二羟环氧苯并芘诱发细胞恶变后基因和蛋白表达差异分析

目的 研究苯并（a）芘代谢产物反式二羟环氧苯并芘（反式-BPDE）诱发人支气管上皮细胞系（16HBE）恶性转化的基因和蛋白表达的改变，探讨苯并（a）芘致癌的分子机制。     

滋养细胞肿瘤P53基因蛋白的检测

本文通过免疫组织化学技术对38例滋养细胞肿瘤组织 P53基因蛋白进行了检测,试图探讨 P53基因与滋养细胞肿瘤的关系。结果38例滋养细胞肿瘤中(葡萄胎10例、恶性葡萄胎10例、绒癌18例),恶葡阳性表达20%(2/10),绒癌阳性表达22.2%(4/18),有3例为高表达,10例良性葡萄胎均为阴性。结果提示:P53基因与滋养细胞肿瘤的生物学行为有密切关系。随着临床的进展,高表达增加。P53的检测可望成为预测滋养细胞肿瘤恶性发展的临床指标。     

高通量实时荧光定量RT-PCR方法验证FL细胞对苯并(a)芘7,8-二氢二醇9,10-环氧化物处理的差异表达基因

目的验证由寡核苷酸微阵列检出的FL细胞对苯并(a)芘7,8-二氢二醇9,10-环氧化物(BPDE)处理的差异表达基因。方法FL细胞分别经0.1%二甲亚砜(DMSO)和0.5μmol.L-1BPDE处理,以高通量实时荧光定量RT-PCR方法(TaqMan(低密度芯片)平行检测185个目标基因的相对表达水平。结果BPDE处理组相对DMSO对照组,51个基因表达有差异,12个基因表达上调,39个基因表达下调,(n=3,P<0.05),涉及细胞增殖,分化,凋亡调控基因,转录调节基因和代谢酶基因等。结论高通量实时荧光定量RT-PCR可迅速对微阵列数据进行验证,差异表达谱有助于研究BPDE的毒理机制和细胞的应答反应。     

苯并（a）芘诱导BEAS-2B细胞恶性转化的不同阶段特征

目的分析致癌物作用后至细胞转化过程中不同代次细胞的表达变化特征,为寻找出可能发生癌变的关键阶段、揭示致癌机制提供科学依据。方法采用永生化人支气管上皮细胞BEAS-2B,分设B(a)P组和DMSO溶剂对照组,染毒时间24h后细胞常规培养,系统观察发生于转化过程中的细胞生物学特性并由血清抗性、锚着独立性生长能力,裸鼠成瘤试验来鉴定细胞的恶性转化情况,建立B(a)P诱导永生化人支气管上皮细胞BEAS-2B恶性转化不同代次的细胞模型。采用细胞遗传学方法分析了不同代次细胞的染色体改变;提取实验组和对照组不同代数细胞的mRNA,用Affymetrix公司U133plus2.0人类全基因组表达谱芯片分析获得差异表达基因,并对差异表达的基因进行筛选。结果实验组细胞在20代后细胞形态不规则的数量开始增多,核浆比例增大,出现病理性核分裂像。第10代时各组细胞血清抗性试验均为阴性;第20代实验组细胞出现血清抗性和倍增时间增长。25代时实验组细胞在软琼脂上可形成小的细胞集落,细胞数十个左右聚集成团,但生长缓慢,不能进一步形成较大的克隆,达不到每个集落大于50个细胞的标准;至第30代时,实验组细胞即能在软琼脂上形成克隆。软琼脂克隆形成细胞经体外扩增培养后生长紊乱,极性消失,可见重叠生长。中途死亡实验组裸鼠两只,常规病理切片检查,有异形上皮细胞聚集成团,已具备癌前转化的特征。从第20代出现染色体核型异常,主要类型有多倍体、染色体断裂、双着丝粒等。得到第1、5、10、20和30代实验组与对照组细胞不同代次细胞表达的基因谱。发现不同代次细胞表达的基因谱有明显不同的特征,且各代之间差异表达的基因也有很大差异,以1、5和30代细胞表达的差异基因为基准对差异基因进行了聚类分析,初步揭示了这些差异基因在不同代次细胞的表达模式和特征。结论从已知的与肿瘤相关的基因变化来看,尽管第5代已有肿瘤相关基因的变化,但以10、20代细胞的变化最明显,提示此阶段可能是癌变的关键阶段,而第10代可能是细胞癌变的早期阶段。     

ALCAM基因敲除对苯并[a]芘恶性转化细胞THBEc1转录组的影响

目的 检测活化白细胞黏附分子(ALCAM)基因敲除后苯并[a]芘(BaP)恶性转化细胞THBEc1转录组的改变,探究ALCAM在BaP致癌中的作用,为进一步阐明BaP致癌机制提供线索.方法 选择两株ALCAM基因纯合敲除THBEc1细胞THBEc1-△ALCAM-c5和THBEc1-△ALCAM-c7及一株敲除对照细胞...     

塞替派诱发人支气管上皮恶性转化成瘤细胞的基因突变

目的　旨在了解转化细胞在成瘤过程中的p15 ,p16 ,p5 3和K ras基因编码序列突变情况。方法　运用逆转录聚合酶链反应 (RT PCR)技术和DNA测序技术。结果　转化各阶段细胞p15和K ras基因编码序列为野生型。在转化进程中 ,各转化细胞均携带与细胞永生化有关的p5 3第 4 7密码子CCG→TCG(脯氨酸→丝氨酸 )转换 ,在此基础上无新的错义突变发生。p16基因在BEAS 2B细胞和BEAS STE细胞中均为野生型 ,在BEAS TTb细胞中第 4密码子发生TCG→TAG(丝氨酸→终止密码子 )碱基转换 ,结果为无义突变 ,第 19,5 2密码子分别发生GAG→AAG (谷氨酸→赖氨酸 )转换 ,GCG→ACG(丙氨酸→苏氨酸 )转换的错义突变。BEAS TTc细胞中第 19密码子发生GAG→AAG(谷氨酸→赖氨酸 )转换的错义突变 ,而没有可检测的BEAS TTa细胞的mRNA存在。结论　p16基因的突变或表达缺失与恶性转化细胞的成瘤过程相关。     

二(噁)(口英)致大鼠肺癌与芳香烃受体介导关系的研究

目的用致癌物四氧二苯二氧杂环已二烯(二,TCDD)和苯并(a)芘(B(a)P)联合诱导构建大鼠肺癌的模型,探讨芳香烃受体(AhR)介导TCDD致肺癌的发生机制。方法选Wistar大鼠80只,随机分为ABCD 4组分别代表施以TCDD、TCDD B(a)P、B(a)P染毒的处理组和未施任何染毒处理的对照组,各组在第1.5、3、4.5和6个月,分批系列宰杀,观察肺部癌变情况。结果结果表明,TCDD染毒组在101 d时出现首例肺癌,累计TCDD用量865.94 ng,致癌率为15%,TCDD B(a)P联合染毒组在81 d时出现首例肺癌,TCDD累计用量622.34 ng,B(a)P累计用量为26.83 mg;致癌率30%,B(a)P染毒C组在161 d时,出现首例肺癌,B(a)P累计用量为87.58 mg,致癌率为5%;对照组未出现肺癌。4组之间比较,差异有统计学意义(P<0.05)。结论TCDD和B(a)P成功地诱发了大鼠肺癌,证实了TCDD既是致癌剂又是促癌剂。     

二羟环氧苯并芘恶性转化细胞miR-10a及其靶基因HOXA1的表达改变

目的 检测二羟环氧苯并芘恶性转化细胞miR-10a表达水平,探讨其与靶基因HOXA1癌基因的关系.方法 以正常支气管上皮细胞为对照组,二羟环氧苯并芘恶性转化细胞为实验组,用茎环结构逆转录引物逆转录,定量PCR检测两组细胞miR-10a表达水平.运用半定量逆转录-聚合酶链反应(RT-PCR)、定量PCR和流式细胞术间接法检测2组细胞HOXA1 mRNA和蛋白表达水平.结果 二羟环氧苯并芘恶性转化细胞miR-10a表达水平是对照组的0.01倍,RT-PCR HOXA1 mRNA是对照组的(3.12±0.23)倍,定量PCR HOXA1 mRNA是对照组的8.75倍,HOXA1蛋白表达是对照组的3.48倍.结论 二羟环氧苯并芘恶性转化细胞miR-10a可能反向调控靶基因HOXA1的表达.     

塞替派诱发人支气管上皮细胞恶性转化

目的观察抗癌药物塞替派诱发永生化人支气管上皮细胞恶性转化过程中相关基因的突变并分析其意义.方法以塞替派作为致癌原诱导永生化人支气管上皮细胞(BEAS-2B),获得转化细胞(BEAS-TE)和克隆化多倍体癌前转化细胞(BEAS-STE).采用PCR-SSCP法检测上述3种细胞p53、p16和Ki-ras 3种基因是否出现点突变,进一步测序确定其突变情况.结果 SSCP结果阳性的有BEAS-TE细胞p53第7外显子,BEAS-STE细胞p53第8外显子以及这二种细胞的p16基因第1外显子;Ki-ras基因第1外显子的结果仅为可疑阳性.测序证明,p53、Ki-ras基因存在多位点的碱基突变,而p16基因仅为单位点的碱基突变.结论塞替派可诱发人支气管上皮细胞p53、Ki-ras多位点的碱基突变和p16的单位点突变.分析前者为塞替派诱导细胞转化过程中发生的重要分子事件,后者是次要分子事件.     

宫颈鳞癌中高危人乳头状瘤病毒和P53 MDM2 P16~(INK4A)表达的关系

目的探讨浸润性宫颈鳞癌(ISCC)中人乳头状瘤病毒(HPV)16/18、31/33感染及P53、MDM2、P16INK4A蛋白的表达,研究各指标的相关性及在宫颈鳞癌发生、发展中的作用。方法利用组织芯片、原位杂交和免疫组织化学SP法对照检测106例ISCC、10例宫颈上皮内瘤变(CIN),10名正常宫颈鳞状上皮(NCE)中HPV16/18、31/33和P53、MDM2、P16INK4A的表达,应用SPSS12.5统计软件分析结果。结果HPV16/18、31/33和P53、P16INK4A蛋白在ISCC的表达高于CIN和NCE组,差异有统计学意义,HPV31/33表达与淋巴结转移相关(P<0.05);P53蛋白与组织学分级、淋巴结转移相关(P<0.05);P16INK4A表达与组织学分级呈正相关(P<0.05);MDM2蛋白与临床各参数均无相关性(P>0.05)。结论宫颈鳞癌的发生发展中高危HPV和突变型P53起重要作用,p53的下游基因MDM2是作为癌基因在宫颈鳞癌恶性进展过程中起作用,而p16INK4A可能不是单纯作为抑癌基因在起作用。     

Benzo(a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells

Although benzo(a)pyrene (BP) induces apoptosis in vitro in murine Hepa1c1c7 cells and in vivo indications of apoptosis in rat lung exist, related cellular mechanisms in human cells are not known. p53 protein participates in several apoptotic processes. We found that BP induces cell death in human MCF-7 breast adenocarcinoma cells at 48 and 72 h but not in human A549 lung carcinoma cells. BP did not induce measurable caspase-3-like protease activity or internucleosomal DNA fragmentation in either cell types. However, procaspase-7 cleavage in MCF-7 cells by BP-treatment indicates activation of caspase-7 meaning that apoptosis is most likely involved in BP-induced MCF-7 cell death. BP-7,8-dihydrodiol-9,10-epoxide (BPDE)–DNA adducts and level of p53 protein increased dose-dependently, but more extensively in MCF-7 cells. Phosphorylation of p53 protein at serines 15, 20, 46 and 392 increased in MCF-7 cells. Increase in phosphorylation at serine 392 was clear already at 24 h by 1 μM concentration of BP. Increase of phosphorylation at other sites occurred only with higher concentrations or at later time points in relation to the increase of p53 protein. These results suggest that serine 392 phosphorylation is the first stabilizing event of p53 associated with BP exposure and subsequent cell death in MCF-7 cells.     

塞替派诱发人支气管上皮细胞恶性转化过程中的基因突变（二）

目的 观察抗癌药物塞替派诱发永生化人支气管上皮细胞恶性转化过程中相关基因的突变并分析其意义。方法 以塞替派作为致癌原诱导永生化人支气管上皮细胞（BEAS－2B）,获得转化细胞（BEAS－TE）和克隆化多倍体癌前转化细胞（BEAS－STE）。采用PCR－SSCP法检测上述3种细胞p53、p16和Ki-ras 3种基因是否出现点突变,进一步测序确定其突变情况。结果 SSCP结果阳性的有BEAS－TE细胞p53第7外显子,BEAS－STE细胞p53第8外显子以及这二种细胞的p16基因第1外显子;Ki-ras基因第1外显子的结果仅为可疑阳性。测序证明,p53、Ki-ras基因存在多位点的碱基突变,而p16基因仅为单位点的碱基突变。结论 塞替哌可诱发人支气管上皮细胞p53、Ki-ras多位点的碱基突变和p16的单位点突变。分析前者为塞替派诱导细胞转化过程中发生的重要分子事件,后者是次要分子事件。     

Influence of estradiol on the benzo(a)pyrene hydroxylase activity induced by hexachlorocyclohexane

Basal BP-hydroxylase activity was measured in male Swiss mice from the age of 3 weeks to 20 months. Maximal enzyme activity was at the age of 5 months. Comparison of the inducibility of BP-hydroxylase by HCH was also investigated in male and female mice of different ages. Male mice showed higher induction of BP-hydroxylase by HCH than females of the same ages. Sterilization of female mice enhanced enzyme induction. Estradiol exhibited competitive inhibition of BP-hydroxylase activity. After treatment with HCH for 8 months, female mice had a lower tumour incidence than males, and this paralleled a lower induction of BP-hydroxylase.     

Benzo[a]pyrene induced p53‐mediated cell cycle arrest,DNA repair,and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus)

The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45β, igfbp‐3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p < 0.05), but gadd45β and igfbp‐3 expression was not significantly changed. Microphotographs revealed enlargement of the cell nuclei and cellular degeneration in males, while atrophy and vacuolization of hepatocytes were observed in females (10 µg/L). These results suggested that BaP induced liver DNA repair and apoptosis pathways and caused adverse pathological changes in rare minnow. The strongly responsive p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979–988, 2017.     

Cell cycle changes mediated by the p53/miR-34c axis are involved in the malignant transformation of human bronchial epithelial cells by benzo[a]pyrene

Characterization of aberrant microRNA (miRNA) expression during carcinogen-induced cell transformation will lead to a better understanding of the role of miRNAs in cancer development. In this investigation, we evaluated changes in p53 function and its downstream target miRNAs in benzo[a]pyrene (BaP)-induced transformation of human bronchial epithelial (HBE) cells. Chronic exposure to BaP induced malignant transformation of cells, in which there were increased levels of mutant p53 (mt-p53) and reduced expression of wild-type p53 (wt-p53) and phosphorylated p53 (p-p53). With acute (12 h) exposure to BaP, p-p53 was increased, and with increasing time of exposure (24 h), the increase in p-p53 at a concentration of 1 μM BaP was followed by a decline with increasing concentrations; wt-p53 and mt-p53 did not change. With prolonged exposure (48 h), p-p53 and wt-p53 decreased, but mt-p53 increased. At different exposure times, the levels of miR-34c were consistent with p-p53. Over-expression of miR-34c resulted in inhibition of the BaP-induced G1-to-S transition and diminished up-regulation of cyclin D. Further, up-regulation of miR-34c or silencing of cylin D prevented BaP-induced malignant transformation. Thus, changes in the cell cycle mediated by the p53/miR-34c axis are involved in the transformation cells induced by BaP.     

U87胶质瘤细胞p53基因全场cDNA克隆及序列分析

目的克隆U87胶质瘤细胞p53基因编码序列,并对其序列进行分析。方法根据NCBI GenBank中p53 cDNA序列设计引物,利用RT-PCR技术从U87胶质瘤细胞总RNA中对p53基因编码序列进行克隆;并通过DNAMAN软件将克隆序列与NCBI GenBank中序列进行比对,分析U87细胞中p53基因编码序列特点。结果经测序验证,成功克隆了p53基因编码序列,通过序列比对发现U87细胞中p53基因72位密码子为精氨酸残基。结论成功克隆了U87细胞中p53基因编码序列,分析发现U87细胞中p53基因表达产生的是72位密码子为含精氨酸残基的p53野生型蛋白,为进一步研究U87细胞中p53基因功能特性及其与胶质瘤的相关性奠定了基础。     

大鼠气管上皮细胞体内—体外转化模型的研究

本研究建立了大鼠气管上皮细胞体内－体外转化模型，大鼠气管内滴注苯并芘，三天后处死大鼠，消化气管上皮细胞，接种于无血清完全培养基。细胞形成集落后，换为选择培养基继续培养五周，统计转化率。结果显示，２５ｍｇ／ｋｇ和５０ｍｇ／ｋｇ的苯并芘可诱导大鼠气管上皮细胞转化及微核增加，用同样方法研究了煤焦沥青提取物，结果表明，剂量为８ｍｇ／ｋｇ和２５ｍｇ／ｋｇ的煤焦沥青提取物能明显诱导大鼠气管上皮细胞转化。     

Prenatal induction of benzo(a)pyrene hydroxylases in mice

1. Benzo(a)pyrene hydroxylase (BPH) activity was measured in homogenates of fetal liver (day 18) or of whole-embryos of mice on day 9, 10 or 12 of gestation after maternal pretreatment with B(a)P on 3 consecutive days. A³H-liberation assay with³H-B(a)P labelled either generally or at the 6-position was used. The values obtained with the embryonic/fetal tissues were compared with those found in maternal liver. 2. Three oral doses of 17.5 mg B(a)P/kg body wt were found to just significantly induce BPH in maternal liver. An induction was observed after pretreatment with 24 mg B(a)P/kg body wt in 9-, 10-or 12-day-old whole-embryos, but the V_max reached was only 10–20% (1% on day 9) of that of adult non-induced liver. The K_m (6-hydroxylation) for all tissues tested were in the same range (600–900 nM). The induction was demonstrable in embryos at tissue levels about one order of magnitude lower than those required for induction in maternal liver. 3. Treatment with 25 mg B(a)P/kg body wt on 3 consecutive days was required to induce BPH in fetal liver on day 18 of gestation. The required B(a)P tissue concentrations were about one half of those necessary for induction in maternal liver. 4. Among a variety of other polycyclic hydrocarbons only chrysene showed an inducing potency similar to that of B(a)P in adult and fetal liver. For all compounds tested there was no correlation found in the inducing potency between adult and fetal liver (e.g. coronene). 5. The doses required to induce BPH in the maternal or fetal liver or in whole embryos of rodents are significantly higher (mg range) than those of usual average human exposure or those taken up by smokers (ng range).Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo(a)pyrene - BPH benzo(a)pyrene hydroxylases - PAH polycyclic aromatic hydrocarbons     

Characterization of the microsomal cytochrome p-450 species induced in rat liver by trans-stilbene oxide

trans-Stilbene oxide differs from the classical inducers of drug-metabolizing enzymes, pheno-barbital and 3-methylcholanthrene, in that it induces the so-called phase II activities, epoxide hydrolase and glutathione S-transferase, to a much larger extent than it induces cytochrome P-450. Nonetheless, the level of cytochrome P-450 in liver microsomes from rats treated with trans-stilbene oxide is increased significantly to twice the control value.The existence of a number of different isozymes of cytochrome P-450 has now been clearly demonstrated and in the present study we have posed the question: What form(s) of cytochrome P-450 is induced by trans-stilbene oxide? A number of criteria including substrate specificity, pattern of benzo(a)pyrene metabolism, sensitivity to inhibitors, substrate binding spectra, ethylisocyanide binding spectra, sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis were used to answer this question. It seems clear that trans-stilbene oxide induces the same form(s) of cytochrome P-450 as phenobarbital.