致癌物诱导大鼠食管上皮细胞转化的研究

非致瘤性的F344大鼠食管上皮细胞RE-149培养于低钙、并含有透析血清及七种生长因子的PFMR-4培养基中。经致癌物(±)反式-7.8-二氢苯并(a)芘-7.8-二醇或甲基硝基亚硝基胍作用后2周,种入不含血清和EGF的选择性培养基中。经致癌物作用的细胞集落转化率增高11～77倍。这种转化集落可以在选择性培养基中连续传代,接种于同种新生大鼠皮下形成鳞状细胞癌。该研究表明,致癌物作用后引起细胞对生长因子依赖性的变化,可能作为选择转化上皮细胞的一个新途径。     

急性髓性白血病细胞体外培养及其生物学特性的研究

目的:研究急性白血病细胞体外培养的方法及其在体外培养过程中生物学特性的变化.方法:分别收集10例急性髓性白血病细胞,用含细胞因子无血清液体培养基进行培养2～4周,并对培养前后细胞进行细胞计数,流式细胞仪检测CD33、CD13、CD34及CD14表面抗原鉴定细胞分化,半固体培养法对培养前后的白血病细胞进行集落形成能力检测.结果: 含细胞因子无血清液体培养基能有效支持AML细胞进行体外短期培养及增殖, 14 d时细胞得到明显增殖,达(25 1±11 7)倍,优于培养前,P<0 05;培养28 d时细胞数较前明显减少,P<0 05.集落形成单位培养14 d后与培养前比较差异无统计学意义,P>0 05;但培养28 d后显著低于培养前,P<0 05.在体外悬浮培养14 d时,CD33、CD13、CD34及CD14的表达率与培养前差异无统计学意义(P>0 05),但至28 d时,CD33、CD13及CD14的表达率高于培养前,而CD34表达率比培养前降低,P<0 05.结论: 含细胞因子无血清液体培养基能有效支持急性髓性白血病细胞短期体外培养并维持其生物学特性.     

无血清培养基中急性非淋巴细胞性白血病细胞的药物敏感试验

在无血清培养基中,41例急性非淋巴细胞性白血病病人中有39例的骨髓单个核细胞生长了白血病集落,有35例病人测定了体外CFU-L对化疗药物的敏感性。结果发现,只有用Ara-C、Ara-C DNR(AD)、VCR Pred(VP)和H VCR Ara-C Pred(HOAP)测试的病例,其体外白血病细胞杀死率和临床化疗结果间存在明显的一致性。作者认为,在CFU-L药物敏感试验中,无血清培养系统可避免血清中未知物质的干扰。     

无血清培养基与完全培养基对体外诱导免疫细胞支持作用的比较

目的：多方面比较无血清培养基AIMV与完全培养基对体外诱导免疫细胞支持作用。方法：分别用AIMV及完全培养基在IFN-γ、IL-2,及抗-CD3单抗存在的条件下培养人外周血单个核细胞,比较细胞的增殖能力、细胞表型、及细胞因子分泌能力,并比较不同培养基培养细胞回输体内后的抑制病毒效果。结果：与完全培养基培养细胞相比,无血清培养基AIMV培养细胞增殖能力与之相当;CD25的表达率增高,表达持续时间延长;细胞因子IFN-γ的分泌时间延长;回输体内后的抑制病毒作用更明显。结论：无血清培养基AIMV用于培养临床治疗用的免疫细胞,综合效果优于完全培养基。     

几种致癌物体外转化大鼠气管上皮细胞致癌的进程

已明确正常细胞与一定量的化学或物理致癌物接触后第一步成为癌前性变异细胞,进而发展为瘤性变体细胞。本文探讨大鼠气管上皮(RTE)细胞与不同致癌剂接触后,从形态学上的转化到细胞获不死性再进展为癌性细胞的过程与致癌剂的性质的关系。 本文用体外研究RTE细胞致癌作用的模式系统,将原代RTE细胞分为三组,分别与致突变浓度的DNA烷化剂N-甲基-N-硝基-N’-亚硝基胍(MNNG)0.3微克/毫升、非致突变浓度的DNA次甲基化剂5-氮杂胞苷(5-aza-C)4毫克分子浓度接触,另一为自发转化组°前二组得～30％的残存细胞,再培养5周能鉴别转化细胞并分离之,得到形态学上转化的变体细胞。后者被称为生长力增强的变体细胞,简称(EGV)。     

在体外软琼脂不贴壁生长和裸鼠体内发瘤的表型差异

体外培养的肿瘤细胞,一个重要的标准是能在适宜宿主体内发生肿瘤。广泛应用的动物是无胸腺裸鼠。但体外实验方法更为简便迅速。这些实验基于肿瘤细胞的一些生长特性,主要是能在软琼脂上贴壁生长。正常细胞和未转化细胞不能贴壁生长。因而,研究细胞贴壁生长能力和在动物体内发生肿瘤的关系,可以证明体外培养的肿瘤细胞生长特性对肿瘤的发生是不是必需的问题。本文报告了一例鳞状上皮癌LT-2细胞系在体外软琼脂上不贴壁生长和能在裸鼠体内发生肿瘤,这两     

裸小鼠高分化食管鳞状细胞癌株(RE25/N)的建立及其生物学特性的研究

大鼠食管癌裸小鼠细胞株在国内外均很少见,本实验室于1986年11月开始,将体外恶性转化的大鼠食管癌细胞系(RE_(25-3))接种于Swiss及NC两种裸小鼠皮下,并传代获得成功,至今已传至14代,该移植瘤具有生长稳定、接种成功率高(100％),潜伏期短及无自发消退等特点。形态学观察:光镜下肿瘤为高分化鳞癌;电镜观察,超微结构符合高分化鳞癌的改变。流式细胞仪(FCM)的DNA分析,其DNA含量的DI值与其在体外培养的母细胞系相似,但细胞周期各期细胞的比例有明显差异。在裸小鼠体内生长的移植瘤,其S期及G_(2+)M期细胞比例明显降低,说明体内生长的移植瘤其生长增殖能力较体外培养的细胞系为低,生长较慢,可能与宿主对移植瘤的作用有关。     

应用TNF基因转染细胞进行的肿瘤实验性免疫疗法

我们应用逆转录病毒载体pLXSN将人IL-2基因插入大鼠和人的肿瘤细胞，探讨了携带外源性IL-2基因的肿瘤细胞的体内外生物学特性及其意义。转基因缃胞在体外培养中的细胞形态、生长特性以及软琼脂集落形成能力未见明显改变，但对杀伤性免疫活性细胞的杀伤更为敏感，与淋巴细胞混合培养的上清中测得更高水平的IFN和TNF活性。大鼠肿瘤细胞WBT插入IL-2基因后，其体内成瘤性明显下降。腹腔接种WBT／IL-2细胞可使大鼠获得抗肿瘤免疫能力以抑制皮下野生型WBT细胞的生匠。携带IL-2基因的人和大鼠肿瘤细胞在裸鼠体内可以成瘤，但生长缓慢。具有高转移能力的Anip973细胞插入IL-2基因后，在裸鼠体内的转移性丧失。结果提示IL-2基因插入肿瘤细胞进行肿瘤基因治疗的有效性、可行性和发展潜能。     

金龙胶囊(JLC)抑制癌细胞转移及复发的实验观察

目的观察点突变重组人β-干扰素（IFN-β）对人肿瘤细胞体内外生长的抑制作用,为其临床应用提供实验资料。方法分别将人结肠癌LoVo、肺腺癌A549细胞置于含200IU/ml,1000IU/ml及5000IU/mlIFN-β培养基中培养24h后,于0.3%琼脂糖培养基中培养10～14d,观察软琼脂中集落形成情况。将裸鼠皮下接种LoVo,A549细胞,72h后分组,每次注射20万IU/kg或100万IU/kg,500万IU/kgIFN-β,每天1次,连续10d,同时设置生理盐水对照组、标准品组和注射用环磷酰胺治疗组。荷瘤后第14天处死小鼠,称取瘤重。以上实验均重复3批。结果在体外,经重组人新型β-干扰素处理的LoVo细胞及A549细胞的集落数均明显低于未经处理的细胞（0.05>P>0.01）,与标准品组无明显差异(P>0.05)。荷瘤裸鼠经治疗后,瘤重随着重组人β-干扰素剂量的升高而降低,即重组人β-干扰素可呈剂量依赖性地抑制LoVo,A549细胞在体内的生长,在疗效方面与标准品间无显著差异(P>0.05)。结论重组人β-干扰素在体内外能明显抑制人结肠癌和肺腺癌的生长。     

生长因子对人支气管上皮细胞和肺癌细胞生长的影响

MCDB151加多种生长因子的无血清培养基已成功用于培养成人和胎儿支气管上皮细胞及肺癌细胞(腺癌和鳞癌)。用该无血清培养基进行集落形成试验的结果表明,牛垂体提取物、胰岛素和氢化可的松可以促进人支气管上皮细胞及肺癌细胞的增殖,表皮生长因子和转铁蛋白却只刺激正常支气管上皮细胞的生长,而对本实验所检测的肺癌细胞无生长刺激作用。本研究所培养的大部分肺癌标本,在MCDB151完全培养基中加2％胎牛血清时,比不加血清时生长更快、更好。在MCDB151无血清培养基中,低浓度的钙有利于人支气管上皮细胞和肺癌细胞的生长。     

Role of spontaneous transformation in carcinogenesis: development of preneoplastic rat tracheal epithelial cells at a constant rate

The rate of spontaneous transformation of normal rat tracheal epithelial (RTE) cells to preneoplastic enhanced growth (EG) variants was estimated in serum-free culture. Spontaneous transformation of RTE cells has previously been observed, but an accurate estimation of the rate of change has not been possible due to the use of serum and feeder cells in the cultures which prevents both unlimited RTE cell proliferation and an accurate determination of the number of cells at risk. RTE cells were plated in serum-free medium and were switched to serum-containing medium at various times during the first 23 days of culture. In serum-containing medium, normal RTE cells cease proliferation, while EG variants continue to proliferate. The fraction of RTE cell colonies which developed into EG variants increased with time to a maximum of 15% when selection was imposed 5 to 23 days after plating. The number of cells per culture also increased during the same time, suggesting a role for cell proliferation in the spontaneous generation of EG variants. In contrast to the time-dependent increases in cell number and the frequency of EG variants, the rate of development of spontaneous EG variants remained constant with time and was estimated to be 7.5 +/- 4.1 X 10(6) variants/cell generation. The rate of spontaneous preneoplastic transformation of normal epithelial cells reported here, the rates of spontaneous progression of preneoplastic and neoplastic cells reported elsewhere, and the association between cell proliferation in vivo and increased cancer risk are consistent with the hypothesis that spontaneous changes play a role in the multistep progression of cells to cancer.     

In vivo and in vitro characteristics of early carcinogen-induced premalignant phenotypes in cultured rat tracheal epithelial cells

The initial stages of neoplastic transformation in respiratory tract epithelial cells were defined and studied by characterizing a series of morphologically transformed cell colonies from carcinogen-exposed rat tracheal epithelial (RTE) cell cultures both in vivo and in vitro. RTE cells were isolated from Fischer 344 rats, plated on collagen-coated dishes, and exposed to 7,12-dimethylbenz[a]anthracene on day 1 for 24 h. Between days 26 and 30, single colonies of morphologically altered cells were isolated and classified into three major classes based on cell density. Following replating, the cells were tested for their ability to grow on various substrates and in various culture media. Generally, Class II and III cells exhibited a higher colony forming efficiency when replated on various substrates. Class III cells appeared to grow better than Class I or II cells in complete medium, while Class I cells grew better in medium without 3T3 conditioning factors. At early passage levels, the population doubling times were longer for Class I cells than for Class II cells. Class III cells had the shortest population doubling times. The various cell lines were also placed into denuded tracheal grafts. Untreated cells produced a normal mucociliary epithelium, while Class I cells produced a simple cuboidal epithelium. Class II and III cells formed a highly atypical and usually malignant epithelia. Inoculation of the three classes of cells into nude mice provided confirming evidence of the benign nature of Class I cell lines and the malignant nature of some Class II cell lines and all of the Class III cell lines.     

Development of a quantitative in vitro transformation assay for kidney epithelial cells.

A quantitative in vitro transformation assay has been developed for the first time using primary rat kidney epithelial (RKE) cells. RKE cells were grown in a 50:50 mixture of 3T3 conditioned medium and DF8 medium composed of Ham's F-12/DMEM supplemented with ferrous sulfate, vasopressin, transferrin, sodium selenite and 10% fetal bovine serum. Colony forming efficiency of cells plated in this medium was high, ranging from 2.4 to 16%. Normal RKE cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) became transformed to a preneoplastic state of enhanced in vitro growth potential and formed large colonies of morphologically altered cells, whereas RKE cells treated with vehicle alone ceased proliferating and/or sloughed off the dish within 4-6 weeks. Relative survival and percent transformation frequency (Tf) of RKE cells exposed to MNNG were inversely proportional and both were dose dependent. MNNG concentration of 0.5, 1.0 and 2.0 micrograms/ml resulted in transformation frequencies of 0.13, 0.37 and 1.1% respectively (n = 4). Morphologically transformed colonies gave rise to cell lines with indefinite growth capacity and neoplastic potential. One of six transformed RKE cell (TRKE) lines injected into nude mice produced adenocarcinomas. This assay represents the first in vitro model for studying mechanisms of chemical transformation of normal kidney epithelial cells and may also be useful as a screen for identifying potential renal carcinogens.     

Changing responsiveness to all-trans retinoic acid of rat tracheal epithlial cells at different stages of neoplastic transformation

Retinoic acid (RA) treatment of rat tracheal epithelial (RTE)cells, pre-exposed to the direct-acting carcinogen N-methyl-N'-nitiro-N-nitrosoguanidine(MNNG), inhibited transformation in a dose-dependent manner.Treatment with RA at concentrations ranging from 3–33nM reduced the MNNG-induced transformation frequencies by 13–81%and in some experiments by >90%. RA treatment for only 3days caused 65–75% inhibition of transformation; treatmentsof longer duration resulted in greater inhibition of transformation.Delaying the onset of RA treatment reduced its effectiveness,but even when RA treatment was delayed for 3 weeks followingMNNG exposure, 60% inhibition still occurred. The inhibitionof transformation appeared to be irreversible. The colony formingefficiency of cells isolated from transformed colonies 5 weeksafter MNNG exposure was drastically reduced when the replatedcells were treated wtih RA either 1 day or 4 days after plating,indicating that RA blocked cell replication. However, cellsisolated from transformed colonies at later times after MNNGexposure were increasingly resistant to the antiproliferativeeffects of RA. The RA concentration causing 50% inhibition (RA-IC₅₀)of colony formation was 0.1–0.3 nM for cells isolatedfrom 3–5-week-old transformed colonies; it increased >100-fold for cells isolated from 12-week-old transformants.Five established RTE cell lines also showed a much increasedresistance to the antiproliferative effects of RA; two of thesecell lines were even slightly stimulated in their colony formingabilty by RA. The RA-ICM of colony forming efficiency of normalRTE cells was also determined and compared to that of cellsisolated from 5-week-old transformed colonies. Since the normalRTE cells require 3T3 feeder layers for clonal growth, bothcell types were grown on feeders. For both cell types, the RA-ICMwas similar (150–300 nM); the requirement for relativelyhigh RA concentrations was attributed in part to the rapid RAmetabolism by the feeder cells. These experiments show thatearly RTE cell transformants are growth-inhibited by RA; however,they increasingly lose their sensitivity to the growth controllingeffects of RA as they progress to a more advanced stage of transformation.The inhibition of tracheal cell transformation by RA is probablydue to the antiproliferative effects of the retinoid.     

Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens

A serum- and feeder cell-free medium has been developed forthe proliferation of rat tracheal epithelial (RTE) cells atclonal density. In this medium, RTE cells continue to proliferatefor several weeks after cells in serum containing medium onfeeder cells have begun to differentiate. The responsivenessof RTE cells to selected hormones and growth factors was determinedusing a clonal growth assay. The colony-forming efficiency (CFE)of RTE cells was reduced >85% when bovine pituitary extractor bovine serum albumin were deleted from the medium and 45– 70% reductions in CFE were observed when insulin, hydrocortisone,epidermal growth factor or cholera toxin were deleted. RTE cellsalso require high concentrations of Ca²⁺ (0.8 mM) for maximalclonal proliferation in this medium. The induction by carcinogensof preneoplastic RTE cell variants resistant to serum-mediatedsquamous differentiation was compared in serum-free medium andin serum-containing medium on feeder cells. N-methyl-N'-nitro-N-nitrosoguanidmewas considerably more cytotoxk and effective as a transformingagent on an equivalent dose basis for RTE cells in serum-freemedium. In contrast, (±)-7B, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene was equally cytotoxic andtransforming under both culture conditions. This serum-freeculture system for primary RTE cells will be useful in studieson the control of normal epithelial cell proliferation and differentiationby defined growth factors and in studies on the cellular changesinvolved in carcinogenesis.     

Increased resistance to transforming growth factor beta accompanies neoplastic progression of rat tracheal epithelial cells

Transforming growth factor beta (TGFß) is an inhibitorof normal epithelial cell growth. To investigate the role ofTGFß in respiratory epithelial cell neoplasia, normal,preneoplastic, tumorigenk and tumor-derived rat tracheal epithelial(RTE) cells were plated in serum-free medium and grown in thepresence of 0–300 pg TGFfß₁/ml. TGFß₁markedly inhibited the formation of colonies by primary RTEcells and some preneoplastic RTE cells. However, tumor-derivedRTE cells were relatively resistant to TGFß₁inducedgrowth inhibition. Resistance to TGFßrinduced growthinhibition, therefore, accompanies neoplastic progression ofRTE cells.     

Cellular oncogene expression in cell lines derived from tumors produced by transformed rat tracheal epithelial cells

Five tumor-derived cell lines established from transformed rat tracheal epithelial (RTE) cells were examined for activated oncogenes using the NIH 3T3 assay, and the expression of 11 cellular oncogenes in the transformed cells was quantitated and compared with expression in normal RTE cells. DNA from the tumor-derived cell lines lacked transforming activity, but expression of several oncogenes (fos, abl, Ki-ras, Ha-ras, and p53) was higher in the transformed cells than in normal RTE cells.     

Changes in stem cell populations of rat tracheal epithelial cell cultures at an early stage in neoplastic progression

The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 micrograms/ml) or solvent. Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 weeks after carcinogen exposure. Most of the RTE cell colonies, which originally formed during the first 7 days of culture, disappeared within 2 weeks after feeder cell removal in control and MNNG-treated cultures. In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (approximately 9%) of the colonies persisted. These percentages remained constant from 3 to 7 weeks. Based on colony size, cell density, and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear:cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear:cytoplasmic ratio). In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology. Their frequency remained unchanged between 3 and 7 weeks of culture. In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 weeks, but that proportion increased steadily between 3 and 7 weeks. These data suggest that, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies. Autoradiographic studies with [3H]thymidine showed that labeling indices in the early "normal" RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90%. In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 18 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly. The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of growth fractions and stem cell compartments in transformed rat tracheal epithelial cell colonies

The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631-4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (approximately 10(-3)). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 X 10(-2) when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was less than 4 X 10(-5) and in colonies of types II to IV, between 5 X 10(-4) and 10(-2). In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct types of rat tracheal epithelial cell colonies represent different stages of the clonal evolution of transformants.