人支气管上皮细胞和肺癌的无血清培养及生物学性质的研究

一、人上皮细胞无血清培养系统的建立绝大多数人类恶性肿瘤起源于上皮细胞。上皮细胞的无血清培养技术的发展，对研究上皮细胞癌变及其机理具有重要意义。从1986年起，我们实验室先后建立了多种上皮细胞无血清培养系统。利用MCDB151加多种生长因子的无血清培养基，已成功培养了     

致癌物诱导人支气管上皮细胞UDS及微核的初步研究

由于存在着种系,器官和细胞间的差异,发展用人靶细胞器官上皮细胞检测致癌物的方法是当前遗传学重要课题之一。我们用MCDB151加多种生长因子的无血清培养基,成功地培养人的多种上皮细胞。本文报道用体外培养人支气管上皮细胞,观察与人肺癌发生有关的几种因素诱导程序外DNA合成(UDS)及微核的效应。实验方法除在微核实验中加入了细胞松驰素B,用以产生双核细胞(代表分裂细胞)进行微核计数外,其它方法均按我们以前发表的方法进行。     

生长因子对气管上皮细胞及肺癌细胞生长影响

绝大多数人类的肿瘤起源于上皮细胞。上皮细胞的培养对癌变机理及遗传毒理学的研究均具有重要的意义。我们采用MCDB151培养基加多种生长因子,已成功地培养     

致癌物和X射线对人支气管上皮细胞的遗传毒理作用

从组织块长出的人支气管上皮细胞培养于MCDB151无血清培养基中,利用非程序性DNA合成(UDS)和微核试验检测化学致癌物和X射线对人支气管上皮细胞的遗传毒理作用。直接作用致癌物MNNG对所测试的7例上皮细胞均可分别引起UDS或微核增高,并具有剂量反应关系。X射线对4例都可引起微核增高。然而间接作用致癌物NNK和BaP引起的反应有明显个体差异。UDS试验中,NNK处理5例中有3例增高,BaP处理的4例中有3例增高。微核试验表明NNK处理的5例中3例为阳性,BaP处理4例中3例为阳性。本实验结果提示,NNK,BaP和X射线可能是人支气管上皮细胞潜在的致癌物。     

人支气管上皮细胞转化过程中凋亡与增殖的改变

目的 观察凋亡、增殖在人支气管上皮细胞转化中的改变,探讨其有关机理。方法 使用末端脱氧核苷酰转移酶标记凋亡法（ＴＤＴ）、Ｂｒｄｕ掺入法、Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ、荧光原位杂交等方法。结果 人永生化支气管上皮细胞Ｙ在无血清培养基连续转代过程中,晚代细胞软琼脂克隆形成率显著增高,并对血清分化产生明显抗性,对表皮生长因子（ＥＧＦ）依赖性有减弱倾向,细胞增殖速度明显加快,顺铂诱导的凋亡发生率显著低     

RhoC及其调节因子RhoGDIβ和RhoGDIγ在肺癌细胞系中的表达

目的:研究ras同系物(ras homologue,Rho)C蛋白及其调节蛋白Rho二磷酸鸟苷解离抑制因子(Rho guanosine diphosphate dissociation inhibitor, RhoGDI)-β和-γ在肺癌细胞中的表达情况.方法:应用Western印迹法和RT-PCR检测人正常支气管上皮细胞和不同人肺癌细胞系中RhoC、RhoGDIβ和RhoGDIγ蛋白及mRNA的表达.结果:RhoC、RhoGDIβ和RhoGDIγ的蛋白及mRNA在人肺腺癌细胞系、人肺巨细胞癌细胞系(高和低侵袭力亚型)和人支气管上皮细胞系中均有表达.3种肺癌细胞系中RhoC、RhoGDIβ和RhoGDIγ的表达均明显高于人支气管上皮细胞系.高侵袭力人肺巨细胞癌细胞系BE1中RhoC和RhoGDIγ的表达均明显高于低侵袭力人肺巨细胞癌细胞系LH7.低侵袭力人肺巨细胞癌细胞系LH7中RhoGDIβ的表达高于高侵袭力人肺巨细胞癌细胞系BE1.结论:高侵袭力人肺巨细胞癌细胞BE1中RhoC和RhoGDIγ的表达较高,低侵袭力人肺巨细胞癌细胞系LH7中RhoGDIβ的表达较高.     

NNK及X-射线诱导大鼠气管上皮细胞转化的研究

本文采用无血清F12培养基培养大鼠气管上皮(RTE)细胞。RTE细胞可在该种培养基中生长,增殖。形成典型的上皮细胞集落。当无血清培养基换成含血清的选择性培养基时,正常细胞停止生长进而退化死亡,而转化的RTE细胞则可以继续生长,成为转化集落。转化集落经多次传代可在裸鼠体内引起肿瘤。大鼠暴露于致癌物NNK或X射线后,收集其RTE细胞,先在体外进行无血清培养然后再进行选择性培养,其转化率明显高于对照组。RTE细胞体内—体外转化实验对于探究人类肺癌发生机制有一定意义。     

无血清培养基与完全培养基对体外诱导扩增CIK细胞及抗肿瘤活性的比较研究

目的:比较无血清培养基AIMV与完全培养基对体外诱导扩增CIK细胞的效果。方法:分别用AIMV及完全培养基加入4种细胞因子(IFNγ、IL2、IL1及OKT3)将脐带血单个核细胞(CBMNCs)诱导成CIK细胞,比较细胞的增殖能力、细胞表型、对肿瘤细胞的增殖抑制作用及其诱导肿瘤细胞凋亡等几个方面。结果:与完全培养基比较,无血清培养基AIMV培养的CIK细胞增殖高峰较晚(14～17d),但增殖倍数高(倍),在细胞表型、对三株肺癌细胞的生长抑制作用及不同效靶比诱导细胞凋亡方面两种培养基培养的CIK细胞差异无统计学意义,P>0.05。结论:无血清培养基AIMV可代替完全培养基。     

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。     

非小细胞肺癌组织CD151蛋白表达临床意义探讨

目的:探讨CD151在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达及意义。方法:免疫组化SP法检测云南省肿瘤医院2006-01-01-2011-12-31收治的78例NSCLC和22例良性肺病变组织中CD151蛋白的表达,并结合临床资料进行分析。RT-PCR法检测正常支气管上皮细胞株BEAS-2B、肺腺癌细胞株A549、云南宣威肺癌细胞株XWLC-05和云南个旧肺鳞癌细胞株YTMLC中CD151mRNA的相对表达量。结果:CD151在NSCLC组织中表达阳性率为57.7%(45/78),高于对照组的18.2%(4/22),χ2=10.72,P=0.001。CD151蛋白表达与性别、年龄、是否吸烟、T分期、TNM分期、肿瘤细胞分化程度以及PS评分无明显相关性,P值均>0.05;与病理分型(χ2=6.312,P=0.043)和淋巴结数(χ2=7.386,P=0.007)有关。生存分析提示,CD151蛋白阳性表达预后差;Cox回归分析表明,CD151蛋白是影响NSCLC预后的独立危险因素,P=0.014。荧光定量PCR结果显示,CD151mRNA在细胞株中的表达为BEAS-2B>A549>XWLC-05>YTMLC。结论:CD151蛋白可以作为判断NSCLC预后的参考之一。CD151mRNA在细胞株A549和XWLC-05中高表达,为进一步研究肺癌高发区特殊环境作用下发病机制以及预防、治疗提供新的思路。     

Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells

BACKGROUND: Because lung cancer is the leading cause of cancer-related death, new approaches for preventing and controlling the disease are needed. Chemoprevention approaches are both feasible and effective. We evaluated the potential of deguelin, a natural plant product, as a lung cancer chemopreventive agent and investigated its mechanism of action. METHODS: The effects of deguelin on proliferation and apoptosis of normal, premalignant, and malignant human bronchial epithelial (HBE) cells were assessed by using the MTT assay, a flow cytometry-based TUNEL assay, and western blot analyses. The effects of deguelin on the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways were assessed by western blot analyses and with adenoviral vectors that expressed constitutively active Akt. RESULTS: Deguelin treatment in vitro at doses attainable in vivo inhibited the growth of and induced apoptosis of premalignant and malignant HBE cells but had minimal effects on normal HBE cells. Levels of phosphorylated Akt (pAkt) were higher in premalignant HBE cells than in normal HBE cells. In premalignant HBE cells, deguelin inhibited PI3K activity and reduced pAkt levels and activity but had mimimal effects on the MAPK pathway. Although overexpression of a constitutively active Akt in premalignant and malignant HBE cells had no effect on growth inhibition mediated by N-(4-hydroxyphenyl)retinamide (4-HPR), a novel chemopreventive retinoid, it blocked deguelin-induced growth arrest and apoptosis. CONCLUSIONS: The ability of deguelin to inhibit PI3K/Akt-mediated signaling pathways may contribute to the potency and specificity of this pro-apoptotic drug. Because both premalignant and malignant HBE cells are more sensitive to deguelin than normal HBE cells, deguelin may have potential as both a chemopreventive agent for early stages of lung carcinogenesis and a therapeutic agent against lung cancer.     

Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium

We tested the ability of serum-free media to support the in vitro growth of human non-small cell lung carcinoma. A medium containing insulin, transferrin, sodium selenite, hydrocortisone, epidermal growth factor, and bovine serum albumin (1 mg/ml) with serum precoating of culture dishes (modified LA medium) supported three previously established cell lines of non-small cell lung cancer and prevented fibroblast proliferation in fresh tumor specimens but did not support long term tumor cell growth from fresh specimens. We added triiodothyronine, sodium pyruvate, and additional glutamine, insulin, and epidermal growth factor to modified LA medium, precoated with fibronectin and collagen instead of serum, and deleted bovine serum albumin, defining a new medium called ACL-3. ACL-3 medium alone supported the short term growth of 10 of 12 cell lines and the soft agarose cloning of 9 of 12 cell lines tested, and ACL-3 supplemented by an optimal concentration of bovine serum albumin (5 mg/ml) supported the long term growth of 10 of 12 cell lines tested. Moreover, we have grown tumor cells for more than 6 months from 11 of 33 (33%) consecutive fresh clinical specimens of human lung adenocarcinoma in ACL-3 with bovine serum albumin. ACL-3 medium provides a defined environment for the study of growth factor requirements of human non-small cell lung cancer and enhances our ability to grow human lung cancer, particularly adenocarcinoma, in vitro.     

Growth stimulation of human breast epithelial cells by basic fibroblast growth factor in serum-free medium

Growth-stimulating activities of basic and acidic fibroblast growth factors (FGFs) toward human breast epithelial cells were examined and compared with the mitogenic activity of bovine pituitary extract (BPE) by the use of a serum-free medium which contained epidermal growth factor, insulin, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, prolactin and prostaglandin. Addition of 1 ng/ml of basic FGF (bFGF) to the serum-free medium significantly enhanced the growth potential of epithelial cells derived from human breast carcinoma, and the number of cells grown for 7 days with bFGF was more than 1 1/2 times higher than that in the serum-free medium containing BPE instead of prolactin and prostaglandin. Growth responsiveness toward bFGF of epithelial cells derived from histologically non-malignant breast tissues was lower than that of carcinoma-derived cells, and the growth-stimulating activity of bFGF was lower than that of BPE, which could significantly enhance the growth potential of the cells. Contrary to bFGF, acidic FGF at 1 ng/ml had no significant effect on the growth potential of breast epithelial cells which had grown out from either carcinoma or non-malignant tissues. The present results suggest that bFGF is a putative growth-stimulating factor for human breast epithelial cells, especially for carcinoma-derived cells, and can substitute at least in part for BPE in serum-free monolayer culture of the cells.     

Identification of genes expressed differentially in an in vitro human lung carcinogenesis model

Lung cancer is the deadliest among all cancers in the US for both men and women. Early detection of premalignant lesions or tumors appears to be a promising approach to reducing the morbidity and mortality from lung cancer because the survival of early stage lung cancer patients is better than that of patients with advanced cancers. We approached the identification of early biomarkers of human lung carcinogenesis, by combining the use of cDNA array and an in vitro human lung carcinogenesis model that consists of normal (NHBE), immortalized (BEAS-2B and 1799), transformed (1198) and tumorigenic (1170-I) human bronchial epithelial (HBE) cells. We hypothesized that certain genes that are expressed differentially among these cells can serve as both biomarkers for early detection and targets for intervention. Nineteen genes were downregulated and six were upregulated in tumorigenic 1170-I HBE cells compared to normal HBE cells (NHBE) using cDNA array. Downregulated genes encode cell-to-cell and cell-to-matrix adhesion-related proteins, calcium-binding proteins and some enzymes or growth factor-related proteins. The functions of upregulated gene were more variable. Similar expression of selected genes was found in different nonsmall cell lung cancer cell lines analyzed by Northern blotting. Furthermore, the differential expression of some genes was also observed by in silico analysis of database of human lung tumors. The differentially expressed genes encode calcium-binding proteins and cell-cell and cell-matrix adhesion proteins. Furthermore, Northern blotting analysis of RNA from different cell lines comprising an in vitro carcinogenesis model including normal, immortalized, transformed, and tumorigenic cells indicated that the expression of subsets of the identified genes changed at different stages of carcinogenesis. These data show that the in vitro carcinogenesis cell system could be useful for discovering potential biomarkers of early and late stages of lung carcinogenesis and targets for chemoprevention.     

Differential responses of normal, premalignant, and malignant human bronchial epithelial cells to receptor-selective retinoids.

Using an in vitro lung carcinogenesis model consisting of normal, premalignant, and malignant human bronchial epithelial (HBE) cells, we analyzed the growth inhibitory effects of 26 novel synthetic retinoic acid receptor (RAR)- and retinoid X receptor (RXR)-selective retinoids. RAR-selective retinoids such as CD271, CD437, CD2325, and SR11364 showed potent activity in inhibiting the growth of either normal or premalignant and malignant HBE cells (IC50s mostly <1 microM) and were much more potent than RXR-selective retinoids. Nonetheless, the combination of RAR- and RXR-selective retinoids exhibited additive effects in HBE cells. As the HBE cells became progressively more malignant, they exhibited decreased or lost sensitivity to many retinoids. The activity of the RAR-selective retinoids, with the exception of the most potent retinoid, CD437, could be suppressed by an RAR panantagonist. These results suggest that: (a) RAR/RXR heterodimers play an important role in mediating the growth inhibitory effects of most retinoids in HBE cells; (b) CD437 may act through an RAR-independent pathway; (c) some of the RAR-selective retinoids may have the potential to be used in the clinic as chemopreventive and chemotherapeutic agents for lung cancer; and (d) early stages of lung carcinogenesis may be responsive targets for chemoprevention by retinoids, as opposed to later stages.     

硫化镍转化人支气管上皮细胞基因差异表达芯片的研究

背景与目的近年研究发现硫化镍(NiS)可引起人支气管上皮细胞(humanbronchialepithelialcell,16HBE)发生恶性转化及转化细胞致癌性,其机制可能与NiS引起基因突变、多种转录因子的异常表达有关。为此,本研究利用NiS恶性转化的体外培养细胞模型,用cDNA微阵列技术研究经NiS转化后的16HBE细胞基因的差异表达,从基因组水平探讨NiS所致细胞恶变的相关基因差异改变。方法分别提取16HBE和经NiS单独处理发生转化的NiS16HBE细胞的总RNA,逆转录合成cDNA并以Cy3dCTP和Cy5dCTP荧光素分别标记制作探针。探针混合后与含4000个人类基因的芯片杂交。以ScanArray4000扫描仪扫描芯片,以GenPixPro3.0软件分析荧光信号,然后对差异表达的基因进行生物学信息分析。结果NiS16HBE细胞样本与16HBE细胞样本比较,呈现差异表达的基因有151个(3.78%),其中81个(53.64%)上调,70个(46.36%)下调。结论NiS的转化作用与应激反应基因、免疫相关基因、DNA合成和修复基因、代谢相关基因、原癌基因和抑癌基因等多类基因的异常表达有关。     

结晶型NiS诱发人支气管上皮细胞系恶性转化

目的:研究结晶型硫化镍(NiS)诱发SV-40 Large T抗原永生化的人支气管上皮细胞系(16HBE)恶性转化作用.方法:体外用不同浓度结晶型NiS多次处理16HBE细胞,软琼脂集落形成和裸鼠成瘤试验鉴定转化细胞的恶性度.结果:细胞培养至35代,发现NiS可诱导16HBE恶性转化.转化细胞排列紊乱,重叠生长,在软琼脂中的集落形成率显著高于对照组(P＜0.01),并呈时间剂量依赖关系;转化细胞在裸鼠体内成瘤,组织学证实为低分化鳞状细胞癌.结论:结晶型NiS有较强的诱导人支气管上皮细胞恶性转化能力；该转化模型为镍致癌机制的分子水平研究提供了理想的研究系统。     

HMGA2在肺癌细胞系中的表达及与转移的相关性

目的:检测在肺癌细胞系中HMGA2的表达及分布情况并分析其与肺癌细胞转移能力间的关系。方法:采用Western blot、RT-PCR方法检测肺癌细胞系A549、BE1、LH7、95-D、95-C中HMGA2蛋白及mRNA的表达并和人支气管上皮细胞系HBE进行对比,结果由计算机图像系统采集并分析。结果:Western blot检测HMGA2蛋白在肺癌细胞系中过表达,而在人支气管上皮细胞系弱表达。而Western blot和RT-PCR结果均显示高转移能力的BE1和95-D肺癌细胞系的表达强于低转移能力的LH7和95-C肺癌细胞系（P<0.05）。结论:HMGA2表达在肺癌细胞中显著高于正常上皮细胞,其表达水平与细胞的转移能力正相关。HMGA2的过表达可能促进肺癌的转移。