ESTABLISHMENT OF CELL CLONE OF LYMPHOMA AND A CELL LINE INFECTED WITH LEUKEMIA VIRUS AND STUDY ON ITS INDUCTED DIFFERENTIATION

Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.     

耐甲氨蝶呤人骨肉瘤细胞株建立及其生物学特性

目的：诱导并建立耐甲氨蝶呤（methotrexate，MTX）的人骨肉瘤细胞株并观察耐药细胞系（Saos-2／MTXl、Saos-2／MTX2）的生物学特性。方法：以MTX为诱导剂，采用逐步增加药物剂量冲击诱导方法，诱导人成骨肉瘤原代Saos-2细胞株，建立不同药物浓度耐药系（Saos-2／MTX1、Saos-2／MTX2）；MTT法检测原代与耐药细胞株对MTX、顺铂（cisplatin，DDP）、阿霉素（doxorubicin，ADM）、异环磷酰胺（ifosfamide，IFO）、表柔比星（epirubicine，EPI）、比柔比星（pirarubicin，THP）、紫杉醇（paclitaxel，PTX）药物敏感性；利用光学显微镜、透射电镜观察细胞形态及超微结构变化；利用细胞生长曲线检测细胞增殖能力；利用流式细胞仪检测细胞周期比例。结果：经178d的诱导，建立Saos-2／MTXl，Saos一2／MTX2耐药细胞株，其对MTX的耐药指数（resisitantdrugindex，RI）分别为原代细胞株的4．87和12．73倍；耐药细胞株对ADM、EPI、THP、PTX产生不同程度的交叉耐药，对DDP敏感；光镜观察Saos-2／MTX1、Saos-2／MTX2细胞体积增大，多核现象较原代Saos-2细胞株明显增加；透射电镜显示Saos-2／MTX1、Saos-2／MTX2细胞株表面突起较原代Saos-2细胞株减少、且核仁增大增多；细胞生长曲线和流式细胞仪检测显示耐药细胞株增殖能力下降，S期细胞所占比例减少（P〈0．05）。结论：本研究建立了耐MTX人成骨肉瘤细胞株Saos-2／MTX1、Saos-2／MTX2，并观察了耐药株细胞的生物学特性，为进一步研究甲氨蝶呤的耐药机制提供了一种实验模型。     

人骨肉瘤甲氨蝶呤耐药细胞系的建立及其生物学特性

目的:诱导并建立耐甲氨蝶呤(methotrexate,MTX)的人骨肉瘤细胞系,观察耐药细胞系(SOSP-9607/MTX)的生物学特性.方法:以MTX为诱导剂,采用逐步增加药物剂量冲击诱导方法,诱导建立人成骨肉瘤耐药细胞系SOSP-9607/MTX.利用光学显微镜观察细胞形态及其结构变化;MTT法检测原细胞株与耐药细胞株对MTX、顺铂(cisplatin,DDP)、卡铂 (carboplatin,CBP) 、阿霉素(doxorubicin,ADM)、异环磷酰胺(ifosfamide,IFO)、紫杉醇(paclitaxel,PTX)的药物敏感性;细胞生长曲线检测耐药细胞增殖能力;利用流式细胞仪检测细胞周期比例.结果:经12个月的诱导,建立SOSP-9607/MTX耐药细胞系,其对MTX的耐药指数(resistant index,RI)为原细胞株的4.63倍;耐药细胞系对ADM、DDP、IFO、PTX产生不同程度的耐药指数,对CBP敏感;光镜观察SOSP-9607/MTX细胞异型性与多核现象较原细胞株明显增加,细胞生长曲线显示耐药细胞株增殖能力下降,流式细胞仪检测表明,G2期细胞减少,G1和S期细胞增加(P<0.05).结论:建立了耐MTX人成骨肉瘤细胞株SOSP-9607/MTX,并观察了耐药株细胞的生物学特性,为进一步研究甲氨蝶呤的耐药机制提供了良好的实验模型.     

Establishment of a human pulmonary adenocarcinoma cell line Lu-YePa and its biological characteristics

This paper presents the establishment of a human pulmonary adenocarcinoma cell line Lu-YePa and its biological characteristics. The primary cells of Lu-YePa cell line were derived from a twenty six year old patient with poorly differentiated adenocarcinoma of the lung. After 36 days' culture, transfer was made and the cells proliferated steadily and rapidly afterwards. The doubling time was 34.6 hours. Mitotic index reached 49.5% on day 5. Chromosome number was subtriploid with the mode of 62. The large submetacentric marker chromosome was presented in 68% of cells. PAS positive granules were found in the cytoplasm. The study of concanavalin A condensation, electron microscopic observation, heterotransplantation and plating efficiency indicates that Lu-YePa cells are concordant with the human pulmonary adenocarcinoma cells in morphology and biological behavior.     

人肝癌顺铂耐药细胞系的建立及其生物学特征

背景与目的：药物耐受性是当前肿瘤研究的热点,国内外普遍应用体外建立的耐药细胞系作为研究模型。为探讨肝癌对顺铂耐药的机理。本研究首次建立了人肝癌顺铂耐药细胞系并研究其生物学特性。方法：采用逐步递增顺铂浓度,间歇作用体外诱导法建立人肝癌顺铂耐药细胞系QGY/cDDP;MTT法测定药物敏感性,光镜,电镜,活细胞计数法,流式细胞仪及染色体分析等方法观察其生物学特征的改变。结果：历时3个月建成人肝癌顺铂耐药细胞系QGY/cDDP,其对顺铂的耐药指数为10.81,并且与5-氟尿嘧啶,表阿霉素等多种抗癌药有不同程度的交叉耐药性;QGY/cDDP的细胞形态及染色体数目发生改变;体外群体倍增时间较亲代细胞延长;细胞周期分析发现其S期与G2/M期细胞减少,G0/G1期细胞增多。结论：QGY/cDDP细胞具有耐药表型,且耐药性能稳定。     

人子宫颈鳞状细胞癌细胞系HCC-0214的建立及其生物学特性

目的 建立人子宫颈鳞状细胞癌细胞系HCC-0214,为子宫颈癌研究提供实验模型。方法 无菌切除人子宫颈癌的手术标本,用组织块贴壁法体外培养,连续传代稳定生长后,绘制细胞生长曲线。采用光镜、电镜观察细胞形态,并进行细胞周期和染色体核形分析。用免疫组化法测定细胞系肿瘤标记物的(ER、PR、Keratine、PCNA)表达情况。结果 组织块贴壁法体外培养获得人子宫颈鳞状细胞癌细胞系HCC-0214(简称H),细胞维持培养16个月,传代131代,生长稳定,群体倍增时间为35．48h,细胞呈上皮镶嵌状贴壁生长,趋向复层生长,无接触抑制。超微结构显示,具有典型的桥粒结构和较多的张力原纤维。染色体数目35—156条,主流范围58—80条(64．8％),结构为人类染色体。细胞的肿瘤标记物(ER、PR、Keratine、PCNA)检测呈高表达,DNA指数为1．931。裸鼠移植瘤组织病理形态学与患者原始肿瘤一致,无血清培养成功。结论 通过组织块贴壁法体外培养建立的人子宫颈鳞状细胞癌细胞系HCC—0214,与原发癌保持相同的生物学特性,体外连续传代16个月以上,细胞形态不变,生长周期恒定,可望作为一个稳定的细胞系。     

人肝癌细胞系EH-H1的建立及其相关生物学特性

目的: 采用原代培养的方法建立一株人肝癌细胞系EHH1,并对其生物学特性进行分析。方法：将取自东方肝胆外科医院诊断为原发性肝细胞癌的组织标本分离成单细胞悬液,用含10%胎牛血清的DMEM培养液进行原代和传代培养,在普通显微镜以及电子显微镜下观察细胞的形态,并绘制细胞的生长曲线。用放射免疫法检测细胞系AFP和HBV的含量,采用染色体G显带方法进行细胞染色体核型的分析,裸鼠皮下接种细胞检测该细胞的成瘤能力。结果：成功建立一株新的人肝癌细胞系,命名为EHH1。EHH1细胞体外连续传代80代以上,细胞形态不变,生长周期恒定在24 h。放射免疫检测EHH1各代细胞HBV均为阴性,AFP分泌微量(0.6 μg/L) 。染色体G带显示,EHH1细胞染色体为非整倍体,以超2倍体为主。该细胞经皮下接种可使裸鼠致瘤(10/10),移植瘤病理组织学类型和分化程度与肝细胞癌一致。结论: 通过原代培养建立的肝癌细胞系EHH1与原发癌保持相似的生物学特性,可望成为一个稳定的细胞系。     

Establishment of a spontaneously transformed human fetal lung cell line and its biological characteristics

A spontaneously transformed human fetal lung cell line (HFLT) was derived from a human fetal lung diploid fibroblast cell line (2BS) by continuous culture. The biological characteristics of this cell line were studied and compared with those of 2BS cells. Accompanying the morphological alteration, the growth rate of the transformed cell was accelerated and the maximum cell density was increased. The anchorage-independent growth was shown by its ability to form colonies in soft agar. The collagen synthesis phenotype of 2BS cells was changed. In addition, the increased chromosome number and the nodule formation after heterotransplantation were pathognomonic of malignant transformation.     

人肝癌细胞系EH-H1的建立及其相关生物学特性

目的: 采用原代培养的方法建立一株人肝癌细胞系EH-H1,并对其生物学特性进行分析.方法:将取自东方肝胆外科医院诊断为原发性肝细胞癌的组织标本分离成单细胞悬液,用含10%胎牛血清的DMEM培养液进行原代和传代培养,在普通显微镜以及电子显微镜下观察细胞的形态,并绘制细胞的生长曲线.用放射免疫法检测细胞系AFP和HBV的含量,采用染色体G显带方法进行细胞染色体核型的分析,裸鼠皮下接种细胞检测该细胞的成瘤能力. 结果:成功建立一株新的人肝癌细胞系,命名为EH-H1.EH-H1细胞体外连续传代80代以上,细胞形态不变,生长周期恒定在24 h.放射免疫检测EH-H1各代细胞HBV均为阴性,AFP分泌微量(0.6 μg/L) .染色体G带显示,EH-H1细胞染色体为非整倍体,以超2倍体为主.该细胞经皮下接种可使裸鼠致瘤(10/10),移植瘤病理组织学类型和分化程度与肝细胞癌一致.结论: 通过原代培养建立的肝癌细胞系EH-H1与原发癌保持相似的生物学特性,可望成为一个稳定的细胞系.     

Establishment of a mouse forestomach carcinoma cell line (MFC) with spontaneous hematogenous metastasis and preliminary study of its biological characteristics

A mouse forestomach carcinoma cell line (MFC) prone to blood-born metastasis to lung was established in vitro. This cell line has been subcultured for more than 132 passages. MFC cells lacked in contact inhibition and, morphologically, appeared as round, polygonal and spindle cells. Ultrastructurally, there were abundant microvilli on the cellular surface and extensive filopodia in the cytoplasm; nuclei showed irregular shape with an increased nucleus-cytoplasm ratio; desmosomes, hemidesmosomes as well as a small number of tonofibrils were observed. The doubling time of MFC cell population was 24.7 hours. The average mitotic index was 32.9% and the maximum was 62% with 70-76 mode. The homotransplant efficiency was 100% (11/11). In morphology, the solid tumors induced were similar to FC tumor, from which the MFC cell line was derived and 81.8% (9/11) developed spontaneous lung metastasis. It is indicated that this cell line still retains the primary tumor's feature of spontaneous hematogenous metastasis. MFC cell line might be very useful in experimental treatment, study of tumor metastatic mechanism and isolating subpopulation.     

人卵巢癌细胞系OV1228的建立及其生物学特性

目的用人卵巢癌组织建立新的卵巢癌细胞系,为探讨卵巢癌细胞的发病机制和治疗药物的筛选提供可靠的材料。方法选用人复发卵巢癌组织,进行肿瘤细胞原代培养,通过克隆稀释法,培养建立卵巢癌细胞系,进行体外传代,并通过细胞形态学、生长动力学以及致瘤性来分析其生物学特性。结果该卵巢癌细胞系已在体外培养生存18个月以上,传代超过80余代,被命名为OV1228。其生物学特性显示：倍增时间为29．39h;软琼脂集落形成率为17．5％;接种至裸鼠后具有较高的致瘤性;染色体核型分析为多倍体,众数为21～61条;透射电镜下可观察到卵巢癌细胞表面有少量短的细胞突起,部分细胞间可见发育比较差的桥粒样结构,具有明显的恶性卵巢癌细胞特征。结论OV1228经体外长期培养后已形成永生化卵巢癌细胞系,并具有明显的恶性特征,为开展人类卵巢癌的基础及临床研究提供了理想的实验材料。     

Establishment of epithelial cell line TBC-27 from a human bladder transitional cell carcinoma and its biological characteristics

The cell line TBC-27, from a human bladder transitional cell carcinoma Grade II, was established and maintained for 8 months through 27 passages. TBC-27 cells have the epithelioid cell morphology during culture. The population doubling time of the 14th generation cells was 27 hours by growth curve. The chromosome karyotype of the 12th generation cells consisted mainly of hypodiploid (mode 40-45). Ultrastructurally, there was a great deal of freeribosome and vesicle in the cytoplasm and a great number of atypical microvilli on the surface of the 21st generation cells. The heterotransplantation test revealed that the xenografted tumor in the immunosuppressed suckling rats was similar to the initial bladder carcinoma in histology.     

一株HGPRT缺陷型小鼠肺癌细胞株的建立及其生物学性质

目的　为研制肺癌疫苗提供适合细胞融合的抗原细胞。方法　用8氮鸟嘌呤(8AG)从小鼠肺癌细胞LA795渐进诱导次黄嘌呤鸟嘌呤转磷酸核糖基酶(HGPRT)缺陷型细胞株ALA9702,并鉴定其生物学性质及成瘤性。结果　ALA9702细胞在20μg/ml8AG培养液中生长了一年,在HAT培养液中不能形成集落,并保持了来源细胞LA795的生物学性质及成瘤性。结论　ALA9702细胞株稳定表现为HGPRT缺陷型小鼠肺癌细胞,为建立有效的特异性肺癌疫苗治疗提供有利的抗原材料     

Hairy cell leukemia: establishment of a cell line and its characteristics

A hairy cell leukemia (HCL) line, ZK-H, was established from peripheral blood of a 69-year-old male patient. The ZK-H cells and the patient's original hairy cells shared the same surface properties; both possessed membrane-bound IgG with kappa light chains and villous surface structures. The ZK-H line carried Epstein-Barr virus (EBV)-determined nuclear antigen, but the patient's fresh leukemic cells lacked this antigen. Morphologically, the ZK-H cells appeared lymphoblastoid and more primitive than the preculture cells. The ZK-H line had a hyperdiploid chromosome constitution of 47 and trisomy no. 2. The presence of membrane-bound immunoglobulin and of B-cell tropic EBV in this cell line provides further evidence for the B-cell nature of HCL in this patient     

高转移胃癌细胞株的建立及其肿瘤干细胞的生物学特征

目的:建立高转移胃癌细胞株并研究其肿瘤干细胞生物学特征.方法:将胃癌细胞株SNU-5接种至裸鼠皮下成瘤后,取肺部转移灶,通过机械分离法并反复接种裸鼠,获取细胞株SNU-5-V12,采用无血清悬浮培养及PKH26染色确定SNU-5-V12细胞中存在肿瘤干细胞.流式细胞术分析SNU-5和SNU-5-V12细胞中CD44肿瘤干细胞标志物表达情况,分选SNU-5和SNU-5-V12细胞中CD44+细胞并进行体外生物学特征研究及裸鼠致瘤实验.结果:建立高转移SNU-5-V12细胞株,SNU-5-V12细胞无血清悬浮培养11d后形成的细胞球体中存在单个PKH26阳性细胞.流式分析显示高转移SNU-5-V12细胞中干细胞标志物CD44比例显著高于SNU-5细胞[(72.9±1.5)％ vs (8.96±1.2)％],且SNU-5-V12的CD44+细胞比SNU-5的CD44+细胞具有更强的自我更新能力[成球率(27.8±1.7)％ vs (20.4±1.0)％,P＜0.01],转移细胞数增加1.64倍[(329.5±7.5) vs (200±2.0)个,P＜0.01],耐药能力也显著增强[IC50:(0.286 vs 0.196)μ∥ml,P＜0.01],裸鼠皮下接种2×102个CD44+ SNU-5-V12细胞2个月2/6裸鼠可致瘤,而CD44+ SNU-5细胞需要接种2×104个细胞2个月时仅1/6裸鼠致瘤.结论:获得高转移胃癌细胞株SNU-5-V12,其CD44+细胞体内外功能显著增强,为胃癌干细胞的靶向治疗提供有价值的细胞模型.     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.     

Establishment of a hairy cell leukemia cell line carrying Tac antigen and phagocytic activity with B-cell characteristics

A hairy cell leukemia cell line designated "Hair-M" was established in a suspension culture derived from the peripheral blood of an 86-year-old Japanese male with a diagnosis of hairy cell leukemia. The Hair-M cells were identified as having prominent hair-like cytoplasmic projections by examination with phase-contrast and scanning electron microscopy. These cells displayed ruffled membranes and stublike microvilli similar to those observed on the surfaces of cells in the peripheral blood of the patient. Immunologic and cytochemical studies on the Hair-M cells confirmed derivation from the clone of the patient's leukemia cells. Although the cultured Hair-M cells had definite B-cell characteristics, such as IgG kappa-chains on the surface and in cytoplasm, they also demonstrated Tac antigen, which is usually expressed on activated T-cells, and myelomonocyte antigens determined by OKM-1 and MCS-1 monoclonal antibodies. Other cell surface markers, including E(-), IgGFc(-), IgMFc(-), C3R(+), Ia-like antigen(+), OKT9(+), OKT10(+), and terminal deoxynucleotidyl transferase(-), were detected; no Epstein-Barr virus-determined nuclear antigen was detected. The karyotype of the Hair-M cells was determined to be 46XY with -11, -14, and two marker chromosomes. The Hair-M cells also had phagocytic activity to rabbit anti-human IgG serum-coated polyacrylamide gel particles.     

肺癌细胞系XWLC-05的建立及其生物学特性研究

背景与目的:云南省宣威地区是我国肺癌高发地区之一,女性肺癌死亡率居全国首位,本研究旨在建立云南宣威女性肺腺癌细胞系,为肺癌高发区的防治研究提供理想的体外实验模型.方法:以手术切除的肺癌组织标本进行原代培养,通过光镜观察、电镜观察、绘制生长曲线、计算倍增时间、双层软琼脂培养、流式细胞仪、染色体及其显带分析、肿瘤标志物检测、异体移植实验及免疫组化检测等对其生物学特性进行分析鉴定,建立细胞系.结果:体外培养细胞生长稳定,细胞形态学、增殖动力学及浸润性生长结果表明该细胞系符合恶性细胞特征,染色体数目分布在55～69之间,众数为60～63;小鼠接种成瘤率为100%,瘤细胞形态与原患者的病理切片相似,命名为XWLC-05(Xuanwei Lung Cancer-05).结论:该细胞系符合建系标准,是一株新建的人肺腺癌细胞系.     

人大细胞肺癌多药耐药株H460/cDDP的建立及其生物学特性

目的：建立人大细胞肺癌多药耐药株H460/cDDP．并对其生物学特性进行测定。方法：以人大细胞肺癌细胞株H460为亲本细胞．采用顺铂大剂量问歇诱导法建立多药耐药细胞株H460／cDDP。光镜下观察其形态学变化；MTT法测定药物敏感性；锥虫蓝拒染实验测定细胞生长曲线、倍增时间；进行染色体核型分析；棵鼠成癌实验测定其体内成瘤活性。结果：历时近6个月建成人大细胞肺癌多药耐药株H460／cDDP．经鉴定其对顺铂的耐药指数为10．21，对5-氟尿嘧啶，多柔比星(阿霉素)、依托泊苷(足叶乙甙)、甲氨蝶呤有不同程度的交叉耐药。H460／cDDP较亲本细胞分裂高峰延迟，倍增时间由20．78小时延长至3646小时．细胞形态改变，染色体变化不突出。经过反复冻存．复苏．上述特性保持稳定．并具有体内成瘤活性，成瘤时间较亲本细胞有所延迟。结论：新建大细胞肺癌多药耐药株H460／cDDP呈中等度耐药、多药耐药表型稳定且具有体内成瘤活性，可用于下游实验。