人鼻咽癌裸鼠移植瘤SUNT—1及其体外培养上皮细胞株SUNE—1细胞遗传学研究

对我所建成的鼻咽低分化癌的Ｅ８病毒核抗原阳性的裸鼠移植瘤ＳＵＮＴ－１及其体外培养上皮细胞株ＳＵＮＥ－１，分别于各代进行细胞遗传学分析，结果显示ＳＵＮＴ－１和ＳＵＮＥ－１癌细胞染色体的数目和结构均有相似的变化；染色体数呈非整倍体改变，并以亚四倍体为主，染色体畸变则以易位、重排、缺乏和等臂染色体为多见并形成较恒定的标记染色体Ｍ１、Ｍ２、Ｍ３、Ｍ５和Ｍ６，涉及３号染色体改变较多。提示进一步研究克隆ＳＵＮ     

骨髓增生异常综合征患者体外CFU-GM、CFU-L培养和细胞遗传学研究

研究了４２例骨髓增生异常综合征患者体外ＣＦＵ－ＧＭ、ＣＦＵ－Ｌ培养和细胞遗传学改变。结果显示，患者ＣＦＵ－ＧＭ明显低于正常对照组（Ｐ＜０．０１），但高于再生障碍性贫血（ＡＡ）（Ｐ＜０．０１），ＣＦＵ－Ｌ高于正常对照组和ＡＡ组（Ｐ＜０．０１）。患者５２．４％存在染色体异常，常见为：＋８、－２２、－Ｘ、－Ｙ、－２０、－７／７ｑ－。ＣＦＵ－ＧＭ减低、ＣＦＵ－Ｌ增高和染色体异常者疗效较正常者差。表明ＣＦＵ－ＧＭ、ＣＦＵ－Ｌ体外培养和细胞遗传学检查可作为ＭＤＳ协助诊断和预测疗效的指标之一。     

原发性肝细胞癌多染色体杂合性丢失研究

研究原发性肝细胞癌（ＨＣＣ）５对染色体３８个位点等位基因杂合性丢失（ＬＯＨ），ＬＯＨ与肝癌的临床病理改变和肝炎病毒的关系。方法：用ＰＣＲ微卫星多态性分析检测肝癌ＬＯＨ。结果发生高频率ＬＯＨ的位点有染色体１ｐ的Ｄ１Ｓ１８６（这，４８．１％）和Ｄ１Ｓ２４３（１ｐ３６．３，５１．６％）；染色体９ｎ２４的Ｄ９Ｓ５４（６１．８％），９这的Ｄ９Ｓ１７４７（５２．％）和ＤＤ９Ｓ１７５２（５１．８％）ＷＵＧ ＨＫ     

人鼻咽癌裸鼠移植瘤SUNT─1及其体外培养上皮细胞株SUNE─1细胞遗传学研究

对我所建成的鼻咽低分化癌的ＥＢ病毒核抗原阳性（ＥＢＮＡ）的裸鼠移植瘤ＳＵＮＴ－１及其体外培养上皮细胞株ＳＵＮＥ－１，分别于各代进行细胞遗传学分析，结果显示ＳＵＮＴ－１和ＳＵＮＥ－１癌细胞染色体的数目和结构均有相似的变化；染色体数呈非整倍体改变，并以亚四倍体为主，染色体畸变则以易位、重排、缺乏和等臂染色体为多见并形成较恒定的标记染色体Ｍ１、Ｍ２、Ｍ３、Ｍ５和Ｍ６，涉及３号染色体改变较多。提示进一步研究克隆ＳＵＮＦ－１和ＳＵＮＴ－１亚株的细胞遗传学和ＥＢ病毒基因存在变化，将为深入探讨鼻咽癌病因提供更多启示。     

抗CD_3/抗CD_(20)微型双功能抗体裸鼠体内分布研究

目的：建立裸鼠皮下人Ｂ－淋巴细胞移植瘤模型,研究抗ＣＤ３／ＣＤ２０。微型双功能抗体在裸鼠体内的分布。方法：采用亲和层析分离纯化本室构建的抗 ＣＤ３／抗 ＣＤ２０微型双功能抗体可溶性表达产物,并用ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ ｂｌｏｔ、分子排阻层析和 ＦＡＣＳ鉴定纯化产物;采用５周龄左右的雌性 ＢＡＬＢ／ｃ－ｎｕ,经４Ｇｙ照射、皮下接种 Ｒａｊｉ细胞建立裸鼠皮下人Ｂ－淋巴细胞移植瘤模型,并于眼底静脉丛注射１２５Ｉ标记的抗ＣＤ３／抗ＣＤ２０微型双功能抗体,测定各组织中１２５Ｉ的分布。结果：雌性ＢＡＬＢ／ｃ－ｎｕ经照射、皮下接种１×１０～７～３×１０～７Ｒａｊｉ细胞／只,６～１４天均开始出现移植瘤,移植瘤细胞膜表面标记与 Ｒａｊｉ相同,且在２４小时时肿瘤部位１２５ Ｉ的分布明显低于心、肝、肾、脾,其比值分别为０．０８、０．１９、０．０６和０．５１,而 ７２小时时肿瘤部位１２５Ｉ的分布则高于心、肝、肾、脾,其比值分别为 ２．３２、９．４６、８．２４和９．５０。结论：抗ＣＤ３／抗ＣＤ２０微型双功能抗体能在裸鼠皮下人Ｂ－淋巴细胞移植瘤部位富集,是一个有望用于Ｂ细胞恶性肿瘤临床治疗的双特异性抗体。     

长春地辛及含春地辛方案治疗晚期恶性肿瘤的二期临床研究

使用长春地辛（ＶＤＳ）和长春碱（ＶＬＢ）治疗恶性肿瘤５６例，结果ＶＤＳ单药有效率为２２．７％，ＶＤＳ联合组有效率４２．１％，ＶＬＢ联合组有效率３５．７％，其中ＶＤＳ＋顺铂（ＤＤＰ）治疗非小细胞肺癌有效率达５０％，稍高于国外相同方案的结果，ＶＬＢ＋ＤＤＰ则为３７．５％，ＶＤＳ和ＶＬＢ的主要毒性为骨髓抑制，白细胞下降（Ｉ＋Ⅱ＋Ⅲ＋Ⅳ）分别为７３．３％和７４．９％，外周神经毒性轻，所有毒性可以耐受。     

长春地辛及含长春地辛方案治疗晚期恶性肿瘤的二期临床研究

使用长春地辛（ＶＤＳ）和长春碱（ＶＬＢ）治疗恶性肿瘤５６例。结果ＶＤＳ单药有效率为２２．７％，ＶＤＳ联合组有效率４２．１％，ＶＬＢ联合组有效率３５．７％，其中ＶＤＳ＋顺铂（ＤＤＰ）治疗非小细胞肺癌有效率达５０％，稍高于国外相同方案的结果，ＶＬＢ＋ＤＤＰ则为３７．５％。ＶＤＳ和ＶＬＢ的主要毒性为骨髓抑制，白细胞下降（Ⅰ＋Ⅱ＋Ⅲ＋Ⅳ）分别为７３．３％和７４．９％，外周神经毒性轻，所有毒性可以耐受。     

尼妥珠单抗对耐多西紫杉醇人肺腺癌细胞株化疗敏感性的调变作用

目的　通过体内外实验研究人源化抗表皮生长因子受体（ＥＧＦＲ）单抗尼妥珠（ｈ-Ｒ３）对耐多西紫杉醇（ＤＴＸ）人肺腺癌细胞株（ＳＰＣ-Ａ１／ＤＴＸ）化疗敏感性的调变作用。方法　免疫组化法及流式细胞仪测定人肺腺癌细胞株ＳＰＣ-Ａ１和ＳＰＣ-Ａ１／ＤＴＸ肺腺癌细胞株表面ＥＧＦＲ的表达强度,突变富集液相芯片法检测其ＥＧＦＲ、Ｋ-Ｒａｓ和ＰＩ３ＫＣＡ基因的突变情况,流式细胞仪检测ｈ-Ｒ３对细胞周期的影响以及ｈ-Ｒ３联合ＤＴＸ对细胞凋亡的影响,ＭＴＴ法检测ｈ-Ｒ３与ＤＴＸ的联合指数（ＣＩ）,裸鼠ＳＰＣ-Ａ１／ＤＴＸ移植瘤模型观察联合组及单药组对裸鼠抑制瘤模型肿瘤的增殖情况并计算瘤重抑制率（ＴＷＩ）。结果　ＳＰＣ-Ａ１细胞株ＥＧＦＲ表达强度为（＋,２１.5３％）,ＳＰＣ-Ａ１／ＤＴＸ细胞株为（＋＋＋,９２.４７％）;ＳＰＣ-Ａ１／ＤＴＸ细胞株的ＰＩ３ＫＣＡ基因外显子２０突变,ＳＰＣ-Ａ１细胞株无突变。ｈ-Ｒ３作用２４ｈ后,ＳＰＣ-Ａ１／ＤＴＸ细胞Ｇ１ 期阻滞较ＳＰＣ-Ａ１细胞更显著（Ｐ＝０.０００２）;ｈ-Ｒ３及ＤＴＸ联合用药后ＳＰＣ-Ａ１／ＤＴＸ细胞株的凋亡率为（２４.７±０.５）％,明显高于ｈ Ｒ３单药组的（１４.５±０.１）％,而单用ＤＴＸ后的凋亡率无显著升高,ＳＰＣ-Ａ１细胞株单药组及联合组凋亡率均有升高（Ｐ＜０.０５）;１００μｇ／ｍｌ及２００μｇ／ｍｌｈ Ｒ３与ＤＴＸ联合对ＳＰＣ-Ａ１或ＳＰＣ-Ａ１／ＤＴＸ细胞株的增殖抑制具有协同作用;联合组对ＳＰＣ-Ａ１／ＤＴＸ肺腺癌荷瘤裸鼠的ＴＷＩ为７１.７％,高于ＤＴＸ组的５２.６％和ｈ-Ｒ３组的３６.９％。结论　ｈ-Ｒ３能明显增加耐ＤＴＸ的人肺腺癌细胞株化疗的敏感性,其机制可能为ｈ-Ｒ３具有Ｇ１期阻滞和逆转耐药细胞凋亡抵抗的作用,并且其疗效与耐药细胞较亲本细胞的ＥＧＦＲ表达增强有关,而耐药细胞的ＰＩ３ＫＣＡ基因突变对疗效的影响不显著。     

重组腺病毒介导反义c-myc基因对人肝癌细胞系的治疗作用

目的：探讨重组腺病毒介导反义ｃ－ｍｙｃ基因（Ａｄ－ＡＳｍｙｃ）治疗人肝癌细胞的作用。方法：观察Ａｄ－ＡＳｍｙｃ对人肝癌细胞系的转导效率,通过细胞生长曲线、克隆形成实验、ＤＮＡ片段化分析、ＲＴ－ＰＣＲ、裸鼠皮下移植瘤治疗实验,分析Ａｄ－ＡＳｍｙｃ对人肝癌细胞系Ｂｅｌ－７４０２、ＱＳＧ－７７０１、ＳＭＭＣ－７７２１和ＨＣＣ－９２０４细胞生长和ｃ－ｍｙｃ基因表达及裸鼠肿瘤生长的抑制作用。结果：Ａｄ－ＡＳｍｙｃ可高效转导人肝癌细胞系,抑制细胞生长;转染细胞克隆形成能力降低,克隆成活率为对照组的５３．９％～６９．１％,ｃ－ｍｙｃ基因表达下降;Ａｄ－ＡＳｍｙｃ处理肝癌细胞,ＤＮＡ凝胶电泳出现明显的梯形条带,瘤内注射Ａｄ－ＡＳｍｙｃ可抑制裸鼠皮下移植瘤生长。结论：重组腺病毒介导的反义ｃ－ｍｙｃ基因转移,有可能成为肝癌基因治疗     

肺癌生物学特性与灌注化疗疗效和生存关系的初步探讨

本文对１７例Ⅲ期肺癌，测定了癌细胞ＤＮＡ含量，ＤＮＡ指数（ＤＩ），细胞增殖核抗原（ＰＣＮＡ）。１７例肺癌均给支气管动脉灌注化疗。小细胞肺癌（ＳＣＬＣ）和非小细胞肺癌（ＮＳＣＬＣ）分别缩小４８．８±３１．０％，３６．８±１７．１％（Ｘ±ＳＥ）。化疗后７例开胸手术６例切除肿瘤。ＳＣＬＣ和ＮＳＣＬＣ的中位生存期分别为３个月和８．５个月。在ＮＳＣＬＣ中，ＰＣＮＡ与肿瘤缩小范围呈负相关（ｒ＝－０．４７）；ＤＮＡ超四倍体百分比和ＤＩ存活时间呈正相关（（ｒ＝０．５１，ｒ＝０．５６）。ＳＣＬＣ则相关性差。结果表明，ＰＣＮＡ、ＤＡＮ和ＤＩ是ＮＳＣＬＣ判定疗效和预后的有用指标。     

具有高转移潜能的人肝癌细胞系的建立及其生物学特性

目的利用裸鼠人肝癌高转移模型（ＬＣＩ－Ｄ２０）的皮下移植瘤组织在体外建立一株具有高转移潜能的人肝癌细胞系（ＭＨＣＣ９７）,并对其一般生物学特性进行观察。方法将分离的瘤细胞制成细胞悬液,用１０％人ＡＢ型血清的高糖ＤＭＥＭ培养液建成该细胞系,采用流式细胞术和染色体Ｇ－显带方法,进行细胞遗传学分析;用ＡＢＣ免疫组化法,观察其肺转移灶中癌细胞甲胎蛋白（ＡＦＰ）表达情况。结果ＭＨＣＣ９７细胞为典型的上皮样细胞,符合一般上皮性恶性肿瘤细胞的病理学特征。该细胞经皮下和肝内接种均可使裸鼠致瘤,并发生肺部转移。肝内接种者,肺转移达１００％（１２／１２）。ＭＨＣＣ９７细胞为异倍体细胞,染色体均为超二倍体,ｉ（１）（ｑ）和ｄｅｒ（４）（ｐｔｅｒ→ｑ３５：：？）等为其标志染色体,未显示有完整Ｙ染色体存在。肺转移灶的癌细胞ＡＦＰ阳性。结论ＭＨＣＣ９７细胞具有与原移植瘤相似的生物学特性。染色体的畸变可能与其发生发展有关     

Suppression of malignancy in human lung cancer (A549/8) times mouse fibroblast (3T3-4E) somatic cell hybrids.

Interspecies hybrid cells were formed by the fusion of two parent cells: 1) the human lung cancer (bronchioloalveolar) line A549/8, which is not contact inhibited, rapidly produces tumors in athymic nude (nu/nu) mice, and forms colonies in agarose, and 2) the mouse fibroblast line 3T3-4E, which is contact inhibited, is nontumorgenic in nuce mice, and does not form colonies in agarose. These hybrid cells were tested 40-50 generations after fusion. The presence of 20 of the 23 different human chromosomes was tested by isoenzyme analysis, and examples of expression of each isoenzyme marker were found in at least some hybrid clones. All 14 independent hybrid clones tested were nontumorigenic in nude mice. Testing of hybrid clones for their ability to form colonies in agarose revealed two distinct phenotypes: agarose (clones forming colonies at 1-4% of the plated cells) and agarose (no colonies formed/10(5) cells tested). These phenotypes were discordant with all human isoenzymes tested. Malignant human lung cancer A549/8 times non-malignant mouse 3T3-4E cell hybrids were nontumorigenic in nude mice; thus malignancy of the bronchioloalveolar lung cancer behaved as a recessive trait. This nontumorigenicity was not accounted for by an absolute loss of the human chromosomes tested, but gene dosage may play a role. In contrast, the ability to clone in agarose was expressed in some hybrids (and thus behaved as a dominant trait); at present, agarose clonability cannot be related to specific human chromosomes.     

Isolation of a metastatic ascitic subpopulation from a human lung adenocarcinoma cell line in nude mice and studies on its biological properties

An ascitic subpopulation (Anip) was isolated from human lung adenocarcinoma cell line AGZY-83-a through serial intraperitoneal passages in nude mice. Anip manifested a much higher metastatic potential than the parent line. Spontaneous lung metastases occurred in 95% of the mice xenografted intraperitoneally, and the diffuse pattern of the metastases in the lung was very impressive. Anip also differed more or less from the parent line in morphology, growth, rate, electrophoretic mobility, platelet aggregating activity and noticeably in the chromosome histogram. The present model may be used in the study of tumor invasion and metastasis, as large amounts of well dispersed metastatic human carcinoma cells could be reproduced by ascites and much information about the biology of invasion and metastasis of human tumor cells in nude mice could be obtained.     

Loss of RAB25 expression in breast cancer

A novel breast cancer cell line (RAO-3) was established by transduction of the Q61L mutant RAS into human mammary epithelial cells that were immortalized with catalytic subunit of telomerase (hTERT). The cells displayed anchorage-independent growth and proliferation, and formed human mammary spindle cell carcinoma when injected into nude mice. Chromosome locus 1q22-23 was partially duplicated and inverted on one of the 3 chromosomes present in the cell line. We report here that mutations of chromosome 1q22-23 locus have resulted in the loss of RAB25 expression in the breast cancer cell line. Transduction of RAB25 into the breast cancer cell line arrests anchorage-independent growth. We have also demonstrated loss of RAB25 in human breast tumor tissue. These data suggest that loss of RAB25 might contribute to tumorigenesis of breast cancer, and RAB25 is likely to be an important factor in the development of breast cancer. RAB25 could be used as biological marker of breast cancer and provides a target for gene replacement therapy.     

lncRNA ZFAS1在肺癌中的表达及其对肺癌细胞生物学功能的影响及机制研究

目的:探究lncRNA ZFAS1在肺癌中的表达及其对肺癌细胞生物学功能的影响,同时通过构建肺癌裸鼠移植瘤模型,探寻其可能存在的分子机制。方法:采用qRT-PCR检测ZFAS1在肺癌组织和细胞系中的mRNA表达水平及荧光原位杂交(FISH)技术检测肺癌组织中ZFAS1的阳性指标;受试者工作特征(ROC)曲线检测血清中ZFAS1在肺癌临床诊断中的灵敏度和特异度;Transwell侵袭实验、细胞划痕实验及Tunel细胞凋亡实验检测上调/下调ZFAS1对NCI-H1299和NCI-H460细胞侵袭、迁移及凋亡的影响;检测肺癌裸鼠皮下移植瘤生长情况;Ki67和PCNA免疫荧光和PCNA免疫组化实验检测裸鼠体内细胞增殖情况。结果:ZFAS1 mRNA表达在肺癌组织和各细胞系中均上调;ROC曲线显示当ZFAS1的相对表达量取值为0.84时,其敏感度和特异度分别为86.7%和76.7%;过表达ZFAS1促进NCI-H1299细胞侵袭、迁移及抑制细胞凋亡,而干扰ZFAS1则抑制NCI-H460细胞侵袭、迁移及促进细胞凋亡;ZFAS1促进裸鼠皮下瘤体生长;ZFAS1在裸鼠皮下肺癌组织中的阳性指标显著增加...     

Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice

A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the over-expression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493–497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.     

Characterization of a continuous cell line (MHH-NB-11) derived from advanced neuroblastoma

A continuous cell line was established from an explanted tumor biopsy obtained from a patient with advanced neuroblastoma, which showed no response to chemotherapy. This cell line MHH-NB-11 retained most properties of the original immature tumor, even after xenotransplantation into nude mice. The cell line consisted of small dense cells with scant cytoplasm and thin; long processes and expressed neuron-specific enolase and synaptophysin, but neither GFAP nor S-100 protein. Karyotyping showed karyograms with 49 to 54 chromosomes, with a modal at 52. Most cells had trisomy 2,7,8,20, but only few structural aberrations were observed. Two of four chromosomes 1 showed a rearrangement of the terminal 1p segment, and all cells had a long HSR on the long arm of one of the chromosomes 13. This region hybridized in situ with the N-myc probe pNB-1. N-myc was amplified 20-fold in this neuroblastoma cell line as determined in Southern blot analysis. This cell line should be a useful tool in vitro or as a xenograft model for neuroblastoma research.