5-溴脱氧尿苷抑制中国仓鼠核仁形成区活性的研究

当培养基中BrdU浓度提高至9—30μg/ml时,会使中国仓鼠二倍体细胞和细胞株wg3h的银染NORs(Ag-NORs)总数明显减少。不同浓度的BrdU对中国仓鼠细胞NORs活性影响的研究表明,中国仓鼠细胞NORs活性受抑制的程度随培养基中BrdU浓度的提高而加强,还与BrdU处理的时间有密切的关系。这些细胞在无BrdU的培养基里继续生长30小时后,它们的NORs活性可以得到恢复。BrdU对中国仓鼠细胞NORs活性的抑制作用很可能是一种毒性效应。本文还对BrdU抑制中国仓鼠细胞NORs活性的机制进行了初步的讨论。     

四细胞杂交克隆的NORs活性和均染区的巨染色体

通过聚乙二醇使3个小鼠细胞与1个中国仓鼠骨髓细胞融合,获得四细胞杂交克隆。在第6世代,该杂种细胞含有100条小鼠染色体和5条仓鼠染色体。本文报道在杂种细胞中双亲NORs活性均受抑制,不仅73.2％的仓鼠NORs活性被抑制,而且还有18.3％的小鼠NORs失去了活性。由于在杂种细胞中,仓鼠第3号染色体上的NORs活性仍保留它原来的91.2％,因此我们的结果表明:不同染色体上18s和28s rRNA基因的转录活性可有明显的差异,这可能与它们所在的染色体结构有关。我们首次报道了杂种细胞中所出现的均染色区巨染色体,并对这条巨染色体进行了G-带、C-带和Ag-NORs的分析。对这条巨染色体与18s和28srRNA基因扩增的关系进行了初步讨论。     

中华按蚊（Anopheles sinensis）细胞系的建立和特征

从中华按蚊初孵幼虫中,建立一株细胞系,命名 AS-684。建系使用我们改良的199培养基及含有10%F 12的 M-M 培养基。原代培养20天形成单层细胞。传代培养,以1:2分种率,相隔4—5天传代1次,连传70代次。细胞系由4种细胞类型组成,以多边形和纺锤形上皮细胞为优势,掺杂少数双核细胞及长梭形细胞。细胞生长,从24小时开始倍增分裂达高峰,对数期二倍增长时间约为16小时。自45代起,培养基中血清含量从20%降至10—15%。4次电镜观察,没有发现支原体和病毒质点。细胞系以二倍体细胞为主,染色体6条(2n=6)。细胞酯酶同工酶分析,酶谱为两条酶带,PI 值为4.75,4.85。各种代次细胞保存于-196℃的液态氮中及4℃普通冰箱中,经多次试验能成功复苏。     

中国仓鼠核仁形成区的活性在仓鼠-小鼠杂交细胞中受抑制

用Polyethyleneglycol(PEG)将中国仓鼠骨髓细胞与小鼠细胞株PG19细胞融合,获得杂交细胞克隆。染色体C-带分析表明,早在细胞繁殖的第4代,杂交细胞就迅速地丢失了17条仓鼠染色体。在杂交细胞中,只有9.4％的仓鼠核仁形成区(NORs)有活性,而且所有这些有活性的NORs都位于仓鼠第3号染色体上,占该号染色体原有NORs总活性的40％,因此我们的结果不仅首次表明在中国仓鼠-小鼠杂交细胞中,绝大部分(90.6％)的仓鼠NORs活性被抑制,而且还发现了位于不同染色体上的仓鼠NORs活性受抑制的程度可有明显的不同,NORs活性很可能与它们所在的染色体的结构有关。小鼠NORs的活性在杂交细胞中没有受到明显的影响。     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

两个癌基因转化细胞系的建立

用BGC-ras~H和v-myc共转染第四代大鼠全胚细胞(REF),BGC-ras~H单独转染NIH3T3小鼠成纤维细胞分别获得两个恶性转化细胞系REF_(4-3)和BGC3T3。其中REF_(4-3)由一早代成纤维样细胞转化成为上皮样的、染色体高度界倍体化并可稳定传代的细胞。两种转化细胞均可接种裸鼠致瘤,成为二个用癌基因转化建立的细胞系。     

BrdU抗性细胞特性的研究——Ⅱ.四个抗性亚系核仁形成区(NORs)活性和DrdU抑制NORs活性机制的探讨

从中国仓鼠细胞株Wg3h中,通过BrdU处理获得了两个抗30μg/ml BrdU和两个抗60μg/ml BrdU的抗性细胞亚系。银染NORs(Ag-NORs)的分析表明,除了一个抗性细胞亚系外,其他3个亚系NORs的活性都明显地被BrdU所抑制,即主要是表现在细胞Ag-NORs数和携带Ag-NORs染色体数的明显减少。不同BrdU抗性亚系NORs活性被抑制的程度可有很大的差异。在培养基里除去BrdU后,抗性细胞被BrdU抑制的NORs活性可以逐步得到恢复,但其恢复的速度显然比Wg3h细胞缓慢得多。抗性细胞NORs活性受抑制,与Wg3h细胞一样,是由于BrdU毒性的缘故。     

人体肝癌细胞染色体的组型分析和G-分带研究

(一)人体肝癌体外原级培养细胞的中期染色体计数表明,第1例肝癌为超二倍体(2.46倍),其他两例为超四倍体(4.28和4.42倍),染色体众数分别为57,111和116个。(二)肝癌细胞染色体组型分析显示,D组和G组等近顶端着丝点染色体比其他各组号减少了,特别在第一例肝癌的超二倍体细胞中,缺少15号染色体的细胞高达90%,大多成为单体状态,甚或缺体。在所有三例肝癌细胞中,15号染色体平均只有全部染色体倍体数的一半。(三)在三例肝癌细胞中,包括超二倍体和超四倍体细胞,E组和F组等中间或近中间着丝点染色体平均比全部染色体倍体数增加两倍。上述这两类染色体数目变化各异,一增一减,导致肝癌细胞的各组号染色体之间相对比例失调。(四)检查分带类型指出,肝癌细胞内一个1号染色体的短臂比另一同源染色体增长一段,在这短臂末端额外多出一条着色较深的带纹,推测是由于G组(21号)染色体易位所形成。相似的现象较少地出现在2号和4号染色体长臂的末端。由于易位,使得染色体之间固有关系改变了。(五)肝癌细胞中出现双着丝点染色体、半环状染色体和微小染色体等,染色体还发生间隙、断裂和缺损等畸变。     

小鼠肿瘤细胞染色体核仁组织者的细胞化学观察

本文报道用Ag-AS染色技术对几种小鼠肿瘤细胞(Ehrlich腹水瘤,肉瘤180,淋巴瘤1号)核仁组织者(NORs)的观察结果,发现肿瘤细胞的NORs即18 S+28 S rDNA或核糖体基因的位置和大小与正常细胞的不同。正常小鼠细胞有3—6条染色体带有银染色的核仁组织者(Ag-NORs),分布位置都紧靠在着丝点下方;而三种小鼠肿瘤细胞都有一个中等大小的近端着丝点染色体,其Ag-NOR的位置移至长臂的中部。小鼠淋巴瘤1号     

中华鲟精巢细胞系的建立和鉴定

为了开展中华鲟(Acipenser sinensis)种质资源保存及其精原干细胞体外培养的研究, 采用蛋白酶消化法, 对中华鲟精巢组织细胞进行原代培养, 建立了中华鲟精巢细胞系(Acipenser sinensis testicular cell line, AST), 经352d传代培养, 已稳定传至80代。中华鲟精巢细胞系形态主要呈类纤维状, 培养基为DMEM, 培养温度为25℃, 最适血清浓度为15%。正常传代的AST细胞冻存、复苏后, 经台盼蓝染色, 约(81.36±1.13)%的细胞具有活性, 复苏后细胞仍生长旺盛。染色体核型分析结果显示, 第30代中华鲟精巢细胞系染色体数目分布在142—310, 众数为264。通过RT-PCR检测发现, 在P0和P1细胞中, Sertoli细胞特异表达基因(amh和gsdf)、Leydig细胞特异表达基因(cyp17a1)和生殖细胞特异表达基因(dazl、dnd和vasa)都有表达, 且表达量与精巢中的相似; 在P15、P30和P60细胞中, 只有amh和vasa基因有微弱的表达, 说明细胞系传代到了后期, 只含有极少量的Sertoli细胞和生殖细胞。通过脂质体转染法将pEGFP-N3质粒转入AST细胞中, 可表达增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)。AST细胞系的建立为中华鲟种质资源的保存、精原干细胞的体外增殖与分化、基因功能等研究提供了重要的实验材料。     

Microsomal epoxide hydrolase activity in human x mouse hybrid cells

Microsomal epoxide hydrolase (E.C.3.3.2.3) activity has been measured in human x mouse hybrid cells prepared from human cells expressing 6-7 x the activity of the mouse cells. Rabbit antihuman and antimouse antisera raised against purified enzymes were used to discriminate between human and mouse enzymes. All twenty five clones examined did not express human enzyme and this correlated with the loss of human chromosome 6 from each cell line. Four hybrids expressed 2-3 x the activity expressed by the mouse cell parent and these all retained more human chromosomes, specifically chromosome 19, than those with low activity. It is concluded that the human gene for epoxide hydrolase may be on chromosome 6 and that other gene products can affect the level of activity expressed by a cell.     

Assignment of two rat dihydrofolate (DHFR) genes to chromosomes 2 and 4

A panel of rat x mouse cell hybrids was used in the chromosomal mapping of the rat dihydrofolate reductase (DHFR) gene. It was determined that the probe hybridized to gene sequences on two different chromosomes (Nos. 2 and 4), possibly representing the active gene and a pseudogene. Hybridization of the DHFR probe to DNA from a methotrexate resistant rat cell line revealed that the gene on chromosome 2 was amplified, but not the gene on chromosome 4. This result was taken to suggest that the active DHFR gene is located on rat chromosome 2 and that the sequence on chromosome 4 is a pseudogene.     

Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy.

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.     

Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Natural history of the Nihon rat model of BHD

Hereditary cancer was first described in the rat by Eker and Mossige in 1954 in Oslo. The Eker rat model of hereditary renal carcinoma (RC) was the first example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal, and has been contributing to the elucidation of renal carcinogenesis. Recently, we found a second hereditary RC model in the Sprague-Dawley (SD) rat, in Japan in 2000, which was named the Nihon rat. The Nihon rat is also an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker rat, which are predominantly of the clear cell type (this type represents approximately 75 % of human RCC), and develop from earlier preneoplastic lesions than the Eker rat. We performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, and found that the Nihon mutation was tightly linked to genes, which are located on the distal part of rat chromosome 10. Finally, we identified a germline mutation in the Birt-Hogg-Dubé gene (Bhd) (rat chromosome 10, human chromosome 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat gene sequence, resulting in a frame shift and producing a stop codon 26 amino acids downstream. Thus, the Nihon rat will contribute to understanding the BHD gene function and renal carcinogenesis.     

Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.

Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis.     

The balance between CD45RChigh and CD45RClow CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RC(high)/CD45RC(low) CD4 T cell ratios. The CD45RC(high) subpopulation predominates in LEW rats, and the CD45RC(low) subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RC(low) CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC(high)/CD45RC(low) CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW x BN) F(2) intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (approximately 17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (approximately 28 cM; LOD score 3.1) and 20 (approximately 40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RC(high)/CD45RC(low) Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.     

Expression of monoamine oxidase A and B activities in mouse cybrids and hybrids

The inheritance of monoamine oxidase (MAO; EC1.4.3.4) was studied in cultured cells using techniques of somatic cell genetics. Cells of a mouse neuroblastoma variant line lacking MAO activity were fused to cytoplasts prepared from a mouse L cell line which expresses MAO activity and is resistant to chloramphenicol (a cytoplasmically inherited trait). The resulting cybrids were resistant to chloramphenicol, but did not recover MAO activity, indicating that the loss of activity in the neuroblastoma parent was not the result of an inherited lesion in a cytoplasmically transmitted gene. This cybrid cells were then fused to rat hepatoma cells expressing both A and B types of MAO activity. A resulting hybrid line, grown in medium containing hypoxanthine, aminopterin and thymidine (HAT) to select cells that had retained HPRT activity and hence the rat X chromosome, expressed both types of activity, but at a reduced level compared to the hepatoma parent. This finding indicates that the genetic lesion in the neuroblastoma cells resulting in loss of MAO activity is not phenotypically dominant, and that both A and B types of activity can be conferred together to neuroblastoma cells which normally express only the A type of MAO activity.