Establishment and Characterization of Human Ovarian Carcinoma Cell Lines

Five human ovarian carcinoma cell lines cultured from primary and metastatic tumors of Korean patients were characterized. These lines were isolated from two papillary serous cystadenocarcinomas, two endometrioid carcinomas, and one malignant Brenner tumor. It was shown that the growth of these cell lines was stable when cultured after at least 20 passages. Population doubling times varied from 40 to 67 hr. All lines showed high viability and were proven by DNA fingerprinting analysis to be unique. Contamination by mycoplasma or bacteria was excluded. In two lines, SNU-8 and SNU-840, an elevated level of CA125 antigen secretion could be detected, whereas CEA was undetectable in all five lines. Four different mutations in functional and highly conserved regions of the p53 gene were identified in three of our five lines (60%), namely in SNU-119, SNU-251, and SNU-563. Included were two missense mutations, one in-frame 3-base-pair deletion, and one out-of-frame 1-base-pair deletion. It is interesting to note that one of these three lines, SNU-251, presented an additional simultaneous nonsense mutation of the BRCA1 gene and missense mutation of the hMLH1 gene. In its lacking both wild-type alleles of the BRCA1 gene, SNU-251 might serve as an unusual and importantin vitromodel for studies related to ovarian carcinoma and the BRCA1 gene. It is thus likely that the establishment and characterization of these permanent human ovarian carcinoma cell lines in continuous cultures can provide useful tools forin vitrostudies related to human ovarian carcinomas.     

人卵巢癌细胞系具有不同转移潜能亚系的分离及体内和体外鉴定

目的:克隆具有不同转移潜能的癌细胞亚系,并对其侵袭转移能力进行体内外鉴定。方法:应用有限稀释法对卵巢癌细胞系SKOV3进行单细胞克隆,共分离出14个克隆亚系,根据细胞电泳率,流式细胞仪检测所得细胞周期,筛选出3个电泳率差异较大的克隆亚系(S1、S6和S14)。并应用体内外试验检测各亚系的生物学特性及其侵袭转移能力。结果:S1为高转移亚系,体外生长快,侵袭人工基底膜细胞数及软琼脂集落形成数显著多于其他二者,裸鼠体内100%成瘤,70%转移;而S14为不转移亚系,体外生长慢,侵袭人工基底膜细胞数及软琼脂集落形成数显著少于其他二者,裸鼠体内仅20%成瘤,无1例发生转移。结论:该系统为来自同一肿瘤母系,遗传背景完全相同,却具有不同转移潜能的异质性克隆,为肿瘤转移机制的研究和克隆肿瘤转移相关基因提供了良好模型。     

Establishment and Characterization of a New HPV-Negative Squamous Cell Carcinoma Cell Line (Yumoto) from the Human Uterine Cervix

A new cell line, Yumoto, derived from a squamous cell carcinoma of the uterine cervix, was established from serially transplanted tumor tissues in nude mice. Monolayer cultured cells were polygonal and formed pavement-like sheet. They showed a piling-up tendency and were devoid of contact inhibition. Electron micrographs demonstrated the presence of microvilli on the cell surface, abundant tonofilaments in the cytoplasm, and the connection with desmosomes. These electron micrographical characteristics of Yumoto cells were consistent with those of squamous cell origin. Yumoto cells were highly tumorigenic in BALB/c nude mice and produced a well-differentiated squamous cell carcinoma of keratinizing type which closely resembled to the original tumor tissues in nude mice. The presence of HPV DNA was examined using polymerase chain reaction and Southern blot analysis, but no known types of HPV DNA could be detected. Exons 2 through 11 of the p53 gene were analyzed by direct DNA sequencing, revealing a homozygous mutation at codon 281 in exon 8, GAC to CAC (Asp→His). Furthermore, physical p53-gene deletion was demonstrated by dual-color fluorescencein situhybridization. This cell line is useful for studying the carcinogenesis of cervical carcinoma and for investigating the biological characteristics of a HPV-negative and mutated p53 squamous cell carcinoma of the uterine cervix.     

人子宫颈癌裸鼠移植瘤株与细胞系的建立及其生物学特性

目的：建立人宫颈癌裸鼠体内瘤株及其相应的体外细胞系。方法：把人宫颈高分化鳞癌手术切除标本移植于裸鼠皮下，生长后行鼠间连续传代。取移植瘤行体外培养，建立相应的体外细胞系。对体内瘤株和体外细胞系进行相关的生物学检测，并行体外细胞系克隆和无血清培养。结果：历时１７个月建成人宫颈高分化鳞癌裸鼠移植瘤株ＨＣＣ－９４Ｖ，瘤株生长稳定，已传２６代，移植成瘤率为９２．９％。光镜、电镜下的形态学特征与患者肿瘤组织一致，染色体多为异倍体；多种肿瘤标记物呈高表达，癌基因蛋白产物呈低表达；人乳头状瘤病毒（ＨＰＶ）１６型呈阳性、１８型呈阴性，与患者肿瘤组织的检测结果一致。体外培养维持传代１６个月，建成人宫颈癌细胞系ＨＣＣ－９４，生长稳定，已传１３１代。电镜下显示典型的桥粒和张力原纤维结构，以及人类肿瘤异常染色体特征。细胞的肿瘤标记物及癌基因蛋白产物呈高表达。ＨＰＶ１６及１８型呈阴性。细胞裸鼠移植致瘤，其组织病理形态学与患者肿瘤组织一致，且ＨＰＶ１６呈阳性。细胞系克隆出了３个亚株，无血清培养成功。结论：ＨＣＣ－９４Ｖ和ＨＣＣ－９４是一株新的宫颈癌裸鼠移植瘤株和细胞系，为宫颈癌的进一步研究提供了理想的材料和模型。     

曲古霉素A对人宫颈癌Hela细胞增殖及凋亡的影响

目的:探讨组蛋白乙酰化酶抑制剂曲古霉素A(TSA)对人宫颈癌Hela细胞增殖和调亡的作用及其机制.方法:不同浓度的TSA处理Hela细胞,应用Western blot法检测细胞内组蛋白H4(Ac-H4)乙酰化水平,荧光定量PCR方法检测p21 mRNA表达,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡率.结果:随着TSA浓度增加,人宫颈癌Hela细胞内Ac-H4乙酰化水平增加(P＜0.05),p21 mRNA表达增加(P＜0.05),细胞增值率减少(P＜0.05),细胞凋亡率增加(P＜0.05).结论:TSA通过提高组蛋白乙酰化水平,增加p21基因表达,抑制人宫颈癌Hela细胞的增殖和诱导细胞凋亡,且呈浓度依赖性.     

卵巢透明细胞癌诊疗及预后

卵巢透明细胞癌(ovarian clear cell carcinoma,OCCC)是卵巢上皮细胞肿瘤中的一种特殊类型,发病率具有明显的地域差异,亚洲发病率居高。临床上以发病率较低、发现时国际妇产科联盟(FIGO)分期较早、治疗及时为其主要的特点。治疗严格按照FIGO分期进行分期手术,并在术后给予适当足疗程的化学治疗、放射治疗或其他治疗方案,但患者预后仍较差。化疗敏感性差是患者预后差的独立危险因素。OCCC患者的5年生存率与患者的肿瘤FIGO分期、术前CA125值是否高于150 U/m L、肿瘤细胞减灭程度、化疗方案的疗程是否足够有关。FIGO分期越早,术前CA125值≤150 U/m L、肿瘤切除干净、术后足疗程的化疗均可以提高患者的5年生存率。     

姜黄素、佛波酯对人卵巢癌细胞株SKOV3细胞生长的影响

目的:通过研究抑癌剂姜黄素(CCM)、促癌剂佛波酯(TPA)对体外培养的人卵巢癌细胞株SKOV3细胞增殖、细胞形态及细胞周期的影响,探讨肿瘤细胞SKOV3增殖抑制的效应机制.方法:采用MTT法测定CCM、TPA不同浓度及不同时间对人卵巢癌细胞株SKOV3增殖的抑制作用,计算细胞生长抑制率.光镜和电镜下观测细胞形态学及超微结构的改变.流式细胞仪检测细胞周期.结果:TPA具有抑制人卵巢癌细胞株SKOV3细胞增殖的作用,呈剂量和时间依赖性,细胞生长停滞于G1/S期.部分细胞出现凋亡形态学改变.TPA抑制人卵巢癌细胞株SKOV3细胞的增殖,呈剂量和时间依赖性.细胞周期发生G1→S期阻滞.结论:CCM、TPA通过抑制细胞增殖,细胞周期发生停滞,显著地抑制卵巢癌细胞SKOV3的体外生长.     

Characterization of Glucose Transport and Glucose Transporters in the Human Choriocarcinoma Cell Line, BeWo

In this study we have characterized 2-deoxyglucose (2DG) transport and hexose transporter expression in the human choriocarcinoma cell line, BeWo. 2DG uptake in BeWo cells displayed saturable kinetics (V(max), 29+/-1.5 nmol/min/mg protein;K(m), 1.5+/-0.02 m m) and was significantly inhibited in the presence of 2-deoxyglucose, mannose and 3- O -methyl glucose (all at a competing concentration of 30 m m) by up to 97 per cent, but not by galactose or fructose. Glucose uptake was not Na(+)-dependent, but was inhibited by cytochalasin B (by approx 85 per cent) indicating that hexose uptake was mediated via a facilitative glucose transport mechanism. Northern and immunoblot analyses revealed that BeWo cells expressed GLUT1 and GLUT5, but not GLUT2 or GLUT3. On immunoblots, GLUT1 migrated as a broad protein band on SDS-gels (average M(r)of 55 kDa) and treatment with N -glycanase resulted in a significant shift in its electrophoretic mobility; the core protein migrating as a 40 kDa band indicating that the carrier was heavily glycosylated. GLUT5 was detected as a discrete 60 kDa band and like GLUT1, the observed immunoreactive signal was lost when using antiserum that had been pre-adsorbed with the antigenic peptide. Our findings indicate that BeWo cells express a facilitative glucose transport system with characteristics broadly similar to those reported in isolated human placental membrane vesicles and that they are likely to serve as a useful experimental system for studying the regulation of placental glucose transport and transporter expression.     

Epithelial Ovarian Carcinoma: Principles of Primary Surgery

Surgery remains the most important facet in the initial management of epithelial ovarian cancer. Initial surgical therapy involves the establishment of the diagnosis, appropriate surgical staging, and primary cytoreductive surgery. For patients with advanced disease, surgical staging of ovarian cancer is obvious, but for apparently early disease (Stage I or II), appropriate surgical staging is extremely important and will result in the upstaging of about one-third of patients (usually to Stage III). The theoretical benefit of initial cytoreductive surgery is the removal of large necrotic tumors with a poor blood supply and the removal of large tumors that are in a slower growth phase, leaving behind tumors that are more sensitive to the effects of chemotherapy. There are multiple clinical studies indicating that "optimal" cytoreduction (removal of all tumor larger than 2 cm) results in improved complete response rates to chemotherapy, improved progression-free and overall survival, and a significant increase in the number of patients who will have a negative second-look surgical reassessment. Recent studies by the Gynecologic Oncology Group have further clarified the role of initial surgery, showing that the "biology" of the tumor is also important and that survival is directly related to residual disease within the following categories: (i) microscopic disease, (ii) optimal disease (2 cm or less in residual diameter), and (iii) suboptimal disease (greater than 2 cm diameter of residual disease). In the latter group (suboptimal disease), there may be a benefit to second attempts at surgical cytoreduction (interval cytoreductive surgery).     

卵巢透明细胞癌70例临床分析

目的:探讨卵巢透明细胞癌的临床特点、预后及与子宫内膜异位症的关系.方法:对70例原发性卵巢透明细胞癌患者回顾性分析其治疗方法、对化疗的敏感性和生存率,以及合并子宫内膜异位症患者对化疗的敏感性和生存率.结果:根据FIGO分期,70例中工期44例,Ⅱ期13例、Ⅲ期13例.70例总的5年生存率为48.97%.患者全部进行手术,其中满意肿瘤细胞减灭术62例,占88.6%(62/70);不满意肿瘤细胞减灭术8例,占11.4%(8/70).两组5年生存率分别为61.52%和7.69%(P＜0.05).术后5例放弃化疗,65例于术后1～2周内进行了以PT、PAC等共3至8个疗程化疗.是否合并子宫内膜异位症对化疗的敏感性和生存率无统计学差异.结论:卵巢透明细胞癌临床特点不同于其他的卵巢上皮性恶性肿瘤,其临床分期早,化疗不敏感,易复发,预后差.伴有子宫内膜异位症与否与预后无相关性.     

逆转录病毒介导的p16基因对人卵巢癌细胞系生长抑制作用的研究

目的研究逆转录病毒介导的ｐ１６基因对人卵巢癌细胞系ＣＡＯＶ３生长与致瘤性的抑制作用。方法应用脂质体介导法,将外源性野生型ｐ１６基因转染不表达ｐ１６的人卵巢癌细胞系ＣＡＯＶ３,对转染后细胞ＤＮＡ、ＲＮＡ和蛋白进行分析并观察其生物学行为变化。结果外源性野生型ｐ１６基因已整合于细胞并获稳定表达,表达有外源ｐ１６基因的细胞生长速度、集落形成率及裸鼠致瘤性均有部分抑制。细胞周期分析可见Ｇ１期增加,Ｓ期下降。电镜下超微结构出现生长抑制特征。结论ｐ１６基因可明显抑制癌细胞生长,在卵巢癌发生、发展的过程中起重要作用。     

白细胞介素2基因转导对卵巢癌细胞系SKOV3免疫原性的影响

目的 探讨白细胞介素２（ＩＬ－２）基因转导对卵巢癌细胞系ＳＫＯＶ３免疫原性的影响。方法 应用流式细胞术,测定ＳＫＯＶ３细胞组织相容性白细胞抗原（ＨＬＡ）ＨＬＡ－ＡＢＣ、ＨＬＡ－ＤＱ及ＨＬＡ－ＤＲ分子的表达,＾５１Ｃｒ－４ｈ释放实验显示ＳＫＯＶ３、ＳＫＯＶ３／Ｎｅｏ、ＳＫＯＶ３／ＩＬ－２细胞对自然杀伤（ＮＫ）细胞和淋巴因子激活杀伤（ＬＡＫ）细胞杀伤的敏感性。结果 ＳＫＯＶ３／ＩＬ－２细胞中ＨＬＡ－Ａ     

原发上皮性卵巢癌组织中多药耐药基因表达及其临床意义

目的探讨多药耐药（ＭＤＲ１）基因表达与原发性上皮性卵巢癌（ＰＥＯＣ）耐药的关系及其临床意义。方法应用半定量逆转录—聚合酶链反应（ＲＴ－ＰＣＲ）技术,对３７例ＰＥＯＣ、１０例正常卵巢上皮组织和２０例正常女性外周血单核细胞（ＰＢＭＣ）新鲜标本ＭＤＲ１基因表达进行了检测;并用四氮唑蓝（ＭＴＴ）法,检测了这３７例ＰＥＯＣ患者的原代癌细胞对临床常用抗癌药物的敏感性。结果ＰＥＯＣ标本中ＭＤＲ１基因阳性表达率为３０％,而正常卵巢上皮组织和ＰＢＭＣ则无ＭＤＲ１基因表达;ＭＤＲ１基因表达与ＰＥＯＣ临床期别及组织学类型无关,而与组织学级别有关,且有随级别升高而升高的趋势;ＭＤＲ１基因阳性表达组与阴性组比较,ＤＡＣＴ、ＡＤＭ、Ｅ－ＡＤＭ、ＶＣＲ和ＶＰ１６对ＰＥＯＣ细胞抑制率差异有显著性,其余则无显著性;ＭＤＲ１基因表达与无进展生存期有关,而与总体生存期无关。结论ＭＤＲ１基因表达与ＰＥＯＣ耐药有关,是抗ＭＤＲ相关药物的主要原因;ＭＤＲ１基因表达的检测,对临床用药有指导意义,对临床疗效判断和患者预后预测有一定价值。     

卵巢癌初始治疗中手术相关若干问题及临床意义

卵巢癌的初始治疗是影响患者预后的最重要因素.早期卵巢癌要进行全面的开腹分期手术,如初次手术未做到全面分期,应在化疗开始前进行再分期手术.晚期卵巢癌要争取在高级别的妇科肿瘤中心进行初次的满意的肿瘤细胞减灭术,特别要重视上腹部手术,力争做到无肉眼残留病灶.新辅助化疗和间歇性肿瘤细胞减灭术可以选择性地应用于部分晚期不适合直接...     

卵巢癌初始治疗中手术相关若干问题及临床意义

卵巢癌的初始治疗是影响患者预后的最重要因素。早期卵巢癌要进行全面的开腹分期手术,如初次手术未做到全面分期,应在化疗开始前进行再分期手术。晚期卵巢癌要争取在高级别的妇科肿瘤中心进行初次的满意的肿瘤细胞减灭术,特别要重视上腹部手术,力争做到无肉眼残留病灶。新辅助化疗和间歇性肿瘤细胞减灭术可以选择性地应用于部分晚期不适合直接手术的患者,或者初次基本手术后仍有残留的患者,但后者的适用指征还需要进一步明确。     

Uterine and Ovarian Conservation in Advanced Small Cell Carcinoma of the Ovary

Background: The decision to recommend removal or conservation of a normal ovary and uterus in a young woman with advanced ovarian cancer is difficult and controversial.Case: A 21-year-old patient with a large-cell variant of small-cell carcinoma of the ovary stage IIIc underwent optimal debulking surgery with preservation of the normal appearing uterus and opposite adnexa followed by aggressive multi-agent chemotherapy. She is menstruating normally and is free of disease, more than 2 years since completion of chemotherapy.Conclusion: In selected cases, conservation of the uninvolved ovary and uterus in patients with advanced-stage, small-cell carcinoma of the ovary may not compromise survival.     

联合检测COX-2及bFGF在卵巢上皮性癌中的表达和意义

目的:探讨环氧化酶-2(COX-2)及碱性成纤维细胞生长因子(bFGF)在卵巢上皮性癌中的表达及意义.方法:采用免疫组化方法检测19例正常卵巢组织及50例卵巢上皮性癌中COX-2与bFGF的表达情况.结果:COX-2及bFGF在卵巢上皮性癌中的表达率分别为76%和78%,明显高于正常卵巢组织,两者在不同病理类型的卵巢上皮性癌中表达均无统计学差异,但在肿瘤的不同FIGO分期及分级中表达有统计学差异.COX-2与bFGF在卵巢上皮性癌中的表达有一致性.结论:COX-2及bFGF与卵巢上皮性癌的分化程度密切相关,检测COX-2及bFGF可作为判断卵巢上皮性癌恶性程度的生物学指标.     

bFGF和Ets-1在原发性卵巢癌中的表达及意义

目的 :检测碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)和E2 6转录因子 (E2 6transformation specific 1,Ets 1)在原发性卵巢癌中的表达 ,为评估卵巢癌的生物学行为提供新的依据。方法 :应用免疫组织化学链霉素抗生物素—过氧化物酶复合法 (streptomycinavidin biotin peroxidasecomplex,SP)检测 32例卵巢癌组织中bFGF和Ets 1的表达 ,观察其与肿瘤组织学分级、临床分期、有无淋巴结转移及相互之间的关系。结果 :卵巢癌组织中bFGF和Ets 1的阳性表达率明显高于癌旁组织 (P <0 .0 0 1) ;bFGF和Ets 1的表达与原发性卵巢癌的FIGO分期和组织学分级密切相关 ;有淋巴结转移患者的bFGF和Ets 1阳性表达率高于无淋巴结转移患者 (P <0 .0 0 5和P <0 .0 1) ;癌组织中bFGF和Ets 1的表达密切相关 ,相关系数rs0 .396 (P <0 .0 5 )。结论 :bFGF和Ets 1的表达与原发性卵巢癌的转移有密切关系 ,检测bFGF和Ets 1的表达可作为了解卵巢癌生物学行为的参考指标