新发现1例人卵巢上皮癌组织移植裸鼠形成的“端着丝粒染色体”恶性细胞系

目的 按照常用肿瘤细胞建系方法,以人卵巢上皮癌组织移植免疫缺陷小鼠,通过原代培养建立永生化细胞系,为人体肿瘤的研究提供体外研究模型。 方法 将1例卵巢浆液性乳头状癌IIIcG3患者手术切除的肿瘤组织接种裸鼠皮下成瘤,通过体外原代培养,建立1株悬浮生长的细胞系。通过光学显微镜、电子显微镜、生长曲线测定、染色体分析、克隆形成实验、双层软琼脂培养、裸鼠接种等,对其生物学特性进行研究。 结果 该细胞系已传至100代以上,命名为WSZ。其生物学特性为：形态学观察细胞呈悬浮球形生长状态;细胞生长增殖旺盛,对数期细胞群体倍增时间约15.8h;染色体为16~135条,以68和69条染色体为多见,染色体分析显示,基本全部为端着丝粒染色体;细胞克隆形成率为89.3%,具有软琼脂集落形成能力;异种移植实验表明10⁶细胞在裸鼠皮下可成瘤, 而100个细胞接种非肥胖型糖尿病/重症联合免疫缺陷（NOD/SCID）小鼠即可形成皮下肿瘤。 结论 WSZ能够在体外长期生长和稳定传代,WSZ是一高度恶性的细胞系。     

人乳头状瘤病毒16型E6/E7基因对人喉鳞癌细胞系表皮生长因子受体和基质金属蛋白酶9表达的影响

表皮生长因子受体（EGFR）和基质金属蛋白酶（MMP）9在肿瘤的侵袭和转移发生过程中发挥着重要作用,其表达上调能导致肿瘤的侵袭和转移潜能增强。人乳头状瘤病毒16（HPV16）为喉鳞癌的主要致病因素,其主要转化活性部位在E6和E7开放读码框架。我们采用脂质体转染技术将携带有HPV16-E6／E7DNA的真核表达质粒导入HPV阴性的Lscc-02喉鳞癌细胞系,使其在细胞中表达,研究其对Lscc-02喉鳞癌细胞系EGFR和MMP-9mRNA及蛋白表达的影响,探讨HPV16-E6／E7基因与喉鳞癌演进及预后的关系。[第一段]     

人类风湿性关节炎滑膜细胞系(RASB)的建立和生物学特性的观察

目的　建立人类风湿性关节炎滑膜细胞永生细胞系。　方法　用重组有HPV16病毒E6 E7基因阅读框架的逆转录病毒载体转染原代培养的人类风湿性关节炎滑膜细胞 ,经G418筛选 ,获取细胞克隆RASB ,并从形态学、生长特性、核型组成、致瘤性和分泌功能等多方面对建系细胞RASB进行生物学观察。　结果　实验和观察证实 ,转化滑膜细胞染色体整合HPV病毒DNA ,表达HPVE6蛋白 ,基本保留了B型滑膜细胞特征形态、细胞骨架和分泌功能 ,倍增时间缩短一半 ,对裸鼠无致瘤性 ,软琼脂培养形成细小集落。已稳定传代大于 10 0代。　结论　建立了能长期体外稳定传代的人类风湿性关节炎滑膜细胞系。此细胞系的建立将为类风湿关节炎致病机理的研究和治疗提供极有意义的体外细胞模型。     

Balb/c裸鼠体内的干细胞致瘤实验

背景:干细胞致瘤性研究是临床应用干细胞安全性所面临的实际问题。因此明确干细胞是否具有致瘤性尤为重要。裸鼠在肿瘤学、免疫学、药品及生物制品的安全性评价领域占据愈来愈重要的地位。目的:将神经干细胞、间充质干细胞移植在Balb/c免疫缺陷裸鼠皮下,检测其是否具有致瘤性。方法:将免疫缺陷Balb/c裸鼠随机分为5组,除空白组外,阳性对照组、阴性对照组、神经干细胞组和间充质干细胞组分别将肝癌细胞HepG2、人视网膜色素上皮细胞、传代至第4代的神经干细胞和间充质干细胞接种于裸鼠皮下,于注射后12周进行解剖检查,观察裸鼠移植部位肿瘤形成情况。同时进行软琼脂克隆形成实验,考察神经干细胞和间充质干细胞在体外软琼脂克隆形成情况。结果与结论:裸鼠致瘤性实验结果显示,空白组、阴性对照组、神经干细胞组、间充质干细胞组在接种12周后,未在裸鼠注射部位形成肿瘤,病理组织学检查未见异常。软琼脂克隆形成实验结果显示,神经干细胞和间充质干细胞在软琼脂阻力介质中未表现出细胞形成克隆的能力。提示短期传代后的神经干细胞、间充质干细胞不具有致瘤性。     

喉鳞状细胞癌患者人乳头瘤病毒感染的检测分析

目的 了解喉鳞状细胞癌患者人乳头瘤病毒感染的情况.方法 对收集到的64例临床诊断喉癌病例的石蜡组织标本应用Luminex及PCR的方法对HPV感染进行基因检测,应用免疫组织化学的方法对HPV16/18E6蛋白进行检测.结果 在64例临床诊断的喉癌病例中,通过Luminex及PCR的方法发现7例病例具有HPV的感染;通过免疫组织化学的方法发现18例病例具有HPV16/18的感染,基因及蛋白检测的总阳性率达到39.1％.结论 此研究发现在喉癌患者中较高的HPV感染率间接说明HPV感染对喉癌发生的重要性,为阐明喉癌的发病机制奠定基础.     

苯并芘诱发人肺癌细胞系裸鼠移植瘤生物学研究

苯并芘诱发人肺癌细胞系裸鼠移植瘤生物学研究高振强郑杰吴秉铨王洁良崔湘林由江峰一、材料和方法１．实验动物移植方法：Ｂａｌｂ／ｃＡ裸鼠，体重２０ｇ，雌雄兼用，无菌饲养。将ＢＴ癌细胞系第２０代细胞皮下接种（１０７个细胞／只），每次接种２～３只支物。待肿瘤长...     

亚硝基吡啶对人乳头状瘤病毒诱导的食管上皮永生化细胞的促癌作用

目的 观察亚硝基吡啶在细胞恶性转化过程中的促癌作用.方法 用HPV18E6E7诱导食管上皮细胞永生化细胞系SHEE,第17代细胞培养在50 ml培养瓶.加入亚硝基吡啶（Nnitrosopiperidine,NPIP）0,2,4,8 mmol/L作用3周.用相差显微镜检查细胞形态,流式细胞仪检测细胞增殖和凋亡;染色体常规制样,检查染色体众数;细胞软琼脂集落形成及接种裸小鼠检查成瘤性;用Western blot检测HPV18表达.结果 当细胞暴露在8 mmol/L NPIP时细胞死亡增加,只剩少量活细胞.换正常培基代替NPIP,经4周后细胞进入增殖状态,细胞出现增生和异型增生.第8周末细胞软琼脂培养有大集落形成,接种裸小鼠成瘤.2,4 mmol/L组细胞倍增时间延长,细胞未能成瘤.8 mmol/L NPIP组染色体众数61～65,对照组56～61.实验组和对照组HPV阳性.结论 NPIP促进人乳头状瘤病毒诱导人胚食管永生化上皮恶性转化,HPV18E6E7和NPIP能协同作用加速食管上皮恶性转化.     

人前列腺癌裸鼠皮下移植瘤模型的建立

目的 建立人前列腺癌裸鼠皮下移植瘤模型.方法 采用人前列腺癌细胞株PC-3细胞接种于裸鼠的颈背部皮下,计算成瘤潜伏期、成瘤百分率,观察移植瘤大体生长情况,绘制生长曲线,并对移植瘤进行病理鉴定.结果 采用人前列腺癌细胞株PC-3细胞皮下接种方式建立移植瘤的平均成瘤潜伏期为24天,成瘤百分率为100％,瘤体积倍增时间为10天左右,移植瘤的形态和功能特性与原发肿瘤基本相似.结论 本动物模型的建立可为前列腺癌的放射免疫显像以及放射免疫治疗提供一个有价值的实验平台.     

人子宫内膜癌细胞系的建立及其生物学特性研究

目的：建立人子宫内膜癌细胞系，为子宫内膜癌体外实验提供研究材料。 方法： 将人子宫内膜癌手术标本移植于BALB/C（nu/nu）裸鼠皮下，成瘤后对移植瘤原代培养，进行传代。 结果： 建立子宫内膜癌细胞系，取名EAC，成功传代至51代.该细胞系生长快，稳定传代，形态学特征符合腺上皮恶性肿瘤细胞特征。染色体检查为超二倍体。雌孕激素受体检查阴性。P53、MDM2表达阴性。致瘤率100%，移植瘤病理学特征与原细胞系相似。 结论： 成功建立人子宫内膜癌细胞系，为开展人类子宫内膜癌基础和临床研究提供了理想实用的模型。     

人食管癌细胞系的建立及其转移相关基因PCNA的表达

目的:建立人食管癌细胞系,并对其肿瘤标志物(caycinoembryanic antigen,CEA)和转移相关基因增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达进行研究。方法:将人食管癌手术标本移植至裸鼠皮下,成瘤后取组织块体外原代培养,光镜和电镜观察细胞形态,测定细胞周期和染色体众数。取部分裸鼠皮下移植瘤接种至裸鼠食管,观察裸鼠体内肿瘤转移特征,采用免疫组化法测定肝转移灶PCNA基因的表达。结果:裸鼠皮下移植瘤组织16d,可形成明显的瘤结节,体外培养获得大量的肿瘤细胞,命名为YXA-1,其形态和超微结构符合鳞状细胞癌的形态学特征,染色体保持人类肿瘤染色体的特点,众数为70～76,细胞中肿瘤标志物CEA。食管癌标本原位移植裸鼠21d时,发生了广泛的肝内侵袭和转移,转移灶中PCNA表达为阳性。结论:人食管癌细胞系YXA-1维持了原发肿瘤的特征,而裸鼠其原位移植模型体现了肿瘤的转移特性,可用于肿瘤的转移研究。     

人乳头状瘤病毒18型E6E7基因诱导人胚食管上皮永生化

目的为研究病毒和肿瘤的关系,用人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因感染胎儿食管上皮,建立一株新的人食管上皮永生化细胞株（ＳＨＥＥ）。方法ＨＰＶ１８Ｅ６Ｅ７腺病毒伴随病毒（ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ））载体的构建;胚胎食管组织培养,ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ感染,继续培养传代。用光镜、电镜检查其形态;聚合酶链反应（ＰＣＲ）、荧光原位杂交（ＦＩＳＨ）检查该病毒片段;用软琼脂培养和裸鼠接种检查致瘤性。结果经过长时间的传代培养,ＳＨＥＥ的表型仍保留原代上皮细胞培养的特征,表现为单层生长和锚锭依赖性生长,在软琼脂培养不形成克隆,接种裸鼠未成瘤。ＳＨＥＥ细胞系电镜检查可见张力原纤维,免疫组织化学检查细胞角质蛋白阳性,证实为鳞状上皮来源。ＦＩＳＨ和ＰＣＲ检测显示有ＨＰＶ１８Ｅ６Ｅ７基因。结论用ＨＰＶ１８Ｅ６Ｅ７基因建立食管上皮永生化细胞株ＳＨＥＥ,支持ＨＰＶ１８可能和食管癌病因有关的观点,可进一步用以研究食管癌的病因和发病机制。     

兔VX2肿瘤细胞系的建立及其生物学特性的观察

目的建立兔VX2肿瘤细胞系,观察其生物学特性。方法采用小块法对兔VX2肿瘤进行原代培养,体外传代观察,传代40次以上对培养细胞进行形态学观察、组织化学染色、细胞周期检查、核型分析、兔及裸鼠移植。结果新建立的兔VX2肿瘤细胞,呈多角形、短梭形,电镜下细胞内可见张力纤维、细胞间可见桥粒,细胞角蛋白阳性,体外连续培养10个月,传代70次以上,细胞倍增时间为34．5h,细胞周期测定G,期为69．3％,G：期为5．6％,S期为25．1％。染色体为亚三倍体核型,众数为58～62条。同种移植成瘤率100％,裸鼠移植成瘤率100％,无支原体污染。结论兔VX2肿瘤细胞系来源于兔鳞状细胞癌,可用于兔（较大动物）的肿瘤实验研究。     

Establishment of a human hepatic adenosquamous carcinoma cell line (KMC-2) and its response to cytokines

A human hepatic adenosquamous carcinoma cell line (KMC-2) was established from a serially transplanted tumor in nude mice (nuKMC-2), which originated from human cholangio-cellular carcinoma and showed histological alteration from adenocarcinoma to squamous cell carcinoma (SCC) along with serial transplantation. KMC-2 cells in monolayer culture proliferated in a sheet-like arrangement with a population doubling time of 29.5 h, whereas the cells in 0.1% collagen gel embedded culture formed a compact and tubular structure with the population doubling time of 35.4 h. The cells secreted carbohydrate antigen 19–9 (CA19–9), tissue polypeptide antigen and SCC-related antigen. The back-transplanted nude mouse tumor exhibited morphologic features of adenosquamous carcinoma resembling those in the original nude mouse tumor. IFN-α, IFN-γ and TNF-α suppressed cell proliferation significantly. Functionally, IFN-γ significantly suppressed CA19–9 secretion, and conversely promoted SCC-related antigen secretion. These findings suggest that KMC-2 is the first human hepatic adenosquamous carcinoma cell line primarily originated from adenocarcinoma; the environmental factors, such as the presence of extracellular matrix and the cytokines influenced the growth, morphology and function of KMC-2.     

Electron microscopic investigation of transplanted squamous cell carcinoma in nude mice

Extirpated specimens of a squamous cell carcinoma from a human thigh were transplanted to the subcutaneous tissue of nude mice. Fourteen days later, the transplanted tumor masses were, again, extirpated from the nude mice. The transplanted chimera of the squamous cell carcinoma as seen with the electron microscope resembled the tumor cells before transplantation. It is concluded that ultrastructural investigation of transplanted chimera from squamous cell carcinoma cases may be useful for examining the site of action and clinical effects of anticancer drugs on this kind of tumor.     

Association between human papillomavirus infection and laryngeal squamous cell carcinoma

The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR‐DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin‐embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti‐human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF₁₀ PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma. J. Med. Virol. 82:1017–1023, 2010. © 2010 Wiley‐Liss, Inc.     

A comparative study of telomerase activity and malignant phenotype in multistage carcinogenesis of esophageal epithelial cells induced by human papillomavirus.

To examine certain characteristics of multistep carcinogenesis, we studied telomerase activity and malignant phenotypes in the immortal, premalignant and malignant stages of esophageal epithelial cells induced by HPV. An immortalized human fetal esophageal epithelial cell line (SHEE) was induced by E6E7 genes of human papillomavirus (HPV) type 18. Cells in the 10th passage, (SHEE10), 31st passage (SHEE31), 61st passage (SHEE61) and SHEE61A which were selected and expanded from anchorage-independent growth colonies of SHEE61, were examined as follows: cell morphology by electron-microscopy; the cell cycle by flow cytometry, telomerase activity by TRAP assay, tumorigenic detection including anchorage-independent growth by soft agar culture and tumor formation by inoculating cells into SCID and nude mice, and detection of HPV18 E6E7 oncoprotein by Western blot. The morphology of the SHEE10 cells exhibited good differentiation, the SHEE60 and SHEE61A cells were relatively poorly differentiated, and the SHEE31 cells were differentiated in two distinct ways. The telomerase was activated in SHEE31, SHEE61 and SHEE61A, but not in SHEE10 cells. SHEE61 and SHEE61A cells were weakened in contact-inhibition and increased in anchorage-independent growth. Inoculated into SCID and nude mice, the cells of the earlier two passages could not develop tumors; the SHEE61 developed one tumor in four SCID mice, but not in nude mice, and the SHEE61A cells developed tumors in both strains of immunodeficient mice. HPV18 E6E7 DNA detection by Western blotting was positive in all cell passages. In the process of carcinogenesis by HPV, the cells of SHEE31 are in an immortalized state with telomerase activity. The fact that SHEE61 cells remained immortalized and also demonstrated anchorage-independent growth, reveals premalignant character; the cells of SHEE61A exhibited malignant transformation with tumor formation in mice. The results revealed that the telomerase activity, anchorage-independent growth and tumor formation in nude mice are the indicators for immortalization, premalignancy and malignancy, respectively.     

A neuroendocrine (Merkel) cell carcinoma with coexisting intraepidermal squamous cell carcinoma of the skin. Its growth accelerated by an extrinsic factor

A case of neuroendocrine (Merkel) cell carcinoma with coexisting intraepidermal squamous cell carcinoma of the skin was studied histologically, immunohistochemically and ultrastructurally as well as with tissue-culture and transplantation into nude mice. The primary tumor found in the lower leg of a 68-year-old Japanese man had remained thumb-sized for five years and, after contusion, had begun to enlarge rapidly up to 5 cm in size during one month. The patient died of metastatic neuroendocrine cell carcinoma nine months after excision of the primary tumor. Histologically the primary tumor was composed of neuroendocrine cell carcinoma extending down to subcutaneous adipose tissue and a small amount of intraepidermal squamous cell carcinoma, not associated with a wide range of necrosis, hemorrhage, granulation tissue or fibrosis. The tumor cells of the former were diffusely positive for neuron-specific enolase. They contained a few secretory granules, 100 nm in diameter. The tumor cells both cultured in media and transplanted into nude mice died two months later. The present case is the first report of Merkel cell carcinoma in which the growth accelerated by an extrinsic factor was proved. Histogenesis of neuroendocrine cell carcinoma with coexisting squamous cell carcinoma is also discussed.     

Noncorrelative c-myc and ras oncogene expression in squamous cell carcinoma cells with tumorigenic potential.

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen-positive cells was not uniform throughout the tumor. Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC-83-01-82, isolated by passage through soft agar, was treated with 50 micrograms/ml of methyl methane sulfonate (MMS). These MMS-converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c-myc and H-ras mRNA expression. Serial passage of the MMS-converted tumors in vivo was accompanied by consistent enhanced c-myc expression. However, the levels of H-ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c-myc and H-ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC-83-01-82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c-myc and ras gene expression.