胞质因子对肿瘤细胞恶性的调控研究——小鼠骨髓瘤细胞与网织红细胞胞质体的胞质杂交实验

本研究选用无细胞核而富含珠蛋白mRNA的正常小鼠网织红细胞与骨髓瘤细胞系中的BW—胸腺淋巴肉瘤细胞系,NS—1浆细胞瘤细胞系和P388淋巴瘤细胞等分别进行胞质杂交的实验,以研究胞质因子调控肿瘤细胞恶性的可能性。实验结果表明三种淋巴瘤细胞在并入网织红细胞胞质体后所形成的杂交细胞呈现下列去恶性化的特征:1.杂交细胞出现组织化学反应及超微结构的形态改变;2.细胞分裂指数及生长速度明显下降;3.作异种移植至裸鼠后丧失发生肿瘤的能力;4.染色体数量明显减少,畸变率下降;5.出现基因产物血红蛋白的联苯胺阳性反应,并在BW—R杂交细胞传20多代过程中持续出现。对胞质体杂交细胞中基因产物血红蛋白的连续表现,染色体的丢失和在异种接种中丧失长瘤能力的可能机理予以讨论。     

细胞遗传学中单个细胞染色体结构畸变的意义探讨

对门诊遗传咨询者（ 咨询组）２６０ 例进行了外周血染色体检查,检出异常核型３０ 例,发生率为１１－５ ％ ,在核型正常者中分析３０５０ 个分裂相,发现仅有单个细胞染色体结构畸变２６ 例,畸变包括易位、倒位、缺失等,发生率为１０－０ ％ 。同时还选择了正常对照（对照组）４８ 人次,检查结果全部核型正常,分析３０５０ 个分裂相,未发现一例单个细胞染色体结构畸变。两组畸变率,经统计学处理,Ｐ＜ ０－０５ ,两者之间有显著差异,说明细胞遗传学中单个细胞染色体结构畸变对临床诊断有显著意义。认为单个细胞染色体结构畸变的发现即证明亲代可能是一种嵌合体,只是比率极小,它不象数目上的异常具有偶然性。文中讨论了其临床意义。     

30例膀胱癌的细胞遗传学研究

本文作者通过输尿管用冷杯活检术取得膀胱癌标本46例,29例用直接法制片,第30例用3天短期培养法,都具有适当的中期分裂相。每一例最少计数4个中期分裂相,最多计数24个。22例进行了染色体显带分析,结果发现在膀胱癌中存在染色体数目和结构畸变,其中19例存在异常克隆(按Rowely标准,鉴定克隆异常,即用显带技术确定至少有两个细胞具有相同的额外染色体或结构重排,或三个细胞都丢失了同一个染色体),主要涉及1号、3号、及11号染色体的改变(分别是36.6%、26.6%、20%)其次是4号、7号、及18号(均为10%)。结     

两种小鼠生精细胞染色体制备方法的探讨与评价

目的：比较改良的体内注射秋水仙素制备法和空气干燥直接制备法制作小鼠生精细胞减数分裂中期染色体 标本的优劣。方法：选用8~12 周龄雄性Balb/c 小鼠,分别采用改良的体内注射秋水仙素制备法和空气干燥直接 制备法进行减数分裂中期染色体制备,并用Giemsa 染色。光镜下观察小鼠生精细胞减数分裂不同阶段分裂相细 胞染色体形态并计数,计算各分裂相细胞的检出率。结果：体内注射秋水仙素制备法和空气干燥直接制备法对生 精细胞减数分裂中期染色体核型的检出率分别为10.37% 和6.46%。秋水仙素处理小鼠的最适浓度是4 mg/kg,体 内注射秋水仙素制备法制得的核型分裂相比空气干燥直接制备法多,直接制备法得到的染色体核型边缘清晰,更 便于计数。结论：体内注射秋水仙素制备法的优势在于核型分裂相多,而空气干燥直接制备法的优势在于染色体 核型边缘清晰,更便于计数,可根据不同实验需求进行选择。     

癌症患者外周血染色体畸变的研究

应用TC199培养基培养外周血淋巴细胞,获取中期分裂的染色体,观察和分析了32例癌症患者及20例正常人外周血淋巴细胞染色体畸变的情况。结果表明,癌患者染色体畸变率及代表畸变率的三个参数:细胞断裂率、染色体断裂率、畸变细胞率均明显高于对照组,t检验有高度显著性差异。染色体畸变的现象说明,在体内有恶性肿瘤存在,血液淋巴细胞染色体是不稳定的,同时也表明,染色体不稳定的增高,可能导致个体对肿瘤易感性的增加。     

单个细胞染色体畸变的遗传学分析及实验对策

目的对外周血中单个细胞染色体畸变的类型及其临床表现进行遗传学分析并探讨相应对策。方法取两种培养基,采用外周血常规培养法,培养72h后收获制片,G显带,进行核型分析,以每30～100个分裂相中找到1个异常染色体核型作为单个细胞染色体畸变的诊断标准,并比较两种培养基中出现单个细胞染色体畸变的比例。结果26例外周血单个细胞染色体畸变的畸变类型可分为平衡易位,不平衡易位,倒位,缺失及性染色体数目异常等;主要临床表现包括性发育异常,不良生育史及病残等;两种培养基中出现单个细胞染色体畸变的比例有显著性差异。结论在遗传咨询中,单个细胞染色体畸变有一定的临床意义,具有这种异常的个体生育后代时可考虑做产前诊断;染色体制备要严格控制条件,使用有正规批号的培养基。     

急性非淋巴细胞性白血病的微核、染色体的不隐定性与突变活性的相互关系——一种假说

在最近研究中,作者发现急性非淋巴细胞性白血病病人的骨髓细胞染色体畸变和微核数之间有相互关系,细胞中期分裂相异常的病人比中期分裂相正常病人的微核数明显增高。在混合有正常和异常骨髓细胞中期分裂相的病人中,细胞中期分裂相异常频率与     

两例具有染色体易位的T-细胞白血病患者其T-细胞抗原受体α-链座位的断裂点

特定的染色体易位在人体肿瘤发生中起着重要的作用。作者观察研究了两例其白血病细胞内含有11号染色体短臂(11P13)和14号染色体长臂(14q13)之间易位的T-细胞急性淋巴母细胞白血病患者(T-ALL).将他们的白血病细胞与缺乏胸苷激酶的小鼠L-细胞进行杂交,然后采用胰蛋白酶处理和吉姆萨染色的分带技术对各体细胞杂种克隆进行核型分析,以检出易位染色体(11P~ 和14~-q),同时又从继代克隆的杂种细胞中提取     

染色体作为癌患者诊断和判断预后指标的研究

应用ＴＣ１９９培养基培养外周血淋巴细胞，获取中期分裂的染色体，观察和分析了７６例癌患者和４０例正常人外周血染色体畸变的情况，对４４例患者进行了化疗前后染色体观测。结果表明，癌患者畸变细胞数、染色体畸变率及代表畸变率的三个参数：畸变细胞率、细胞断裂率、染色体断裂率均明显高于对照组，随着治疗后病情的变化，染色体的畸变与临床病情程度有明显的平行关系。作者认为，测定外周血淋巴细胞染色体情况，可作为癌患者诊断和判断预后的指标。     

荧光原位杂交新技术在恶性血液病中的研究进展

荧光原位杂交（ＦＩＳＨ）是以非放射性物质标记核酸探针，根据核酸杂交原理，在间期核和分裂中期染色体上检测特异性ＤＮＡ序列的一种新技术。由于它克服了放射性原位杂交（ＲＩＳＨ）的一些弊端，具有快速、准用、灵敏、经济等优点，现已应用于肿瘤生物学、产前诊断和基因突变等诸多领域。因恶性血液病常规制备染色体有较大难度，成功率不高，检出率较低等问题。而ＦＩＳＨ技术既可以分析中期核染色体，又能用于间期染色质，故该技术对恶性血液病研究有着特殊的应用价值，可发现一些新的染色体畸变包括染色数目、结构变化以及用以往一些常规细胞遗传学技术不能确认的微小染色体异常。现就ＦＩＳＨ在恶性血液病中的研究进展作一简要综述。     

淋巴瘤细胞与网织红细胞融合——胞质杂交细胞基因产物血红蛋白表达的观察

本实验选用富含有血红蛋白特征面无核的正常小鼠网织红细胞,与小鼠淋巴瘤细胞株融合,对杂交细胞基因产物血红蛋白进行联苯胺组织化学染色、荧光抗体和生化测定。结果与电镜和流式细胞光度计的实验结果一致,表明珠蛋白基因产物血红蛋白住杂交细胞中,能够表达并成为融合细胞的标志。联苯胺阳性细胞数在HAT选择性培液筛选后数量增加。BW×R胞质杂交细肥在传代过程中,仍继续出现血红蛋白。文中对杂交细胞血红蛋白表达及其在传代过程中,在一部分细胞中丢失的可能机理进行了讨论。     

Early Phase Karyotype Analysis of Chromosome Segregation After Formation of Mouse–Mouse Hybridomas with Chromosome Painting Probes

FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 × 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine–azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine–antidigoxigenin antibodies and fluorescein–avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%.     

人早幼粒白血病细胞与小鼠网织红细胞融合的胞质杂交细胞形态学观察

本研究对HGPRT缺失的人早幼粒白血病细胞突变株(HL-60-AR)与小鼠网织红细胞融合所形成的胞质杂交细胞,进行了光镜和电镜的形态学观察。所得结果显示:胞质杂交细胞在短期培养过程中,可见大量细胞呈现核固缩和核偏位,甚至出现排核现象;长期培养的胞质杂交细胞沿不同途径向较成熟的方向分化。本文并讨论了网织红细胞胞质因子对人早幼粒白血病细胞分化途径的影响。     

敌枯双对雄性小鼠生殖细胞分裂比率及精原细胞染色体畸变的影响

本文研究丁敌枯双对BALB/C雄性小鼠生殖细胞分裂比率及精原细胞染色体畸变的影响。将实验小鼠随机分为三组,即实验组(敌枯双组),阳性对照组(环磷酰胺组)和阴性对照组(双蒸水组)。结果发现:敌枯双能明显诱发精原细胞多倍体率和裂隙率增加,抑制终变期/中期Ⅰ和中期Ⅱ细胞的减数分裂比率,促进精原细胞有丝分裂比率。实验结果还提示:在遗传毒理检测中亚急性实验是必要的,并对实验结果进行了初步讨论。     

杂交瘤研究中染色体分析的方法及其意义

目的探讨细胞同步化染色体高分辨技术检测杂交瘤细胞染色体的可行性。方法选择小鼠骨髓瘤SP-2/O细胞株、人淋巴母细胞CKM-8细胞株、人骨髓瘤KM细胞株、抗小鼠胰腺癌鼠-鼠杂交瘤细胞株,体外培养24～48h。对4种细胞株分别进行同步化处理,加入5-氟脱氧尿嘧啶核苷和尿嘧啶培养16～18h后,加入5-溴脱氧尿嘧啶核苷,并在收集细胞前加入秋水酰胺;收集细胞进行离心、低渗处理、固定、再固定,制备染色体滴片,应用胰蛋白酶-Giemsa染液对染色体进行G显带处理。每例标本以30个以上分裂象做众数分析,按照《人类细胞遗传学国际命名体制》(ISCN 1978)对细胞株染色体进行G显带分析。结果 4种细胞株的染色体均较长、分散良好、形态较好。SP-2/O细胞株、CKM-8细胞株、KM细胞株、抗小鼠胰腺癌鼠-鼠杂交瘤细胞株的染色体数目分别为63、46、42、105,其中抗小鼠胰腺癌鼠-鼠杂交瘤细胞染色体数目接近小鼠淋巴细胞和小鼠骨髓瘤细胞染色体数的总和,且多数为端着丝点染色体。结论细胞同步化染色体高分辨技术检测杂交瘤细胞染色体具有良好的效果。     

Further evidence for the aneuploidogenic properties of chelating agents: induction of micronuclei in mouse male germ cells by EDTA.

A micronucleus assay based on cytogenetic analysis of early spermatids (Tates et al.; Mutation Research 121:131-138, 1983) was applied to determine if the chelating agent ethylenedinitrilotetraacetic acid (EDTA) may induce aneuploidy in mouse meiotic cells. Previous results indicated aneuploidogenic activity of EDTA in Drosophila female germ cells (induction of chromosome loss; Zordan et al.: Environ Mol Mutagen 15:205-213, 1990). In the same study, a standard aneuploidy test based on chromosome counting in mouse secondary spermatocytes failed however to show aneuploidogenic properties of EDTA in mouse somatic and germ cells. In the present study the effects of two clastogens, adriamycin (ADM) and mitomycin C (MMC), and of the aneuploidogenic agent chloral hydrate (CH) were also evaluated. All compounds were tested at a single dose level and at two time intervals corresponding to the treatment of diakinesis/metaphase I/metaphase II spermatocytes. The clastogenic potential of the compounds under study was also evaluated, by chromosomal aberration analysis in mouse spermatogonia, in an independent set of experiments. The results obtained indicate that ADM, CH and EDTA are able to induce micronuclei at meiosis. On the contrary, only ADM and MMC induced chromosomal aberrations in mouse spermatogonia. Therefore, the most probable origin of micronuclei produced by CH and EDTA is whole chromosome lagging. These results provide further evidence for the aneuploidogenic properties of these chelating agents.     

Clastogenicity of PvuII and EcoRI in electroporated CHO cells assayed by metaphase chromosomal aberrations and by micronuclei using the cytokinesis-block technique

The clastogenicity of two restriction endonucleases with almost equal cutting frequencies: PvuII, generating blunt-ended DNA double-strand breaks (dsb) and EcoRI, generating cohesive-ended dsb, has been measured after treatment of electroporated CHO cells with these enzymes. Chromosome damage was assessed by the micronucleus cytokinesis-block technique, and for certain electroporation voltages by analysis of metaphase preparations. As has been found in previous studies, PvuII was found to be more effective in causing chromosomal damage than EcoRI, indicating the greater importance of blunt-ended dsb in chromosome aberration induction. These findings also validate the use of the micronucleus cytokinesis-block technique for evaluating chromosomal damage from restriction endonucleases. The results show a biphasic induction of micronuclei in binucleate cells as a function of time after treatment. This pattern is interpreted as indicating variable sensitivity of cells to restriction endonucleases at different stages of the cell cycle. The micronucleus data show that late collection times (40-48 h after treatment) give higher frequencies than short times. Both micronucleus and metaphase aberration data indicate that voltages in excess of 260 V are more efficient in porating cells than lower voltages and, as a result, lower restriction endonuclease concentrations could be used.     

Increased amplification proximal to the MLL gene in acute myeloid leukemia

Amplification of the chromosomal region 11q23 encompassing the MLL gene has been observed in patients with acute myeloid leukemia (AML). Patients with this abnormality often have a poor response to chemotherapy and short survival. We have studied the regions flanking the MLL gene using 8 bacterial artificial chromosome (BAC) probes and dual color fluorescence in situ hybridization (FISH) analysis. Two contigs of BAC probes flanking the MLL gene, were constructed by standard primer walking and BAC end sequencing. For comparison, metaphase chromosome preparations were also hybridized with a commercial MLL specific probe. Metaphase chromosomes were prepared from the bone marrow sample of an 80-year old female patient with AML-M1 and the cytogenetic aberration der(11)hsr(11) (q23). FISH analysis on metaphase chromosomes showed amplification on the homogeneously staining region (hsr) and marker chromosomes for both the MLL gene and the BAC probes. Using dual color FISH, probes proximal to MLL showed greater amplification than those distal to MLL, as represented by additional red signals on both metaphase and interphase chromosomes. Ratios of the copy numbers of the BAC probes relative to the chromosome 11 centromere copy number confirmed a higher copy number for probes proximal to MLL. These results suggest that other gene(s) proximal to MLL could be the target gene(s) of amplification in this case and not the MLL gene as previously assumed.     

Chromosome instability in lymphocytes from two patients affected by three sequential primary cancers: the role of fragile sites

The chromosomal aberration rate and the expression of fragile sites induced by aphidicolin were evaluated in metaphase chromosomes obtained from peripheral blood lymphocytes of two untreated patients with multiple primary cancers. Spontaneous aberrations of chromosome number and structure and chromosome fragility were compared with controls with the use of the same methods. Chromosomal aberration rates and expression frequencies of fragile sites were significantly higher in the patients than in normal control subjects. In the patients, all but one structural chromosome aberration involved at least one fragile site. Our results suggest that fragile sites may be unstable regions of the human genome, which might play an important role in the genetic instability associated with cancer predisposition.