometrium epithelial cell,then approaching a traditional Chinese drug which is good for fertilization and embryo culture in vitro,and searching the right time of admoveaturing,at last estabilishing a reasonable sequential co-culture system.
To studying the effect of right Traditional Chinese drug on fertilization and embryo culture in vitro.The first stage is to approaching a safe method which can culture mouse endometrium epithelial cell,and getting purity endometrium epithelial cell.The second stage is to approaching a traditional Chinese drug which is good for fertilization and embryo culture in vitro,and searching the right time of admoveaturing through observing the effect of three traditional Chinese drug admoveaturing on different time on fertilization and embryo culture in vitro.The last stage is to integrating the result before, and observing the effect of new sequential co-culture system on fertilization and embryo culture in vitro.
The results indicate that:
(1) The mouse endometrium epithelial cells could be isolated by using enzyme digestion,But the purity ,the vigor and the proliferation velocity of cell was influenced by the enzyme. At the same time, the purity ,the vigor and the proliferation velocity of cell was not influenced by using the method of turnover uterus tissue.This method was used for providing somatocyte to studying sequential co-culture.
(2) The effect of 0.5mg/L ligustrazine and 4mg/L baicalin on fertilization and embryo culture in vitro was not significant(P>0.05),but they could delay the development of embryo after 2-cell stage. 0.1mg/L berberine could protect oocyte on the process of fertilization and make the rate of oocyte be significantly elevated(49.5% control group 7.5%).but it was bad for culturing of fertilized ovum (P<0.05).
(3) The rate of 2-cell development arrest of embryo which cultured in co-culture system is lower than the control group(P<0.05).
引文
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