Objectives. In order to identify the relationship between telomerase and the biological effect of radiation injury,and investigate the role of human telomerase catalytic subunit gene (hEST2) reverse transcriptase(RT) ...Objectives. In order to identify the relationship between telomerase and the biological effect of radiation injury,and investigate the role of human telomerase catalytic subunit gene (hEST2) reverse transcriptase(RT) segment in the expression of telomerase activity. Methods. Tumor HeLa cells, KB cells and A431 cells were employed to measure the change in telomerase activity after 60Co- ray irradiation at RNA level and protein level. Quantitative PCR and Northern blotting were used to determine the expression of hEST2 RT segment that encodes seven motifs of the human telomeres, a PCR- based telomeric repeat amplification protocol (TRAP)was used to assay telomerase activity after exposure to radiation. Results. Both of telomerase activity and the expression hEST2 RT segment were decreased with increasing dosage of radiation. In addition, testing the expression of motifs domain is similar to the measurement of telomerase activity. Conclusion. The detection of the hEST2 RT segment by Northern blotting and quantitative PCR are new methods for testing telomerase activity. Furthermore, radiation can cause a dose- dependent decrease in telomerase activity. The effect of radiation on telomerase is one possible reason for the death of cancer cells after irradiation.展开更多
By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation be...By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation between the expression level of hTERT mRNA and the prognosis of hydatidiform mole, and to evaluate the clinic value of quantitative determination of hTERT mRNA in the diagnosis of hydatidiform mole. Methods： A real-time fluorescent quantitative RT-PCR （FQ RT-PCR） assay based on TaqMan fluorescence methodology and the Light-Cycler system was used to quantify the full range of hTERT mRNA copy numbers in 30 samples of hydatidiform mole and the neoplasia of hydatidiform mole. The normalized hTERT （NhTERT） was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100x （hTERT/GAPDH） ratio. Based on the prognosis of the hydatidiform mole, the patients were divided into two groups： the experimental group and the control group, to compare the telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells. Results： hTERT mRNA was both expressed in the peripheral blood mononuclear cells and pathological tissues in the mole experimental group and the control group. In the mole experimental group, the values were 6.31±0.32 and 6.24±0.44, respectively, and there was no significant difference between them （P〉0.05）. But in the control group the values were 1.21±0.65 and 1.40±0.61, respectively, and there was no significant difference between them （P〉0.05） The values in experimental group was significantly higher than those in the control group （P〈0.01）. Conclusion： Quantitative determination of hTERT mRNA by FQ RT-PCR is a rapid and sensitive method, hTERT in peripheral blood mononuclear cells may have potential use as a biomarker for the early detection of the prognosis of the hydatidiform mole.展开更多
文摘Objectives. In order to identify the relationship between telomerase and the biological effect of radiation injury,and investigate the role of human telomerase catalytic subunit gene (hEST2) reverse transcriptase(RT) segment in the expression of telomerase activity. Methods. Tumor HeLa cells, KB cells and A431 cells were employed to measure the change in telomerase activity after 60Co- ray irradiation at RNA level and protein level. Quantitative PCR and Northern blotting were used to determine the expression of hEST2 RT segment that encodes seven motifs of the human telomeres, a PCR- based telomeric repeat amplification protocol (TRAP)was used to assay telomerase activity after exposure to radiation. Results. Both of telomerase activity and the expression hEST2 RT segment were decreased with increasing dosage of radiation. In addition, testing the expression of motifs domain is similar to the measurement of telomerase activity. Conclusion. The detection of the hEST2 RT segment by Northern blotting and quantitative PCR are new methods for testing telomerase activity. Furthermore, radiation can cause a dose- dependent decrease in telomerase activity. The effect of radiation on telomerase is one possible reason for the death of cancer cells after irradiation.
基金Supported by the Natural Science Foundation of Shaanxi Province (2005C265)
文摘By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation between the expression level of hTERT mRNA and the prognosis of hydatidiform mole, and to evaluate the clinic value of quantitative determination of hTERT mRNA in the diagnosis of hydatidiform mole. Methods： A real-time fluorescent quantitative RT-PCR （FQ RT-PCR） assay based on TaqMan fluorescence methodology and the Light-Cycler system was used to quantify the full range of hTERT mRNA copy numbers in 30 samples of hydatidiform mole and the neoplasia of hydatidiform mole. The normalized hTERT （NhTERT） was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100x （hTERT/GAPDH） ratio. Based on the prognosis of the hydatidiform mole, the patients were divided into two groups： the experimental group and the control group, to compare the telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells. Results： hTERT mRNA was both expressed in the peripheral blood mononuclear cells and pathological tissues in the mole experimental group and the control group. In the mole experimental group, the values were 6.31±0.32 and 6.24±0.44, respectively, and there was no significant difference between them （P〉0.05）. But in the control group the values were 1.21±0.65 and 1.40±0.61, respectively, and there was no significant difference between them （P〉0.05） The values in experimental group was significantly higher than those in the control group （P〈0.01）. Conclusion： Quantitative determination of hTERT mRNA by FQ RT-PCR is a rapid and sensitive method, hTERT in peripheral blood mononuclear cells may have potential use as a biomarker for the early detection of the prognosis of the hydatidiform mole.
