Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells

INTRODUCTION Nickel is a widely distributed occupational and environmental pollutant. Epidemiological studies demonstrated that the incidence of respiratory tract cancer is correlated with worksite exposure to nickel. Nickel is also a potent carcinogen in laboratory animals. The International Agency for Research on Cancer (IARC) has classified nickel compounds as confirmed human carcinogens[1]. However, the mechanism of nickel carcinogenesis remains unknown. Insoluble nickel compounds…     

BPDE诱发16HBE恶性转化过程中eIF3 p36的表达变化

目的探讨二羟环氧苯并芘（BPDE）诱发人支气管上皮细胞（16HBE）恶性转化过程中不同阶段真核生物蛋白翻译启始因子（elF3 p36 mRNA）表达水平的变化，为进一步阐明BPDE的分子致癌机理提供线索。方法应用RT—PCR和FQ—PCR方法，检测并分析BPDE诱发16HBE恶性转化不同阶段的eIF3p36mRNA表达量的变化。结果相对于非转化对照细胞，BPDE诱发恶性转化16HBE不同阶段细胞（转化细胞和成瘤细胞）的eIF3p36mR-NA基因表达水平均显著高于对照组（P〈0．01或P〈0．05），其中BPDE-转化细胞的elF3 p36平均表达量分别是对照细胞的2．3～5．1倍：而BPDE-转化细胞与BPDE-成瘤细胞的elF3 p36平均表达量相比，差别无显著性（P〉0．05），提示elF3 p36的异常表达量与BPDE诱发16HBE细胞恶变程度之间存在一定的正向关系。结论BPDE在诱发16HBE细胞恶变过程中，存在明显的elF3 p36的异常表达现象，其表达水平与细胞的恶变程度密切相关，这可能是BPDE诱发人细胞肿瘤的重要分子致癌机理之一。     

镉和镍恶性转化16HBE上调基因eIF3 p36的克隆与序列

目的分析镉和镍恶性转化16HBE（人支气管上皮细胞）成瘤细胞株中翻译起始因子eIF3 p36基因序列的变化情况，探讨镉、镍重金属的致癌分子机制。方法取正常对照16HBE细胞、氯化镉（CAC12）和结晶型硫化镍（NiS）恶性转化的16HBE细胞，用有限稀释法分别建立单细胞克隆株，通过逆转录聚合酶链反应（RT—PCR）获得目的基因翻译起始因子eIF3 p36的eDNA，将目的基因产物连接到T载体中，转化、扩增重组质粒。提取大肠杆菌中的重组质粒进行DNA测序。将三组DNA测序所得到的基因序列进行对比分析。结果镉、镍转化组各细胞株（各10株）翻译起始因子的eIF3 p36 cDNA测序结果与正常对照细胞（10株）比较未发现基因的突变位点；所有镉、镍转化细胞和正常对照细胞株eIF3 p36 cDNA序列与Genbank数据库进行同源性分析，相似性比值均为100％。结论实验中镉、镍恶性转化16HBE细胞中翻译起始因子eIF3 p36上调未见基因序列改变，提示在镉、镍致癌中可能涉及到表遗传机制。     

应用基因芯片技术筛查肺癌变相关基因的研究

目的 使用基因芯片技术研究肺鳞癌及化学致癌物诱导入气管上皮细胞恶性转化的癌变相关基因。方法 应用含4096条人类全长基因的cDNA芯片分别检测6例肺鳞癌和6例正常肺组织的基因表达谱;并用上述芯片研究苯并(a)芘代谢产物BPDE[anti-Benzo(a)pyrene diolepoxide,BPDE]和结晶型硫化镍所诱导的人支气管上皮细胞恶性转化与正常的人支气管上皮细胞系(16HBE)在基因表达谱上的差异;将3类标本共同的差异表达基因确定为肺癌变的相关基因。结果 在肺鳞癌／正常肺组织之间、BPDE诱导的恶性转化细胞／正常16HBE细胞之间及硫化镍诱导的恶性转化细胞/正常16HBE细胞之间发现差异表达基因分别为171条、143条和151条。通过比较,发现89条与肺癌变相关的共同基因,其中表达显著增加的基因39条,它们是：癌基因6条;细胞周期相关基因4条;细胞增殖基因6条;肿瘤转移基因8条;神经内分泌基因3条;耐药基因1条;凋亡抑制基因1条;氧化基因1条;其他基因9条。表达显著下降的基因50条,它们是：抑癌基因7条;DNA修复基因11条;抗氧化基因1条;GST基因家族3条;细胞骨架基因3条;凋亡诱导基因2条;信号传导基因5条;细胞因子及其受体基因5条;细胞代谢基因7条,细胞外基质基因1条;其他基因5条。结论 cDNA基因芯片技术能有效地研究基因的表达谱,筛查出肺癌变相关基因。     

Malignant transformation and abnormal expression of eukaryotic initiation factor in bronchial epithelial cells induced by cadmium chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.     

Alterative Expression and Sequence of Human Elongation Factor-15 during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ(human EF-1δ p31) during malignant transformation of human bronchial epithelial cells induced by cadmium chloride (CdCI2) and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells (16HBE) induced by CdCI<.2> at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-1δ mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-1δ cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl<.2> was elevated (P<0.01 or P<0.05). Compared with their corresponding non-transformed cells, the overexpression level of EF-18 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed cells and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl<.2>. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl<.2>, but is not correlated with DNA mutations.     

三种镍化合物诱发转化细胞恶性度的鉴别与分析

目的 :利用已建立的三种镍化合物体外转化细胞进行裸鼠体内成瘤实验研究 ,比较它们恶性度的差异。方法 :三种镍化合物转化细胞接种BALB/C裸鼠 ,进行肿瘤细胞和转化细胞的透射电镜及扫描电镜观察。结果 :硫化镍 ,氯化镍 ,硫酸镍分别诱发的转化细胞都能在BALB/C裸鼠皮下形成肿瘤 ,病理组织学检查确定为纤维肉瘤。经扫描电镜与透射电镜观察瘤细胞形态与转化细胞有一定差别。结论 :本文进一步说明了三种镍化合物所诱发的细胞转化为恶性转化 ,且都具有体内致瘤性。     

人鼻咽癌癌基因及其产物的研究

Oncogene of nasopharyngeal carcinoma (NPC) by means of external origin DNA transfection experiment and its gene products by immunohistochemical method have been studied. These DNAs were isolated from human primary poorly differentiated NPC tissues and were transfected into NIH/3T3 mouse fibroblasts to induce the foci of the morphologically transformed cells in the culture, while DNAs of normal placenta tissues failed to do so. The DNAs were extracted from the primary and secondary transformed cells to analyse human sequence with human Alu sequence probe. The human sequence has been detected in the DNAs of the primary and secondary transformed foci cells, while none of the human sequence was detected in the DNAs of the control. The results indicated that human transforming sequences had been integrated into transformed cells. The malignant properties of the transformed foci cells were evidenced by tumorigenic experiment of nude mice. The transformed foci cells were inoculated subcutaneously in the nude mice and induced fibrosarcoma in vivo. The tumorigenic rate was 87.5%. It was further demonstrated that DNAs from human NPC possessed carcinogenicity and induced malignant transformation. The primary result revealed that the transforming gene of NPC may be homologue to Ha-ras oncogene. The expression of Ha-ras gene products-p21 has been studied in human NPC tissues. The primary results showed a positive expression of p21 in human NPC tissues by immunohistochemical method. The positive rate was 90.4%.     

Down-regulation of mitotic checkpoint in transformed human embryo lung fibroblasts induced by N-methyl-N’-nitro-N-nitrosoguaridine

Background Mutations in mitotic checkpoint genes have been detected in several human cancers, which exhibit chromosome instability. We wanted to know whether mutation of hBubl could occur in transformed human embryo lung fibroblasts (HELF) cells induced by a chemical carcinogen. Methods HELF cells were transformed by N-methyI-N‘-nitro-N-nitrosoguaridine (MNNG), and three flasks of transformed HELF cells (named as T1, T2, and T3) were selected as amplifiers, and mutations of hBubl in these transformed cells were analyzed by PCR-SSCP and sequencing. Results It was found that any one of three transformed cell lines exhibited aneuploidy with a low mitotic checkpoint function. Subsequent PCR-SSCP and sequence analysis showed an AGT to CGTor ATT mutation at codon 80 in hBubl gene in T1 cells with a resultant change in amino acid sequence. Conclusion Our study demonstrated that the mitotic checkpoint genes could be targets of MNNG.     

核糖核酸酶抑制因子与人乳腺癌细胞恶性增殖的相关性研究

目的:探讨核糖核酸酶抑制因子(ribonuclesae inhibitor,RI)的表达与人乳腺癌细胞MCF-7、MDA-MB-231、SKBR-3恶性增殖之间的关系。方法:四甲基偶氮唑蓝(MTT)法检测细胞增殖情况,流式细胞术检测细胞周期分布,RT-PCR检测RI基因表达水平,细胞免疫化学技术检测RI蛋白表达情况。结果:人乳腺癌细胞MCF-7细胞增殖速度、S期所占比例均小于人乳腺癌细胞MDA-MB-231、SKBR-3(P<0.05~P<0.01),且MCF-7细胞中RI基因表达水平、蛋白表达水平明显高于MDA-MB-231、SKBR-3细胞(P<0.01)。结论:RI与人乳腺癌细胞恶性增殖生长有一定的相关性。     

肺癌诊断基因的初步筛选

目的 筛选肺癌细胞恶性转化不同时期差异表达的基因，以期用于肺癌的诊断。方法 应用抑制消减杂交技术(SSH)、测序、cDNA芯片(cDNA Miemarray)、northern blot。结果 利用SSH建立了永生化人支气管上皮细胞(BEP2D)恶性转化不同时期差异表达基因的cDNA文库，其中，A消减文库(BEP2D细胞的cDNA为tester，α粒子照射BEP2D细胞后35代恶性转化细胞R15Hp35的cDNA为driver)有416个克隆，B消减文库(α粒子照射BEP2D细胞后20代转化细胞R15Hp20的cDNA为tester，BEP2D和R15Hp35细胞的cDNA混合后为driver)有301个克隆，C消减文库(R15Hp35细胞的cDNA为tester，BEP2D细胞的eDNA为driver)有586个克隆。对文库中107个克隆单向测序发现：19个克隆在GenBank中没有查到对应的同源序列，其它88个克隆共代表了76个不同的已知基因。然后，将3个文库中全部克隆的cDNA制作成cDNA芯片，用该芯片筛选了15例肺癌组织、5例肺癌旁组织和其他8种癌组织(肝癌、胃癌、食管癌、乳腺癌、白血病、子宫内膜癌、脑神经角质瘤和结肠癌)中mRNA的表达差异。结果，获得肺癌组织高于肺癌旁组织表达的eDNA26个，肺癌旁组织高于肺癌组织表达的31个，2者高于其他8种癌组织的分别为：肺癌旁组织中63个，肺癌组织中87个。将这208个具较大差异表达的基因重新制作成cDNA芯片，再选用临床上同一鳞癌病人鳞癌和鳞癌癌旁组织各1例与该芯片杂交，发现这些筛选出的基因的确在癌组织和癌旁组织中存在较大差异。结论 这些初步筛选出的差异表达基因有望成为肺癌诊断的侯选基因。     

17-AAG对MCF-7细胞增殖的抑制作用及对STAT3和VEGF表达的影响

 目的观察17-AAG对人乳腺癌MCF-7细胞株STAT3、VEGFmRNA及蛋白表达水平的影响。方法体外培养MCF-7细胞,分别给予不同剂量及不同作用时间的17-AAG,采用四甲基偶氮唑盐微量酶反应比色法（MTT法）检测细胞增殖能力;RT-PCR和Westernblot法检测各组细胞STAT3和VEGFmRNA及蛋白的表达水平。结果0.165～10.000mg/L17-AAG作用24h、48h对MCF-7细胞有显著的抑制作用,且有明显的时间、剂量依赖性;1.0、2.0、3.0、5.0mg/L17-AAG作用48h后,各组细胞STAT3和VEGFmRNA及蛋白表达水平均下调,且具有浓度依赖性。结论17-AAG对人乳腺癌MCF-7细胞的增殖具有明显的抑制作用,并能抑制STAT3和VEGFmRNA及蛋白的表达,且具有剂量依赖性。     

烹调油烟冷凝物诱导永生化人支气管上皮细胞恶性转化的研究

 目的用烹调油烟冷凝物（COFC）诱导正常永生化人支气管上皮细胞（BEP-2D）慢性恶性转化,构建恶性转化的细胞模型,以探讨COFC 的危害。方法通过MTT 法确定诱导物的染毒剂量,以COFC 处理细胞,用血清抗性实验和锚着独立生长实验鉴定细胞恶性转化的倾向和恶性特征,比较对照组和实验组抗血清促分化的能力和集落形成率,并将细胞接种于裸鼠,观察其成瘤情况。结果以50 μg/mL的COFC对细胞进行染毒,传至第20 代细胞,与对照组相比,其抗血清促分化的能力差异均有统计学意义（ P< 0.01）;传至第35 代,转化细胞的锚着独立生长实验克隆形成率差异均有统计学意义（ P< 0.05）,并接种裸鼠成瘤,病理检查结果为低分化鳞癌。结论COFC 可诱导正常永生化人支气管上皮细胞发生恶性转化,提示烹调油烟对人体具有致癌致畸作用。     

CⅡTA基因在五种人类恶性血液病细胞株细胞中的表达及意义

目的:探讨5种血液病细胞株细胞的HLA-Ⅱ类抗原表达,以及对IFN-γ诱导HLA分子表达的反应性与MHCⅡ类分子反式激活因子 (CⅡTA) 表达的关系.方法:采用流式细胞术和免疫组化法检测肿瘤细胞HLA分子及CⅡTA 蛋白的表达,RT-PCR检测肿瘤细胞CⅡTA基因表达.混合淋巴细胞反应检测肿瘤细胞刺激外周血T细胞反应的能力.结果:肿瘤细胞HLAⅡ类分子表达与CⅡTA表达一致; 结构型或诱导型表达CⅡTA的肿瘤细胞,经IFN-γ作用后其HLAⅠ、Ⅱ类抗原表达增高;IFN -γ诱导后仍不表达CⅡTA的肿瘤细胞,其对IFN-γ促HLAⅡ表达的作用不反应.Jurkat诱导后刺激T细胞表达高水平的IL-2 mRNA.结论:某些恶性血液病细胞株细胞对IFN-γ不能诱导HLA分子表达与CⅡTA诱导型表达缺陷有关,表明CⅡTA参与调控肿瘤细胞 HLAⅠ、Ⅱ类抗原表达,可能在肿瘤免疫逃逸中起重要作用.     

GPC3-WNT-JNK通路介导脂多糖诱导肺局部细胞炎症

目的：探讨GPC3在脂多糖（LPS）诱导的气道上皮细胞局部炎症微环境中的重要作用,并以GPC3-WNT通路介导的调控机制为核心进一步研究LPS诱导的肺局部炎症分子机制。方法：LPS经气道滴入小鼠后模拟肺损伤模型,检测肺泡灌洗液及肺组织中GPC3表达情况以及其表达位置。培养人肺16HBE细胞,LPS刺激16HBE细胞建立急性炎症细胞,予以RT-PCR检测其细胞中GPC3、TNF-α和TGF-β的mRNA表达情况,予以Western Blot检测GPC3、TGF-β和TNF-α蛋白表达情况。加入外源性GPC3刺激16HBE细胞,建立GPC3高表达细胞模型,检测炎症因子TNF-α和TGF-β mRNA的表达情况。不同浓度GPC3-WNT通路抑制剂处理细胞,观察上述指标变化。结果：LPS诱导ALI小鼠模型显示GPC3表达水平显著提高。此外,LPS在体外也产生了GPC3表达升高的现象。同时,外源性GPC3蛋白刺激支气管上皮细胞产生多种炎症因子,可能提示GPC3具有促炎作用。此外,GPC3诱导支气管上皮细胞系炎性细胞因子的产生被WNT-JNK通路的抑制剂阻断,提示ALI/ARDS过程中GPC3参与肺局部的潜在信号通路。结论：LPS刺激支气管上皮细胞的过程中存在一条线性信号通路即GPC3-WNT-JNK。这一发现可能为ALI/ARDS的治疗和生物标志物的开发提供一个新的分子靶点和分子机制。     

hPDGF-A/hBD2双基因共表达腺病毒载体的构建及表达

目的 制备人血小板源性生长因子-A(human platelet-derived growth factor A, hPDGF-A) 和人β防御素2(human beta defensins, hBD2)双基因共表达腺病毒载体并观察其在大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BMSCs)中的表达.方法 将hPDGF-A和hBD2通过内部核糖体插入位点(internal ribosome entry site, IRES) 连接后,以同源重组的形式插入腺病毒表达载体,由人胚肾293 细胞包装,获得有感染能力的重组腺病毒颗粒.重组病毒感染BMSCs后,用RT-PCR检测重组腺病毒的表达.结果 ①成功构建穿梭质粒pAdTrack-hPDGF-A-IRES2-hBD2.②成功构建重组腺病毒质粒pAdeasy-hPDGF-A-IRES2-hBD2.③293细胞包装获得有感染能力的重组腺病毒.④转染的BMSCs高表达hPDGF-A和hBD2.结论 构建了hPDGF-A/hBD2双基因共表达腺病毒载体,证实其转染BMSC后的表达.     

人δ珠蛋白基因启动子区CAAT盒C→T突变对其转录的影响

目的研究人δ珠蛋白基因启动子区-64位C→T点突变对其转录调控的影响。方法将人δ基因启动子区-64位C突变为T后,克隆于去除强启动子CMV的真核基因表达载体pcDNA3.1穴-雪/Myc-HisA中,分别将含野生型CAAT盒和突变型CAAT盒的人δ基因转染鼠红白血病(MEL)细胞,经DMSO诱导细胞分化,利用半定量RT-PCR技术检测人δ基因转录的变化。结果δ基因启动子区CAAT盒C→T点突变后,含突变型CAAT盒的人δ基因的转录水平是含野生型CAAT盒的2.2倍。结论在mRNA水平直接证实δ基因启动子区CAAT盒的缺陷是影响其转录的重要原因。     

DNA损伤检测点介质1和p53结合蛋白1在人食管癌细胞系TE-1.TE-13.Eca109中的表达及意义

目的 研究DNA损伤检测点介质1(MDC1)和p53结合蛋白1(53BP1)在人食管癌细胞系中的表达及意义.方法 采用半定量RT-PCR、免疫细胞化学法、间接免疫荧光法和Western blotting检测MDC1、53BP1mRNA和蛋白在人食管癌细胞系TE-1.TE-13.Eca109的表达水平.结果 RT-PCR结果显示MDC1、53BP1mRNA在所检测的人食管癌细胞系中均有表达;免疫细胞化学法、间接免疫荧光法和Western blotting均在人食管癌细胞系中检测到MDC1、53BP1的蛋白表达.结论 首次证实了MDC1、53BP1在食管癌细胞系中的表达.推测MDC1、53BP1可能和食管癌细胞的放射敏感性有关.