小鼠早期胚胎发育期间TGF—β免疫组织化学定位

The distribution of transforming growth factor beta-1 (TGF-beta-1) in the early developing mouse embryos between day 1 and day 12 of gestation was examined by immunohistochemical techniques. Polyclonal rabbit antiserum raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta-1 from human platelets was used. The following results were obtained: 1. Embryonic cells of early cleavage stages (2, 4 and 8 cells) and late morulae showed positive immunofluorescent reaction without any difference in staining intensity (Plate I, Figs. 1-4). 2. Marked staining of blastocysts in toto or sections with anti-TGF-beta-1 antibodies by either immunofluorescence or immunoperoxidase reaction was also observed. Inner cell mass (ICM) cells and trophoectoderm cells were both reacted, but more intense staining was found in primary endoderm cells differentiated from ICM cells adjacent to blastocoele (Plate II, Fig. 5). 3. Scattered granules stained strongly with immunoperoxidase reaction were present in embryonic ectoderm and visceral endoderm surrounding the forming mesoderm which was only slightly stained (Plate II, Fig. 6). 4. Intense immunoperoxidase staining was also present in mesoderm of visceral yolk sac of day 8 and day 10 embryos (Plate II, Fig. 7). 5. During the formation of somites, neural tube and limb bud, remarkable staining was found in mesenchyme, individual cells of somites, mucous layer of gut tubes, heart and limb buds (Plate III, Figs. 8-10). No significant staining was seen in neural cells per se except the inner surface of neural tube. The results of present studies indicate that abundant TGF-beta-1 is present in preimplantation mouse embryos including cleavage, morulae and blastocyst stages. In postimplantation embryos, TGF-beta-1 appears to play an important role in the differentiation of endoderm and mesoderm, particularly in the development of extraembryonic tissues, and in later morphogenetic and histogenetic events involving mainly mesoderm or mesenchyme cells.     

小鼠细小病毒非结构基因转基因小鼠的建立

为探讨小鼠细小病毒（ＭＶＭ）非结构蛋白在ＭＶＭ感染中的抗肿瘤作用,酶切表达质粒ｐＵＬＢ３２３８获取该非结构基因,通过显微注射法接种入小鼠受精卵内制备转基因鼠。共注射受精卵７２０枚,选取存活受精卵２２５枚植入假孕小鼠输卵管,产仔１４只。转基因小鼠尾部组织ＰＣＲ法ＤＮＡ检测证明,其中４只整合靶基因。整合转基因的４只Ｇ０代小鼠与正常Ｃ５７ＢＬ／５Ｊ小鼠配种均可生产整合靶基因的小鼠。ＲＴ－ＰＣＲ法ｍＲＮＡ     

中国小型猪胚胎生殖细胞的培养和建系

首次报道分别从28天和27天中国小型猪胚胎生殖嵴的原始生殖细胞培养和建立了两株胚胎生殖细胞系(embryonic germ cells,EG cells),各命名为BPEGl和BPEG2。根据它们的生长形态、细胞专一性标志以及体内、外分化等特征,证明了它们具有多能性的特性。这些猪EG细胞具有潜在的应用前景。     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

人多潜能胚胎生殖细胞的分离和培养（简报）

To establish human pluripotent embryonic germ (EG) cell lines, human primordial germ cells (PGCs) of embryos aborted in 5-9 week were cultured on inactive mouse STO fibroblast feeder. The medium contained human leukemia inhibitory factor (hLIF), human basic fibroblast growth factor (hbFGF) and forskolin. The EG cells could be passaged continuously until 12 generations. Most cells were positive in alkaline phosphatase staining and expressed cell surface antigen SSEA-3 and pluripotent marker Oct-4. These EG cell populations that retained normal karyotype could form embryoid body in culture and differentiate further into neuron-like cells, mucous epithelial cells, epithelial cells and other types of the cells spontaneously. These results indicated the cell clones derived from human PGCs resemble pluripotent EG cells from mouse PGCs in appearance or nature.     

体外小鼠胚胎性癌细胞BEC—B_7建系和特性

BEC—B_7胚胎性癌细胞 (Embryonal Carcinoma cells,简称EC细胞)是来自129/SV纯系小鼠自发睾丸畸胎癌,经同系小鼠腹腔传至第七代的血性腹水畸胎癌细胞,建株于1981年9月。培养方法是在无菌条件下取出含癌细胞的血性腹水,经自然沉降取得的EC细     

小鼠胚胎性癌细胞研究的一些近况

胚胎性癌细胞(Embryonal Carcinoma,简称EC细胞)是畸胎癌的恶性干细胞,它既有恶性生长性质,又有类似于早期正常胚胎细胞的多能性,能分化为神经、皮肤、肌肉、软骨和腺管等组织。EC细胞注射到同系小鼠腹腔后,可在腹水中分化并聚积成结构类似于早期胚胎的细胞群,称为拟胚体。     

B7-2胚胎性癌细胞与其分化细胞的周期特性

本文用~3H-thymidine标记B7-2EC细胞及其经HMBA诱导的分化细胞。根据连续标记和脉冲标记核百分率,计算和分析这两种细胞的细胞周期各时相特点。结果表明,未分化EC细胞的周期是18小时,其中G_1期1.7小时,S期13.6小时;分化细胞的周期是30小时,G_1期8.6小时,S期17.1小时。因此,B7-2分化细胞与未分化的相比,主要是G_1期和S期时相相应拉长。     

两株克隆F9细胞株的初步比较研究

我们用有限稀释法从F9细胞克隆得到了F9-1和F9-3两个克隆亚株,实验结果表明它们在生长行为,分化发育潜能RA诱导提高AKP活力和合成AFP等方面都存在着差异。F9-1细胞是以团块贴壁生长为主,F9-3细胞是以单个和少数细胞的团块悬浮生长为主。将F9-1细胞接种到129/SV-ter小鼠皮下,长出的瘤组织的切片表明,除了EC细胞组成的未分化的神经管样结构外,还含有分化的腺管组成。经RA处理后的F9-1细胞,长出的瘤块中腺管增多并具有分泌功能。不管RA处理与否。从F9-3细胞长出的瘤块中几乎全由未分化的EC细胞构成。F9-1细胞经RA处理72小时,AKP酶活力明显增加,比相应对照组细胞的酶活力提高2—3倍;而F9-3细胞经RA处理后酶活力无明显提高。F9-1细胞经RA处理72小时,AFP免疫酶染色呈现细胞质阳性反应,对照组呈阴性或弱阳性;而F9-3细胞经RA处理后,与不处理的对照组细胞一样,细胞质的AFP免疫酶染色都呈阴性反应。以上结果表明,F9-1克隆株是一株对RA反应比较敏感的EC细胞,能被RA诱导分化,提高AKP活力并诱导合成AFP。F9-3克隆株是一株对RA不敏感的EC细胞,不能诱导分化,AKP酶活力不增加,也不能合成AFP,这些特性似乎更接近于无能EC细胞。     

体外小鼠胚胎性癌细胞BEC-B7建株和特性

恶性畸胎癌干细胞又叫做胚胎性癌细胞(简称EC细胞),是一种来自生殖细胞或早期胚胎细胞的恶性癌细胞,不仅具有类似于早期胚胎细胞分化多能性,而且能在一定条件下失去恶性生长性质,分化为各个胚层的正常组织。EC细胞是开展哺乳类胚胎早期发育遗传以及癌变和癌细胞分化研究较好的实验材料。EC细胞在体外可以大量培养生长,较之正常胚胎细胞更容易获取。我们在建立体内腹