瘦素对小鼠着床前胚胎发育影响的体外研究

目的　探讨瘦素在体外对小鼠着床前胚胎发育的影响。方法　 1收集小鼠 2 -细胞胚胎 ,在不同剂量瘦素的 CZB培养液中进行体外培养 ,观察胚胎发育情况 ,并进行胚胎移植 ,观察着床率 ;2收集小鼠 2 -细胞胚胎在空白 CZB培养液中体外培养至桑葚胚期 ,再分别置入不同剂量瘦素的 CZB培养液中进行体外培养 ,观察胚胎进一步发育情况。结果　瘦素能提高 2 -细胞胚胎体外发育的质量、发育率和胚胎着床率。当瘦素剂量为 10 ng/ ml时平均胚胎形态学评分 (AES)为 16 .0 8± 0 .37,剂量为 5 0 ng/ ml时 AES为 17.5 7± 0 .4 2 ,两者间差别有统计学意义(P<0 .0 5 )。但剂量进一步增加 ,促进作用不再递增。瘦素对桑葚胚体外进一步发育无显著影响。结论　瘦素参与了小鼠着床前胚胎的发育 ,能提高胚胎发育质量、发育率 ,有利于胚胎的进一步着床。     

IVF-ET周期中单原核胚胎的形态学及体外发育潜能

摘 要: 【目的】 探讨IVF-ET周期中单原核(1PN)胚胎形态学及继续体外培养潜能,指导其临床利用价值?【方法】 对受精后16 ~ 18 h观察到的486个1PN胚胎进行体外培养,观察其原核大小?极体及卵裂情况;通过囊胚序贯培养法将其中374个1PN胚胎培养至囊胚期,观察囊胚形成情况?【结果】 374个1PN胚胎共形成囊胚179个(47.86%),其中优质囊胚41个(22.91%)?行囊胚培养的374个1PN胚胎原核直径为(27.64 ± 3.30)μm,大于未达囊胚培养标准的81个胚胎的(26.22 ± 3.38)μm,以及对照组2PN胚胎(24.08 ± 1.85)μm(P 0.05)?IVF来源的1PN胚胎原核直径为(27.80 ± 3.44)μm大于ICSI组(26.77 ± 2.29)μm(P 0.05)均高于后者?不同极体数目(不确定?2PB?1PB)的1PN胚胎间原核大小无统计学差异(P > 0.05),但其优质囊胚率有统计学差异(39.39%,20.00%,17.65%;P < 0.05)?D3评分级别越优?卵裂球数目越多者,其囊胚形成率(P < 0.05)和优质囊胚率(P < 0.05)越高?【结论】 对于IVF来源的1PN(2PB)胚胎,可通过对其形态学和胚胎发育潜能进行初步筛选,冷冻发育良好的1PN囊胚,在无可利用的2PN胚胎情况下,可选择进行移植?     

不同浓度表皮生长因子对小鼠2-细胞胚胎体外发育的影响

目的:研究不同浓度表皮生长因子(ep iderm al growth factor,EGF)对ICR小鼠2-细胞胚胎体外发育的影响。方法:在mKSOM培养液中添加不同浓度(0.1,1.0,10和100 ng/m l)的EGF对小鼠2-细胞胚胎进行体外培养72 h、96 h,观察囊胚发育率、孵化率和胚胎细胞数的变化。结果:实验组加入四种不同浓度EGF胚胎培养72 h后,其囊胚率分别为93.8%、92.4%、91.8%、91.2%;96 h后其孵出率分别为61.1%、61.8%、63.0%、60.1%,均显著高于对照组(79.9%、43.1%,P<0.05),囊胚细胞数分别为73.1±13.4、73.8±7.0、74.3±12.2、77.6±8.2,与对照组(72.1±6.9)比较,差异无显著性(P>0.05);四个实验组间差异亦无显著性(P>0.05)。结论:EGF浓度在0.1,1.0,10和100 ng/m l范围内可提高体外培养鼠胚的囊胚率与孵出率;胚囊细胞数无变化;不同浓度EGF对早期鼠胚未见有毒性作用。     

氨基酸浓度对昆明小鼠胚胎体外发育的影响

目的探讨培养液中氨基酸成分对植入前昆明小白鼠胚胎发育的影响,摸索植入前胚胎体外培养氨基酸的最佳浓度。方法从超排昆明小鼠输卵管中取出的630枚受精卵均分为7组,分别在经添加不同浓度Eagle's氨基酸的去牛血清白蛋白KSOM培养液(mKSOM、mKSOM 1/16AA、mKSOM 1/8AA、mKSOM 1/4AA、mKSOM 1/2AA、mKSOM AA、mKSOM 2AA)中培养,对比观察各组胚胎体外发育的过程。用卡方检验分析添加各浓度氨基酸的培养液培养效率的差别,并将8细胞发育率和囊胚发育率分别与相应的氨基酸浓度进行曲线拟合分析。结果与未添加氨基酸组对比,添加氨基酸组胚胎发育率高,8细胞胚和囊胚的形成率与培养液中氨基酸的浓度关系均符合三次模型,在氨基酸为1/2浓度时8细胞胚和囊胚的形成率均达到最高。结论氨基酸对胚胎的体外发育有促进作用,但含有高浓度氨基酸的培养液培养效果反而有所下降,可能是由于高浓度的氨基酸影响胚胎的代谢和渗透压的改变,且氨基酸分解及胚胎代谢所产生的铵对胚胎有毒性作用。     

KSOM培养基营养成分调整对小鼠植入前期胚体外发育的影响

目的囊胚发育率低和发育迟缓是继““2-细胞阻滞““解决后小鼠胚胎体外培养中面临的两大问题.本实验通过对 KSOM培养基营养成分的调整,观察对胚胎发育的影响,优化培养基的构成.方法采用微滴培养法培养昆明小鼠单细胞胚胎,以胚胎发育到囊胚的比例、孵出比例以及注射hCG后120 h囊胚全细胞数作为判断培养效果的标准,比较 KSOM在葡萄糖、牛血清白蛋白、氨基酸3种营养物质不同浓度下对小鼠植入前胚体外培养的效果.结果在未加氨基酸时葡萄糖、牛血清白蛋白的变化对注射hCG后120 h囊胚形成率、囊胚全细胞数影响不大,添加氨基酸后,囊胚发育速度提高,部分孵出和全部孵出比例提高,囊胚全细胞数增大明显,且葡萄糖、牛血清白蛋白与氨基酸对囊胚的发育有共同促进的作用.结论通过添加适量的氨基酸、葡萄糖、BSA改良KSOM培养基能很好的促进小鼠植入前胚体外发育.     

KSOM培养基营养成分调整对小鼠植入前期胚体外发育的影响

目的 囊胚发育率低和发育迟缓是继“2-细胞阻滞”解决后小鼠胚胎体外培养中面临的两大问题。本实验通过对KSOM培养基营养成分的调整,观察对胚胎发育的影响,优化培养基的构成。方法 采用微滴培养法培养昆明小鼠单细胞胚胎,以胚胎发育到囊胚的比例、孵出比例以及注射hCG后120h囊胚全细胞数作为判断培养效果的标准,比较KSOM在葡萄糖、牛血清白蛋白、氨基酸3种营养物质不同浓度下对小鼠植入前胚体外培养的效果。结果 在未加氨基酸时葡萄糖、牛血清白蛋白的变化对注射hCG后120h囊胚形成率、囊胚全细胞数影响不大,添加氨基酸后,囊胚发育速度提高,部分孵出和全部孵出比例提高,囊胚全细胞数增大明显,且葡萄糖、牛血清白蛋白与氨基酸对囊胚的发育有共同促进的作用。结论 通过添加适量的氨基酸、葡萄糖、BSA改良KSOM培养基能很好的促进小鼠植入前胚体外发育。     

Effects of Malachite Green on the Development of Mouse Embryo In Vitro

Objective To observe effects of malachite green （MG） on the development of mouse embryo in vitro.Methods Two-cell mouse embryos were obtained and exposed to different concentrations of MG （0 ng/ml, group A, the control; 10 ng/ml, group B; 100 ng/ml, group C; 1 000 ng/ml, group D）. The 2-cell mouse embryo assay was used to determine the 2-cell embryo development to the blastocyst. In addition, at the same concentrations of MG, the human sperm motility assay was used to evaluate the influence of MG on sperm viability.Results At the 8-cell stage, groups C and D showed inhibitory effects when compared with the control （P〈0.01）. Rate of the blastocyst in groups C and D was lower than that of the control （P〈O.O1）. In the sperm motility assay, significant changes in motility after 24 h of incubation in groups C and D were observed compared with the control （P〈0. 01）. However, no differences were found on 24 h sperm motility between group B and the control （P〉0.05）. Conclusion MG would have deleterious effects on the development of mouse embryo, and the effects were dose-related. The sperm motility assay could also detect high levels of MG in culture medium.     

氨基酸浓度对昆明小鼠胚胎体外发育的影响

目的探讨培养液中氨基酸成分对植入前昆明小白鼠胚胎发育的影响,摸索植入前胚胎体外培养氨基酸的最佳浓度.方法从超排昆明小鼠输卵管中取出的630枚受精卵均分为7组,分别在经添加不同浓度Eagle‘s氨基酸的去牛血清白蛋白KSOM培养液(mKSOM、mKSOM 1/16AA、mKSOM 1/8AA、mKSOM 1/4AA、mKSOM 1/2AA、mKSOM AA、mKSOM 2AA)中培养,对比观察各组胚胎体外发育的过程.用卡方检验分析添加各浓度氨基酸的培养液培养效率的差别,并将8细胞发育率和囊胚发育率分别与相应的氨基酸浓度进行曲线拟合分析.结果与未添加氨基酸组对比,添加氨基酸组胚胎发育率高,8细胞胚和囊胚的形成率与培养液中氨基酸的浓度关系均符合三次模型,在氨基酸为 1/2浓度时8细胞胚和囊胚的形成率均达到最高.结论氨基酸对胚胎的体外发育有促进作用,但含有高浓度氨基酸的培养液培养效果反而有所下降,可能是由于高浓度的氨基酸影响胚胎的代谢和渗透压的改变,且氨基酸分解及胚胎代谢所产生的铵对胚胎有毒性作用.     

应用序列培养系统培养鼠囊胚的效果观察

目的比较序列培养和非序列培养支持鼠囊胚发育和分化的效果。方法应用Q-one培养基培养鼠合子48h后,移至Q-three培养基中继续培养48h;对照组应用Q-one培养基完成从合子至囊胚期的培养。结果序列培养组囊胚发育率及囊胚孵出率均明显高于非序列培养组,分别为89％和12％;38％和0。在序列培养中镜下扩张态囊胚形成好。结论序列培养系统有利于鼠胚从早期胚胎向囊胚的进一步发育和分化。     

表皮生长因子对小鼠早期胚胎体外发育影响的研究

目的：探讨表皮生长因子对小鼠植入前胚胎体外发育的影响。方法：用添加不同浓度EGF(epidermal growth factor)的CZB培养液对小鼠早胚进行体外培养，观察囊胚发育率和胚胎细胞数的变化。结果：HCG(human chorionic gonadotropin)注射后138h，0．1ng／ml EGF添加组扩张囊胚率、孵化囊胚率显著高于其他各组。100．0ng／ml EGF添加组扩张囊胚率和孵化囊胚率显著低于其余各组，且囊胚细胞死亡较多。结论：EGF通过刺激囊胚期细胞分化而促进胚胎体外发育。但高浓度EGF抑制胚胎体外发育并使早期胚胎受损。     

Effect of the P1 Medium and the ECM Medium on Embryo Quality in IVF

Objective To investigate the effect of the glucose-free preimplantation stage one （P1） medium and the ECM medium on embryo development quality in IVF. Methods The patients with ≥4 zygotes of 2PN were studied. A total of 201 retrieval cycles were included in a prospective randomized study. Each patient was herself control. Half of zygotes of 2PN were transferred into ECM medium （group A） and half into P1 medium （group B）for further culture. Embryo development was evaluated on the day of embryo transfer. The efficacy of ECM was compared with P1 as culture medium for the development of preimplantation embryos. Results No statistically significant differences were noted between the two groups regarding embryo-cleavage rate （97.13% vs 97.55%） and rate of normal-cleaving embryos （58.29% and 58.37%）. The rate of top-quality embryos was statistically higher in group A than in group B （27.59% vs 19.75%, P〈O.05）. Embryo quality, as assessed by morphological parameters （the amount of detached anuclear fragments 〉30%）, was better in group A than in group B （19.86% vs 21.75%）, however, there was no statistically significance. Both the rate of good-quality embryos （47.95% vs 46.17%） and available embryos （63.22% vs 61.19%） were higher in group A than in group B, but there was also no statistically significance. Conclusion The ECM medium may be associated with a better embryo quality compared with the P1 medium.     

Effects of Placental Isoferritin on the Mouse Embryo Development in vitro

To investigate the effect of placental isoferritin （PLF） on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell 8-cell and morula （P〉0.05）. PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst （P〈0.05）. PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics （P〉0.05）. It was concluded that PLF at the concentration of 10-- 100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantion blastocysts.     

TGF-β1改善体外培养小鼠胚胎种植率及其作用机制的研究

目的 探讨TGF-β1在体外对小鼠胚胎发育及胚胎种植的影响及其作用机制.方法 ①收集小鼠2-细胞期胚胎,在含不同剂量TGF-β1的M16培养液中进行体外培养,观察胚胎发育情况,并进行胚胎移植,观察着床率;②测定移植后小鼠外周血及胚胎种植部位母胎界面IL-10/INF-γ水平.结果 ①1 ng/mL和10 ng/mL TGF-β1能提高2-细胞期胚胎体外发育速率、质量和胚胎着床率.其中TGF-β1 10 ng/mL时作用最显著,TGF-β1浓度过高对体外胚胎发育不利;②M16+TGF-β1 10 ng/mL组体外培养囊胚移植后母胎界面INF-γ低于M16组,差异有统计学意义[(30.89±11.31) pg/mL vs. (43.23±18.09) pg/mL,P<0.05].两组间IL-10水平无明显差异.结论 ①小鼠胚胎体外培养基中加入适宜浓度TGF-β1可提高2-细胞期胚胎体外发育速率、质量和胚胎着床率;②TGF-β1可能通过促进体外胚胎发育速率和质量,改善胚胎和内膜发育同步性,促进母胎界面Th1/Th2平衡向Th2偏移而促进胚胎着床.     

成纤维生长因子2对人乳腺癌细胞MCF7骨涎蛋白表达的影响

【目的】研究成纤维生长因子2（FGF2）对人乳腺癌细胞MCF7骨涎蛋白（BSP）表达的影响。【方法】将MCF7细胞随机分为5组：空白对照组、FGF2（10 ng/ml）刺激3、6、12和24 h组,分别应用实时荧光定量PCR和免疫印迹技术检测BSP mRNA和蛋白表达水平。【结果】各实验组均有BSP的mRNA表达,其表达水平在FGF2刺激后呈时间依赖性增加,6 h增强最为明显。BSP蛋白表达量也呈时间依赖性增加,12 h增强最为明显。【结论】乳腺癌细胞MCF7组成性表达BSP,在FGF2作用下BSP表达水平呈时间依赖性增强。     

未见原核来源囊胚复苏移植的临床价值

目的 分析未见原核（0PN）来源囊胚在复苏周期中的临床应用价值。方法回顾分析接受体外受精-胚胎移植（IVF-ET）助孕治疗后,进行囊胚复苏移植的345例患者,48例解冻移植0PN来源囊胚患者为0PN组,297例解冻移植2PN来源囊胚患者为2PN组。移植后对患者临床妊娠进行随访分析。结果435枚0PN卵裂胚与2 891枚2PN卵裂胚比较,两者的囊胚形成率无明显差异（72.87% vs 73.88%,P>0.05）,0PN卵裂胚的可利用囊胚率（52.64% vs 46.90%）和优质囊胚率（27.13% vs 22.28%）均高于2PN卵裂胚,差异有统计学意义(P<0.05);0PN组的胚胎着床率（59.21 % vs 52.72%）、临床妊娠率（68.75 % vs 63.64%）、流产率（18.18 % vs 14.29%）、抱婴率（54.17 % vs 53.54%）均高于2PN组,但差异无统计学意义（P>0.05）。结论IVF-ET助孕治疗后,无2PN来源卵裂胚和囊胚的患者,可谨慎选择0PN来源囊胚进行复苏周期移植。     

肝细胞生长因子对骨髓间充质干细胞迁移的影响

目的研究肝细胞生长因子（HGF）对大鼠骨髓间充质干细胞（BMSCs）迁移的影响。方法采用Percoll密度梯度离心法分离BMSCs,进行表面抗原表达及分化鉴定。使用Dunn chamber装置研究BMSCs的定向迁移。并且研究PI3K抑制剂LY294002对HGF诱导BMSCs迁移的影响。结果分离培养的BMSCs呈CD29、CD90、CD106阳性表达,CD34、CD45阴性表达,BMSCs可诱导分化为成骨细胞及脂肪细胞。Dunn chamber迁移实验显示,外槽加入不同浓度的HGF,BMSCs迁移速率没有变化,迁移效率随着HGF浓度的增加而增加,50ng/ml与100ng/mlHGF均显著提高了细胞迁移效率;内外槽同时加入50ng/mlHGF,迁移速率与迁移效率均没有变化;经30μmol/LLY294002预处理1h后,HGF诱导的BMSCs的定向迁移受到抑制。结论 HGF能够趋化BMSCs的定向迁移,PI3K信号通路参与介导这一过程。     

Extended Culture of Early Stage Embryos in Frozen-thawed Cycles

Objective To investigate the impact of extended culture of early stage embryos on pregnancy outcome of frozen embryo transfer （FET）. Methods The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between zygote and cleavage embryos which cultured to cleavage stage or extended cultured to blastocysts. Results A total of 425 zygote embryos in 67 cycles were thawed. After thawing, the survival rate was 94.4% and with an average transfer of 2.8 embryos, the clinical pregnancy rate was 55.2% （37/67）. In 222 FET cycles, totally 1 270 cleavage stage embryos were thawed and the overall survival rates were 80.3%. With an average transfer of 2.7 embryos, the clinical pregnancy rate was 55.4% （123/222）. A significantly lower percentage of degenerated embryos were found for zygotes （5.6%） than that for cleavage stage embryos （19.7%） （P〈0.01）. The clinical pregnancy rate was 53.4% （116/217） in the group of transfer at cleavage stage, while the clinical pregnancy rate was 61.1% （44/72） in the group of transfer at blastocyst stage （P〉0.05）. Conclusion Although the clinical pregnancy rate was not different between patients with freeze-thaw zygote and cleavage stage embryo transfer, higher survival rate for zygote was shown compared with that for cleavage stage embryo. However, the present studies did not demonstrate that extended culture thawing embryos to blastocyst could achieve favor clinical outcome.     

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

Clenbuterol hydrochloride is a strongβ2 receptor agitator,and it was used toprevent and treat bronchial asthma.However,it is seldom used today because of itsstrong side-effect to the heart[1 ] .Clenbuterol hydrochloride also helps in the forma-tion of bo…     

生长激素、肝细胞生长因子和烟酰胺对人胎胰岛细胞的增殖作用

目的研究生长激素(GH)，肝细胞生长因子(HGF)和烟酰胺(NIC)对体外培养的人胎胰岛细胞的增殖作用及其交互作用。方法采用La(2^7)正交设计法在体外培养的人胎胰岛细胞的各组中分别加入不同浓度及组合的GH，HGF和NIC，培养48h后，收集各孔细胞，DTZ染色，计数。结果GH，HGF和NIC均起主要作用，HGF和NIC的交互作用不可忽视，最佳的生长因子组合及适配浓度为GH(100ng／ml)HGF(25ng／ml)NIC(100mmol／L)。结论GH，HGF和NIC均能促进体外培养的胰岛细胞的增殖，且组合GH(100ng／ml)HGF(25ng／ml)NIC(100mmol／L)的作用最大。     

人输卵管上皮细胞共培养和表皮生长因子对小鼠受精卵体外发育的影响

目的：通过人输卵管上皮细胞共培养体系及表皮生长因子（EGF）作用,探讨输卵管上皮及EGF对受精卵体外发育的影响。方法：建立人输卵管上皮细胞共培养体系,将小鼠雌雄原核（2PN）受精卵随机分为3组,即对照组、实验组Ⅰ和实验组Ⅱ。结果：（1）小鼠受精卵在对照组中单独培养,发育至2细胞、4细胞、8细胞及囊胚期的比率分别为：89．3％、48．7％、34．7％和29．3％。（2）小鼠受精卵在实验组Ⅰ中培养,发育至2细胞,4细胞、8细胞及囊胚期的比率分别为：86．3％、80．6％、75．4％和66．3％。（3）小鼠受精卵在实验组Ⅱ中培养,发育至2细胞、4细胞、8细胞及囊胚期的比率分别为：82．5％、76．3％、70．1％和62．7％。结论：（1）人输卵管上皮细胞共培养体系和EGF对小鼠早期胚胎克服体外发育中的阻断有帮助。（2）人输卵管上皮细胞共培养体系及EGF可以提高小鼠受精卵发育至多细胞及囊胚期的胚胎数。