第一次减数分裂期小鼠卵母细胞的化学去核

过免疫荧光染色法确定了小鼠第一次减数分裂的进程,在此基础上分别用脱羧秋水仙碱和放线菌酮处理成熟培养的小鼠卵母细胞,抑制其纺锤体极间微管的延长和降低成熟促进因子的浓度,使其核物质随极体全部排出.结果表明:(1) 卵母细胞从有腔卵泡中分离时处于生发泡期,可见核仁;成熟2h内则全部发生生发泡破裂;培养5h,90%到达第一次减数分裂的前中期;培养8h,则33.3%处于中期;71.4%的细胞在成熟12h后发育为后期;第二次减数分裂的中期则于成熟培养15h发生.(2) 于成熟的不同时间取卵母细胞-卵丘细胞复合体进行去核处理后发现,体外成熟培养5h(即卵母细胞处于第一次减数分裂前中期时进行处理),其去核率最高,达85.6%;成熟5h之前各组随成熟培养时间的延长而去核率升高,之后各组则降低.(3) 比较卵母细胞-卵丘细胞复合体与自然裸卵去核率,后者效率为56%,显著低于前者(p＜0.05),因此该方法在卵母细胞-卵丘细胞复合体的完全去核率与传统的物理去核方法相当.     

克隆绵羊遇到的问题及其分析

本研究以体外成熟不同时间的绵羊卵母细胞为胞质受体,对去核、融合、不同激活方法、卵裂率、桑/囊胚率和克隆胚受胎率等作了研究与分析.观察妊娠受体母羊的状态、胎儿体内发育情况,分析影响胎儿发育的原因.结果显示,成熟培养18h、22h、24h卵母细胞的盲吸去核率无显著性差异(84.9%,86.6%,84.3%,p>0.05),但显著高于26h组的去核率(71.7%).离子酶素联合cycloheximide(CHX)对重构胚进行激活,卵裂率显著高于A23187联合6-DMAP的激活方法(84.6%vs 63.2%,p<0.05),但二者处理对桑/囊胚率发育无显著差异(25.5%vs18.7%,p>0.05).作为胞质受体,成熟20h的卵母细胞与成熟24h和26h的卵母细胞相比,不利于重构胚的体外发育,桑/囊胚率差异显著(12.3%vs 25.3%,23.4%,p<0.05).分别以美丽奴、杜泊肉羊的体细胞为供体细胞进行克隆生产,90天以后的妊娠率为10%-17%,发育到期率为10%以上.出生羔羊的体重普遍较大.     

牛卵母细胞不同时间的成熟培养可提高核移植效率

：比较了成熟培养不同时间的卵母细胞的成熟率、去核率及用于核移植的效率,认为成熟培养21 h的卵母细胞虽然有较高的成熟率,但不适于采用盲去法去核.成熟培养19 h的卵母细胞核移植效率略高于成熟培养17 h.因此认为采用盲去法去核操作大批卵母细胞时,19 h的体外成熟培养时间是比较适宜的.     

促减数分裂甾醇（MAS）对猪卵母细胞体外成熟及其胚胎发育能力的影响

利用促减数分裂甾醇(MAS)合成代谢过程中的抑制剂AY9944累积FF-MAS的原理,在猪卵母细胞体外成熟过程中添加AY9944,间接地研究了内源性MAS对猪卵母细胞体外成熟质量的影响.猪卵丘卵母细胞复合体(cumulus oocyte complexes,COCs)培养在NCSU23成熟培养液中,并添加不同浓度(0,10,20,40μmol/L)的AY9944培养44h.培养结束后,成熟的卵母细胞进行孤雌激活和以胎儿成纤维细胞为核供体重构胚胎,分别于48和144h观察胚胎发育情况,统计卵裂率和囊胚率及囊胚/2-细胞胚比率.结果如下:(1)随着AY9944添加浓度的增加,退化的卵母细胞增多,40μmol/L AY9944处理的退化卵显著,卵母细胞成熟率显著下降.(2)成熟培养液添加20和40μmol/L AY9944处理的孤雌激活胚胎的囊胚形成率和2-细胞胚发育到囊胚的比率显著增加(P<0.05).(3)以对照组(0μmol/L AY9944)和20μmol/L AY9944处理的卵母细胞为胞质受体,发现20μmol/L AY9944处理的克隆胚的发育能力囊胚率和2-细胞胚发育到囊胚的比率有所提高,但无显著差异(P>0.05).以上结果表明,猪卵母细胞体外成熟过程中添加AY9944提高了猪卵母细胞体外成熟的胞质质量,胚胎的发育能力提高.     

细胞松弛素B、温度、离心处理对猪卵母细胞OPS玻璃化冷冻的影响

主要探讨使用CB预处理和不同温度冷冻保护剂对猪卵母细OPS玻璃化冷冻保存的影响,并对使用不同温度冷冻保护剂下,探讨离心猪卵母细胞后的OPS冷冻、解冻的效果.结果表明,体外成熟培养12h后,将经7.5μg/mL细胞松弛素B(CB)处理的猪卵母细胞进行OPS玻璃化冷冻保存.解冻后继续体外成熟培养,猪卵母细胞的形态正常率和成熟率与对照组无显著差异(P>0.05),但是与对照组相比,经冷冻前经CB预处理的卵母细胞其体外受精胚胎的卵裂率、桑葚胚和囊胚率显著提高(P<0.05).此外,使用预平衡至39℃的冷冻保护剂能够达到较好的解冻效果,卵母细胞成熟率较高,但离心并没有促进冷冻后猪卵母细胞体外受精胚胎发育率.     

猪体外成熟卵母细胞激活方法的比较

研究了离子霉素/6-二甲氨基嘌呤(DMAP),乙基汞硫代水杨酸钠/二硫苏糖醇(DTT)激活猪体外成熟卵母细胞的最佳条件,比较研究了电脉冲和化学刺激对猪体外成熟卵母细胞的激活效率.结果表明,用15μmol/L离子霉素处理猪体外成熟卵母细胞10,20,30,40,50,60和70min,其囊胚发育率差异不显著,以40min时的囊胚发育率最高.用15,20和25μmol/L离子霉素处理猪体外成熟卵母细胞,其囊胚率无显著差异.用100,200和300μmol/L乙基汞硫代水杨酸钠分别激活猪体外成熟卵子,发现100μmol/L乙基汞硫代水杨酸钠中处理30min组的卵裂率和囊胚率最高.电脉冲与两种最佳化学激活组的比较研究表明,电激活组的囊胚率最高,但囊胚细胞数低,相反,乙基汞硫代水杨酸钠/二硫苏糖醇激活组的囊胚细胞数最高,囊胚率却较低.     

卵细胞体外成熟(IVM)在小鼠生物净化中的应用

目的 在小鼠生物净化中,从超排后未排卵的卵巢,取卵泡内未成熟卵采用卵母细胞体外成熟(in vitro maturation,IVM)使之在体外成熟并具备受精能力,以提高卵子利用率和作为常规促超排失败的一种补救措施。方法 在小鼠生物净化中,取注射PMSG和HCG后未排卵卵巢,在实体显微镜下划破卵泡挑选卵丘卵母细胞复合物(COCs),置于成熟液滴中在体外发育成熟。同时以注射PMSG和HCG后正常超排卵、只注射PMSG后取未成熟卵体外成熟和注射PMSG和HCG后取疑似成熟卵和裸卵体外成熟作为对照。经体外受精、体外胚胎发育后,将2-细胞胚移植到受体输卵管,使其在受体内发育成为成熟的个体。结果 注射PMSG和HCG后未排卵组(A组),卵细胞体外成熟率为87.0%±3.2%,二胞率为55.1%±12.3%,囊胚率为23.1%,移植41枚二细胞胚至2只受体鼠,出生5只幼崽,产仔率12.2%。只注射PMSG,48 h后取未成熟卵组(B组),体外成熟率为83.9%±3.9%,二胞率为51.8%±9.3%,囊胚率为38.5%±13.9%。注射PMSG和HCG正常超排组(C组),其二胞率为78.9%±0.6%,囊胚率为78.0%±3.8%。注射PMSG和HCG未排卵卵巢,取裸卵和疑似成熟卵(D组),体外成熟培养0 h,6 h和16～18 h,其成熟率、二胞率和囊胚率与其它三组相比均较低且有极显著性差异。A组和B组与正常对照C组相比,二胞率均有显著性差异,囊胚率均有极显著性差异。结论 卵母细胞体外成熟(IVM)可以作为小鼠生物净化中促超排失败的一种补救措施,并且可以提高珍稀品系小鼠的卵子利用率。     

体外培养时间和表皮生长因子对牦牛卵母细胞成熟及孤雌胚胎体外发育的影响

研究在成熟液中添加表皮生长因子（Epidermal growth factor,EGF）以及成熟时间对牦牛卵母细胞体外成熟及孤雌胚胎体外发育的影响,以确立牦牛卵母细胞体外成熟的最佳体系.结果表明,成熟液中添加EGF组的成熟率明显高于未添加组（P＜0.05）,并且牦牛卵母细胞的成熟率随着EGF添加的浓度增加也不断上升;随着体外培养时间的延长,成熟率逐渐增加,培养24 h后成熟率趋于稳定;胚胎体外培养液中添加EGF能显著提高孤雌胚胎的8-细胞和囊胚形成率（P＜0.05）.由此推测：在体外培养22-24 h,且添加40 μg/mL EGF最有利于牦牛卵母细胞体外成熟;胚胎培养液中添加40μg/mLEGF能显著提高牦牛孤雌胚胎的体外发育能力.     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育的比较

目的对MII期水牛卵母细胞进行人工诱导激活,可以间接判断体外成熟卵母细胞质量的优劣,并且,卵母细胞的充分激活也是提高核移植效率的关键因素之一。比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较。结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%,17.7%)明显高于体外成熟21 h或24 h的囊胚发育率(12.3%,13.8%);Ion联合6_DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同培养条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚(21.7%,13.0%)。     

瘦素对猪卵母细胞体外成熟及克隆猪妊娠率的影响

体细胞克隆猪在人类医学、基础科学研究和畜牧业生产方面均具有很大的应用潜力.为了提高体细胞克隆猪的效率,首先比较了两种基础成熟液NCSU-23和TCM199的体外成熟效果,然后在TCM199培养液的基础上,较系统地研究了添加不同浓度的瘦素对猪卵母细胞成熟、孤雌激活胚胎和克隆胚胎的体外发育以及克隆胚胎体内发育的影响.结果表明:成熟液中添加100ng/mL或200ng/mL瘦素对猪卵母细胞的核成熟没有显著影响(P>0.05);对孤雌激活卵裂率和克隆胚胎卵裂率也没有显著影响(P>0.05);但却显著提高了孤雌激活胚胎的囊胚率和囊胚细胞数,并且显著提高了克隆胚胎的囊胚率;当添加量为100ng/mL时,克隆囊胚的细胞数显著升高.另外,研究表明,瘦素很可能具有提高克隆胚胎移植妊娠率和妊娠到期率的作用.     

EGF、IGF-Ⅰ对牛卵母细胞体外成熟的影响

在牛卵母细胞体外成熟培养液中添加不同量的EGF、IGF-Ⅰ,观察其对牛卵母细胞体外成熟的影响并确定成熟液中添加这两种生长因子的最佳浓度.在卵母细胞成熟培养液中分别添加1 ng/m l、10 ng/m l、50 ng/m l、100 ng/m l、200 ng/m l的EGF,10 ng/m l、50 ng/m l、100 ng/m l、150 ng/m l、200 ng/m l的IGF-Ⅰ,分析成熟培养22 h后的卵母细胞成熟率.发现添加有EGF实验组的成熟率分别是36.73%、42.18%、56.18%、53.67%、56.50%,同对照组相比都较对照组的成熟率(34.04%)高,且50 ng/m l、100 ng/m l、200 ng/m l实验组的成熟率同对照组差异显著(P<0.05).IGF-Ⅰ实验组的成熟率分别是38.46%、46.94%、58.73%、55.00%、58.70%,50 ng/m l、100 ng/m l、150 ng/m l、200 ng/m l都较对照组的成熟率(38.60%)要高,100 ng/m l、150 ng/m l、200 ng/m l实验组同对照组相比差异显著(P<0.05).将50 ng/m l的EGF、100 ng/m l的IGF-Ⅰ共同添加在成熟培养液中得到的成熟率为75.00%,同对照组(36.54%)相比差异极显著(P<0.01).在牛卵母细胞体外成熟培养液中添加EGF、IGF-Ⅰ会提高卵母细胞的成熟率,并且随着浓度的增加,这种作用将达到饱和,其最佳作用浓度为50ng/m l EGF、100 ng/m l IGF-Ⅰ.     

Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

Cloning pigs by somatic cell nuclear transfer （SCNT） has wide applications in basic research, human medicine and agricultural production. To improve cloning efficiency, the effect of two basic maturation media, NCSU-23 and TCM199, was compared, and TCM199 was selected for the following experiments with leptin. We systematically studied the effects of leptin supplementation on oocytes in vitro maturation （IVM）, in vitro development of parthenogenetically activated （PA） and SCNT embryos and in vivo development of SCNT embryos after embryo transfer （ET）. The results showed that supplementation of 100 or 200 ng/ml leptin into the maturation medium did not greatly affect nuclear maturation of oocytes, or cleavage rates of PA and SCNT （P 〉 0.05）. Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium, and the number of cells in PA blastocysts was also improved （P 〈 0.05）. The number of cells in blastocyst of SCNT was improved, when 100 ng/ml leptin was added （P 〈 0.05）. Furthermore, supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate inpig cloning.     

MAPK regulates cell cycle progression in pig oocytes and fertilized eggs

Oocytes collected from prepubertal gilt ovaries were matured in vitro (IVM), and fertilized in vitro (IVF) or electrically activated. Phosphorylation of mitogenactivated protein kinase (MAPK) was detected with SDS-PAGE and Western blotting, and translocation of ERK₂ was observed with immunofluorescent cytochemistry. We found that the quantity of MAPK kept unchanged during oocyte maturation. There was no phosphorylated MAPK in porcine oocytes at the germinal vesicle (GV) stage; a little MAPK was phosphorylated at 20 h of IVM; a high level phosphorylation was detected at 30 h, while MAPK phosphorylation decreased at 36 h; and then MAPK phosphorylation increased again to the peak level from 40 to 60 h. ERK2 translocated from the peripheral cytoplasm to inner cytoplasm and nuclear area during oocyte maturation. There was nearly no phosphorylated MAPK at 18 and 20 h of electrically activated oocytes, but phosphorylation increased at 22 h. There was no phosphorylated MAPK at 12 h of IVF, while phosphorylation resumed at 16 h. These results suggest that MAPK may play an important regulatory role in MI-MII transition, pronucleus formation and the initiation of the first mitosis in pig eggs.     

牛体外受精技术研究的现状与展望

系统综述了牛体外受精技术研究的现状及存在的问题．并对今后的研究方向和前景提出了展望。牛卵母细胞可在不含血清的成熟培养液中获得受精及胚胎发育能力，促卵泡素、促黄体素及上皮细胞生长因子均对这一过程具促进作用。肝素是迄今最为有效的精子获能处理方法，但在受精过程中对精子获能起决定作用的是卵丘细胞和卵母细胞本身。提高受精质量可明显提高受精卵的胚胎发育能力。体外受精卵可通过改善培养条件或与体细胞进行复合培养发育到囊胚阶段．但后者更为稳定可靠。目前，牛卵母细胞的体外受精分裂率可达８０％，囊胚发育率达４０％左右．两枚鲜胚的移植妊娠率可达５０％～６０％．而两枚冻胚的移植妊娠率仅３０％～５０％。因此，尚需进一步提高体外受精胚胎的活力和改进胚胎的冷冻保存技术。     

牛胚胎体外生产技术的应用研究

利用屠宰牛卵巢抽出的卵母细胞，以卵裂率和囊胚发育为标准，对牛胚胎体外生产过程进行了简化试验，结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００－２００枚／平皿）可代替加卵丘细胞标准密度培养，其卵裂率和囊胚发育率没有显著变化，体外授精时间可从成熟培养后２２小时延长至２７小时，卵裂率和囊胚发育率均没有显著变化。卵母细胞体外成熟后可以不经洗涤直接移入受精滴，原成熟培养皿内残留孵丘细胞再培养４８小时形     

Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%-3.1%, P < 0.05) and fetus mouse fibroblast cells (2.1%-3.7%, P < 0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.     

骨质疏松性骨伤患者术前下肢深静脉血栓筛查及影响因素分析

目的探讨影响骨质疏松性骨伤患者术前下肢深静脉血栓发生的相关因素.方法选取690例骨质疏松性骨伤患者,入院后24 h内行双下肢静脉超声筛查和D-二聚体浓度检测,并采取常规血栓防治流程;收集患者年龄、性别、骨折部位、合并内科基础疾病情况,分析与下肢深静脉血栓发生的相关性.结果股骨颈骨折患者中术前出现下肢深静脉血栓41例(13.85%),明显低于股骨转子间骨折患者(75例,19.04%),差异具有统计学意义(P0.05);男性患者下肢深静脉血栓发生率明显高于女性患者,差异具有统计学意义(P0.05);不同年龄段患者术前下肢深静脉血栓发生率之间差异无统计学意义(P0.05).骨折后24 h内下肢深静脉血栓发病率较高,19例(46.34%)股骨颈骨折患者出现下肢深静脉血栓,39例(52.00%)股骨转子间骨折患者出现下肢深静脉血栓,之后下肢深静脉血栓发病率明显降低.骨折后24 h内D-二聚体浓度300 ng/m L的患者下肢深静脉血栓发生率均明显高于≤300 ng/m L的患者,差异具有统计学意义(P0.05).股骨颈骨折患者中术前合并低蛋白血症、贫血的患者下肢深静脉血栓发生率明显高于未合并者,差异具有统计学意义(P0.05);股骨转子间骨折患者中术前合并心律失常、电解质紊乱、低蛋白血症、贫血的患者下肢深静脉血栓发生率明显高于未合并者,差异具有统计学意义(P0.05).结论骨质疏松性骨伤患者术前下肢深静脉血栓发生率较高,应从急症诊断开始给予预防措施,避免危险因素,以降低术前下肢深静脉血栓发生率.     

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

Somatic cell nuclear transfer has been succeeded in procedures of nuclear transfer. One is single nucleartransfer, the other is serial nuclear transfer. Viable animals have been cloned in different species using both me-thods[1—6]. Different nuclear recipients and donors wereused in serial nuclear transfer, namely, transferring thenuclear of reconstructed embryo into enucleated MⅡoocytes[7], transferring the nuclear of reconstructed em-bryos at one cell stage into enucleated zygote[4] and t…