肌肽对小鼠卵母细胞体外成熟和胚胎发育的影响

目的探讨肌肽对小鼠卵母细胞体外成熟和胚胎发育的影响。方法获取小鼠卵丘-卵母细胞复合体(cumulus-oocyte complexes, COC), 分别用含10 μmol/L、30 μmol/L、50 μmol/L、100 μmol/L、200 μmol/L肌肽的体外成熟培养液培养18 h, 不含肌肽的培养液设为对照组。根据卵母细胞成熟率、卵裂率和囊胚形成率确定作用的最优浓度。通过检测活性氧(reactive oxygen species, ROS)水平、总谷胱甘肽(total glutathione, T-GSH)含量、皮质颗粒分布、线粒体分布和线粒体拷贝数进一步验证肌肽的作用。结果肌肽浓度为100 μmol/L时, 卵母细胞成熟率[69.23%(135/195)]、卵裂率[66.06%(72/109)]和囊胚形成率[48.61%(35/72)]最高, 与对照组[44.44%(84/189)、38.67%(29/75)、20.69%(6/29)]相比, 差异具有统计学意义(P<0.001、P<0.001、P=0.010), 故选定100 μmol/L作为肌肽作用的最优浓...     

1,2-二氯乙烷对体外培养SW620细胞增殖和细胞周期及凋亡的影响

目的 探讨1,2-二氯乙烷(1,2-DCE)对体外培养细胞周期、凋亡和增殖能力的影响.方法 不同浓度1,2-DCE染毒0.5或1h,噻唑蓝(MTT)法检测活细胞相对数和相对活力,流式细胞术(FCM)分析细胞周期和凋亡情况.结果 MTT法检测发现,随1,2-DCE染毒剂量的增加和染毒时间的延长,细胞相对存活率逐渐降低.与二甲亚砜(DMSO)组比较,染毒0.5 h,25、75、100、125、150、175、200 μmol/L 1,2-DCE组的细胞相对存活率降低,差异均有统计学意义(P＜0.05或P＜0.01);染毒1h,75、100、125、150、175、200 μmol/L 1,2-DCE组的细胞相对存活率降低,差异均有统计学意义(P＜0.05或P＜0.01);与0.5 h各组比,175μmol/L组染毒1h的细胞相对存活率降低,差异有统计学意义(P＜0.01);增殖曲线经标准化拟合后发现,染毒0.5 h,1,2-DCE的最适IC50为89.41 μmol/L,95％的可信区间为85.23～93.79μmol/L;染毒1h,1,2-DCE的最适IC50为87.68 μmol/L,95％的可信区间为83.71～91.82 μmol/L.FCM法检测发现,与对照组比较,1,2-DCE染毒1h,25、50、100、150、200 μmol/L组的G0/G1期比例降低,25、50、100 μmol/L组的S期比例降低,25、50、100、150、200 μmol/L组的G2/M期比例升高,差异均有统计学意义(P＜0.05或P＜0.01);但各组均不引起细胞凋亡.结论 1,2-DCE能够抑制体外培养SW620细胞增殖,使细胞周期停滞在G2/M期,但不诱导细胞凋亡.     

叶酸对非洲爪蟾卵母细胞体外成熟的影响及作用机制

目的 探讨叶酸( folic acid,FA)对非洲爪蟾卵母细胞体外成熟的影响及作用机制.方法 将健康卵母细胞移入含不同浓度FA(分别为125 μmol/L、250 μmol/L、500 μmol/L和1 000 μmol/L)的ND96培养液中体外培养,同时设立阴性对照组和孕酮阳性对照组.加FA后每隔1h观察1次细胞生发泡破裂(germinal vesicle breakdown,GVBD),记录发生GVBD的卵母细胞数量,计算GVBD率,同时用10％三氯乙酸溶液固定后,切开进行确认.观察6h后收集各组卵母细胞,采用蛋白免疫印迹实验(Western Blotting)检测CyclinB2,Mos,p-ERK1/ERK1蛋白的表达.结果 除FA 125 μmol/L、250 μmol/L、500 μmol/L、1 000 μmol/L组2h时与空白对照组GVBD率之间差异无统计学意义,其余各记录点的GVBD率与空白对照组间差异均有统计学意义(均有P＜0.05);Western Blotting分析显示,FA和孕酮处理后卵母细胞的p-ERK1,CyclinB2,Mos蛋白表达水平明显升高.结论 FA可促进爪蟾卵母细胞体外成熟过程中GVBD发生,提示FA对爪蟾卵母细胞的成熟具有促进作用,其作用机制可能与干扰成熟促进因子和丝裂原活化蛋白激酶有关.     

添加两种不同培养成分对人未成熟卵体外成熟培养的影响

目的:研究添加两种不同培养成分对人未成熟卵母细胞体外成熟培养(IVM)的效果。方法:选择接受ICSI治疗的77名不孕患者共收集未成熟卵177枚,随机分A组(激素组)33例,添加0.075 IU/ml FSH及0.075 IU/ml LH至基础IVM培养液中;B组(成熟卵泡液组)44例,50%基础IVM培养液与50%人成熟卵泡液配成。将两组收集的未成熟卵母细胞放入不同培养液中培养48 h,每24 h倒置显微镜下观察卵母细胞的形态;对MII卵进行ICSI,ICSI操作后继续培养,ICSI后16～20 h进行受精观察,24及48 h分别进行卵裂观察及胚胎评分。结果:①B组未成熟卵母细胞48 h成熟率和MI期卵母细胞48 h成熟率显著高于A组,差异有统计学意义(P<0.05),两组培养液GV期卵母细胞24和48 h成熟率比较差异无统计学意义(P>0.05)。②B组2PN卵裂率显著高于A组,差异有统计学意义(P<0.05),两组2PN受精率及可利用胚胎率差异无统计学意义(P>0.05)。结论:人成熟卵泡液内可能含有一些未知的有利于卵母细胞成熟的因子,深入研究卵泡液的成分,进一步明确其促卵母细胞成熟机制对开发新的IVM培养液、改善IVM的临床结局有重要意义。     

人脐带血清用于人未成熟卵母细胞体外成熟培养并获得妊娠的观察

目的:以人脐带血清作为卵母细胞体外成熟培养(IVM)培养液的主要成份开展IVM技术研究,旨在为IVM临床妊娠率的提高,IVM技术的进步探索一条新路。方法:47例、47个周期的PCOS患者接受了IVM技术的治疗。未成熟卵母细胞分别入IVM培养液Ⅰ(含人成熟卵泡液,卵泡液组)和IVM培养液Ⅱ(含脐带血清,脐带血清组)行体外成熟培养,分析人脐带血清对IVM结果影响。结果:脐带血组:15周期,160枚未成熟卵母细胞,体外成熟过程中,成熟率、优质胚率及临床妊娠率分别为93.75%(150/160)、50.00%(60/120)和40.00%(6/15),已分娩4例,另2例处于妊娠阶段;卵泡液组:32周期,未成熟卵母细胞349枚,成熟率、优质胚率分别为77.08%(269/349)和23.77%(53/223),胚胎移植31周期,获得7例临床妊娠,妊娠率为22.58%(7/31),目前已分娩4例,3例流产。成熟率和优质胚率方面,脐带血组高于卵泡液组。结论:人脐带血清作为IVM液的主要成份相对于人成熟卵泡液,将更加有助于未成熟卵母细胞的体外成熟培养、改善胚胎质量,并获得较好的妊娠结局。     

抗氧化物原花青素对人未成熟卵母细胞体外成熟的影响

目的探讨抗氧化物原花青素对卵胞浆内单精子注射(intracytoplasmic sperm injection,ICSI)周期中人类未成熟卵母细胞体外成熟(in vitro maturation,IVM)、受精及胚胎发育的影响。方法选取2019年1月至2020年12月在成都市妇女儿童中心医院行ICSI治疗周期321例,共获得未成熟卵母细胞837枚,随机分为对照组和原花青素组两组,原花青素组在人未成熟卵母细胞IVM培养液中添加原花青素,对照组不添加原花青素。未成熟卵母细胞在两组培养液中培养24 h,观察两组的成熟率、受精率、卵裂率、可利用胚胎率以及囊胚形成率,并通过荧光染料Hoechst33342对胚泡进行染色,检测囊胚的总细胞数。结果无论是GV期还是MI期卵母细胞,原花青素组的成熟率、可利用胚胎率及囊胚形成率均高于对照组,差异有统计学意义(P 0.05),但受精率和卵裂率两组间比较差异无统计学意义(P0.05);原花青素组孵化囊胚细胞总数均高于对照组,差异有统计学意义(P 0.05)。结论添加原花青素能提高人类未成熟卵母细胞的体外成熟率和胚胎发育潜能。     

超排卵周期未成熟卵母细胞体外培养及其激活效应

目的:探讨超排卵周期中获得的未成熟卵母细胞的利用价值及人工辅助激活能否改善源于此类卵母细胞的胚胎的发育结局。方法:对226枚超排卵周期中获得的未成熟卵母细胞行体外成熟(IVM)培养,发育至MⅡ期卵母细胞行卵胞浆内单精子显微注射(ICSI)授精,受精后卵母细胞随机分成非激活组和激活组,非激活组卵母细胞ICSI后不作任何处理直接移入G-1中培养,激活组卵母细胞ICSI后入7%无水乙醇作用6 min(即人工辅助激活),比较两组受精、卵裂、优质胚胎、囊胚及优质囊胚形成情况。结果:未激活组和激活组体外成熟率分别为83.9%(94/112)和82.5%(94/114)、受精率分别为80.9%(76/94)和76.6%(72/94)、优质胚胎率分别为14.3%(10/70)和25.0%(18/72),囊胚形成率分别为8.6%(6/70)和16.7%(12/72),两组间差异无统计学意义(P>0.05),但卵裂率激活组(100%,72/72)高于未激活组(92.1%,70/76),差异有统计学意义(P<0.05),优质囊胚率激活组(83.3%,10/12)明显高于未激活组(0%,0/6),差异有统计学意义(P<0.01)。结论:超排卵周期中的未成熟卵母细胞经体外培养成熟可获得比较满意的受精率及早期胚胎发育潜能,人工辅助激活技术为超排卵周期中获取的未成熟卵母细胞的充分利用提供一种新的思路     

巯基乙酸对小鼠MⅠ／MⅡ期卵母细胞进程及结构的影响

目的 探讨巯基乙酸(TGA)对卵母细胞体外成熟的影响.方法 采用小鼠卵母细胞体外成熟和免疫荧光染色法,观察巯基乙酸处理后小鼠MⅠ/MⅡ期卵母细胞的成熟进程和形态结构.结果 对照组、0.005 mg/ml TGA组和0.020 mg/ml TGA组生发泡破裂率分别为48.67%、62.67%和56.00%,第一极体排出率分别为26.47%、65.96%和45.24%,0.005 mg/ml处理组第一极体排出率高于对照组和0.020 mg/ml组,差异有统计学意义(P＜0.05).随着TGA处理剂量的增加,卵母细胞纺锤体和染色体形态结构异常的细胞比例和异常程度增加,0.020 mg/ml TGA处理组与对照组有显著性差异.结论 巯基乙酸可促进卵母细胞体外减数分裂的重启和成熟,但中期卵母细胞的纺锤体及染色体有结构缺陷,对后续胚胎发育可能会产生潜在影响.     

BDNF对小鼠卵母细胞体外成熟及发育能力的影响

目的:观察脑源性神经营养因子(BDNF)对小鼠未成熟卵的体外成熟及发育能力的影响。方法:以α-MEM为基础培养液,添加不同浓度(0、1、5、10 ng/ml)的BDNF以及FSH、FBS培养小鼠未成熟卵,并进行体外受精,观察卵母细胞成熟率、受精率和胚胎发育至囊胚的能力,了解不同培养条件下BDNF对卵母细胞发育能力的影响。结果:当体外成熟培养液中含有FSH和10%的FBS时,与体外成熟对照组比较,BDNF虽然不影响卵母细胞的成熟率和受精率,但BDNF 5 ng/ml组的囊胚形成率(75.00%)显著高于体外成熟对照组(56.63%),而接近体内成熟组(76.92%);当培养液中仅含FBS时,各组间卵母细胞成熟率和受精率没有差异,但与对照组比较,BDNF显著提高囊胚形成率;当培养液中不含FBS、FSH时,虽然无囊胚形成,但BDNF显著提高了卵母细胞的受精率。结论:BDNF能促进小鼠未成熟卵胞质的发育,提高卵母细胞的发育能力。     

蛋白酶体抑制剂对人晶状体上皮细胞的增殖抑制作用

目的 探讨蛋白酶体抑制剂MG132对SRA01/04细胞增殖的影响.方法 以不同浓度MG132处理SRA01/04细胞36 h,通过MTT微量比色法检测MG132对SRA01/04细胞活力的影响,通过流式细胞术（FCM）检测MG132对SRA01/04细胞凋亡及细胞周期的影响,通过AnnexinV/FITC-PI双染法观察MG132对SRA01/04细胞的影响.结果 随着浓度的提高（0、0.1、0.5、1.0、2.5、5.0、10.0 μmol/L）,MG132对SRA01/04细胞增殖的抑制作用逐渐增强.36 h时,半数有效抑制浓度IC50为2.50μmol/L.FCM检测细胞凋亡结果：2.5、5.0 μmol/L的MG132作用于SRA01/04细胞36 h,细胞早期凋亡率分别为（6.55±0.35）%和（13.75±3.18）%,而对照组为（0.75±0.21）%,差异有统计学意义（P＜0.01）.2.5、5.0 μmol/L MG132处理SAR01/04细胞48 h,G0/G1期细胞比例分别为（73.42±3.10）%,（80.95%±3.83）%,而对照组为（42.57±0.64）%,差异有统计学意义（P＜0.01）;S期细胞比例分别为（17.40±1.50）%,（19.57±1.29）%,而对照组为（49.44±1.36）%,差异有统计学意义（P＜0.01）.免疫荧光镜下,MG132诱导SRA01/04细胞的早期凋亡.结论 蛋白酶体抑制剂MG132诱导细胞凋亡、影响细胞周期来抑制SRA01/04细胞的增殖.蛋白酶体抑制剂可起到防治后发性白内障的作用.     

叔丁基对苯二酚对亚砷酸钠致Chang liver细胞毒性的影响

目的 探讨叔丁基对苯二酚(tert-butylhydroquinone,tBHQ)对亚砷酸钠(NaAsO_2)致Chang liver细胞毒性的影响.方法 Chang liver细胞培养48 h后分别以20、40、60和80 μmo/L的NaAsO_2染毒24和48 h,作为NaAsO_2单独作用组.以5和20μmol/L的tBHQ预处理Chang liver细胞24h,以40和60μmol/L的NaAsO_2染毒24和48h,作为tBHQ预处理组;对照组处理同NaAsO_2:单独作用组.每个浓度设3个复孔.用Alamar Blue法检测细胞活力.结果 NaAsO_2单独作用24和48 h组Alamar Blue还原率显著下降,与对照组比较,差异有统计学意义(P<0.05);且NaAsO_2单独作用48 h组的Alamar Blue还原率均显著低于24 h组,差异有统计学意义(P＜0.01).5 μmol/L的tBHQ预处理组与对应的60 μmol/L NaAsO_2单独作用24h组相比,显著提高了Alamar Blue还原率(P＜0.01);20 μmol/L的tBHQ预处理组的Alamar Blue还原率均显著高于相对应NaAsO_2单独作用24 h组(P＜0.05).5 μmol/L的tBHQ预处理组的Alaraar Blue还原率均显著高于相对应NaAsO_2单独作用48h组(P＜0.01);20μmol/L的tBHQ预处理组与对应的40μmol/L NaAsO_2单独作用48h组相比,显著提高了Alamar Blue还原率(P<0.01).结论 tBHQ能够降低NaAsO_2致Chang liver细胞的毒性,增强细胞对NaAsO_2毒性的抵抗能力.

Abstract：

Objective To study the antagonism of text-butylhydroquinone (tBHQ)to NaAsO_2 induced cytotoxicity in Chang liver cells in vitro.Methods Chang liver cells were exposed to NaAsO_2(0,20,40,60 and 80μmol/L)for 24 and 48 hours,or Chang liver cells were treated with tBHQ(5 and 20 μmol/L),then exposed to NaAsO_2(40 and 60 μmol/L)for 24 and 48 hours.The conditions of control group was the same as NaAsO_2 group.Alamar Blue was used to evaluate the viabifity of cells.Results Chang liver cells were exposed to NaAsO_2 for 24 and 48 hours,Alamar Blue reduction rates decreased significantly and Alamar Blue reduction rates of 48 hours group were lower than 24 hours group (P<0.01).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group was higher than 60 μmol/L NaAsO_2 group for 24 h(P<0.01);Alamar Blue reduction rate of 20μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 24 h(P＜0.05).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 48 h(P＜0.01);Alamar Blue reduction rate of 20 μmol/L tBHQ pretreatment group was higher than 40 μmol/L NaAsO_2 group for 48 h(P<0.01).Conclusion tBHQ can decrease the cytotoxieity induced by NaAsO_2 in Chang liver cells,increase the resistance to the cytotoxieity induced by NaAsO_2 in Chang liver cells.     

In vitro maturation of oocytes from non-stimulated common wombats

Assisted reproductive techniques, such as in vitro oocyte maturation in conjunction with in vitro fertilisation, may be used as a tool to manipulate reproduction. Using the common wombat as a model for the critically endangered northern hairy-nosed wombat, the present study examined whether oocyte maturation could be achieved under field conditions. At the time of collection, no oocytes were at the metaphase II (MII) stage (0/42). After 60 h culture using the submarine incubation system, 34% of oocytes (24/70) matured to telophase/MII, as indicated by the presence of a polar body. The proportion of oocytes that reached MII was higher for oocytes collected from follicles >2 mm in diameter compared with follicles <2 mm (40% v. 22%, respectively). The presence of cumulus cells alone did not influence the maturation potential. Oocytes without cumulus cells collected from follicles >2 mm in diameter had the highest maturation rate (58%). Maturation was not affected by the reproductive status of the common wombat or a delay of up to 5 h before oocyte collection. In conclusion, the present study demonstrated that oocytes collected from non-stimulated common wombats can mature to MII in culture.     

不同形态砷化合物对非致瘤性人源性肝细胞的毒性作用

目的探讨不同形态砷化合物对非致瘤性人源性肝(QSG7701)细胞的毒性及氧化应激作用。方法将处于对数生长期的QSG7701细胞分别暴露于亚砷酸钠(砷浓度为1、5、25、100μmol/L)、砷酸钠(砷浓度为10、25、100、500μmol/L)及MMA和DMA(砷浓度均为100、500、1 000、2 000μmol/L)培养24 h,并设对照(含10%胎牛血清的RPMI-1640培养基)。采用四甲基偶氮唑盐(MTT)法测定细胞活性;检测细胞培养液中乳酸脱氢酶(LDH)释放率、谷草转氨酶(AST)活力、总超氧化物歧化酶(T-SOD)活力和丙二醛(MDA)含量。结果一定浓度的As~(Ⅲ)(≥1μmol/L)、As~Ⅴ(≥10μmol/L)、MMA(≥100μmol/L)和DMA(≥2 000μmol/L)均能显著降低QSG7701细胞的存活率,差异有统计学意义(P0.05);且随着As~(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的存活率呈逐渐降低的趋势。经计算,QSG7701细胞暴露于As~(Ⅲ)、As~Ⅴ、MMA和DMA 24 h的IC_(50)分别为170.89、863.73、2 235.67、4 045.31μmol/L,对QSG7701细胞生长的抑制作用依次为As~(Ⅲ)As~ⅤMMADMA。在当As~(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA中的砷浓度为1 000、2 000μmol/L和DMA中的砷浓度为2 000μmol/L时,QSG7701细胞的LDH释放率均高于对照组,差异有统计学意义(P0.05);另外,当As~(Ⅲ)浓度为1~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA和DMA中的砷浓度为100~2 000μmol/L时,QSG7701细胞培养液中的AST活力均高于对照组;且随着As~(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的LDH释放率和细胞培养液中的AST活力呈上升的趋势。与对照组比较,当As~(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L时,QSG7701细胞中的MDA含量均较高,而T-SOD活力均较低;与对照组比较,当MMA和DMA中的砷浓度均为500~2 000μmol/L时,QSG7701细胞中的MDA含量均较高;而当MMA中的砷浓度为100~2 000μmol/L和DMA中的砷浓度为100、500μmol/L时,QSG7701细胞中的T-SOD活力均较低,差异有统计学意义(P0.05)。结论在本实验条件下,4种砷化合物均可不同程度地损伤肝细胞膜,破坏肝细胞的氧化平衡状态,产生氧化应激并导致脂质过氧化,对人肝细胞QSG7701的毒性作用依次为As~(Ⅲ)As~ⅤMMADMA。     

In vitro maturation of porcine oocytes with retinoids improves embryonic development

In the present study, the effects of retinoid metabolite administration during in vitro maturation (IVM) on oocyte maturation, parameters of in vitro fertilisation (IVF) and embryo development were examined. Varying concentrations of 9-cis retinoic acid (RA; 0, 5, 50 and 500 nm; Experiment 1) and all-trans retinol (ROH; 0, 125, 1250 and 12 500 nm; Experiment 2) were included in the maturation medium. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters or for 7 days to assess embryo development and quality. In Experiment 1, the oocyte maturation rate to metaphase II was significantly decreased (P < 0.001), with values below 5%, in the presence of the highest concentration of RA (500 nm). However, 5 and 50 nm RA had no effect compared with control. Treatment with 5 nm RA improved the blastocyst development rate (P < 0.001). In Experiment 2, the oocyte maturation rate did not differ between 125 and 1250 nm ROH treatment groups and control. However, treatment with 12 500 nm ROH was deleterious because no matured oocytes were observed following the treatment. The penetration rate was lower in the group treated with 1250 nm ROH compared with the 125 nm ROH-treated and control groups, but the blastocyst formation rate did not differ among the three groups. In conclusion, 5 nm RA in the IVM medium significantly increased the blastocyst formation rate, suggesting that RA may play an important role during IVM.     

Differential effects of linoleic and alpha-linolenic fatty acids on spatial and temporal mitochondrial distribution and activity in bovine oocytes

Using specific stains and confocal microscope imaging, the patterns of mitochondrial distribution, mitochondrial inner membrane potential and reactive oxygen species (ROS) levels during bovine oocyte maturation were determined in the presence or absence of physiological concentrations of linoleic acid (LA; 100μM) or α-linolenic acid (ALA; 50μM). Mitochondrial distribution in control oocytes at 0h was mainly peripheral and changed to a diffused pattern after 1h of culture; this was maintained up to 24h. Mitochondrial clusters were observed during the early hours of maturation (1-4h); the majority of these were arranged in perinuclear fashion. LA supplementation resulted in: (1) delayed redistribution of the mitochondria from a peripheral to a diffuse pattern and a decreased percentages of oocytes showing perinuclear mitochondrial clusters, (2) decreased mitochondrial inner membrane potential at 1 and 24h compared with the control and (3) higher ROS levels, associated with a lower nuclear maturation rate. In contrast, ALA supplementation had no effect on mitochondrial distribution and activity and decreased ROS levels compared with the control; this was associated with an increased nuclear maturation rate. In conclusion, LA induced alterations in mitochondrial distribution and activity as well as increasing ROS levels, which mediate, at least in part, the inhibitory effect on oocyte maturation.     

In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode's medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mM), lactate (10 mM) and/or pyruvate (0.5 mM) to mT (37-74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.     

α-亚麻酸对高糖损伤LLC-PK_1细胞的保护作用及其机制探讨

目的建立猪肾近曲小管上皮细胞(LLC-PK1)的高糖损伤模型,观察α-亚麻酸(ALA)对高糖损伤LLC-PK1细胞的保护作用并探讨其作用机制。方法 CCK-8试剂盒测定葡萄糖对LLC-PK1细胞增殖的影响,流式细胞术测定不同浓度ALA干预高糖损伤LLC-PK1的凋亡率和活性氧(ROS)含量。结果高糖环境可以抑制体外培养的LLC-PK1细胞的增殖,形成体外高糖损伤模型;经适当浓度(50～100μmol/L)的ALA干预后,前干预组和持续干预组细胞的凋亡率显著低于阳性对照组(P<0.05);当ALA浓度为10～100μmol/L时,持续干预组LLC-PK1细胞内ROS含量显著低于阳性对照组(P<0.05),当ALA浓度为50μmol/L时,前干预组LLC-PK1细胞内ROS含量显著低于阳性对照组(P<0.05)。结论高糖损伤LLC-PK1模型为研究DN肾小管上皮细胞的防治干预提供了良好的体外研究平台,ALA有望成为预防肾小管高糖损伤的保护剂,减少活性氧的产生可能是ALA保护肾小管上皮细胞的作用机制之一。