MNNG

摘要： 目的HP联合MNNG处理人食管上皮(HEEC)细胞,观察其对细胞增殖及周期的影响。方法CCK8法测定MNNG的IC_(50)值,确定染毒浓度;HP以感染复数(MOI)=100∶1(细菌∶细胞=100∶1)为实验浓度;分为对照组、HP处理组、MNNG处理组、HP+MNNG联合处理组(联合组),并分别在处理后1、6、12 h测定细胞增殖能力、细胞周期及相关周期蛋白表达的变化。结果MNNG IC_(50)值为40.90μmoL/L,本研究以1/2 IC_(50)值为实验浓度。对照组和MNNG组细胞形态无明显变化,在6 h和12 h后HP处理组和联合组细胞形态有较大改变,主要表现为细胞质紊乱不均匀,出现空泡样变化等。在经过1、6、12 h处理后,各组增殖速度为联合组MNNG处理组<对照组(F_(1 h趋势)=1089.3,P<0.001;F_(6 h趋势)=322.78,P<0.001;F_(12 h趋势)=50.01,P<0.001)。各处理组随着时间的延长,细胞增殖速度变缓(F_(HP)=370.44,P<0.001;F_(MNNG)=65.01,P<0.001;F_(联合)=98.46,P<0.001)。各组细胞在不同时间处理后G0/G1、S、G2/M细胞周期差异均有统计学意义(P<0.05)。随着染毒时间的延长,HP组、MNNG组和联合组均呈现G2/M期阻滞逐渐增大(F_(HP)=238.76,P<0.001;F_(MNNG)=182.47,P<0.001;F_(联合)=58.43,P<0.001),且同一时间为联合组大于HP和MNNG单独处理组(F_(1 h)=32.73,P<0.001;F_(6 h)=70.37,P<0.001;F_(12 h)=125.41,P<0.001)。染毒12 h后与对照组相比,其余3组细胞Cyclin B1蛋白表达量均显著升高,差异有统计学意义(P均<0.001),以联合组Cyclin B1表达最高,差异有统计学意义(P均<0.001)。结论HP及MNNG均能抑制HEEC细胞的增殖并改变其固有的细胞周期,联合作用表现更为明显,周期蛋白Cyclin B1表达升高,细胞周期G2/M期阻滞增大。

摘要： 目的:探讨应用N-甲基-N′-硝基-N-亚硝基胍(N-Methyl-N′-Nitro-N-Nitrosoguanidine, MNNG)配合复合因建立大鼠胃癌模型的方法。方法:将50只雄性Wistar大鼠随机分为对照组和实验组,每组25只。对照组给予生理盐水灌胃,实验组以100 mg·L^(-1) MNNG为起始浓度,之后每10周增加25%,直至浓度为150 mg·L^(-1)的递进浓度方式进行灌胃。对照组大鼠普通饲料正常进食,实验组给予高盐饲料并配合饥饱失常(饥1天,饱1天)喂养。从第16周至第40周,每隔4周分别从不同组随机选3只大鼠取胃组织,HE染色观察胃组织病理损伤变化;在20周称大鼠的体质量;统计大鼠的存活率。结果:病理结果显示:实验组大鼠胃组织第24周开始观察到肠化生,28~30周出现不同程度的异型增生,30~40周开始呈现胃癌的进展过程,总诱变率为80.95%;在20周时,与对照组比较,实验组大鼠体质量极显著降低(P<0.01);对照组大鼠的存活率为88.0%,实验组为76.0%。结论:梯度浓度MNNG灌胃配合高盐饲料、饥饱失常等复合因素可建立大鼠胃癌模型。

摘要： 目的:探讨人乳头状瘤病毒(HPV)全长基因转染与N-甲基-N'-硝基-N-亚硝基胍(MNNG)染毒联合作用对食管正常上皮Het-1A细胞恶性转化的影响,为食管癌的发病机制提供理论依据.方法:以Het-1A细胞为靶细胞,设4组,包括空载体转染对照组、转染HPV-18组(HPV)、MNNG染毒组(MNNG)、HPV与MNNG联合作用组(HPV+MNNG),其中MNNG染毒剂量为2μmol/L,每代染毒1次,每次24 h;选取第10代、20代及35代细胞进行形态学观察和功能学试验.采用CCK-8法检测各代各组细胞增殖活力,流式细胞术检测各代细胞周期分布,用增殖指数表示细胞的增殖状况.并采用Annexin V-APC/PI双染法检测细胞凋亡,通过侵袭迁移能力以及软琼脂集落形成试验检测细胞恶性转化表型,最后通过裸鼠成瘤试验观察恶性转化细胞在体内的增殖状况.结果:随着染毒代数增加,HPV+MNNG组与MNNG组细胞形态发生了明显的改变,连接松散、细长、伴伪足.CCK-8检测结果与细胞周期检测结果一致,与对照组比较,HPV+MNNG组和MNNG组的细胞增殖活性出现了先升高再下降最后升高的趋势(P均＜0.05),且HPV+MNNG组细胞变化更明显.与MNNG单独作用组比较,HPV+MNNG联合作用组细胞的抗凋亡能力增强(P＜0.05).侵袭和迁移试验结果显示HPV+MNNG组穿膜细胞数均显著高于其他各组(P＜0.05),MNNG组次之;软琼脂集落形成试验中,HPV+MNNG组细胞集落形成率[(30.8±0.2)％]高于MNNG组[(27.1±0.3)％](P＜0.05).HPV+MNNG组与MNNG组裸鼠成瘤率均为100％,瘤体大小无显著性差异.HPV单独作用组与对照组裸鼠未成瘤.结论:HPV与MNNG联合作用可致食管正常上皮细胞Het-1A恶性转化,使细胞增殖能力、抗凋亡能力、侵袭迁移能力及非锚定独立生长能力均显著增强,参与并促进恶性转化进程.

摘要： Objective It is to observe the effect of Huatan Xiaoyu prescription (HTXY) on the expression of bcl-2, bax, mTOR and LKB1 in rats with precancerous lesions of gastric cancer ( PLGC) .Methods 100 rats were randomly divided into normal group (17 cases) and model group (83 cases).Normal group was given normal diet , the model was built using the N-methyl-N’-nitro-N-nitrosoguanidine ( MNNG) combined with Hunger and satiety method .The successful model rats were ran-domly divided into model group , low, middle and high dose HTXY group , and Vitacoenzyme group .The normal group and model group were given the same amount of normal saline , and each administration group were given the corresponding medi-cine for once a day, for a period of 12 weeks.At the end of 37 weeks, all rats were sacrificed and the gastric antrum and gas-tric tissue were taken , the pathological changes of gastric mucosa in rats were observed under microscope ; the expression of bcl-2, bax, mTOR and LKB1 protein in gastric mucosa were detected by immunohistochemistry .Results The incidence rate of gastric precancerous lesions in model group was 84.6%, the incidence rate of gastric precancerous lesions in each administra-tion group was significantly lower than that in the model group (P0.05).Conclusion HTXY is effective in the treatment of rats with PLGC by down-regulation the expression of bcl-2, mTOR and up-regulation the expression of Bax and LKB 1.%目的：观察化痰消瘀方对胃癌前病变大鼠胃黏膜bcl－2、bax、mTOR、LKB1表达的影响。方法将100只大鼠随机分为正常组17只和造模组83只。正常组给予正常饮食，造模组采用N－甲基－N’－硝基－N－亚硝基胍（ MNNG）联合饥饱失常法造模。将造模成功大鼠随机分为模型组、化痰消瘀低剂量组、化痰消瘀中剂量组、化痰消瘀高剂量组和维霉素组。正常组和模型组给予等量生理盐水灌胃，各给药组分别给予相应药物灌胃，1次／d，连续12周。37周末处死所有大鼠，取胃窦及胃体组织，镜下观察各组大鼠胃黏膜组织病理变化，计算胃癌前病变发生率；免疫组化法检测胃黏膜组织中bcl－2、bax、mTOR、LKB1蛋白表达情况。结果模型组胃癌前病变发生率为84．6％，各给药组胃癌前病变发生率均明显低于模型组（P均＜0．05）。模型组大鼠bcl－2、mTOR蛋白表达水平明显高于正常组（P均＜0．05），bax、LKB1蛋白表达水平明显低于正常组（P均＜0．05）。各给药组bcl－2、mTOR蛋白表达水平明显低于模型组（P均＜0．05），且化痰消瘀方中高剂量组明显低于维霉素组（ P均＜0．05）；各给药组bax、LKB1蛋白表达水平明显高于模型组（ P均＜0.05），但化痰消瘀方低、中、高剂量组与维霉素组比较差异均无统计学意义（P均＞0．05）。结论化痰消瘀方可逆转胃癌前病变的发生，作用机制可能与其能下调bcl－2、mTOR蛋白的表达，上调bax、LKB1蛋白的表达有关。

摘要： OBJECTIVE:To explore the influence of the high expression of DNMT1 on MNNG-induced malignant transformation of Kazakh esophageal epithelial cell and to provide theoretical basis for pathogenesis of esophageal cancer.METHODS:Normal esophageal cells in culture were derived from Kazakh esophagus epithelium.MTT and clonal assays were conducted to determine the malignant transformation doses of MNNG.DNMT1 overexpression cells and the normal epithelial cells were exposed to MNNG for one hour.MTT and soft agar colony forming experiment were performed to observe the formation of cell colony under different exposure conditions and different multiplying rate.Nude mouse tumorigenicity assay was conducted to detect malignantly transformed cells.RESULTS:After treatment with MNNG (1.50 × 10-5 mol/L),cells with high expression of DNMT1 were able to form colonies in soft-agar medium after the 27th generation in vitro.These cells also formed local mass in nude mice but pathological evaluation showed that the local mass had disorderly structure and without squamous cell carcinoma morphology.On the other hand,innoculation of epithelial cells with high expression of transformed cells induced tumors in nude mice which showed squamous cell carcinoma morphology.CONCLUSION:A malignant transformation model of Kazakh esophageal epithelial cell with high DNMT1 expression was successful established.In addition,the model showed that DNMT1 high expression could promote induction of malignant transformation by MNNG.%目的:利用已构建的哈萨克族食管上皮DNMT1高表达细胞模型,探讨DNMT1高表达在MNNG诱导食管上皮细胞恶性转化中的作用,为研究食管癌发病机制提供理论依据.方法:以MNNG为诱导剂,MT法和克隆形成实验确定MNNG的恶性转化剂量;对哈萨克族食管上皮DNMT1高表达细胞和正常上皮细胞采用隔代染毒的方式进行染毒,每次染毒1h,软琼脂集落形成实验观察不同染毒和传代次数的细胞集落形成情况;裸鼠成瘤实验观察哈萨克族食管上皮DNMT1高表达细胞及其转化细胞的恶变程度.结果:MTT去和克隆形成实验发现MNNG转化浓度为1.50×10-5 mol/L,软琼脂集落形成实验发现染毒14次第27代的哈萨克族食管上皮DNMT1高表达细胞在软琼脂上有细胞集落形成,并且随着MNNG染毒剂量、次数和细胞传代次数的增加,出现细胞形态不规则等恶性转化特点,接触抑制逐渐消失,耐受性逐渐增强,生长速度逐渐增加,而正常上皮细胞未出现阳性结果,细胞固缩死亡.接种哈萨克族食管上皮DNMT1高表达细胞的裸鼠出现局部肿块,但病理鉴定仅为结构紊乱,并未出现鳞癌,而接种转化细胞的裸鼠出现肿瘤,经病理鉴定为鳞癌.结论:成功构建了哈萨克族食管上皮DNMT1高表达细胞恶性转化模型,DNMT1高表达可促进MNNG致细胞恶性转化,加快其恶变速率和恶变程度.

摘要： 目的:探讨叶酸在N-甲基-N-硝基-N-亚硝基胍(MNNG)影响哈萨克族食管上皮细胞DNA甲基转移酶1(DNMT1)活性及表达中的作用,为食管癌的防治提供理论依据.方法:采用3因素3水平(3×3)析因设计将体外培养的哈萨克族食管上皮细胞分别暴露于不同浓度叶酸(0.000、0.400、0.800 μg/mL)和MNNG(0.000、0.750、1.500μg/mL)的培养液中作用48、72和96 h后,采用ELISA法检测DNMT1酶活性;采用RT-PCR的方法检测DNMT1mRNA表达水平;采用Western blot法检测DNMT1蛋白的表达水平.结果:不同浓度叶酸、MNNG作用细胞不同时间后,在固定MNNG浓度和时间因素下,随着叶酸浓度的增加DNMT1酶活性、DNMT1 mRNA和蛋白表达水平均逐渐降低(P均＜0.01);固定叶酸浓度和时间,随着MNNG浓度的增加DNMT1酶活性、DNMT1mRNA和蛋白表达水平均逐渐升高(P均＜0.01);固定MNNG和叶酸浓度,随着干预时间的延长DNMT1酶活性、DNMT1 mRNA和蛋白表达水平均逐渐升高(P均＜0.01);叶酸浓度与MNNG浓度之间、MNNG浓度与作用时间之间均存在交互作用(P＜0.01).不同叶酸浓度与不同MNNG浓度之间、不同叶酸浓度与不同作用时间之间、不同MNNG浓度与不同作用时间之间差异均有统计学意义(P＜0.05).结论:叶酸浓度降低对MNNG影响哈萨克族食管上皮细胞DNMT1酶活性及表达有一定的激发作用,对食管癌的发生有一定的促进作用,而保持充足的叶酸则对MNNG的损害起到一定的保护作用.

摘要： Objective To investigate the role of folic acid in MNNG-induced proliferation,cell cycle and ap-optosis of Kazakh normal esophageal epithelial cell.Methods Using 3 factors 3 level (33)factorial design, Kazak normal esophageal epithelial cells were treated with different concentrations of folic acid (0.000, 0.400 and 0.800 μg/mL)and MNNG (0.000,0.750 and 1.500 μg/mL),cultured 48,72 and 96 h,MTT assay was used to measure cell proliferation,and cell cycle and apoptosis rate were detected by flow cytom-etry and Annexin V-FITC/PI staining.Results With decreasing of folic acid concentration and increasing of MNNG concentration:Morphogenesis of Kazakh normal esophageal epithelial cell had significant chan-ges,number of the dead cells were gradually increased,nuclei ruptured in varying degrees,cytoplasm was uneven and cell integrity was destroyed.Cell sensitivity to MNNG damage were increased significantly, particularly on 96 h;Kazakh normal esophageal epithelium inhibition rate increased,which there was a significant difference (P <0.05);Proportion of G0 +G1 phase,S phase and G2 +M phase of cells were sig-nificantly different (P <0.05),cell cycle arrest in S and G2 + M phase;Apoptosis rate of Kazakh normal esophageal epithelium cell were increased,which there was a significant difference (P <0.05).Conclusion Re-duction of folic acid concentrations promoted MNNG-induced Kazak normal esophageal epithelial cell injury.%目的：探讨叶酸在 N-甲基-N-硝基-N-亚硝基胍（MNNG）致哈萨克族（哈族）食管正常上皮细胞增殖、细胞周期及凋亡中的作用。方法采用3因素3水平（33）析因设计将体外培养的哈族食管正常上皮细胞暴露于不同浓度叶酸（0．000、0．400、0．800μg／mL）和 MNNG（0．000、0．750、1．500μg／mL）的培养液中作用48、72和96 h后，采用噻唑蓝（MTT）法检测叶酸及 MNNG 对哈族食管上皮增殖的影响，流式细胞术检测细胞周期和细胞凋亡。结果随着叶酸浓度的降低和 MNNG 浓度增加，哈族食管正常上皮细胞形态发生了明显的变化，死细胞数目逐渐增多，细胞核出现不同程度的破裂，胞质不匀和细胞完整性损伤，细胞对 MNNG 损害的敏感性明显增加，96 h 时尤为严重；哈族食管正常上皮细胞的抑制率升高，差异有统计学意义（P ＜0．05）；哈族食管正常上皮细胞 G0＋G1期、S 期和 G2＋M 期细胞比例不同，差异有统计学意义（P ＜0．05），细胞周期阻滞在 S 期和 G2＋M 期；哈族食管正常上皮细胞的凋亡率升高，差异有统计学意义（P ＜0．05）。结论叶酸浓度降低对 MNNG 致哈族食管正常上皮细胞损伤有一定的促进作用。

摘要： Objective To discuss the expression of Sonic hedgehog blocking antibody (Shh Ab) in peripheral blood mononuclear cells(PBMCs) of anti-gastric cancer cells. Methods Healthy human PBM-Cs were centrifugated by Ficoll density gradient (GES-1 cells were treated by nitrite amides) and gastric cancer cells were co-cultured with MC. To observe the expression and semi-quantitative analysis of Shh and Gli-1 by RT-PCR, Shh blocking antibodies were added in the co-culture system, and the expression of CD3, CD5, CD69 was assayed by flow cytometry. Results RT-PCR showed that Gli-1mRNA in MC+Shh Ab cell group was 0.284 5±0.002 5, lower than the MC cell group 0.516 7 ± 0.010 9 (P<0.05). Flow cytometry showed Shh blocking antibodies promoted the expression of CD3 and CD69. Shh Ab enhanced cell killing PBMCs for MC. Conclusion Shh Ab can promote activation of PBMCs, and enhances anti-carcinogenic effect of nitrite amides.%目的探讨Sonic hedgehog阻断抗体(Shh Ab)对外周血单个核细胞(PBMCs)抗胃癌MC细胞作用的表达。方法 Ficoll密度梯度离心法分离正常人PBMCs,并与胃癌MC细胞(GES-1细胞经亚硝酰胺类化合物处理)建立共培养体系；RT-PCR观察Shh、Gli-1基因的表达并进行半定量数据分析；于共培养体系中加入Shh阻断抗体,流式细胞术检测CD3、CD5、CD69分子表达。结果RT-PCR结果显示Gli-1mRNA在MC+Shh Ab细胞组值为0.2845±0.0025,低于MC 细胞组的0.5167±0.0109(P<0.05)；流式细胞检测Shh Ab可促进CD3、CD69分子表达,对CD5分子没有显著影响；Shh Ab增强PBMCs对MC细胞的杀伤。结论 Shh Ab可促进PBMCs化,增强PBMCs抗亚硝酰胺类化合物致癌机制的作用。

摘要： 通过N-甲基-N'-硝基亚硝基胍、8-氮鸟嘌呤、6-巯基鸟嘌呤紫外线照射的联合作用,试图建立C6细胞的次黄嘌呤鸟嘌呤磷酸核糖基转移酶缺陷株,为建立大鼠垂体促性腺激素杂交瘤细胞株奠定基础,但最终未能获得预期的能够被HAT培养基筛选的HGPRT-缺陷株,提示C6细胞株的HGPRT基因非常稳定,诱变方法难以使其突变缺失.%This study tries to product rat gloma cells hypox anthine-guaninep hosphoribosyl transferase deficiency lines by combined effects of N-methyl-N'- nitro-nitrosoguanidine,8- azaguanine,6-thioguanine and ultraviolet irradiation in order to establish rat pituitary gonadotropic hormone hybridoma cell lines in future,but wo are not able to gain the expectant glioma HGPRT cell lines which can be sieved by hypoxan thine-aminop terinthymidine selective medium. The experiment indicate that the HGPRT gene of C6 cells is too stabile to be induced deficient on this experiment conditions.