Objective: To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods: A mouse lung adenocarcinoma cell clone 12 derived from its parent cell ...Objective: To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods: A mouse lung adenocarcinoma cell clone 12 derived from its parent cell line LM2, which had been transduced with normal human genomic DNA, was previously reported. Compared with LM2, the metastatic potential of clone 12 was very much decreased. Clone 12 was used in this study to amplify the human DNA fragments by Inter Alu PCR technique. The human DNA fragments obtained were then transfected into LM2 cells and their malignant phenotype was tested in vitro and in vivo, and compared with that of the untransfected LM2 cells.Results Three human DNA fragments of 700, 500 and 300 bp were isolated. DNA sequencing revealed that the 700bp fragment does not show homology with hitherto reported genes and was accepted by the Genbank (pt712 U67835). In vitro proliferation and colony formation in soft agar of the 700 bp fragment-transfected LM2 cells were significantly inhibited as compared to the untransfected LM2 cells. Upon subcutaneous inoculation to syngeneic T739 mice, the 700bp-transfected LM2 cells grew more slowly and smaller tumors developed compared to the untransfected ones. Moreover, lung metastasis was not found in 6 of 10 mice inoculated with the 700bp-transfected LM2 cells, while it was found in 9 of 10 mice inoculated with the untransfected LM2 cells. The difference was statistically significant (P<0.001). The frequency of lymph node metastasis was also statistically different between the 2 groups of mice.Conclusion The newly isolated 700bp human DNA fragment may be a metastasis suppressor gene of malignant tumor.展开更多
目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预...目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。展开更多
Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patien...Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences展开更多
Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be sui...Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method （method A）, traditional phenol-chloroform method （method B）, and TianGen kit method （method C）. Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C （P〈0.01）. PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 （DDX4） and copy number variations （CNVs） than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reli- able results and could be an optimal technique for extracting sperm DNA for methylation assay.展开更多
In this work, an electrochemical sensor was fabricated for determination of an anthracycline, doxorubicin(DOX) as a chemotherapy drug in plasma based on multi-walled carbon nanotubes modified platinum electrode(Pt/MWC...In this work, an electrochemical sensor was fabricated for determination of an anthracycline, doxorubicin(DOX) as a chemotherapy drug in plasma based on multi-walled carbon nanotubes modified platinum electrode(Pt/MWCNTs). DOX was effectively accumulated on the surface of modified electrode and generated a pair of redox peaks at around 0.522 and 0.647 V(vs. Ag/Ag Cl) in Britton Robinson(B-R) buffer(p H 4.0, 0.1 M). The electrochemical parameters including p H, type of buffer, accumulation time, amount of modifier and scan rate were optimized. Under the optimized conditions, there was a linear correlation between cathodic peak current and concentration of DOX in the range of 0.05–4.0 μg/m L with the detection limit of 0.002 μg/m L. The number of electron transfers(n) and electron transfer-coefficient(α) were estimated as 2.0 and 0.25, respectively. The constructed sensor displayed excellent precision, sensitivity, repeatability and selectivity in the determination of DOX in plasma. Moreover, cyclic voltammetry studies of DOX in the presence of DNA showed an intercalation mechanism with binding constant(K_b) of 1.12×10~5L/mol.展开更多
To investigate the genotoxicity and reveal the potential toxicological mechanisms of Hexabromocyclododecane （HBCD）, human breast cells HBL-100 were exposed to a sequence of HBCD concentrations （0, 5, 10, and 50 mg/...To investigate the genotoxicity and reveal the potential toxicological mechanisms of Hexabromocyclododecane （HBCD）, human breast cells HBL-100 were exposed to a sequence of HBCD concentrations （0, 5, 10, and 50 mg/L） for 24 h. With a series of zymology and molecular biology methods, we found that HBCD induced dose-dependent oxidative stress on HBL-100 DNA. As revealed in q RT-PCR, activated prognostic factor ATM down-regulated tumor suppressor gene BRCA1 and prompted DNA repair genes h OGG1 and h MTH1 expression in lower concentrations of HBCD （〈 10 mg/L）. However, DNA repair were inhibited as well as cell proliferation rate by higher concentrations of HBCD （50 mg/L）. The results inferred that the genotoxicity of HBCD was dose-dependent and related to DNA repair pathway.展开更多
The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitrowith the DNA-damaging agents, methyl methanesulfonate （MMS） or ...The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitrowith the DNA-damaging agents, methyl methanesulfonate （MMS） or hydrogen peroxide （Hz02）, and then embedded in agarose gel on glass sl ides. The slides were immersed in alkaline solution （〉pH 13） for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period （1 min） of the alkaline DNA unwinding time.展开更多
DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in diseases, including human cancers. In recent years, our understanding on DNA methylation with human cancers has made si...DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in diseases, including human cancers. In recent years, our understanding on DNA methylation with human cancers has made significant progress, which was facilitated by stunning development in the analysis of the human methylome of multiple cancer types. In this review, recent developments in the characterization of aberrant DNA methylation involved in human cancers development were discussed with special emphasis on the mechanisms of aberrant DNA methylation in human cancers. We also summarize the recent treatment strategy for human cancers with de-methylation drugs.展开更多
objective: To study the effects of human genome DNA on the cultured spinal cord neurons of em bryonic mouse. Methods: The human genome DNA was added to the culture medium of the spinal cord neu rons of embryonic mouse...objective: To study the effects of human genome DNA on the cultured spinal cord neurons of em bryonic mouse. Methods: The human genome DNA was added to the culture medium of the spinal cord neu rons of embryonic mouse. Eight days later, MTT assay, NSE immunocytochemical staining and image analy sis were proformed to examine the viabilities and the neurites lengths of the neurons. Results: The neurite length of the experimental group was significantly Ionger than that of the control group, but no marked dif ference was found between the viabilities of the neurons of the experimental groups and that of the control ones. Conclusiou: Human genome DNA has no effects on the viabilities of the cultured neurons but can pro mote the neurite growth.展开更多
基金the National Natural Science Foundation of China (No. 39370761 ).
文摘Objective: To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods: A mouse lung adenocarcinoma cell clone 12 derived from its parent cell line LM2, which had been transduced with normal human genomic DNA, was previously reported. Compared with LM2, the metastatic potential of clone 12 was very much decreased. Clone 12 was used in this study to amplify the human DNA fragments by Inter Alu PCR technique. The human DNA fragments obtained were then transfected into LM2 cells and their malignant phenotype was tested in vitro and in vivo, and compared with that of the untransfected LM2 cells.Results Three human DNA fragments of 700, 500 and 300 bp were isolated. DNA sequencing revealed that the 700bp fragment does not show homology with hitherto reported genes and was accepted by the Genbank (pt712 U67835). In vitro proliferation and colony formation in soft agar of the 700 bp fragment-transfected LM2 cells were significantly inhibited as compared to the untransfected LM2 cells. Upon subcutaneous inoculation to syngeneic T739 mice, the 700bp-transfected LM2 cells grew more slowly and smaller tumors developed compared to the untransfected ones. Moreover, lung metastasis was not found in 6 of 10 mice inoculated with the 700bp-transfected LM2 cells, while it was found in 9 of 10 mice inoculated with the untransfected LM2 cells. The difference was statistically significant (P<0.001). The frequency of lymph node metastasis was also statistically different between the 2 groups of mice.Conclusion The newly isolated 700bp human DNA fragment may be a metastasis suppressor gene of malignant tumor.
文摘目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。
文摘Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences
基金supported by the National Natural Science Foundation of China(No.81370755)
文摘Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method （method A）, traditional phenol-chloroform method （method B）, and TianGen kit method （method C）. Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C （P〈0.01）. PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 （DDX4） and copy number variations （CNVs） than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reli- able results and could be an optimal technique for extracting sperm DNA for methylation assay.
基金the research council of Gachsaran Branch, Islamic Azad University, Iran for supporting this project under Grant no. 25518
文摘In this work, an electrochemical sensor was fabricated for determination of an anthracycline, doxorubicin(DOX) as a chemotherapy drug in plasma based on multi-walled carbon nanotubes modified platinum electrode(Pt/MWCNTs). DOX was effectively accumulated on the surface of modified electrode and generated a pair of redox peaks at around 0.522 and 0.647 V(vs. Ag/Ag Cl) in Britton Robinson(B-R) buffer(p H 4.0, 0.1 M). The electrochemical parameters including p H, type of buffer, accumulation time, amount of modifier and scan rate were optimized. Under the optimized conditions, there was a linear correlation between cathodic peak current and concentration of DOX in the range of 0.05–4.0 μg/m L with the detection limit of 0.002 μg/m L. The number of electron transfers(n) and electron transfer-coefficient(α) were estimated as 2.0 and 0.25, respectively. The constructed sensor displayed excellent precision, sensitivity, repeatability and selectivity in the determination of DOX in plasma. Moreover, cyclic voltammetry studies of DOX in the presence of DNA showed an intercalation mechanism with binding constant(K_b) of 1.12×10~5L/mol.
基金The work was supported by grants from the National Nature Science Foundation of China (No. 30271155) China national key basic research and development program (No. 2022CB512908).
基金supported by the National Natural Science Foundation of China(No.41406088)The open fund of Key Laboratory for Ecological Environment in Coastal Areas,State Oceanic Administration(201506)
文摘To investigate the genotoxicity and reveal the potential toxicological mechanisms of Hexabromocyclododecane （HBCD）, human breast cells HBL-100 were exposed to a sequence of HBCD concentrations （0, 5, 10, and 50 mg/L） for 24 h. With a series of zymology and molecular biology methods, we found that HBCD induced dose-dependent oxidative stress on HBL-100 DNA. As revealed in q RT-PCR, activated prognostic factor ATM down-regulated tumor suppressor gene BRCA1 and prompted DNA repair genes h OGG1 and h MTH1 expression in lower concentrations of HBCD （〈 10 mg/L）. However, DNA repair were inhibited as well as cell proliferation rate by higher concentrations of HBCD （50 mg/L）. The results inferred that the genotoxicity of HBCD was dose-dependent and related to DNA repair pathway.
文摘The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitrowith the DNA-damaging agents, methyl methanesulfonate （MMS） or hydrogen peroxide （Hz02）, and then embedded in agarose gel on glass sl ides. The slides were immersed in alkaline solution （〉pH 13） for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period （1 min） of the alkaline DNA unwinding time.
文摘DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in diseases, including human cancers. In recent years, our understanding on DNA methylation with human cancers has made significant progress, which was facilitated by stunning development in the analysis of the human methylome of multiple cancer types. In this review, recent developments in the characterization of aberrant DNA methylation involved in human cancers development were discussed with special emphasis on the mechanisms of aberrant DNA methylation in human cancers. We also summarize the recent treatment strategy for human cancers with de-methylation drugs.
文摘objective: To study the effects of human genome DNA on the cultured spinal cord neurons of em bryonic mouse. Methods: The human genome DNA was added to the culture medium of the spinal cord neu rons of embryonic mouse. Eight days later, MTT assay, NSE immunocytochemical staining and image analy sis were proformed to examine the viabilities and the neurites lengths of the neurons. Results: The neurite length of the experimental group was significantly Ionger than that of the control group, but no marked dif ference was found between the viabilities of the neurons of the experimental groups and that of the control ones. Conclusiou: Human genome DNA has no effects on the viabilities of the cultured neurons but can pro mote the neurite growth.
