结晶型硫化镍诱发细胞恶性转化过程中基因组总体DNA甲基化的改变

Objective To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells.Methods 16HBE Cells were treated with crystalline NiS at 0.25,0.50,1.00 and 2.00 μg/cm2 for 24 h and three times at total.DAC treatment was given at 3 μmol/L for 72 h.5-mC immunofluorescence and SssI emthyltrasferase assay methods were applied to investigate if the hypomethylation of genome DNA involved.Results The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically.By the SssI methylase assay, an average of (81.9 ± 7.3 )% methylated CpG were found in negative control cells.By contrast, ( 77.9 ± 6.2) %, ( 75.3 ± 6.8 ) %, ( 59.5 ± 4.9 ) %, ( 67.4 ±5.1 ) % methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25,0.50,1.00and 2.00 μg/cm2 which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively.The ANOVA analysis results showed that there was a significant difference in the 5 groups above ( F = 124.95,P ＜0.01 ).The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00were significantly decreased compared with the negative control group(t values were 7.64,4.89 respectively,P ＜0.01 ).For methylated CpG, (46.2 ±4.1 ) % and (43.6% ±4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group(t values were 12.79,13.56 respectively, P ＜ 0.01 ).Conclusion Genomic DNA methylation levels were decreased during NiS induced malignant transformation.     

结晶型硫化镍诱发细胞恶性转化过程中基因组总体DNA甲基化的改变

Objective To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells.Methods 16HBE Cells were treated with crystalline NiS at 0.25,0.50,1.00 and 2.00 μg/cm2 for 24 h and three times at total.DAC treatment was given at 3 μmol/L for 72 h.5-mC immunofluorescence and SssI emthyltrasferase assay methods were applied to investigate if the hypomethylation of genome DNA involved.Results The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically.By the SssI methylase assay, an average of (81.9 ± 7.3 )% methylated CpG were found in negative control cells.By contrast, ( 77.9 ± 6.2) %, ( 75.3 ± 6.8 ) %, ( 59.5 ± 4.9 ) %, ( 67.4 ±5.1 ) % methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25,0.50,1.00and 2.00 μg/cm2 which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively.The ANOVA analysis results showed that there was a significant difference in the 5 groups above ( F = 124.95,P ＜0.01 ).The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00were significantly decreased compared with the negative control group(t values were 7.64,4.89 respectively,P ＜0.01 ).For methylated CpG, (46.2 ±4.1 ) % and (43.6% ±4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group(t values were 12.79,13.56 respectively, P ＜ 0.01 ).Conclusion Genomic DNA methylation levels were decreased during NiS induced malignant transformation.     

1,4-苯醌暴露下小鼠骨髓细胞p15基因表达和甲基化状态

目的 探讨1,4-苯醌(1,4-BQ)暴露下,体外原代培养的小鼠骨髓细胞中抑癌基因p15的mRNA表达及启动子区的甲基化状态.方法 体外分离小鼠骨髓细胞,测定0、0.1、1.0、5.0、10.0、20.0、40.0 μmol/L的1,4-BQ对小鼠骨髓细胞活力的影响,最终确定以0、0.1、1.0、10.0 μmol/L的1,4-BQ染毒骨髓细胞24 h;实时荧光定量聚合酶链反应(PCR)检测p15基因mRNA的表达变化;重亚硫酸盐测序法(BSP)检测启动子区域CpG岛的甲基化状态.结果 1,4-BQ对小鼠骨髓细胞具有剂量依赖的毒性作用,其LC50为8.3 μmol/L(95%CI:4.6～10.6 μmol/L).与对照组(100.0%)比较,10.0、20.0、40.0μmol/L 1,4-BQ染毒组小鼠的骨髓细胞生存率(35.9%、35.8%、30.5%)均明显降低,差异有统计学意义(P＜0.01).10.0μmol/L染毒组p15 mRNA表达为对照组的43%,与对照组比较,1.0和10.0 μmol/L染毒组p15基因mRNA表达量明显下降,差异有统计学意义(P＜0.05或P＜0.01).BSP测序结果显示,染毒组和对照组相同,p15基因启动子区域CpG岛上56个甲基化位点仍保持非甲基化状态.结论 1,4-BQ暴露可导致原代培养的小鼠骨髓细胞中p15基因的mRNA表达量下降,但启动子区域CpG岛的甲基化状态未受影响,提示甲基化不参与1,4-BQ介导的p15基因表达下调.

Abstract：

Objective To detect the expression and the CpG island methylation status of tumor suppressor gene p15 after exposure to 1,4-benzoquinone (1,4-BQ) in primary cultivated C57BL/6J mouse bone marrow cells in vitro. Methods The mouse bone marrow cells were isolated in vitro. The effect of 0, 0.1, 1, 5,10, 20, and 40 μmol/L 1,4-BQ on cell viability (CKK-8) was detected. 0, 0.1, 1, 10 μmol/L 1,4-BQ were used to intoxicate the mouse bone marrow cells for 24 h; Real-time PCR was employed to analyze the mRNA expression level of p15; The bisulfite sequencing PCR (BSP) was used to look into the methylation status of CpG islands in p15 promoter region. Results 1,4-BQ exhibited dose-dependent toxicity to mouse bone marrow cells, and the LC50 was 8.3 μmol/L (95% CI: 4.6-10.6 μmol/L). The mRNA expression of p15 in 10 μ mol/L group was only equivalent to 43% of control group. Compared with control group, the decrease of p15 mRNA expression in 1 and l0 μ mol/L concentration were obvious, and the differences had statistical significance(P＜0.05 or P＜0.01 ). BSP sequencing results were same between the exposure groups and control group, the 56 CpG sites on CpG islands remained in the state of unmethylated. Conclusion mRNA expression of p15 gene decreases after exposure to 1,4-BQ, but the CpG islands menthylation status in promoter is not affected, suggesting that methvlation does not participate in 1,4-BQ-mediated p15 gene expression decrease, other effect mechanisms still need to be investigated.     

胆囊癌及胆囊腺瘤中p53蛋白异常表达的研究

Objective To inviestigate the expression of gall bladder cancer and its relationship with eaneeration. Methods Immunohistoehemistry techniques are used to detect the expression of p53 protein in 36 eases of gall bladder cancer and 23 adenoma. Results p53 protein were founded positive in 19 cases (52.78%) of gall blodder cancer, in 5 cases (21.74%) of adenoma. Conclusion There is close relationship between the over expression and the cancemtion of gall bladder, p53 gene mutation and over expression are in the early stage of gall bladder cancer, and play an important role in the cancemtion of adenoma.     

Diagnostic value of vascular endothelial growth factor combined with β-human chorionic gonadotropin for suspected ectopic pregnancy

Objective To explore the diagnostic values of vascular endothelial growth factor (VEGF) and p -human chorionic gonadotropin( β -hCG) for early ectopic pregnancy. Methods The serum levels of VEGF and β-hCG were measured in 109 patients with suspected ectopic pregnancy by ELISA and RIA respectively, receiver operating characteristic (ROC) curvers were established, the diagnostic values of VEGF and β -hCG for ectopic pregnancy were analyzed. Results One hundred and nine patients with suspected ectopic pregnancy were diagnosed in the end: 62 cases with ectopic pregnancy and 47 cases with intrauterine pregnancy. The levels of VEGF in ectopic pregnancy were significantly higher than those in intrauterine pregnancy (P ＜ 0.05), but the levels of β -hCG were significantly lower than those in intrauterine pregnancy (P ＜ 0.05). The areas under ROC curves of VEGF were 0.87, the areas under ROC curves of β -hCG were 0.71. In VEGF ＞ 140 ng/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 75.8% (47/62) and 80.9% (38/47) respectively. In β-hCG ＜ 1200 U/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 64.5%(40/62) and 61.7%(29/47) respectively. The sensitivity and specificity of combined detection of VEGF and β -hCG were 80.6%(50/62) and 57.4%( 27/47) respectively. Conclusions The diagnostic values of VEGF are better than those of β -hCG. The combined detection of VEGF and β -hCG can increase the rate of early diagnosis and shorten observation time.     

兔颈动脉损伤后再狭窄与p53蛋白关系的研究

Objective To observe the effect of p53 protein on smooth muscle cell(VSMC)in rabbit artery balloon injury.Methods Restenosis model of carotid artery after balloon injury was established in rabbits.30 rabbits were divided into 2 groups,the sham group(n = 6)and the vascular injury group(n = 24).With H.E.staining and automatic image analysis system,we investigated artery morphology alteration and measured the area of arterial intima and media.The expressions of p53 protein were detected by immunohistochemical analysis.Results With H.E.staining and automatic image analysis and immunohistochemistry,the results showed that the expression of p53 was significantly reduced and the intima area was increased in model group compared with the sham group(P ＜0.01).But the expression of p53 in media was remarkably reduced compared with intima(P ＜ 0.01).Conclusions The possible mechanism of preventing arterial restenosis in the balloon injury might be related with p53,which may be through inhibiting neointimal proliferation in arterial restenosis .     

聚腺苷二磷酸核糖聚合酶1在苯并(a)芘诱导人支气管上皮细胞DNA甲基化改变中的作用

目的 观察苯并(a)芘[benzo(a)pyrene,B(a)P]诱导体外细胞DNA甲基化水平改变,探讨聚腺苷二磷酸核糖聚合酶1[poly(ADP-ribose)polymerase 1,PARP1]在该过程中的作用.方法 以1.0、2.0、5.0、10.0、15.0、30.0 μmol/L浓度B(a)P分别处理人支气管上皮细胞(16HBE)及其PARP1缺陷细胞(16HBE-shPARP1)72 h.采用免疫荧光和高效毛细管电泳检测其基因组DNA整体甲基化水平改变,同时动态监测PARP1和DNA甲基转移酶1(DNA methyltransferases 1,DNMT1)表达的变化.结果 16HBE和16HBE-shPARP1细胞基因组整体甲基化百分比(mCpG%)分别为(4.04±0.08)%和(9.69±0.50)%.经5-氮杂脱氧胞苷(DAC)处理72 h后,mCpG%值分别下降为(3.15±0.14)%、(6.07±0.54)%.经B(a)P染毒72 h后,16HBE细胞基因组mCpG%值[B(a)P浓度由低到高]分别为(5.10±0.13)、(4.25±0.10)、(3.91±0.10)、(4.23±0.27)、(3.70±0.15)、(3.08±0.07);16HBE-shPARP1细胞基因组mCpG%值(浓度由低到高)分别为(10.63±0.60)、(13.08±0.68)、(9.75±0.55)、(7.32±0.67)、(6.90±0.49)、(6.27±0.21).两种细胞不同处理组间mCpG%差异有统计学意义(F值分别为61.67、60.91,P值均＜0.01).16HBE细胞各剂量组[B(a)P浓度由低到高]PARP1基因mRNA相对表达水平分别为对照组的141.0%、158.0%、167.0%、239.0%、149.0%、82.9%,差异均有统计学意义(t值分别为11.45、17.32、32.24、33.44、20.21、9.87,P值均＜0.01);16HBE-shPARP1细胞各剂量组(浓度由低到高)PARP1基因mRNA相对表达水平分别为对照组的169.0%、217.0%、259.0%、323.0%、321.0%、256.0%,差异有统计学意义(t值分别为9.06、15.92、22.68、26.23、37.19、21.15,P值均＜0.01).当B(a)P染毒剂量达5.0 μmol/L后,16HBE细胞各剂量组(浓度由低到高)DNMT1基因mRNA相对表达水平分别为对照组的125.0%、162.0%、275.0%、233.0%,差异有统计学意义(t值分别为12.74、24.92、55.11、59.07,P值均＜0.01);当B(a)P染毒剂量达2.0 μmol/L后,16HBE-shPARP1细胞各剂量组(浓度由低到高)DNMT1基因mRNA相对表达水平分别为对照组的135.0%、151.0%、180.0%、229.0%、186.0%,差异有统计学意义(t值分别为23.82、40.17、32.69、74.85、46.76,P值均＜0.01).结论 B(a)P诱导的16HBE细胞基因组整体甲基化水平降低可能是其恶性转化过程中早期重要的分子事件,PARP1可通过抑制DNMT1的酶活性来调节B(a)P诱导的16HBE细胞DNA甲基化水平,这种效应可因PARP1的缺失而缓解.

Abstract：

Objective To investigate DNA methylation variation in human cells induces by B (a)P, and to explore the role of PARP1 during this process. Methods The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B ( a ) P ( 1.0, 2. 0, 5.0, 10. 0, 15.0, 30. 0 μ mol/L ) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP1 and DNMT1 were monitored dynamically. Results The percentage of methylated DNA of overall genome ( mCpG% ) in 16HBE and 16HBE-shPARP1 cells were separately (4. 04 ±0. 08) %and (9. 69 ±0. 50)%. After being treated by 5-DAC for 72 hours,mCpG% decreased to (3.15 ±0. 14)%and (6. 07 ± 0. 54 ) %. After both being exposed to B (a) P for 72 hours, the mCpG% in 16HBE group ( ascending rank ) were separately ( 5. 10 ± 0. 13 ), ( 4. 25 ± 0. 10 ), ( 3.91 ± 0. 10 ), ( 4. 23 ± 0. 27 ),(3.70 ± 0. 15 ), ( 3.08 ± 0. 07 ); while the figures in 16HBE-shPARP1 group ( ascending rank ) were respectively (10.63 ±0.60), (13.08 ±0.68), (9.75 ±0.55), (7.32 ±0.67), (6.90 ±0.49) and (6. 27 ±0. 21 ). The difference of the results was statistically significant ( F values were 61.67 and 60. 91,P＜ 0.01 ) . For 16HBE group, expression of PARP1 and DNMT1 were 141.0%, 158.0%, 167.0%,239. 0%, 149. 0% ,82. 9% and 108. 0%, 117.0%, 125.0%, 162. 0% ,275. 0% ,233.0% comparing with the control group, whose difference also has statitical significance (t values were 11.45,17. 32,32. 24,33.44,20.21 and 9. 87 ,P ＜ 0. 01 ). For 16HBE-shPARP1 group, expression of PARP1 and DNMT1 were 169.0% ,217.0%, 259.0%, 323.0% , 321.0% , 256.0% and 86.0% , 135.0% , 151.0% , 180.0%,229. 0%, 186. 0% comparing with the control group,with statitical significance (t values were 9. 06,15. 92,22. 68,26. 23,37. 19 and 21.15, P ＜ 0. 01 ). When the dose of B (a) P reached 5.0 μmol/L, the mRNA expression of DNMTI in 16HBE group (ascending rank) were 125.0%, 162. 0% ,275.0% ,233.0% times of it in control group, with statistical significance ( t values were 12. 74,24.92,55. 11,59. 07, P ＜ 0. 01 );while the dose of B(a) P reached 2.0 μmol/L, the mRNA expression of DNMT1 in 16HBE-shPARP1 group were 135.0%, 151. 0%, 180. 0% ,229.0%, 186. 0% of the results in control group, and the differences were statistically significant ( t values were 23. 82,40. 17,32. 69,74. 85,46. 76, P ＜ 0. 01 ). Conclusion The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.     

Study on the incidence of β-Thalassemia and genotypes among children under 7 year-olds in Nanning, Liuzhou and Baise areas, Guangxi province

Objective To conduct research of β-Thalassemia incidence and genotypes on children below 7 years of age in Nanning, Liuzhou and Baise areas, Guangxi province. Methods A total of 2261 children aged below 7 in Nanning, Liuzhou and Baise areas were studied. Venous blood was detected by routine blood test, hemoglobin analysis and β-Thalassemia genotyping. Results Among 2261 samples, 125 showed high level of HbA2 and were diagnosed as β-Thalassemia (5.53%). Genotypes of the patients were classified as: 59 cases with β-globin gene eondon (CD) 41-42 mutation, 33 cases CD17 mutation, 18 cases with TA TA box nt-28 mutation, 7 with IVS-Ⅱ-654 mutation, 3 with CD43 mutation, 3 with HbE mutation, one with CD71-72 and TATA box nt-29 mutation, respectively. The genotyping frequencies of β-Thalassemia were as follows: 47.20% for CD41-42 mutation, 26.40% for CD17 mutation, 14.40% for TATAbox nt-28 mutation, 5.60% for IVS-Ⅱ -654 mutation, 2.40% for CD43 mutation, 2.40% for HbE mutation, 0.80% for CD71-72 mutation and TATAbox nt-29 mutation respectively. Conclusion This study on children in the area with high incidence of β-Thalassemia reflected the incidence and characteristics of genotypes in this area. Our data also provided evidence for the development of a program on genetic counseling and prevention for thalassemia.     

三甲基氯化锡对亚慢性染毒大鼠肾氢钾ATP酶活力及蛋白与基因表达的研究

Objective To study the activity、protein and gene expression of renal HK-ATPase (HKA) in rats subchronicly exposed to trimethyltin chloride (TMT). Methods In subchronic toxic test (14-week), 55 female SD rats (age, 6 weeks) were divided randomly into 5 groups: control, low, medium, high and super high dosage, respectively, which drank water with TMT of 0, 8.20, 32.81, 131.25 and 262.50 μg·kg-1 ·d-1 for 14 weeks.Then serum K+ levels were measured; the activities of H K-ATPase (HKA) in kidneys were detected by the method of determinenate phosphorus content;Western Blot assay and real-time PCR were used to exam the protein and mRNA expression levels of HKA in kidneys, respectively. Results The serum K + level in super-high dosage group was (5.6±0.4) mmol/L, which was significantly lower than that [(6.9±0.3) mmol/L] in control group (P＜0.01).The HKA enzymatic activity of kidneys in low and super high dosage groups was 4.50±1.45 and 4.55±0.72 μmolPi·mg prot-1h-1, respectively, which were significantly lower than that (6.55±0.77 μmol Pi ·mg prot-1h-1) in control group (P＜0.05). Conclusion When rats were exposed subchronicly to TMT, the renal HKA activity could reduce, but the expression levels of HKA protein and mRNA did not decrease.     

错配修复基因hMLH1和hMSH2甲基化及突变与地方性砷中毒关系

[目的]探讨错配修复基因hMLH1和hMSH2启动子区CpG岛甲基化及第12外显子（exon12）突变与燃煤污染型地方性砷中毒发生发展乃至癌变的关系。[方法]以砷中毒患者110例为病例组,按临床诊断分为轻、中、重度组;按皮肤病理诊断分为非癌变组和癌变组。采用甲基化特异性聚合酶链反应（MSP）法检测其中105例砷中毒患者和82例对照人群外周血中hMLH1和hMSH2基因启动子区的甲基化情况;采用聚合酶链反应-单链构象多态性分析（PCR-SSCP）检测110例砷中毒患者和110例对照人群外周血中hMLH1和hMSH2基因exon12突变情况。[结果]①轻、中、重度组砷中毒患者hMSH2基因甲基化阳性率分别为11.76%、16.28%和32.14%,均明显高于对照组,重度组亦明显高于轻度组（P〈0.05或P〈0.01）;癌变组患者hMLH1和hMSH2基因甲基化阳性率分别为11.11%和27.78%,均明显高于对照组（P〈0.05）;hMLH1和hMSH2基因甲基化阳性率均随临床病情和皮肤病变程度加重而增高（P〈0.05或P〈0.01）。②病例组和对照组均未发现hMLH1和hMSH2基因exon12突变。[结论]错配修复基因hMLH1和hMSH2启动子区甲基化是砷中毒发生发展乃至癌变的早期分子特征,亦可能是导致错配修复功能缺陷的主要方式之一。     

p53-Bax线粒体凋亡通路DNA甲基化与胆管癌病理生物学的关系

目的探讨p53-Bax线粒体凋亡通路抑癌基因启动子区的甲基化状态与胆管癌病理生物学行为的关系。方法采用甲基化PCR检测36例胆管癌患者癌组织和癌旁组织中抑癌基因甲基化诱导静止基因（TMS1／ASC）、死亡相关蛋白激酶（DAPK）和p14启动子区甲基化状态,并对p53基因外显子5～8进行DNA测序,分析以上基因的突变与胆管癌生物学行为的关系。结果24例（66．7％）胆管癌患者癌组织中至少有1个抑癌基因启动子区存在甲基化,p14、DAPK和TMS1／ASC的甲基化率分别为25％、30．6％和36．1％。5例（13．9％）患者的癌旁组织中存在抑癌基因启动子区甲基化,其中TMS1／ASC启动子区甲基化3例（8．3％）,DAPK启动子区甲基化2例（5．6％）。DNA测序显示,22例（61．1％）胆管癌患者癌组织中p53基因外显子5～8存在突变。其中14例（38．9％）p53基因外显子突变伴1个以上抑癌基因启动子区甲基化,与胆管癌的病理类型、浸润深度、分化程度显著相关（P〈0．05）。抑癌基因非甲基化及p53突变阴性组（4例）术后1、2、3年的生存率分别为70％、43％、28％,抑癌基因甲基化及p53突变阳性组（14例）术后1、2、3年的生存率分别为28％、5％、0％,前者的生存率显著高于后者（X^2=9．060,P=0．03）。结论p53-Bax线粒体凋亡通路中抑癌基因启动子过甲基化是胆管癌组织中常见的分子事件,癌旁组织中TMS1／ASC和DAPK基因甲基化程度虽然较低,但可能对胆管癌有早期诊断意义。p53突变伴抑癌基因甲基化与胆管癌的病理生物学行为有关,趋向于较高的恶性程度和较低的生存率。     

食管癌P53基因第5外显子突变分析

目的　分析不同地区食管癌组织中 p5 3基因第 5外显子的突变谱。 方法　采用PCR扩增产物纯化后直接DNA序列测定技术对陕西省西安市 4 2例和河南省林州市 4 3例食管癌标本 p5 3基因第 5外显子突变情况进行检测。结果　西安和林州市食管癌标本中 p5 3基因第 5外显子突变率分别为 14 3% (6 /42 )和 18 6 % (9/43) ,两地突变率比较差异无统计学意义 (P >0 0 5 )。西安 6个突变位点中 4个为点突变 ,2个为缺失突变 ;林州 10个突变位点中9个为点突变 ,1个为插入突变。西安市有 4例突变发生在 12 6～ 12 8位点 ,林州市仅有 2例发生在该区域 (2 /10 ) ,但两者相比差异无统计学意义 (P >0 0 5 )。结论　西安市食管癌 p5 3基因第 5外显子的突变位点相对集中 ,林州市突变位点比较分散 ,可能与该地区多种危险因素的暴露有关。     

聚合酶链反应-单链构象多态法在研究新疆地方性砷中毒患者p53基因突变中的应用

目的 探索砷致癌的基因多态性,为进一步研究砷对人体健康作用的远期影响提供科学依据.方法 于2001年采集新疆奎屯123团4例经病理诊断为皮肤癌的砷中毒患者的6份皮肤癌石蜡包埋组织及其4份血液组织;采集奎屯128团2例经病理诊断为皮肤癌的砷中毒患者和2例癌前砷中毒患者的血液组织;采集对照区新疆奎屯125团2例健康居民的血液组织.采用聚合酶链反应-单链构象多态(PCR-SSCP)银染术测定p53基因外显子(exon,E)5～9突变情况.结果 p53基因中,仅E6、E7发生突变.凝胶电泳成像显示,p53基因E6、E7有异常条带、条带缺失及条带上移现象.结论 砷对p53基因有致突变作用,突变位于E6、E7.     

人宫颈癌及慢性宫颈炎组织内P_(53)基因变异的研究

本文应用多聚酶链反应—单链构象多态性(PCR-SSCP)分析,对人宫颈癌(cervical carcinoma)和慢性宫颈炎(chronic cervicitis)组织内的P_(53)基因5-6、7-8、9外显子(exon)变异进行研究。结果表明:35例宫颈癌组织中8例出现了异常电泳带,总变异率22.86％(8/35)。其中,5-6外显子2例,变异率为5.71％(2/35);7-8外显子5例,变异率为14.29％(5/35),是变异率最高的基因片段;9外显子1例,变异率为2.86％(1/35)。8例变异标本中3例P_(53)5-6、7-8外显子同时发生突变,占变异标本的37.5％(3/8),占宫颈癌组织总标本的8.57％(3/35)。17例慢性宫颈炎组织P_(53)5-6、7-8、9外显子均未出现异常。提示:PCR-SSCP技术能有效地检出宫颈组织P_(53)基因5-9外显子变异的情况;P_(53)5-9变异是人宫颈癌发生发展过程中的一个重要事件和生物学行为;宫颈癌组织P_(53)变异多发生在5-8外显子,有不同外显子同时变异的现象;慢性宫颈炎组织无P_(53)5-6、7-8、9外显子变异现象,说明该病的发生发展与抑癌基因P_(53)外显子变异无关。     

食管癌与p53突变和人乳头状肉瘤病毒感染

目的探讨p53基因突变与人乳头状肉瘤病毒16（HPV16）感染在食管癌发病中的作用及相互关系。方法应用病例-病例研究方法进行分析，采用聚合酶链反应（PCR）、单链构像多态性（SSCP）、测序等分子生物学技术对110例食管癌组织标本中p53基因的突变与HPV16感染进行了检测。结果110例食管癌组织标本p53基因突变率为49．1％，其中外显exon5—6，exon7，exon8—9的突变率分别为19．1％，27．3％，17．2％。食管癌组织HPV16的检出率为49．1％。吸烟患者p53基因突变率（61．4％）明显高于非吸烟患者p53基因突变率（48．2％），差异有统计学意义；淋巴结转移患者p53基因突变率（65．2％）明显高于无淋巴结转移患者p53基因突变率（37．5％）；HPV16阳性者中，p53突变率是40．7％，HPV16阴性者仅为57．1％，两者差异无统计学意义。结论HPV16感染可能是食管癌高发区的危险因素；p53基因突变与吸烟、淋巴结转移有明显相关性；HPV16感染和p53基因突变可能是2个独立的事件。     

巢式PCR法在燃煤型砷中毒患者p16基因启动区的异常甲基化检测中的应用

目的 检测燃煤型砷中毒(地砷病)患者p16基因启动区的异常甲基化情况,探讨p16基因启动区的异常甲基化在地砷病癌变中的作用.方法 采用巢式甲基化特异性PCR法(nMSP)对117例地砷病患者(参考地方性砷中毒诊断标准将其分为轻度中毒组、中度中毒组、重度中毒组)、63例内对照人群和70例外对照人群p16基因启动区的甲基化情况进行了检测.结果 砷中毒病例组甲基化阳性率为39.32%,内对照组阳性率为12.70%,外对照组阳性率为4.29%;其中病例组中轻度、中度和重度组阳性率分别为24.00%,48.89%和54.55%.病例组与外对照组和内对照组比较,差异有统计学意义(P＜0.01).外对照组、内对照组、轻度、中度和重度砷中毒组甲基化阳性率经趋势x2检验,差异有统计学意义(P＜0.01).结论 p16基因启动区的异常甲基化在地砷病人的癌变过程可能起重要作用.     

石蜡包埋的石棉肺癌组织中p53基因突变的分析方法

为了利用石蜡包埋的病理组织来研究石棉肺癌的基因突变情况，选用１０例石蜡包埋的石棉肺癌组织进行ＰＣＲ－ＳＳＣＰ分析，检测其抗癌基因ｐ５３的第５、第７和第８外显子的突变情况。经银染检查，发现４个病例的ｐ５３基因的第７或第８外显子片段呈突变阳性。用放射性自显影的ＰＣＲ－ＳＳ－ＣＰ方法分析这１０例病例，检测到７个病例的ｐ５３基因第７或第８外显子片段呈突变阳性。用两种方法检测均未发现这１０个病例的第５外显子呈突变阳性。银染ＰＣＲ－ＳＳＣＰ检测可检出放射性自显影ＰＣＲ－ＳＳＣＰ分析结果的６０％，二者的结果相符合。因而，简便、灵敏而且危害性小的银染检测方法，可以代替放射性自显影用于ＰＣＲ－ＳＳＣＰ分析，研究石棉肺癌的基因突变情况。     

石棉肺癌组织中p53基因突变的PCR-SSCP及部分序列分析研究

为研究石棉致癌的分子机理，本研究首次直接分析石蜡包埋的石棉肺癌病例肺组织中抗癌基因ｐ５３的突变情况。利用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）技术分析１０例石棉肺癌病例，检测到７例（８个片段）存在ｐ５３基因的突变，突变位点位于其第５、７和８外显子区内，突变率为７０％。对其中２例进行ＰＣＲ产物直接序列分析，发现其基因一级结构分别发生Ｃ→Ａ和Ｔ→Ａ转换。     

砷接触工人尿甲基砷酸水平与P53基因损伤的关系

[目的]观察砷职业暴露人群尿甲基砷酸水平与P53基因损伤的关系,深入认识砷的遗传毒性。[方法]选取砒霜厂95名砷接触工人作为暴露组,另选对照组55人,采集外周血和晨尿。用氢化物发生原子吸收分光光度法检测尿中各形态砷化合物和总砷含量,并计算一、二级甲基化指数。实时荧光定量PCR扩增人群外周血淋巴细胞P53基因外显子5和8,通过循环阈值推算损伤后扩增效率,间接计算损伤指数。[结果]暴露组工人尿中无机砷（iAs）、甲基砷酸（MMA）、二甲基砷酸（DMA）均明显高于对照组;暴露组工人二级甲基化指数明显低于对照组;暴露组工人P53基因外显子5和8的损伤指数均明显高于对照组;尿中砷一级甲基化指数与P53基因第5外显子损伤指数存在明显的正相关,尿中砷二级甲基化指数与P53基因第5外显子损伤指数存在明显的负相关。[结论]职业砷暴露工人二级甲基化指数明显增加,体内存在较多甲基砷酸,可能是P53基因外显子5损伤的主要原因。