昆明小鼠原核胚在不同培养液中的体外发育

目的优化昆明小鼠原核胚胎体外培养系统,提高胚胎发育率.方法小鼠经超排获得原核期胚胎,制备小鼠输卵管上皮共培养系统,使用M16、CZB和KSOM培养液进行体外培养,并对体内和体外发育的囊胚细胞计数.结果在KSOM和CZB中添加胎牛血清能显著提高胚胎囊胚发育率(14.71%对85.71%;6.45%对10.81%);输卵管上皮共培养可以提高胚胎的卵裂率和囊胚发育率,同时提高胚胎质量和同步发育,小鼠胚胎在KSOMFBS中囊胚发育率达85.19%,显著高于CZB和M16.结论在小鼠输卵管上皮共培养条件下,KSOMFBS能够很好支持昆明小鼠原核期胚胎体外发育.     

共培养体系在牛核移植胚体外发育培养中的应用

采用电融合法构建牛体细胞核移植重构胚,分析共培养细胞类型、传代次数、细胞冻-融以及蛋白质添加物(BFF和FBS)对牛体细胞核移植胚体外发育的影响,探讨胚胎体外共培养的条件,以建立优化的共培养体系。结果表明与非共培养组相比,共培养组重构胚的囊胚发育率以及胚胎细胞数显著增加(P<0.05),而输卵管上皮细胞共培养组同颗粒细胞共培养组相比胚胎细胞数显著增加(P<0.05),更适合做共培养细胞;随着共培养细胞传代次数的增加重构胚囊胚发育率及胚胎细胞数显著下降(P<0.05),共培养细胞在冷冻处理后重构胚的囊胚率和胚胎细胞数都显著下降(P<0.05);BFF较FBS更能促进牛核移植胚的囊胚发育率(P<0.05)。表明应用新鲜原代输卵管上皮细胞进行牛核移植胚胎的共培养,并在SOFaa添加10?F能够有效促进核移植胚胎的体外发育。     

牛体外受精卵在两种共同培养系统中发育的研究

为了使牛体外受精卵能通过体外早期发育阻滞期,我们建立了卵丘细胞单层(A)和输卵管上皮单层(B)两种共同培养系统。A1共同培养实验是在本实验室进行的,A2和B共同培养实验均在日本岗山大学农学部动物繁殖学研究室进行,牛输卵管组织的分离使用0.76％EDTA—PBS溶液,共同培养系统均使用含10％小牛血清的TCM—199(Earle's salts)作为培养液。培养的卵泡卵母细胞体外成熟率为100％,体外受精率为99—100％。在A1共同培养实验中,越过阻滞期发育到16—细胞以上的胚胎占卵裂胚的35.7％,与A2共同培养实验中越过阻滞期的发育率(40.1％)无显著差异(P<0.05)。A1和A2共同培养实验,在卵裂基础上得到的桑椹胚和囊胚发育率分别为23.7％和27.9％。每百枚培养的卵母细胞,在A1共同培养实验中可获得桑椹胚和囊胚15.1枚,在A2共同培养实验中可获桑椹胚和囊胚的20.5枚。B共同培养实验中桑椹胚和囊胚发育率为54.1％,显著高于A1或A2共同培养实验的相应发育率(P<0.001),使用B共同培养系统每百枚培养的卵母细胞可以获得37枚桑椹胚和囊胚。     

小鼠四倍体早期胚胎基因组甲基化模式

利用小鼠抗5-甲基胞嘧啶(5MeC)单克隆抗体检测了体外培养小鼠四倍体早期胚胎的基因组甲基化模式。结果表明: 利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程, 在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样, 呈现高度甲基化状态; 在细胞核开始融合的时候, 甲基化水平急速下降, 在细胞核完全融合的时候甲基化水平达到最低点; 随着胚胎继续分裂, 胚胎甲基化水平逐渐增加, 在桑葚胚期甲基化水平最高; 但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别, 这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。因此, 小鼠体外培养四倍体胚胎的甲基化模式是不正常的, 这可能是四倍体小鼠难以发育到妊娠足月的原因之一。这是对小鼠四倍体早期胚胎基因组甲基化模式的首次报道。     

小鼠原核期受精卵体外培养系统的筛选

为了筛选出最佳的小鼠原核期受精卵的体外发育培养系统,分别进行了四个试验。试验I:在体外分别用自配的M16、mM16、KSOM、mKSOM、CZB进行体外发育培养,进而筛选出一种最佳的体外培养系统;试验Ⅱ、Ⅲ、Ⅳ分别:探讨了血清、PVA、rhLIF对小鼠胚胎发育的影响。结果,试验I中胚胎发育到2-细胞的比率差异不明显,但是在mM16和mKSOM中,发育到4-细胞的比率94.7%,90.7%(91/96;78/86)和发育到桑椹胚/囊胚的比率分别为78.1%,67.4%(75/96;58/86)均明显高于其他三种培养液;试验II用10?S代替M16中BSA时,胚胎的发育率均下降,即使在mM16中桑椹胚/囊胚率仅为4.8%(12/35)与对照组M16(40.5%)差异显著(p<0.05);试验Ⅲ用PVA取代mM16和mKSOM中的BSA其体外发育率显著下降,胚胎均无一例发育到桑椹胚/囊胚;试验Ⅳ:rhLIF能提高胚胎在体外的发育率可使mM16培养的胚胎囊胚率、囊胚脱出率分别达到84%(47/56)、39.2%(22/56)。结论:在不添减其他成分前提下,只在M16中添加0.1mMolEDTA、0.5mMol牛磺酸、1000IU/mlrhLIF便可获得84%的囊胚率,同时证明在M16或mM16添加血清都会降低其体外发育率;PVA还不能有效的取代mM16、mKSOM中的血清。     

兔输卵管上皮细胞解除大鼠早期胚胎发育阻滞的研究

本研究利用兔输卵管上皮细胞,与异种动物大鼠的受精卵共培养,结果大量出现突破２细胞发育阻滞的现象。体外受精卵和体内受精卵的２细胞发育阻滞突破率分６２％和７３％;利用ＲＯＥＣ条件培养液培养大鼠的体外受精卵,２细胞发育阻滞的突破率达６８％,且能顺利发育至桑椹胚和囊胚。将ＲＯＥＣ直接培养在含＾３５Ｓ－甲硫氨酸的ＣＺＢ培养液中,经ＳＤＳ－聚丙烯酰胺凝胶电泳及放射自显影,发现在该条件培液中出现了分子量分别为１     

胰岛素对小鼠胚胎印迹基因Igf2/H19甲基化水平和表达的影响

探讨胰岛素对小鼠早期胚胎体外发育影响的分子机理.将小鼠2细胞胚胎培养于KSOM+0.25 μg/ml胰岛素培养基内,发育至桑椹胚和囊胚.提取桑椹胚和囊胚的总RNA和DNA.实时定量PCR分析,实验组桑椹胚Igf2 mRNA表达是对照组的4.7倍,H19表达是对照组的5.7倍;实验组囊胚Igf2 mRNA表达是对照组的1.8倍,而H19表达量是对照组的2.3倍;BSP测序法分析Igf2/H19印迹控制区的甲基化水平,实验组桑椹胚、囊胚的甲基化率分别为7.3%和32.3%,分别比对照组下降86.4%和35.4%.结果显示,胰岛素降低植入前胚胎Igf2/H19印迹控制区DNA甲基化的水平,从而使Igf2和H19基因表达升高.     

Scriptaid处理可提高近交系五指山猪克隆胚胎的发育能力和克隆效率

为探讨一种新型低毒的组蛋白去乙酰化酶抑制剂Scriptaid处理克隆胚胎时对其发育能力和克隆效率的影响,本研究以近交系五指山小型猪胎儿成纤维细胞为供体细胞进行体细胞核移植构建重构胚胎,重构胚胎激活后培养在添加Scriptaid不同浓度(0～300 nmol/ L)的胚胎培养液中培养不同的时间(0～36 h),观察克隆胚胎的卵裂率和囊胚率,评价克隆胚胎体外的发育能力.实验结果发现100 nmol/L Scriptaid处理24 h组克隆胚胎的囊胚发育率(30.4%)较对照组(17.5%)显著提高,P<0.05.将100 nmol/L Scriptaid处理24 h组克隆胚胎和对照组胚胎分别移植到4头受体母猪中,进一步观察其体内的发育能力.处理组克隆胚胎的受体在平均窝产仔数和克隆效率(分别为5头,2.4%)均显著高于对照组(分别为1.5头,0.7%),P<0.05.以上结果表明,100 nmol/L Scriptaid处理24 h近交系五指山小型猪克隆胚胎,有利于提高克隆胚胎的发育能力和克隆效率.     

兔输卵管上皮细胞解除大鼠早期胚胎发育阻滞的研究

本研究利用兔输卵管上皮细胞(ROEc), 与异种动物大鼠的受精卵共培养,结果大量出现突破2细胞发育阻滞的现象。体外受精卵和体内受精卵的2细胞发育阻滞突破率分别为62%和73%;利用ROEC 条件培养液培养大鼠的体外受精卵,2细胞发育阻滞的突破率达68%,且能顺利发育至桑椹胚和囊胚。将ROEC直接培养在含~(35)S-甲硫氨酸的CZB 培养液中,经SDS-聚丙烯酰胺凝胶电泳及放射自显影,发现在该条件培液中出现了分子量分别为135Kd,68Kd,55Kd,51Kd 和44Kd 由ROEC 分泌的多肽,其中68 Kd 区带最显著,而135 Kd区带非常弱。用(125)~I-酰化剂标记示踪发现,与ROEC 共培养24 h 的大鼠胚胎透明带上含有68Kd 蛋白,并发现55 Kd 蛋白与透明带结合的痕迹。这些蛋白很可能就是胚胎发育调控由母型向合子型过渡并突破早期发育阻滞的关键因子,在功能上似无种属特异性。     

IVF-20培养液用于大鼠体外受精的实验研究

目的探讨人用体外受精液用于实验大鼠体外受精的可行性,为实验大鼠的胚胎保种提供参考。方法选用人体外受精IVF-20培养液作为大鼠精子获能和受精培养液,对SD、Wistar、GK、F344等四种不同品系的实验大鼠进行体外受精;用mR1ECM培养液对体外受精所得胚胎进行体外培养实验。同时,将所得2-细胞胚胎移植给经假孕处理的受体鼠。结果四个品系大鼠体外受精后的卵裂率分别可达83.2%、72.6%、87.8%和71.6%。体外受精卵裂胚能够在体外进一步发育,SD大鼠囊胚发育率为16.7%,与体内受精胚胎在体外培养的囊胚发育率(24.5%)差异不显著。80枚2-细胞胚胎移植给2只受体鼠后均妊娠成功,产下正常仔鼠10只,产仔率12.5%。结论用于人体外受精的IVF-20培养液同样适合于实验大鼠精子的体外获能和受精,可以在多种品系获得较高的卵裂率,所得卵裂胚能够在体外发育到囊胚,并且所得卵裂胚在移植给受体鼠后能够产下正常仔鼠。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.