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.     

猪克隆胚胎激活方法优化与转人溶菌酶基因克隆猪的生产

为了提高转基因克隆效率和获得转人溶菌酶基因克隆猪,研究了不同电激活参数和化学辅助激活方法对猪克隆胚胎和孤雌胚胎体外发育的影响.结果发现:电场强度会显著影响克隆胚胎的融合率和体外发育能力(P<0.05),电脉冲次数对克隆胚胎体外发育促进作用不显著(P>0.05),而相同电激活条件下克隆胚胎和孤雌胚胎的体外发育能力变化趋势不同;电激活后再利用放线菌酮+细胞松弛素B(CHX+CB)处理4 h能显著提高克隆胚胎的囊胚率(P<0.05),而用二甲基氨基嘌呤(6-DMAP)处理没有提高克隆胚胎囊胚率(P>0.05),但6-DMAP或CHX+CB处理均可显著提高孤雌胚胎的囊胚率(P<0.05).上述结果表明,最佳的孤雌激活条件并不一定是克隆胚胎的最佳激活条件.本研究中猪克隆胚胎的最佳激活方法为1.6 kV/cm、100μs、2次直流电脉冲间隔100μs,再辅以CHX+CB处理4 h.利用优化的激活条件成功获得了乳腺特异表达人溶菌酶的转基因猪,为猪转基因育种奠定了基础.     

化学激活和季节对克隆猪出生率的影响

为了解化学激活对克隆猪出生效率的影响, 研究了体外成熟的猪卵母细胞被激活恢复减数分裂到克隆猪出生的整个过程. 首先研究了电激活(Ele), Ele+CHX+CB和Ele+6-DMAP 3种激活方法对猪卵母细胞孤雌激活(parthenogenetic, PA)和核移植(nuclear transfer, NT)重构胚体外发育的影响. 比较了Ele或Ele+CHX+CB激活方法对克隆猪出生效率的影响. 实验中单独列出了PA胚的扩张囊胚率或NT优质囊胚率来代表囊胚的质量. 结果表明: 化学联合激活提高了PA的囊胚率和扩张囊胚率, 但对PA胚的卵裂率和囊胚细胞数没有显著影响(P>0.05). Ele+6-DMAP对PA胚的囊胚率和扩张囊胚率有显著提高(P<0.05), 但对NT胚的囊胚率和优质囊胚率没有提高甚至降低了NT胚的发育. Ele+CHX+CB虽然提高了NT胚的囊胚率(P<0.05), 但对优质囊胚率没有影响. Ele+CHX+CB激活方法使克隆猪出生率有所提高但不显著. 本文还研究了季节对猪孤雌发育和克隆猪出生率的影响. 结果表明, 春季收集的猪卵母细胞使用3种激活方法卵母细胞的孤雌囊胚率均高于冬季收集的猪卵母细胞的囊胚率. 春季和冬季进行移植, 克隆猪出生率没有区别. 综上, 在PA中获得的结果与NT胚中获得的结果不一定完全匹配, 说明孤雌激活和重构胚的激活机制还是有区别的. 化学联合激活虽然能提高囊胚率, 但它的作用相当于降低囊胚形成的门槛, 却不是从本质上改变囊胚发育能力, 因此不能显著提高克隆猪出生效率. 春季收集的卵母细胞在体外培养中的发育能力好于冬季收集的卵母细胞, 但季节对克隆猪出生率没有显著影响.     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚     

猪体细胞核移植重构胚的体外发育

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚率达到11．7％,囊胚率为6．7％,显著高于成纤维细胞重构胚（P＜0．05）。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导G0/G1期,抽吸法／解剖法采集卵母细胞,体外培养33－44h,将卵丘细胞放至去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6－DMAP激活处理,体外培养6d。研究表明,卵母细胞采集方法、激活液中细胞松驰素（CB）、激活程度并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,体外能发育至囊胚。     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

体细胞的组织来源及培养代数对猪核移植重构胚发育的影响

本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后,将经血清饥饿(0．5?s)培养2-9d、0．1mg/L Aphidicolin (APD)培养 0．5?S培养2-9d或一般培养法(10?S)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞,直接注射到去核的卵母细胞质中,或注射到卵周隙中。再经电融合(100V/mm,30μs,电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6- DMAP激活处理,体外培养6天。耳皮成纤维细胞和颗粒细胞经0．1mg/L APD 0．5?S培养处理后的重组胚卵裂率,均高于血清饥饿和一般培养处理的同种供体细胞(P＜0．01)。卵丘细胞、颗粒细胞经0．1mg/L APD 0．5?S处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P＜0．05)。以猪颗粒细胞为核供体时,电融合法的重构胚分裂率显著高于胞质内注入法(P＜0．05),但囊胚发育率无显著差异(P＞0．05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞,其具有正常二倍染色体的细胞比例均无显著差异(P＞0．05);以这2种细胞不同培养代数做供体进行核移植时,各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P＞0．05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定;(2)0．1mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果,血清饥饿培养则无明显效果;(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞．核移植后都能得到体细胞克隆的囊胚,但前者的效果略优于后者,且其核移植效果不受供核细胞培养代数的影响;(4)电融合核移植胚胎的发育率高于胞质内直接注入法,但两者的总体效率相近。     

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。     

培养基中能量底物对猪胚胎发育的影响

旨在探讨丙酮酸和乳酸对猪(Susscrofa)胚胎早期发育的影响,将NCSU-23培养基中的5.56mmol/L葡萄糖替换为0.2mmol/L丙酮酸、5.7mmol/L乳酸,并将此培养基命名为mNCSU-23。根据实验设计,孤雌胚及核移植胚转移到mNCSU-23或NCSU-23中培养。激活第2天统计孤雌胚及核移植胚中的5～8细胞胚胎数。激活第6天统计孤雌胚及核移植胚囊胚形成率及囊胚细胞数。实验结果表明,mNCSU/NCSU处理组的5～8细胞胚胎数及囊胚数显著高于对照组(P0.05);单纯使用mNCSU培养猪胚胎时,囊胚率最低,发育结果最差(P0.05)。本研究证实,在体外培养前两天,用乳酸和丙酮酸代替培养基中的葡萄糖对胚胎发育有利。     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56.7%,发育至桑椹胚达11.7%、孵化囊胚率为6.7%,显著高于成纤维细胞组成的重构胚(p<0.05)。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至GO或G1期,抽吸法/解剖法采集卵母细胞,体外培养33或44 h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理,体外培养6天,结果表明,卵母细胞采集方法、激活液中细胞松弛素(CB)并不影响重构胚的发育(以卵龄44h的卵母细胞为受体);而以电脉冲结合6-DMAP激活处理能提高重构胚发育能力(以卵龄33 h的卵母细胞为受体)(p<0.05)。本研究显示,以电脉冲结合6-DMAP激活卵丘细胞重构胚,能在体外发育至囊胚     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

Dietary phytoestrogens and cancer: in vitro and in vivo studies

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16α-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Trichinella spiralis and Trichinella pseudospiralis induce collagen synthesis by host fibroblasts in vitro and in vivo

Immunoperoxidase staining of muscle infected with Trichinella spiralis for murine collagen types I and IV provided both qualitative and quantitative evidence of extensive synthesis of both types of collagen by fibroblasts in infected muscle compared to that seen uninfected muscle. Moreover, fibroblasts in muscle infected with T. pseudospiralis, a nonencapsulating species, showed significantly less staining for both types of collagen compared to muscle from mice infected with T. spiralis. Analysis of collagen composition of isolated nurse cells using an ELISA specific for either type I or type IV murine collagen suggested that of these 2 types of collagen, only type IV basement membrane collagen is found in Trichinella capsular collagen. Excretory/secretory products of T. spiralis and T. pseudospiralis induced extensive synthesis of exclusively type IV collagen by 3T3 murine fibroblasts in vitro.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10⁶ cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10⁶ cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.