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。     

高效建立129/ter、C57BL/6J小鼠胚胎干细胞系的方法学探讨

小鼠胚胎干细胞 (ＥＳ细胞 )是从小鼠囊胚内细胞团(ＩＣＭ)分离出来的、在体外培养过程中可维持未分化状态、正常二倍体核型及无限增殖能力 ,具有多能性或全能性的细胞系[1～ 3 ] 。ＥＳ细胞广泛应用于克隆动物制作、转基因动物生产、动物医学模型建立、真核细胞基因表达与调控的研究、细胞分化机制的探索、人及哺乳动物基因功能的研究以及细胞、组织、和器官的修复与移植研究。胚胎干细胞的研究和应用已成为生命科学研究的热点和前沿领域之一[4～ 8] 。自第一株 12 9小鼠ＥＳ细胞系建立以来 ,人们在生物学和医学等多个领域进行了广泛深入的…     

从C57BL/6J小鼠胚胎中分离ES细胞系(英文)

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｓ）是来源于小鼠早期胚胎的细胞系,它可以在体外大量培养而不失去其发育的多潜能性。ＥＳ细胞不仅可以用来制作转基因动物,而且能够作为载体进行基因打靶等多种研究。目前,国际上常用的胚胎干细胞系都是来源于１２９小鼠的胚胎。因而,有必要探讨用其它品系小鼠建立ＥＳ系。１９９１年,Ｌｅｄｅｒｍａｎｎ等人首次报道从Ｃ５７ＢＬ／６Ｊ小鼠胚胎中建成了ＥＳ细胞系,但是没有对建立的细胞系进行特性分析。国内柴桂萱等人虽然做了特性分析,但是他们所建细胞系的细胞直径较大,生长速度较慢,不同于常见的ＥＳ细胞系。我们从Ｃ５７ＢＬ／６Ｊ品系小鼠胚胎中共分离了四个ＥＳ细胞系,分别命名为ＣＥ１、ＣＥ２、ＣＥ３、ＣＥ４（Ｆｉｇ．１ａ＆ｂ）。这四个细胞系核型正常率均达到７０％以上。我们检查了ＣＥ２细胞的分化能力,当将ＣＥ２细胞注入同基因型小鼠的皮下后,获得的畸胎瘤（Ｆｉｇ．３）组织切片检查的结果表明：该细胞能够分化成多种组织（Ｆｉｇ．２）。我们也研究了ＥＳ细胞的嵌合能力,用ＩＣＲ小鼠胚胎作为受体胚胎,采用囊胚显微注射法构建嵌合鼠。在幸存的幼鼠中我们获得了来源于ＣＥ２细胞的嵌合鼠（Ｔａｂｌｅ１,Ｆｉｇ．４）。综     

小鼠孤雌胚胎干细胞的建立及其向运动神经元分化的初探

文章采用小鼠的孤雌囊胚建立胚胎干细胞系,探究其向运动神经元分化的可能,为临床治疗以及研究基因组印记与神经分化的的关系提供理论基础。结果表明:卵母细胞孤雌激活率达到93.26%,成功建立了8个孤雌胚胎干细胞系,建系率达到23.53%。克隆表达多潜能标记Oct4及细胞表面标记SSEA-1,有高水平的碱性磷酸酶活性,在细胞第10代和第30代时核型分析检测显示为正常的40条染色体。体内、外均分化出三胚层来源的细胞。联合应用全反式维甲酸(RA)、音猬因子(Shh)及细胞外基质,小鼠孤雌胚胎干细胞可被诱导表达运动神经元的标志性标记HB9、Olig2。     

体内微环境对B7-2EC克隆细胞株分化的影响

在发育生物学领域中,微环境对早期胚胎纽胞的分化,对造血系统干细胞的分化都能产生影响,这已是人所共知的事实。小鼠胚胎性癌细胞(EC细胞)是一种研究细胞恶变和细胞分化很好的材料,它可以在某些品系小鼠中自发地产生,也可由小鼠早期胚胎细胞或原始生殖嵴细胞经异位移植而获得,但移植的位置不同,生瘤率也不同。EC细胞一般分为无能和多能性两种:无能EC细胞如F9在体内接种后不能分化为各种体细胞,保持着癌细胞恶性生长的特点,而多能EC细胞接种到     

B7-2胚胎性癌细胞诱导分化过程中的表型转变

胚胎性癌细胞(简称EC细胞)作为一类肿瘤(畸胎瘤)的干细胞近年受到广泛的重视,从胚胎学、肿瘤学和分子生物学等许多学科领域都应用它作为实验材料,离体诱导分化研究是其中的一个方面。B 7-2 EC细胞是我们从129品系小鼠的自发睾丸畸胎瘤中分离克隆得到的一株多能EC细胞,它在同种同基因小鼠     

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。     

两个可进入种系的ES细胞系的建立

从１２９／ｔｅｒ小鼠中建立了９个ＥＳ细胞系,它们在核型、生长速度、体内外分化能力等方面显示了各自不同的特点。通过囊胚显微注射法,将ＥＳ细胞注入Ｃ５７ＢＬ／６Ｊ胚胎中,制作了嵌合体,并通过对嵌合体后代毛色的观察,判断了嵌合体生殖细胞的组成。结果表明,ＥＳ细胞系ＭＥＳＰＵ２１、ＭＥＳＰＵ２２都具有很强的种系嵌合能力。比较这两个细胞系与其他细胞系,证明一个好的ＥＳ细胞系必须具备核型正常、生长速度快、体外     

胚胎癌细胞体外诱导分化

小鼠胚胎癌细胞(embryonal carcinomacells,简称EC细胞)与早期多能性胚胎细胞具有许多生物学共性。目前已分离和克隆了许多EC细胞株,其中大多数移植到同系宿主体内后,形成癌块中除了EC细胞外,常混杂有各种类型的分化组织,这种癌块称为畸胎癌     

BALB/c小鼠胚胎干细胞系建立的方法学探讨

以小鼠胚胎成纤维细胞（ME）为饲养层,以大鼠心脏细胞条件下培养基（RH－CM）为ES细胞培养基,全面、详尽地对BALB／c小鼠ES细胞的纱和培养方法进行了探讨,成功地建立了一套建立和培养BALB/c小鼠ES细胞系的新方法。这一培养条件不但有效地维持了ES细胞的未分化状态和正常二倍体核型,而且维持了其作为能性胚胎干细胞的一系列特征。实验设计了两种离散方法和两种浓度的消化兴,用来离散增殖的ICM和ICM离散后出现的ES集落。两种离散方法即“一次离散法”,和“多次离散法”,两种浓度的消化液即0．25％。Trypsin－0．04％。EDTA和．005％。Trypsin-0.888％EDTA;同时对ICM离散时机、RH－CM在BALB／c小鼠ES细胞建系和培养中作用进行了探讨。结果表明：在低浓度消化液作用下,采用“多次离散法”离散增殖4天的ICM和ES集落的方法建立BALC／c小鼠的ES细胞系是理想的;从细胞形态、集落形态、增殖生长能力、核型检测、碱性磷酸酶测定以及体内外分化能力表明,所建立的9个BALB/c小鼠ES细胞系符合小鼠胚胎干细胞的一系列特征。     

维生素A酸受体γ基因表达及其对ES细胞分化和凋亡的影响

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.     

Karyotype Analysis of 5 Species of Polygonatum Mill.

The present paper reports the chromosome numbers and karyotypes of five species in Polygonatum from Anhui of China. The materials used in this work are listed in Table 1, Photomicrographs of somatic metaphase and karyograms of the five species of Polygonatum in Plate 1, 2, 3, the idiograms in Fig. 1-11 and a comparison of the karyotype of them is provided in Table 2. The results are shown as follows: 1. Polygonatum odoratum (Mill.)Druce Two materials were examined. One from Mt. Huangshan, Anhui, has 2n= 16 = 10m (3sc)+ 6sm (Plate 1 :A, B). The idiogram is shown in Fig. 1. The chromosomes range in length from 2.85 to 8.85 μm, with the total length 48.63μm and the ratio of the longest to the shortest 3.11, The karyotype belong to Stebbins’(1971) 2B. The two chromosomes of the first pair have arm ratios 1.01 and 1.29 respectively, and The first pair has one chromosome carrying a satellite attached to the short arm, showing heterozyosity .The chromosome num ber of 2n= 16 in P. odoratum and its karyotype are reported for the first time. The other from Langyashan, Chu - xian, Anhui, is found to have 2n = 18 = 10m (Isc)+2sm+6st(2sc) (Plate 1: C, D). The idiogram is shown in Fig. 2. The chromosomes range in length from 2.43 to 8.29μm, with the total length 46.67µm and the ratio of the longest to the shortest 3.41. The karyotype is also of 2B. In a somatic chromosome complement the 2nd pair have one chromosome carrying a satellite attached to the long arm, showing heterozygosity. 2. Polygonatum filipes Merr. Two materials were examined. One from the Huangshan, Anhui is found to have two cytotypes: 2n= 16 and 2n=22. This paper reports one of them. The karyotype formula is 2n=22=8m+8sm(2sc)+6st(Plate 3: Q, R). The idiogram is shown in Fig. 3. The chromosomes range in length from 2.55- 5.85μm, with the total length 45.01 μm and the ratio of the longest to the shortest 2.29. The karyotype belongs to 3B. The other material from the Fangchang, Anhui, is shown to have four cytitypes: 2n= 14, 2n= 16, 2n=20 (Plate 3: W) and 2n=22. This paper reports two of them. Type I: the karytype formula is 2n=14=10m+4sm (Plate 3: S, T). The idiogram is shown in Fig. 5. The chromosomes range in length from 2.59 to 7.61μm, the total length 37.44μm and the ratio of the longest to the shortest is 2.94. the karyotype belongs to 2B. Type II :The karyotype formula is 2n=16=8m+4sm+4st (Plate 3: U, V). The idiogram is shown in Fig. 4. The chromosomes range in length from 2.65 to 8.21 μm, the total length 46.01 μm and the ratio of the longest to the shortest 3.10. The karyotype belongs to 2B. The chromosome numbers of 2n=20, 2n= 14 and 2n=22, and karyotype of 2n= 14 and 2n=22 in P. filipes are reported for the first time. 3. Polygonatum cytonema Hua Two materials were examined. One from the Langyashan, Chuxian, anhui, is found to have 2n = 18 = 8m (2sc)+ 6sm+ 4st (Plate 2: K, L). The idiogram is shown in Fig. 7. The chromosomes range in length from 3.41 to 9.21 μm, the total length 56.34μm and the ratio of the longest to the shortest is 2.70. The karyotype belongs to 2B. The other material from the Huangshan, Anhui, has two cytotypes: 2n=20 and 2n= 22. Type I: The karyotype formula is 2n= 20= 8m+ 6sm+ 6st (Plate 2: M, N). The idiogram is shown in Fig. 8. The chromosomes range in length from 1.75 to 5.03μm, with the total length 32. 91μm and the ratio of the longest to the shortest 2. 87. The karyotype is also of 2B. Type II: The karyotype formula is 2n=22=6m+ 8sm+4st+ 4t (Plate 2: O, P ). The idiogram is Shown in Fig. 10. The chromosomes range in length from 1.75 to 4.95 μm, with total length 35.05μm and the ratio of the longest to the shortest 2.83. The karyotype brlongs to 3B. 4. Polygonatum desoulayi kom. The material from Xuancheng, Anhui, is found to have karyotype 2n = 22 = 10m (2sc) + 6sm (lsc) + 6st ( Plate 2. I, J). The idiogram is shown in Fig. 6. The chromosomes range in length from 1.86 to 5.61μm, with the total length 41.98μm and the ratio of the longest to the shortest 3.02. The karyotype is also of 3B. The first pair has one chromosome carrying a satellite attached to the long arm, showing heterozygosity. The chromosome number and karyotype of Chinese material are reported for the first time. 5. Polygonatum verticillatum (L.) All. The material from the Langyashan, Chuxian, Anhui is found to have two cytotypes. Type 1: the karyotype formula is 2n = 18 = 2m+ 2sm+ 10st+ 2t+ 2T (Plate 1: G, H). The idiogram is shown in Fig.9. The chromosomes range in length from 1.86 to 4.03μm, with total length 28.28μm and the ratio of the longest to the shortest 2.17. The karyotype classification belongs to 3B. Type II: The karyotype formula is 2n=24=6m+4sm+12st+2T (Plate 1: E, F). The idiogram is shown in Fig. II. The chromosomes range in length from 2.01 to 5.03μm, with total length 41.36μm and the ratio of longest to shortest 2.50. The karyotype is also of 3B. The chromosome numbers and karyotypes of Chinese material are reported for the first time.     

A Chromosomal Study of Eight Species in Allium Sect. Rhiziridium G. Don in China

The present paper reports the chromosome numbers and karyotypes of eight species of Sect. Rhiziridium in Allium (Liaceae). The materials were all collected from their natural populations in east Inner Mongolia, China. The karyotype analysis is made on the basis of Li et al. (1985).The results are as follows (for chromosomes parameters, voucher specimens and localities, see Table 1 and Plate 1--2 the idiograms of the eight species in Fig. 1): (1) Auium leucocephalum Turcz. The somatic chromosome number and karyotype of this species is 2n=16=12m=2sm+2st (2SAT), in Stebbinsl(1971) kayotype classification, which belongs to 2A (Plate 1: 1; Fig. 1: 1). The range of chromosome relative length varies between 8.90--15.55%. Two small satellites are attached to the short arms of the 8th pair of chromosomes. (2) A. strictum Schrader has 2n (4x) =32=16m+4sm+12st, belonging to 2B (Plate 1: 2 & Fig. 1: 2). Satellites were not observed., and the range of chromosome relative length is between 3. 67-11.00%. (3) A. ramosum L. 2n=16=14m+ 2st (2SAT), belonging to 2A (Plate 1: 3 & Fig. 1: 3), Two small satellies are attached to the short arms of the 8th pair of chromosomes. The range of chromosome relative length is between 9.17-16.39%. The chromosome number and karyotype of this species are in accordancewith those reported by Li et al. (1982) with the material from Jinshan, Beijing. (4) A. bidentatum Fisch. ex Prokh. 2n (4x) =32=24m+4sm+4T, belonging to 2B (Plate 1: 4 & Fig. 1: 4). Satellites were not observed. A small median B-chromosome was found in root-tip cells of the population growing in sandy soil, and it is the first discovery (Plate 2: 9). The species has terminal chromosomes, which are seldom seen in Sect. Rhiziridium. The range of chromosome relative length is between 3.32—9.06%. (5) A. tenuissimu L. 2n=16= 10m+4sm+2st(2SAT), belonging to 2B(Plate 1:5 & Fig. 1:5). Two large satellites are attached to the short arms of the 8th pair of chromosome. The range of chromosome relative length is between 8.27--17.56%. (6)A. anisopodium Ledeb. 2n = 16 = l2m +2sm + 2st (2SAT), belonging to 2A (Plate 2:7 & Fig. 1: 7). Two large satellites are attached to the short arms of the 8th pair of chromosomes. In somatic cells of some plants of this species, a small submedian B-chromosome was found (Plate 2: 10, 11). The range of chromosome relative length is between 8.05-17.08 %. (7) A. anisopodium Ledeb. var. zimmermannianum (Gilg) Wang et Tang 2n (4x)=32=24m+4sm+4st( 4SAT), belonging to 2A (Plate 1: 6 & Fig. 1: 6). Four large satellites are attached to the short arms of the 15 and 16th pairs of chromosomes. The range of chromosome relative length is between 4.45--8.35%. This variety is similar to A. anisopodium Ledeb. in morphological characters, and their karyotype formulas are also very similar. The present authors consider that the variety is an allotetraploid derived from A. anisopodium Ledeb. (8) A. condensatum Turcz. 2n=16=14m+2st (2SAT), belonging to 2B (Plate 2:8 & Fig. 1:8). Two. small satellites are attached to the short arms of the 6th pair of chromosomes. In a few individuals of this species median (M) B-chromosome was discovered, and the number is stable (Plate 2: 12). The range of chromosome relative length is between 7.64--17.07%. In short, the chromosome numbers of the species studied in the present work are found to be 2n=16 or 32, and the karyotypes belong to 2A or 2B, highly symmetrical. The karyotypes of Chinese materials of these species are mostly reported for the first time. Threespecies have B-chromosomes.     

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.     

Report on Karyotypes of 10 Species of Liliaceae (s. l.) From Qinling Range

The karyotypes of 10 species of the Liliaceae from the Qinling Range are reported as follows. I. Polygonatum Mill. (1) P. odoratum ( Mill. ) Druce was found to have the karyotype 2n=20=12m+8sm ( Plate 3, Fig. I), which belongs to Stebbins’ (1971) karyotype classification 2B. The chromosomes range from 3.88 to 11.26μm in size. Table 2 shows the karyotypes and number fundamentals (N.F.) of 13 materials from 12 different localities. The N. F. of these materials can be classified into two groups: N.F. =36 and N.F.=40, besides one (N.F. =38) from Beijing. N. F. =36 covers all the materials with 2n= 18 which have relatively symmetrical karyotypes ( all consisting of m and sm chromosomes), one with 2n=20 (10m+6sm+4st) and one with 2n=22 (14m+8st). N.F. =40 include four materials with 2n= 20 (all of m and sm chromosomes ) and 3 with 2n= 22 (10m+ 8sm+ 4st). ￥ It is considered that there are two original karyotypes, 2n= 18 with N. F. = 36 and 2n= 20 with N.F. =40, which are relatively symmetrical. All the more asymmetrical karyotypes with some st chromosomes have probably evolved from the symmetrical karyotypes without st chromosomes by centric fission. (2) P. zanlanscianense Pamp. has the karyotype 2n=30=18m(2SAT) + 4sm+ 6st+ 2t (Plate 1, Fig. 1) which belongs to 2C. The chromosomes range from 2.16 to 9.76μm. ￥ II. Asparagus filicinus Buch.-Ham. ex D.Don. The karyotype of this species is 2n = 16= 8m(2SAT )+ 6sm + 2st (Plate 1, Fig. 1 and Table 3 ) , which belongs to 2B. The chromosomes range from 2.33 to 5.30μm. Most species in Asparagus, including A.Filicinus, are reported to have basic number x= 10, and therefore 2n= 16 is a new chromosome number for A.filicinus. EL-Saded et.al.(1972) gave a report of n=8 for A. stipularis from Egypt, while Delay (1947) reported 2n = 24 for A. trichophyllus and A. verticillatus, Sinla(1972 ) gave a report of 2n=48 for A.racemosus. It is certain that there are two basic numbers in the genus Asparagus. III. Cardiocrinum giganteum (Wall.) Makino was found to have the karyotype 2n=24=4m+8st+12t (Plate 1, Fig. 1 ), which belongs to 3B. The chromosomes range from 8.71 to 20.24μm. IV. Smilax discotis Warb. was shown to have the karyotype 2n=32=4m+22sm+4st (2SAT)+2t (Plate 1, Fig. 1 and Table 3), which belongs to 3C. The first pair is much longer than others. The chromosomes range from 1.79 to 9.21μm. The chromosome number and karyotype of S. discotis are both reported for the first time. V. Reineckia carnea (Andr.) Kunth is of the karyotype 2n=38=28m+10sm (Plate 2, Fig. 1 ), which belongs to 2B. The chromosomes range from 5.65 to 12.75μm. VI. Tupistra chinensis Baker was found to have the karyotype 2n=38=25m+ 13sm (Plate 2, Fig. 1), which belongs to 2B. The chromosomes range from 8.11 to 23.82μm. A pair of heterozygous chromosomes is arranged at the end of the idiogram. The eighth pair possesses an intercalary satellite. Huang et al. (1989) reported the karyotype of T. chinensis from Yunnan as 2n = 38 = 24m+ 14sm without any intercalary satellite. Nagamatsu and Noda (1970) gave a report on the karyotype of T. nutans from Bhutan, which consists of 18 pairs of median to submedian chromosomes and one pair of subterminal chromosomes. And one pair of submedian chromosomes possess intercalary satellites on their short arms. VII. Rohdea japonica (Thunb) Roth. was found to have the karyotype 2n=38=30m+6sm+2st ( Plate 2, Fig. 1), which belongs to 2B. The chromosomes range from 7.94 to 18.29μm. Nagamatsu and Noda (1970) reported that the karyotype of R.japonica from Japan was the same as that of Tupistra nutans from Bhutan. But we have not discov ered any chromosome with an intercalary satellite. VIII. Hosta Tratt. (1) H. plantaginea (Lam.) Aschers was shown to have 2n=60. The 60 chromosomes are in 30 pairs,which can be classified into 4 pairs of large chromosomes (7.32- 8.72μm ), 3 pairs of medium-sized ones (4.72-5.60μm), and 23 pairs of small ones (1.40-3.64μm), (Plate 3 ,Table 4 ). The karyotype of H. plantaginea is reported for the first time. (2) H. ventricosa (Salisb.) Stearn was counted to have 2n=120, The 120 chromosomes are in 60 pairs, which can be classified into 8 pairs of large chromosomes (7.00- 8.40μm ), 6 pairs of medium-sized ones(4.40- 6.15um ), 46 pairs of small ones (1.20- 3.85μm), (Plate 3, Table 4). Based on the karyotypes of H. plantaginea and H. ventricosa, the latter is probably a tetraploid in the genus Hosta. Kaneko (1968b) gave a report on the karyotype of H. ventricosa, which is of8 pairs of large chromosomes, 4 pairs of medium-sized and 48 pairs of small ones.     

Immuno-electron microscopical studies on the retinal basement membrane of chick embryo

Retinal basement membrane (RBM), also called inner limiting membrane of retina, is constituted by extracellular matrix. It was reported that neurite outgrowth of a neuron was closely related to extracellular matrix, particularly the laminin. In this laboratory RBM was used as the optimal substrate for retinal cells in culture. We have studied the surface of RBM and its relation to neurite outgrowth by scanning electronmicroscopy and immunogold transmission electronmicroscopy. RBM could be separated by mechanical disruption of the retina mounted between 2 adhesive substrata (membrane filter and poly-L-lysine coated glass). The surface of RBM studied was the side of RBM facing the optic fiber layer and ganglion cell layer. Small particles densely distributed on surface of RBM (Plate I, Fig. 1 and 2) were shown to be chrysanthemum-like structures with radiative arms under the scanning electronmicroscopy (Plate I, Fig. 3 and 4). The radiative arms of RBM of 12-day old chick embryo (E 12) were more in number and longer in length than that of the 6-day old chick embryo (E 6). The axons of ganglion cell from E 6 retinal strip extended out very well on RBM (Plate I, Fig. 5). Growth cone was active with filopodia. The chrysanthemum-like structures changed to ball-particles when the RBM was cultured for 24 hr. Some of ball-particles lay over the growth cone, and some beside it. Over and beside the nerve fiber could also be seen some ball-particles. When many neurites grew on RBM, a lot of ball-particles were shown to be displaced and piled up (Plate I, Fig. 6). The whole amount RBM labeled by indirect immunogold staining of Müller glial cell could be observed by transmission electronmicroscopy. The gold particles wer located at the chrysanthemum-like structure of E 6 RBM (Plate II, Fig. 7) and E 12 RBM (Plate II, Fig. 8). It was suggested that those structures were the end foot of Müller glial cells. Staining of PBS control or mouse serum control was negative (Plate II, Fig. 9 and 10).(ABSTRACT TRUNCATED AT 250 WORDS)     

LIF基因转染的ES细胞生长与分化特性的研究

我们将人D型LIF cDNA以正反两种方向分别克隆到载体pKCR 3,并引入neo~r基因,构建成pSVLD( )和pSVLD(-)质粒,按磷酸钙沉淀法分别转染ES-5胚胎干细胞,经G418和不同浓度LIF条件培液共同筛选、Nor-thern和Southern分析以及ES-5细胞集落分化抑制能力测定,建立了过度表达分泌LIF的ESL( )细胞株和表达外源反义LIF RNA的ESL(-)细胞株。我们发现,ESL( )A2细胞能够在无外源LIF常规培液下至少传13代以上,仍能正常生长和传代,并保持与ES-5细胞同样的体外生长的特征性形态,以及具有干细胞特点和发育多潜能性,表明过度表达LIF确实能使ES细胞完全脱离对外源LIF条件培液的依赖性;而表达反义LIP RNA的ESL(-)细胞对培液中LIF浓度的依赖性明显升高,也更易分化,说明ES细胞内源LIF基因的表达水平虽低,但对于抑制ES细胞的分化仍可能是必需的。形态学观察发现,体外悬滴培养中经10~(-6)mol/L RA诱导后,过度表达LIF并未产生抑制ESL( )A2细胞分化的现象,和亲本ES-5细胞比较,也未发现其明显改变了10~(-6)mol/L RA对ESL( )A2细胞诱导分化的方向;而相同条件下,表达外源反义LIF RNA,则使ESL(-)B5细胞更易于向形态明确的细胞包括成纤维样和梭样细胞分化。上述细胞株的建立,提供了一个研究在不添加LIF的常规培液中生长的ES细胞或表达外源反义LIF RNA的ES细胞的生长分化的模型。     

Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages

Mouse embryonic stem (ES) cells are widely used in early development studies and for transgenic animal production; however, a stable karyotype is a prerequisite for their use. We derived 32 ES cell lines of outbred mice (129 × BALB (1B), C57BL × 1B, and DD × 1B F1 hybrids). Pluripotency was assessed by utilizing stem-cell-marker gene expression, teratoma formation assays and the formation of chimeras. It was shown that only 21 of the 32 ES cell lines had a diploid modal number of chromosomes of 40. In these lines, the percentage of diploid cells varied from 30.3 to 78.9 %, and trisomy of chromosomes 1, 8 and 11 was observed in some cells in 16.7, 36.7 and 20.0 % of the diploid ES cell lines, respectively. Some cells had trisomy of chromosomes 6, 9, 12, 14, 18 and 19. In situ hybridization with an X chromosome paint probe revealed that 7 of the 11 XX-cell lines had X chromosome rearrangements in some cells. Analysis of the methylation status of the Dlk1-Dio3 locus showed that imprinting was altered in 4 of the 18 ES cell lines. Thus, mouse ES cell lines are prone to chromosome abnormalities even at early passages. Therefore, routine cytogenetic and imprinting analyses are necessary for ES cell characterization.     

DMSO 对悬浮培养的东北红豆杉细胞凋亡和紫杉醇合成的影响

研究了不同浓度的DMSO对悬浮培养的东北红豆杉(Taxus cuspidata)细胞的增殖能力、细胞活性以及紫杉醇合成和释放等方面的影响,同时应用荧光指示剂双染法检测了细胞凋亡的发生情况.结果显示2%的DMSO处理能显著降低细胞活性,抑制细胞的增殖能力,使细胞核内DNA含量减少,培养中、后期在荧光显微镜下可见部分细胞核出现典型的凋亡形态,同时伴有紫杉醇产量的明显增加;对照组及1%以下浓度组未出现上述改变.结果表明一定浓度的DMSO能诱导细胞凋亡,促进细胞紫杉醇合成能力的提高.