化学激活在小鼠生精细胞体外受精中的应用

发布时间：2018-05-04 18:35

本文选题：生精细胞 + 圆形精子　；参考：《安徽医科大学》2012年硕士论文

【摘要】：第一部分小鼠单倍体精子细胞显微注射方法学研究目的：探讨改良持卵针的显微操作系统在小鼠精子细胞体外授精中的应用价值。方法：通过反复锻烧将常规持卵针制作成喇叭形开口状，制成改良持卵针，辅以独特的显微注射操作手法，对小鼠生精细胞体外培养所获单倍体圆形精子进行卵胞浆内显微注射（ROSI），计算ROSI后30min昆明小白鼠卵母细胞的存活数和存活率，并与常规窄口持卵针行ROSI后的卵母细胞存活率进行比较。结果：分别采用常规和改良后的持卵针进行小鼠ROSI，每种方法各注射320个卵母细胞。改良持卵针组ROSI后30min的卵母细胞存活率显著高于未改进持卵针组（P0.05）。结论：改良持卵针制备简单，，易于操作，ROSI后卵母细胞存活率高，且不需依赖piezo系统，在小鼠单倍体精子细胞体外受精的研究中有较高的临床应用价值。第二部分化学激活在小鼠圆形精子体外受精中的应用目的：通过对生精细胞体外培养所获圆形精子显微注射后的小鼠卵母细胞进行单一和联合化学激活，寻找小鼠圆形精子体外受精的最佳激活方案。方法：选取乙醇（ethanol，EH）、钙离子载体A23187(calcium ionophore A23187，CIA)、离子霉素（ionomycin，Ion）、氯化锶（strontium chloride，SrCl2）、放线菌酮（cycloheximide，CHX）及6-二甲基苯胺嘌呤(6-dimethylaminopurine，6-DMAP)作为外源性化学激活剂，采用不同浓度的单一化学激活剂对ROSI后的卵母细胞进行不同作用时间激活，观察其受精率、卵裂率和桑囊胚形成率。根据激活不同的途径，联合两种最佳激活浓度和激活时间的单一激活剂对ROSI后的卵母细胞进行联合激活，比较不同联合方案对卵母细胞的激活效果；并以成年小鼠睾丸内圆形精子ROSI作为对照，比较体内精子细胞和体外培养精子细胞的受精能力及胚胎发育潜能。结果：（1）单一激活时，各激活剂的最佳方案分别为①乙醇：7%、6min；②CIA：5μmol/L、5min；③Ion：5μmol/L、5min；④6-DMAP：2mmol/L、2h；⑤SrCl2：10mmol/L、1.5h；⑥CHX：10μg/mL、1.5h，其中10mmol/L SrCl2作用1.5h激活效果最佳，显著优于CHX方案，但与其它各组无显著性差异；（2）联合激活时，乙醇+6-二甲基苯胺嘌呤组桑囊胚率（29.63%）显著高于其它各组，以及除SrCl2以外所有单一激活方案（P0.05），为最佳联合激活方案；（3）对成年小鼠睾丸中圆形精子ROSI后的卵母细胞进行乙醇+6-二甲基苯胺嘌呤激活后，其正常受精率、卵裂率及桑囊胚率与体外培养所获圆形精子间均无显著性差异。结论：单一激活方案中10mmol/L SrCl2单独作用1.5h，联合方案中7%EH作用6min+2mmol/L6-DMAP作用2h对体外培养所获圆形精子ROSI后的卵母细胞激活效果最佳；体外培养所获圆形精子ROSI后受精率、胚胎发育潜能与成年小鼠睾丸内圆形精子间无显著性差异。
[Abstract]:Study on microinjection of haploid spermatozoa in mice Objective: to study the application value of modified microoperating system of egg holding needle in mouse sperm cell insemination in vitro. Methods: the conventional egg holding needle was made into a horn shaped opening by repeated burning, and an improved egg holding needle was made with a unique microinjection technique. Haploid round spermatozoa obtained from mouse spermatogenic cells in vitro were microinjected into oocyte cytoplasm to calculate the survival number and survival rate of 30min Kunming mouse oocytes after ROSI. The survival rate of oocytes was compared with that of conventional narrow needle holding oocyte after ROSI. Results: mice ROSIs were treated with conventional and modified oocyte holding needles. 320 oocytes were injected into each method. The survival rate of 30min oocytes in the modified oocyte holding needle group was significantly higher than that in the unmodified egg holding needle group after ROSI. Conclusion: the modified oocyte holding needle is simple to be prepared, easy to operate and has high survival rate of oocytes, and does not depend on piezo system, so it has high clinical application value in the study of in vitro fertilization of mouse haploid sperm cells. Application of Chemical Activation in in Vitro fertilization of Mouse Round sperm Aim: to find out the best method of in vitro fertilization of round spermatogenic cells by single and combined chemical activation of mouse oocytes obtained by microinjection of spermatogenic cells. Methods: ethanolus, A23187(calcium ionophore A23187 CIAA, ionomycin Ionn, Strontium chlorideum chloride, cycloheximidede CHX and 6-dimethylaminopurine 6-DMAPs were used as exogenous chemical activators. Different concentrations of single chemical activator were used to activate oocytes after ROSI for different time. The fertilization rate, cleavage rate and blastocyst formation rate were observed. According to the different ways of activation, two kinds of single activator of optimal activation concentration and activation time were combined to activate oocytes after ROSI, and the effects of different combination schemes on oocyte activation were compared. The fertilization ability and embryonic development potential of spermatozoa in vivo and in vitro were compared with round spermatozoa ROSI in adult mouse testis. Results under the single activation, the best scheme for each activator was 1 ethanol 7: 7 and 2CIA: 5 渭 mol / L 5 min Ion: 5 渭 mol / L 5min 46-DMAP: 2mmol / L 2hmol / L 5SrCl 2: 10mmol / L 1.5hl 6CHX% 10 渭 g mLL = 1.5h. among them, 10mmol/L SrCl2 had the best activation effect for 1.5h, which was significantly better than the CHX scheme, but had no significant difference with other groups. The mulberry blastocyst rate of alcohol 6-dimethylaniline group was significantly higher than that of other groups. The normal fertilization rate of oocytes after ROSI was activated by alcohol 6-dimethylaniline in adult mouse testis, and all the single activation regimen (P0.05G, except SrCl2) was the best combination. There was no significant difference between cleavage rate and blastocyst rate and round spermatozoa in vitro. Conclusion: 10mmol/L SrCl2 alone in single activation regimen and 7%EH in combination with 6min 2mmol/L6-DMAP for 2 h have the best effect on oocyte activation after ROSI obtained from round sperm in vitro, and fertilization rate after ROSI in vitro. There was no significant difference between embryonic development potential and round spermatozoa in testis of adult mice.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

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