小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。     

小鼠胚胎干细胞与四倍体胚胎的嵌合

The oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos.The embryos were placed twenty at a time between two platinum electrodes laid 1 mm apart in 0.3M mannitol in the electrode chamber.The blastomeres were fused by a short electric pulse(80V for 50μsec) applied by a pulse generator.Fusion of blastomeres was usually completed in 20-60minutes.After 25 hours of culture,most of the tetraploid embryos developed to the four-cell stage.Zonae pellucidae of 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode‘s buffer.The embryos were plated on an ES cell layer,After 40 hours of coculture,248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipinets.Ten recipients were pregnant.but no live fetuses were born.Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and seven abnormal fetuses were obtained.The results demonstrate that ES cells derived from C57BL/6 mice are pluripotential to a certain extent.     

奶牛γ干扰素基因的高效表达及活性测定

经刀豆素(conA)刺激诱导奶牛外周血淋巴细胞,应用RT-PCR方法从其总RNA中对奶牛γ干扰素基因cDNA进行扩增,然后将特异性片段连接到pMD18-T载体,测序结果表明,与已知序列同源性为100%。然后将特异性片段连在pRLC载体上进行表达,经SDS-PAGE分析,原核表达产物为16kDa的重组蛋白,占菌体总蛋白的42%,表达产物以包涵体形式存在。经7mol/L盐酸胍的变性液溶解及0.5mol/L盐酸胍复性液处理,表达产物进行脱盐、凝胶层析纯化,细胞病变抑制法结果表明,重组牛IFN-γ具有较高的干扰素活性,约为6.0×105U/mg。     

油菜素内酯对陆地棉体细胞胚胎发生的影响

0.01ppmBR能使陆地棉Coker201,312两品种分化产生胚性愈伤组织,并有效地保持该种愈伤组织的生活力和胚胎发生能力。0.01ppmBR 0.5 ppmIAA促进Coker201,312两品种体细胞胚胎发生,0.01ppmBR 0.05ppm 2,4—D能诱导所有供试品种产生疏松黄绿色愈伤组织,2,4—D用量逐步降低或除去后,一些品种便分化产生胚性愈伤组织或体细胞胚状体。     

小鼠胚胎干细胞定向血管内皮和造血细胞分化体系的建立

目的:建立小鼠胚胎干细胞体外定向分化为血管内皮细胞和造血细胞的体系,并验证诱导后2种细胞的表面分子特征。方法:以小鼠胚胎成纤维细胞为饲养层,首先在无血清培养基StemPro中加入骨形态发生蛋白4(BMP4)、激活素A、碱性成纤维细胞生长因子(FGF-Basic)和血管内皮细胞生长因子(VEGF),诱导小鼠胚胎干细胞系R1/E 4 d后形成拟胚体;再将拟胚体消化后与OP9-DL1基质细胞共孵育,分别用干细胞因子(SCF)、VEGF和SCF、FLt3、白细胞介素3(IL-3)诱导向内皮和造血2个方向分化,并以CD31、CD45、CD144、Kit、CD201作为表面标志,流式检测诱导后细胞的表面分子特征和诱导效率;诱导10 d后免疫组化染色,进行内皮细胞的形态学鉴定。结果:诱导分化10 d后,免疫组化染色观察到多个内皮管状结构,流式检测CD31^+的内皮细胞比例为1.35%±0.05%,进一步分析CD31^+CD144^+CD45^-群体,有3.0%±0.2%的细胞表型为Kit^+CD201^+,提示该部分细胞可能是处于分化上游的内皮干祖细胞;CD45^+的造血细胞比例为35.0%±0.5%,其中0.35%±0.05%的细胞表达Kit和CD201,提示该部分细胞可能是处于分化上游的造血干祖细胞。结论:本研究将胚胎干细胞诱导为内皮细胞和造血细胞,并且能诱导出具有内皮、造血干祖细胞分子特征的细胞,可作为理想的体外诱导分化体系。     

胚胎和成体神经干细胞分化机制的研究

神经干细胞的分化调控一直是发育神经生物学的重要研究课题。综述了调节胚胎和成体神经干细胞分化的细胞内在因素和外部环境因素,初步探讨了胚胎和成体神经干细胞分化机制的差异。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

干扰素基因表达调控研究进展

干扰素基因表达调控研究进展孙劲综述方企圣审（中国预防医学科学院病毒研究所,北京１０００５２）近年来,有关于扰素（ＩＦＮ）分子生物学的研究以及基因工程方法生产干扰素取得很大进展。本文将有关ＩＦＮ蛋白的诱导和基因调控机理方面最新进展作一综述。ＩＦＮ蛋白的...     

胚胎胸腺移植配合蛇毒抗癌素治疗晚期肝癌

目的探讨生物效应调节剂,联合蛇毒制剂治疗晚期肝癌近期疗效。方法采用人胚胸腺组织或细胞移植方法,配合口服蛇毒抗癌素胶囊和（或）腹腔内注射蛇毒抗癌素注射液（以下简称治疗组）与５－Ｆｕ、ＭＭＣ及ＦＴ－２０７三联（以下简称对照组）对晚期肝癌进行随机分组治疗,通过对临床症状改善、免疫功能变化以及生存期长短等项目观察判断疗效。结果对晚期肝癌治疗,治疗组疗效优于对照组,也优于单纯应用胸腺移植治疗晚期肝癌的治疗效果。结论人胚胸腺组织或细胞移植及蛇毒抗癌素胶囊或其注射液抗癌效果是确切的,联合应用比二者单用效果更佳。     

Conceptus interferons and maternal recognition of pregnancy

Ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1) are secreted by ovine and bovine conceptuses for a restricted period when the conceptus acts to block regression of the corpus luteum. Both are considered to be antiluteolytic, acting locally on the endometrium to prevent the production of the uterine luteolysin prostaglandin F2 alpha. Molecular cloning of cDNAs for oTP-1 and bTP-1 has shown that both are structurally related to alpha interferons (IFN-alpha s). In particular, they resemble members of the 172-residue long IFN-alpha II (or IFN-omega) subfamily. Both proteins possess the antiviral and antiproliferative properties of IFN-alpha s and appear to bind IFN-alpha receptors. A novel and previously unsuspected role for IFNs in early pregnancy is apparent.     

Conceptus development in large white and prolific Chinese Meishan pigs

Large White (LW) and Meishan (MS) gilts were killed on Days 8, 10, 11, 12, 14 and 30 of gestation. Mean diameters (mm) for MS and LW conceptuses, respectively, were: Day 8, 0.45 and 0.69; Day 10, 2.7 and 1.9; Day 11, 5.3 and 2.7, with the differences among days being affected by breed (P less than 0.01). Variation in diameter among conceptuses from LW gilts was greater (P less than 0.01) than that for MS gilts on Days 8-11, respectively: Day 8, 20 and 46%; Day 10, 29 and 38%; and Day 11, 22 and 44%. Conceptuses had elongated in 3 of 5 MS and 1 of 4 LW gilts on Day 11, 6 and 6 MS and 2 of 4 LW gilts on Day 12 and all gilts of both breeds on Day 14. These results indicate that conceptuses of MS gilts develop more rapidly and more uniformly between Days 8 and 14 of gestation. Overall, embryonic survival for Days 8-12 for gilts not having elongated conceptuses was 90.2% for MS and 73.2% for LW gilts (P less than 0.01). On Day 30 of gestation, embryonic survival was also higher (P less than 0.01) for MS (89%) than LW (55%) gilts. However, embryonic weight, crown-rump length, placental length, allantoic fluid volume, amniotic fluid volume, as well as total glucose, fructose and protein in allantoic fluid were not affected by breed. Placental weight was greater (P less than 0.01) for LW gilts. Uterine development at Day 30 of gestation, based on total length and weight of uterine horns, width of uterine horns, total endometrial surface area and total endometrial weight was greater (P less than 0.01) for LW gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conceptus elongation in cattle: Genes, models and questions

In ruminants, more than 30% of the embryonic loss observed after artificial insemination has an early origin that is coincident with the marked elongation of the conceptus that occurs before implantation. During this developmental phase, physiological interactions are established between the conceptus and the uterus which are essential for the establishment of pregnancy and the elongation process. Our molecular knowledge of elongating conceptuses in cattle has long been focused on its analysis in view of its interactions with the uterus with the elongating stages being defined, like the uterus stages, by days post insemination or conception. The gene clusters reported so far indicate important pathways, some being shared by the non-elongating conceptuses of other mammals. However, to identify the key components of the elongation process - that could be specific to ungulates - new models are needed. Somatic nuclear transfer could be one of them as it provides complementary insights on differentiation beyond the blastocyst stage. Nonetheless, other models are necessary to convert gene lists or networks in elongating phenotypes. This review partly summarizes information on these topics, but data on the impact of the uterus on the elongation process or on the differentiation of the embryonic tissues are reviewed elsewhere.     

Conceptus influences the distribution of uterine leukocytes during early porcine pregnancy

Pregnancy in humans and rodents is associated with dramatic changes in leukocyte populations within the uterus. In these species, recruitment of leukocytes, mostly natural killer (NK) lymphocytes, accompanies decidualization of endometrial stroma even in the absence of pregnancy. In the pig, a nondecidualizing species, the predominant lymphocytes in the pregnant uterus are T and/or NK cells, but their distribution relative to embryonic attachment sites has not been reported. The objective of this study was to compare the abundance of leukocytes in porcine endometrium in contact with trophoblast with that between attachment sites during the early postattachment period. Uteri were recovered on Days 15-17 (n = 4), 18 and 19 (n = 4), 21 and 22 (n = 5), and 25-27 (n = 2) of gestation and from cycling pigs during the luteal phase (Day 15; n = 3). Leukocytes were identified in uterus obtained at versus between attachment sites using an antibody reactive with all leukocytes (CD44). In all pregnant animals, leukocytes were diffusely scattered throughout the endometrial stroma but were rare or absent in the luminal epithelium. Leukocyte density was approximately 3-fold greater in endometrium in contact with conceptuses than in endometrium between attachment sites throughout the early postattachment period. Leukocyte density during the luteal phase was similar to that between attachment sites, suggesting that leukocyte recruitment was a localized response to the embryo. The ability of an individual porcine conceptus to recruit maternal leukocytes to the adjacent stroma may be a vital step in early placental development and embryo survival.     

Conceptus development after vascular occlusion of the middle uterine artery in the pig

The middle uterine artery of gilts was occluded unilaterally or bilaterally from Days 25 to 70 after mating. The results showed that vascular occlusion of one (N = 7) or both (N = 6) middle uterine arteries during mid-pregnancy markedly reduced, compared with sham-operated controls (N = 7), development of the conceptuses and decreased peripheral oestrogen (oestrone + oestradiol-17 beta) concentrations in maternal blood.