Expression of LIF,VEGF, CD57 and CD68 after the transfer of rat embryos to mouse uteri

The high failure rate of interspecific preganacy is a major obstacle to the successful interspectific cloning of mammals,To in vestigate the reasons for the failure of inter-specfic pregnancy between rats and mice,we transferred rat blastocysts into mouse uteri on the third day of pseudopreg -nancy (D3),oure previous study showed that intact rat embryos could still be observed in mouse uteri on D9.In the present study ,we found that expression of CD57 and CD68 increased significantly at the maternal -fetal interface fol-lowing the transfer of rat embryos,Similarly ,Leukaemia inhibitory factor(LIF) expression increased ,but vascular endothelial growth factor(VEGF) expession decreased,In a co-culture system ,the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs,These results indicate that an increase in the immunological rejection response and a decrease in the in vasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.     

人输卯管组织与小鼠胚胎共培养的初步观察

为了检测人输卵管组织共培养系统对胚胎早期体外发育的影响,将超排得来的小鼠2细胞期胚胎与人输卵管组织共培养,或以人输卵管组织培养后的条件培养液进行培养。培养液为Ham's F10+10％FCS,并以其作为对照培养液。实验结果显示:无论是组织共培养或是条件培养液共培养,胚胎体外发育达囊胚率,胚胎逸出透明带率及胚胎的发育速度都较F10对照组高。其中输卵管壶腹部组织共培养及其条件培养液共培养对胚胎发育的促进作用较峡部大,而且其条件培养液共培养是以组织培养一周内所得的条件培养液效果最好。组织共培养和条件培养液共培养发育的胚胎正常分裂,没有差别,形态也均正常。实验提示人类输卵管上皮可能分泌某种因子能促进胚胎发育。     

Replacement of inner cell mass in mouse

The intra- or inter-strain reconstituted blastocysts were produced by replacing the inner cell mass of Kunming mouse blastocysts with that of Kunming or C57BL/6 mouse strain blastocysts. A total of 192 intra-strain reconstituted blastocysts were transferred into 17 pseudopregnant Kunming mice, and 2 reconstituted embryos were developed into term: while 115 inter-strain reconstituted blastocysts were produced, analysis of the reconstituted blastocysts showed that the morphology and cytoskeleton srtucture of the blastocysts were not different from those of normal blastocysts, however, no viable offspring was obtained after embryo transfer for these inter-strain reconstituted blastocysts. The results demonstrated that the intra-strain reconstituted blastocysts could normally develop into term, whereas the inter-strain reconstituted blastocysts possessed less developmental potential as the intra-strain reconstituted blastocysts. This study may give light to solve the problem of low implantation rate and placenta abnormality in mammal cloning.     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

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.     

Effect of matrix metallo-proteinase-26 (MMP-26) during embryo implantation in the mouse

Matrix metalloproteinase-26 (MMP-26, endometase and matrilysin-2), a novel member of the MMPs family, is detected not only in the placenta and uterus, but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. However, the function of MMP-26 in the reproductive system has never been reported. Expression of MMP-26 in mouse embryos and the function of the MMP-26 antibody during mouse embryo implantation was examined for the first time by injecting the uterine horn, immunohistochemistry,in situ hybridization, co-culture of mouse blastocysts and uterine monolayer epithelial cells, Western blot, RT-PCR, Northern blot and zymography. Our results show that there is strong expression of MMP-26 mRNA and protein in the mouse embryo. Furthermore, the MMP-26 antibody dramatically inhibited mouse embryo implantation and significantly inhibited adhesion and outgrowth of mouse blastocysts onin vitro uterine monolayer epithelial cells. At the same time, the MMP-26 antibody inhibited the expression of integrin αV mRNA and protein in a dose-dependent manner. These data suggest that MMP-26 may play a role in some of the tissue-remodeling events associated with the invasion of the endometrium by trophoblast cells and facilitate successfully embryo implantation.     

The expression and function of VEGF at embryo implantation “window” in the mouse

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays a critical role in angiogenesis. Recent reports indicated that VEGF was closely involved in embryo implantation and embryonic vasculogenesis. However, very little information is available about the detailed expression and function of VEGF at implantation “window”. In this work, VEGFs were primarily present on uterine epithelial cell monolayer and blastocysts including the outgrew trophoblasts at implantation window. VEGF antibodies decreased the number of mice embryos implanted and the percentage of blastocysts with attachment and outgrowth in a co-culture model in a dose-dependant manner. These findings demonstrate that VEGF is one of the essential cytokines for embryo implanta-tion in mouse. VEGF may act as a local mediator to regulate the maternal-fetal interaction, and facilitate blastocyst implantation.     

Secretion and regulatory mechanism of leptin during pregnancy in the golden hamster （Mesocricetus auratus）

Leptin, a 16 kDa polypeptide mainly secreted by white adipose tissue, has important function on the regulation of food intake and energy expenditure[1]. High concentration of leptin in the circulation suppresses food- intake, increases energy expenditure and therefore decreases the body weight; Accordingly, low concentration of leptin in the circulationstimulates food intake, decreases energy expenditure andincreases the body weight[1]. Normally, leptin level in thecirculation correlates with …     

Role of αVβ3 integrin in embryo implantation in the mouse

Integrin, a heterodimeric adhesive molecule composed of α and β subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the “implantation window” stage, α Vβ 3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that α Vβ 3 integrin was clearly expressed in the mouse blastocyst. Injection of α Vβ 3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, α Vβ 3 integrin antisera at 1︰100 and 1︰200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of α Vβ 3 integrin antiserum was dosage/dilution-dependent. Thus, α Vβ 3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at “implantation” stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.     

Experimental chimaeras--removal of reproductive barrier between sheep and goat

Following reciprocal embryo transfer between sheep and goats, the embryo of the foreign species is able to induce early pregnancy but the embryos do not survive beyond the first weeks of gestation. Similar results have been obtained from hybridization experiments on sheep and goats. While the causes for early embryonic death of hybrid eggs from donor sheep are unknown, the reciprocal event has been associated with immunological implications. As a means of overcoming the reproductive barrier between sheep and goats, we have transferred interspecific chimaeric embryos. We report here the birth of a goat kid from a sheep mother.     

组织芯片分析CD44v6和PCNA在口腔鳞癌及癌前病变中的表达及意义

目的：研究CDOv6和PCNA的表达与口腔鳞癌的发生、发展的关系。方法：应用组织芯片技术,通过免疫组织化学SP法分别检测一张组织芯片上117例组织样本中CD44v6、PCNA蛋白表达情况。结果：CD44v6在正常口腔黏膜组织和癌前病变呈阳性表达;癌组织中CD44v6的表达明显,但其表达程度随肿瘤恶性程度的增高而降低,PCNA的表达程度随着增生细胞和癌分级增加而升高。结论：在正常口腔黏膜上皮、增生上皮以及鳞癌中这两种蛋白的表达之间呈负相关。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点。     

Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction

Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.     

小鼠胚胎移植技术优化及对巨细胞病毒净化研究

目的 建立小鼠巨细胞病毒净化方法,以获得无MCMV感染的小鼠。方法 利用胚胎移植技术,通过使用不同激素、不同发情周期注射激素、受体鼠品系、不同的移植方法、不同时期胚胎移植,以及移植胚胎数量等对比试验,优化了净化条件。利用优化的胚胎移植净化方法,对供体鼠进行了净化。结果 SIGMA生产的激素,在10 IU剂量下超排得到的可用胚胎数量约为17枚/只;在小鼠发情间期超排得到的可用胚胎最多,超排的可用胚胎数约为23枚/只;选取C57雄性小鼠与ICR雌性小鼠交配的子一代作为受体鼠,产仔率达44.5%;输卵管移植较子宫移植效果好,产仔率为40.64%;移植2细胞胚胎的妊娠率为80%,明显优于单细胞和8细胞;受体鼠移植24枚胚胎时,产仔率达到了43.75%。利用优化的小鼠巨细胞病毒胚胎移植净化方法,净化得到了无MCMV感染的小鼠。结论 建立了小鼠巨细胞病毒净化方法,为获得无MCMV感染的小鼠种群提供了可靠的保障。     

Monoclonal mice generated by nuclear transfer from mature B and T donor cells

Cloning from somatic cells is inefficient, with most clones dying during gestation. Cloning from embryonic stem (ES) cells is much more effective, suggesting that the nucleus of an embryonic cell is easier to reprogram. It is thus possible that most surviving clones are, in fact, derived from the nuclei of rare somatic stem cells present in adult tissues, rather than from the nuclei of differentiated cells, as has been assumed. Here we report the generation of monoclonal mice by nuclear transfer from mature lymphocytes. In a modified two-step cloning procedure, we established ES cells from cloned blastocysts and injected them into tetraploid blastocysts to generate mice. In this approach, the embryo is derived from the ES cells and the extra-embryonic tissues from the tetraploid host. Animals cloned from a B-cell nucleus were viable and carried fully rearranged immunoglobulin alleles in all tissues. Similarly, a mouse cloned from a T-cell nucleus carried rearranged T-cell-receptor genes in all tissues. This is an unequivocal demonstration that a terminally differentiated cell can be reprogrammed to produce an adult cloned animal.     

有丝分裂原体活化成人外周血自然杀伤细胞CD69表达的研究

目的 :利用自然杀伤细胞 (NK)的早期活化标记CD69的表达探讨NK细胞体外活化的规律 ,促进这类细胞的临床应用 方法 :用Ficoll-Hypaque梯度离心法分离出正常成人外周血单个核细胞 (PBMC) ,分别在两种不同的有丝分裂原 ,植物血凝素 (PHA ,2 0 μg/mL)和佛波醇酯 (PDB ,10 - 7mol/L)存在的条件下进行培养 ,于 0、2、12、2 4h收获细胞后进行双色免疫荧光标记 ,以流式细胞术对NK细胞的CD69分子表达情况进行分析 结果 :NK细胞在PHA、PDB刺激 2h后CD69的表达明显上升 ,而且随时间的延长CD69的表达率逐渐增加 ,2 4h分别达到 ( 84 .96± 9.2 4 ) %、( 91 85± 2 .94 ) % ;PDB刺激组CD69的表达率在不同的时间点均比PHA组高 结论 :体外条件下 ,PHA、PDB均能快速上调NK细胞CD69表达 ,而且PDB( 10 - 7mol/L)作用明显强于PHA( 2 0 μg/mL)     

SPF 金黄地鼠种群标准化管理与质量控制

摘要: 金黄地鼠( Golden hamster; Mesocricetus auratus) 又称叙利亚地鼠,金黄色,成年个体体质量120-150 g,染色体 22 对。目前世界各国饲养的不同品系的金黄地鼠,都是1930 年在叙利亚发现的同胞后代,由于同一祖先,遗传学上血缘相近。金黄地鼠被广泛用于微生物学,肿瘤学,内分泌学,组织移植等的研究,此外还被大量用于制造乙型脑炎疫苗和狂犬疫苗。成都公司长期使用金黄地鼠制造乙型脑炎减毒活疫苗,本公司2003 年底建立SPF 金黄地鼠核心种群, 2004 年3 月建立规模化生产种群, 13 年来累计生产SPF 金黄地鼠180 万只,供应乙脑减毒活疫苗生产150 万只,累计生产乙型脑炎减毒活疫苗2. 3 亿人份,创造产值21 亿元人民币,其中乙脑疫苗出口创汇9 000 万美元。为了长期保持SPF 金黄地鼠种群遗传稳定性和质量稳定性,我们建立了一整套标准化的饲养管理制度和质量控制体系, 10 多年来,无论是公司自检,还是送中检院国家专业实验室检测或是送国外专业实验室检测,均未检测出需要排除的病原微生物和寄生虫, 2013 年我公司的乙脑减毒活疫苗制品成功通过WHO 预认证,作为关键原材料的SPF 金黄地鼠质量也得到了WHO 检查官的认可。     

Cytological mechanisms of interspecific incrossability and hybrid sterility between Oryza sativa L. and O. alta Swallen

Oryza sativa and O. alta belong to AA and CCDD genomes in Oryza, respectively. Interspecific repro-ductive isolation limits the transfer of favorable genes from O. alta into O. sativa. The cytological mechanisms of interspecific incrossability and hybrid sterility between O. sativa and O. alta were studied systematically in this paper. We indentified two cytological causes of interspecific incrossabil-ity. First, we observed embryo sac incompatibility that caused fertilization barriers of variable severity such as non-fertilization, fertilization stagnation and egg cell single-fertilization. Second, we observed hybrid inviability, the major cause for incrossability, apparent from hybrid embryo developmental stagnation and embryo abortion. Hybrid sterility included both embryo sac sterility and pollen sterility. The hybrid embryo sac was completely sterile and exhibited mainly embryo sac degeneration. Hybrid pollen was also sterile and mainly typical abortive. Hybrid sterility was mainly caused by severely ab-normal meioses of megasporocytes and pollen mother cells; it is the most important abnormality, being chromosome sterility. Several methods are suggested to overcome the interspecific reproductive iso-lation between O. sativa and O. alta.     

南阳黄牛CD14基因扩增及序列分析

根据GenBank发表的荷斯坦奶牛CD14基因的序列设计引物,通过PCR方法对南阳黄牛的CD14基因进行分段扩增并测序,拼接后得到包含CD14完整编码区以及5’端和3’端非编码区的2 969 bp的全序列。序列分析结果表明:南阳黄牛CD14基因开放阅读框全长966 bp,共编码321个氨基酸,碱基组成分别为A(18.4%)、T(18.4%)、C(32.9%)、G(30.2%),编码区与荷斯坦奶牛CD14基因相比发生了2个碱基突变,没有引起氨基酸的改变。在5’端和3’端存在较长的非编码区,与荷斯坦奶牛CD14基因相比发生了7个碱基突变。南阳黄牛的CD14基因与荷斯坦奶牛、水牛、绵羊、山羊、猪、猕猴、大猩猩、人、小鼠的同源性依次降低,分别为99.8%、98.2%、96.8%、92.8%、83.5%、79.8%、79.7%、79.5%、71.9%。进化树所得的聚类结果与传统的分类结果一致。通过蛋白质结构预测,发现南阳黄牛CD14没有跨膜结构域。     

昆明小鼠胚胎移植技术的研究

目的研究不同时期胚胎对胚胎移植成功率的影响,以提高胚胎移植率。方法采用经超数排卵的KM雌鼠与正常615雄鼠交配,取受精卵,分别将1-细胞胚、2-细胞胚和桑椹胚、囊胚分别移入发情的假孕KM雌鼠的输卵管和子宫中,妊娠产仔。结果移入1-细胞胚、2-细胞胚、桑椹胚和囊胚的假孕母鼠妊娠率分别为36.36%、42.86%、10%、27.27%,产仔率分别为46.58%、38.46%、58.33%、60%。结论2-细胞胚的移植可提高小鼠的胚胎移植成功率,子宫移植可提高小鼠的产仔率。