NEOPLASTIC TRANSFORMATION OF HUMAN EMBRYONIC NASOPHARYNGEAL EPITHELIAL CELLS INDUCED BY N,N'-DINITROSOPIPERAZINE (DNP) IN VITRO

This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.

THE STUDY ON RELATED GENES IN THE NEOPLASTIC TRANSFORMATION OF IMMORTALIZED HUMAN FETAL TRACHEAL FIBROBLAST CELLS INDUCED BY IRRADIATION

In this study, we investigated the genes related to the transformation of immortalized human fetal tracheal fibroblast cell line induced by alpha particles by means of differential display mRNA method. The result revealed that there were 23 DNA fragments that were expressed intensively in alphaSHTF cells (SHTF cells forming clone on agar after irradiated by alpha particles emitted by 238Pu) only and not in SHTF (SV40-immortalized human fetal tracheal fibroblast) cells. Northern dot confirmed two fragments, C17-5, C23-1 which showed intensive mRNA expression in alphaSHTF cells, but not in SHTF cells. The length of the C17-5 fragment was 310bp. Searching in BLAST database revealed that the C17-5 fragment might be an unknown sequence.

Liver cell adenoma with malignant transformation:A case report

A 57-year-old woman was referred to our hospital because of a liver mass detected by computed tomography.She had taken oral contraceptives for only one month at the age of thirty.Physical examination revealed no abnormalities,and laboratory data,including hepatic function tests,were within the normal range,with the exception of elevated levels of those serum proteins induced by the absence of vitamin K or by raised levels of the antagonist (PIVKA)-Ⅱ (3 502 AU/ml). Abdominal ultrasonography revealed a hyperechoic mass measuring 10×10 cm in the left posterior segment of the liver.Because hepatocellular carcinoma could not be completely excluded,this mass was resected.The tumor consisted of sheets of uniform cells with clear cytoplasm, perinuclear eosinophilic granules and round nuclei.These histological findings were consistent with liver cell adenoma. Background hepatic tissue appeared normal.After resection of the tumor,serum PIVKA-Ⅱ fell to within the normal range. An area of hepatocellular carcinoma (HCC) with a mid- trabecular pattern was immunohistochemically found,which was positive for PIVKA-Ⅱ.Sinusoidal endothelial cells were CD34-positive,containing scattered PIVKA-Ⅱ positive cells. This tumor was therefore finally diagnosed as liver cell adenoma with focal malignant transformation to HCC.

A novel system for Agrobacterium-mediated transformation of wheat(Triticum aestivum L.) cells

A new approach for transforming the cultured cells of wheat(Triticum aestivum L. cv. Ganmai 8) was developed using Agrobacterium tumefaciens. The features of the optimum procedure were: (a) both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA) cells for approximately 16 h; (b) the gyratory magnetic field condition was used during cocultivation; (c) the cocultivating period and selecting condition were modified; (d) the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium. Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT II and NOS genes, located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo, in transformed wheat cell colonies by adopting the techniques of dot blot, ndPAGE or high voltage paper electrophoresis. Integration of the foreign genes into wheat genome was confirmed by Southern blot hybridization. Moreover, a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.

Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.

Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87

AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

Alterations in Retinoic Acid Receptors in Non-Small Cell Lung Cancer and Their Clinical Implications

The nuclear retinoic acid receptor may play a critical role in the process of lung carcinogenesis. Alteration or loss of nuclear retinoic acid receptors (RARs) has been associated with progression in premalignant and malignant tissues and it is associated with malignant transformation in human cells. Vitamin A derivates, such as retinoic acid, have emerged as adjuvant to therapy in several types of cancer with favorable effects. Retinoic acid regulates the expression of target genes through the binding and activation of RARs, inhibiting growth proliferation. Diverse studies have evaluated different retinoids alone or in combination with chemotherapy in lung cancer, from which results have been controversial with benefits observed only in the subpopulation with high levels of triglycerides. Additionally, several large randomized trials using retinoids to prevent tobacco-related cancer have failed;due to the latter the use of retinoids in clinical trials remains controversial. However they could reduce the risk of cancer development in non-smokers. There is evidence that retinoids have different effects on lung cancer;still the identification of biomarkers could determinate their benefits as preventive or therapy agents. This review describes the RAR alterations during the development of Non-Small Cell Lung Cancer and sets out the importance of several cancer treatments with retinoid compounds.

番木瓜胚性细胞悬浮系高效遗传转化体系的建立

【目的】建立番木瓜高效遗传转化体系,为番木瓜基因功能研究和重要农艺性状改良提供新的技术支撑。【方法】以紫晖番木瓜胚性细胞悬浮系(embryogenic cell suspensions,ECS)为遗传转化受体,利用植物表达载体pCAMBIA1301和农杆菌介导法进行遗传转化,对抗生素浓度筛选、侵染时间、继代培养、抗性胚的诱导与萌发以及植株再生整个过程进行探索,最后获得抗性再生植株。【结果】通过设置不同浓度的头孢霉素和潮霉素处理,观察ECS细胞状态,筛选、确定头孢霉素和潮霉素最适处理质量浓度分别为200 mg·L^(-1)和5 mg·L^(-1)。工程菌和ECS共培养侵染2 d后转到含有头孢霉素和潮霉素的液体筛选培养基上进行继代培养,继代周期为14 d。经GUS染色验证,表明继代3次后的ECS几乎全部为转化细胞。将以上ECS转移到液体胚诱导培养基中进行培养,2个月后可获得大量球形体细胞胚,且GUS组织染色为蓝色。将球形体细胞胚转到半固体成熟培养基上培养,2个月后可获得成熟子叶期体细胞胚。子叶期体细胞胚在萌发培养基上光培养30 d后,可获得再生芽。任意选取再生芽进行GUS染色,均可染成蓝色。抗性再生芽经促根培养可成功获得再生植株。利用PCR检测抗性再生植株,可以确定GUS基因已经整合到番木瓜基因组中。【结论】成功建立了一种以番木瓜ECS为转化受体的农杆菌介导的高效遗传转化体系。在该技术体系中,经农杆菌侵染后的ECS继代筛选3次后,几乎全部为转化细胞,这些转化细胞经体胚诱导、成熟、萌发和生根过程可成功获得再生植株,抗性体胚得胚率为43.65%,抗性体胚萌发率为73.26%,植株再生率为80.55%,大大提高了番木瓜遗传转化效率。该体系为番木瓜基因功能研究和分子育种提供了新的途径。

白藜芦醇通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞EMT过程中的作用及其机制

目的探讨白藜芦醇(RSVL)通过Hippo-YAP信号通路在TGF-β1诱导胃癌细胞上皮间充质转化(EMT)过程中的作用及其机制。方法选取胃癌细胞SGC-7901为研究对象,首先将其随机分为空白组、RSVL低剂量组(5μM)、RSVL中剂量组(10μM)、RSVL高剂量组(20μM),通过细胞增殖(MTT)实验确定RSVL浓度,然后对细胞进行转染,分为si-NC组、TGF-β1+10μM RSVL组、TGF-β1+si-YAP组、TGF-β1+pcDNA3.1-YAP组、TGF-β1+10μM RSVL+pcDNA3.1-YAP组。采用MTT、细胞侵袭(Transwell)、划痕实验,分别检测细胞的增殖、侵袭和迁移;采用蛋白质印迹法(WB)和荧光定量PCR检测E-钙粘连蛋白(E-cadherin)、神经型钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)、Snali 1、HIP-PO/Yes相关蛋白(YAP)和mRNA。其次,选取24只裸鼠,将其分为模型组、RSVL组(10μM)、RSVL+pcDNA3.1组、RSVL+pcDNA3.1-YAP组,每组各六只。计算小鼠肿瘤的重量和体积;采用免疫组化检测Ki67蛋白。结果RSVL对细胞增殖有抑制作用(P<0.05);与si-NC组相比,其余各组侵袭细胞数、迁移率较低(P<0.05);与si-NC组相比,其余各组E-cadherin蛋白及mRNA表达较高,N-cadherin、Vimentin、Snali 1蛋白及mRNA以及YAP蛋白表达较低(P<0.05);与Model组相比,其余各组小鼠肿瘤的重量和体积均较低(P<0.05);与Model组比较,其余各组Ki67染色强度均显著减弱。结论RSVL可以通过抑制YAP相关通路的激活,来发挥抑制肿瘤细胞SGC-7901上皮间充质转化、增殖、迁移、侵袭的作用。

超表达CsCslE1_4基因对烟草细胞壁成分的影响

类纤维素合酶E(CslE)基因与纤维素合成相关,决定着细胞形状以及大小,并在植物生长发育阶段起着机械支撑和抵抗病原物侵染的作用。为研究CsCslE1_4基因对植物细胞壁的重要组成成分的影响,应用转基因烟草研究CsCslE1_4基因的基本功能,通过农杆菌介导法遗传转化烟草,经鉴定得到18株阳性植株,结合定量表达以及细胞壁组成成分测定。结果表明,与野生型相比,转基因株系茎的纤维素含量是野生型的1.43~5.48倍,叶中纤维素含量除株系CslE2外,其余株系纤维素含量是野生型的1.08~2.49倍,大部分转基因株系半纤维素含量是野生型半纤维素含量的1.18~2.22倍,但果胶含量变化趋势与纤维素和半纤维素含量变化趋势相反,木质素含量没有明显的变化。CsCslE1_4基因可以促进纤维素合成,这为后续解析基因功能以及抗逆性提供依据。

肿瘤微环境中间充质干细胞的相关生物学特性分析

目的探讨大鼠骨髓间充质干细胞(BMSCs)在肿瘤微环境中是否获得肿瘤细胞的相关生物学特性而导致恶性转化。方法通过6孔板结合Transwell小室建立间充质干细胞和大鼠C6胶质瘤细胞的共培养体系,以共培养组为实验组,另设单独培养的间充质干细胞作为对照组。于相差显微镜下观察培养后两组细胞的形态学改变;采用流式细胞术(FCM)分析细胞周期变化;采用荧光定量PCR和免疫荧光法检测间充质干细胞mdm2、p53mRNA水平及共培养后两组细胞中p53突变蛋白和mdm2癌蛋白的表达。结果共培养后实验组细胞表现为胶质瘤细胞样形态。细胞周期检测结果显示共培养7d后,对照组G1期细胞占90.48%±6.62%,实验组占82.22%±2.74%,两组间差异无统计学意义(P>0.05);对照组S期细胞占4.66%±4.16%,实验组占7.35%±1.93%,两组间差异亦无统计学意义(P>0.05)。定量PCR及免疫荧光检测显示,实验组p53mRNA水平明显降低,为对照组的0.24倍(P<0.05);部分实验组细胞(24.8%)中表达突变型p53蛋白,而对照组无突变p53蛋白表达(P<0.05)。实验组mdm2mRNA及其蛋白的表达水平均明显高于对照组(P<0.05)。结论肿瘤微环境使大鼠骨髓间充质干细胞获得了肿瘤细胞的相关生物学特性。

IL-6对M1型巨噬细胞向肿瘤相关M2型巨噬细胞转化过程的诱导作用

目的共培养小鼠M1型巨噬细胞RAW264·7与乳腺癌细胞4T1,观察白介素6(IL-6)在M1型巨噬细胞向肿瘤相关M2型巨噬细胞转化过程中的作用。方法实验共分为以下3组:A组(4T1细胞),B组(RAW264·7细胞),C组(RAW264·7与4T1以1∶4比例混合培养)。流式细胞技术检测M2型巨噬细胞比例,随后加入IL-6特异性抑制剂激活素A(ACTA,25μg/ml)或重组IL-6(rIL-6,10μg/ml)后再行检测。RT-PCR检测M1、M2型巨噬细胞功能相关细胞因子及IL-6、IL-6Rα的mRNA表达量。ELISA法检测3组细胞上清中IL-6的含量,随后改变两种细胞的混合比例后再行检测。结果RAW264·7和4T1细胞共培养后,M2型巨噬细胞比例显著增加,添加ACTA后该比例显著下降,而添加rIL-6后该比例增加。与B组比较,C组的M2型巨噬细胞相关细胞因子(CCL22、CCL2、YM1、PDGF-c、HIMP、MMP-9、IL-10)mRNA表达水平明显增高,M1型巨噬细胞相关因子(IL-12、IL-15、IL-18)mRNA表达水平明显降低。IL-6mRNA在A组呈微量表达,在B组无表达,在C组表达明显增高,而IL-6RαmRNA在3组中均呈高表达。与A、B组比较,C组细胞上清液中IL-6含量明显增加(P均<0·05);以不同比例将两种细胞混合后发现RAW264·7细胞数量的改变显著影响了共培养细胞上清中IL-6的水平。结论将4T1和RAW264·7细胞共培养后,前者能促进后者分泌IL-6,并诱导其向肿瘤相关M2型巨噬细胞转化。IL-6在转化过程中发挥重要作用,阻断其作用可以遏制转化。

二亚硝基哌嗪诱发转基因小鼠鼻咽上皮细胞恶性转化的研究

本文报道化学致癌物二亚硝基哌嗪（DNP）诱发转基因小鼠鼻咽上皮细胞体外恶性转化。转化细胞表现为形态学改变，生长寿命延长，停泊非依赖性生长，染色体异常，接种裸鼠后形成肿瘤。电镜观察这些细胞呈低分化鳞癌细胞特征，实验结果表明，体外培养的携带EB病毒潜伏膜蛋白瘤基因的转基因小鼠鼻咽上皮细胞在小鼠肝微粒体酶（S9mix）活化下，对DNP的转化具有一定的敏感性，

NNK诱发人支气管上皮细胞恶性转化及氧化损伤机理研究

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