肉苁蓉对猪体外培养胚胎早期发育影响效果的研究及其机理初步探讨

【作者】 杨志蓉；

【导师】 甘世祥； 冯书堂；

【作者基本信息】 贵阳中医学院 ， 中医基础理论， 2007， 硕士

【摘要】 为探讨肉苁蓉在对猪胚胎体外生长发育的影响效果,本研究以猪胚胎为实验材料,设计三个试验:肉苁蓉水提液对猪胚胎早期发育体外培养效果的的筛选试验;在不同时期添加肉苁蓉水提液对猪胚胎早期体外发育的影响;肉苁蓉水提液对猪早期体外发育胚胎中NO浓度含量的影响。以期为提高体外培养胚胎发育率和扩大中医药的应用范围开拓一条新的途径。试验一:以NCSU-23+0.4%BSA基础液为对照组,添加不同浓度肉苁蓉水提液为试验组,初步筛选出四个浓度后再进一步筛选出最佳浓度。试验结果:肉苁蓉水提液的最佳浓度为50μg/ml,其卵裂率和囊胚率分别为85.12±2.9%和31±3.0%,显著高于对照组的76.73±3.2%、24.89±4.3%(P<0.05),试验各组之间无显著性差异。结果表明:在基础培养液中添加肉苁蓉水提液能有效促进胚胎体外生长发育。试验二:胚胎在不同发育时期所需营养物质不同,为了能明确在胚胎发育的哪个时期加入肉苁蓉水提液效果更好,本试验在胚胎发育的三个时期加入肉苁蓉,比较其在何时期加入效果更好。试验结果:在胚胎培养的2-4cell期添加肉苁蓉水提液,其囊胚率达到48.72±6.6%,高于对照组囊胚率25.08±5.4%,两者之间有极显著差异(P<0.01)。而且,也比在体外成熟阶段(IVM)和胚胎培养(IVC)阶段加入的效果要好,二者之间差异显著(P<0.05)。结果表明:在胚胎培养的2-4cell期添加肉苁蓉能更有效的促进胚胎体外发育的能力。试验三:本试验基于对NO前期研究的基础上,对培养到囊胚阶段的胚胎中NO含量进行了测定,观察添加中药是否对胚胎NO含量产生影响。试验结果:经过体外培养到囊胚阶段,胚胎细胞中NO含量均较高,对照组和试验组分别为26.33±1.90和20.30±0.95。但添加肉苁蓉水提液后其胚胎细胞中NO含量明显降低,与对照组有显著性差异(P<0.05)。结果表明:肉苁蓉的水提液在一定程度上清除了胚胎在体外培养过程中所产生的自由基,优化了NO在细胞信号通路中的双相调节作用,从而减少了NO的生成,提高了胚胎体外发育率。以上结果说明:肉苁蓉水提液对猪胚胎体外生长发育的影响效果显著。更多还原

【Abstract】 The investigation studied the effect of Cistache deserticola Y.C,Ma using the embryos of pig as explant.Three experiments were set: the effect of Cistache deserticola Y.C,Ma of different concentration on embryos of pig development in vitro ; the effect of Cistache deserticola Y.C,Ma on the embryos early development of pig in different phases;the effect of Cistache deserticola Y.C about NO concentration on embryos early development of pig in vitro .It expect of deploitation a new approach on improve ratio of embryos development of pig in vitro and extend the application range of Chinese traditional medicine.Experiment 1 used the NCSU-23+0.4%BSA as control,supplemented with Cistache deserticola Y.C,Ma of different concentration as tests.After several replicated,4 concentrations were selected to further selection. The result showed that the best concentration of Cistache deserticola Y.C,Ma is 50μg/ml which cleavaged embryos and blastocyst were 85.12±2.9%、31±3.0% was much higher than the control(76.73±3.2%、24.89±4.3%)(P<0.05),there were no big difference about test groups(P>0.05). The NCSU-23+0.4%BSA supplemented with Cistache deserticola Y.C,Ma significant improved the development of embryos in vitro.Experiment 2 The embryos needed different nutritious substance in different phases .In order to definitude which phase was better than other phases when it access Cistache deserticola Y.C,Ma,the experiment used the NCSU-23+0.4%BSA as control,supplemented with Cistache deserticola Y.C,Ma of three phases of embryos development in vitro.The result showed that when supplemented with Cistache deserticola Y.C,Ma of two_four cell phase was best.it’s blastocyst(48.72±6.6%)was much Higher than the control(25.08±5.4%)(P<0.01),and it better than IVM and IVC(P<0.05).It is concluded that the NCSU-23+0.4%BSA supplemented with Cistache deserticola Y.C,Ma on two_four cell stage can effective promote the development of embryos in vitro.Experiment 3 The experiment was mensurated NO concentration of embryos until they were developmented at blastocyst stage. Observe the effect of Cistache deserticola Y.C.Ma on embryos of pig about NO concentration.The result showed that the embryos had much NO when they were cultured at blastocyst stage,the control and test were 26.33±1.90 and 20.30±0.95 respectively,but the supplemented with Cistache deserticola Y.C,Ma was lower than control(P<0.05).This indicate that basal culture medium supplemented with Cistache deserticola Y.C.Ma can eliminate free oxygen radical(FOR) which were produced by the embryos in vitro culture and it also can optimize bidirectional adjust effect of NO in cellular signal acess that enhance the ratio of development in vitro.更多还原