Research For The Human Perripheral Blood Mitochondrial DNA4977bp Deletion And Ionizing Radiation

The article include two parts contents.Part 1 Research for methods for extraction of the mitoehondrial DNA from hmnan peripheral bloodObjective To investigate a simple and efficient method for extraction of the mitoehondrial DNA(mtDNA) from hmnan peripheral blood. Methods The mtDNA from peripheral blood was extracted by the methods of mtDNA kit, some document and high intensity salt precipitation. The acquired mtDNA was detected by ultraviolet spectrophotometry, agar gel electrophoresis and PCR amplification. Results The mtDNA output of three methods have obvious difference. The result of ultraviolet spectrophotometry was that the mtDNA output of high intensity salt precipitation method was 9.5788±0.3852 to be the highest; the document method was 4.3786±0. 1495 to be less; the mtDNA kit was 0.7408±0. 1801 to be the least. Agar gel electrophoresis also displayed that high intensity salt precipitation method shows the lightest strap. The three mtDNA output as template to carry out PCR amplification, the aim strap of 533bp all was obtained, but, the strap of mtDNA kit method was dim. Conclusion The output of acquired mtDNA from hmnan peripheral blood by high intensity salt precipitation method was highest. The quality of that was controllable and the product of PCR amplificationwas stabile. So, the high intensity salt precipitation have better application value than other methods.Part 2 Research for the dose-effect relation for the human peripheral blood mitochondrial DNA 4977bp deletion and ionizing radiationObjective Cloning of the fragment of the mitochondrial DNA4977bp deletion in human peripheral blood induced by ionizing radiation to detect the correlation for the mitochondrial DNA (mtDNA)4977bp deletion induced by ionizing radiation and radiation biodosimetry. Methods 20ml human peripheral blood samples are collected from a healthy younthful donor who was not radiated resently and are devided into 10 shares. Each share are irradiated by ¹³⁷ Csγray which radiation dose is 0.2,0.5,1.0,2.0,3.0,4.0,5.0,6.0,8.0,10.0 Gy. In addition, collecting a 67 age old-man peripheral blood 2ml as positive comparison. After 2 hours, mtDNA is extracted from peripheral blood. The fragment of mtDNA 4977bp deletion was amplified from mtDNA in nested-PCR methods. At the same time, fragment of related stable domain of mtDNA sequence also amplified to represent total mtDNA. Aimed-fragment separated by agar gel electrophoresis and analyzed by gel image analytical system to gain gray scale value of the mtDNA 4977bp deletion fragment and reference fragment representing total mtDNA. Results 1,The method of mtDNA extracted from human peripheral blood is high efficiency. 2,The fragment of mtDNA4977bp deletion induced by ionizing radiation has the same sequences as the expected gene fragment. However,the non-irradiated sample wasn't amplified differential PCR products. 3,The fragment of mtDNA4977bp deletion was obtained by the nested-PCR methods. By comparating agar gel electrophoresis image and gray scale value, we obtained that gray scale value showed increasing tendency with radiation dose increase in 0.2～3.0Gy. The value had great change and defected regulation when radiation dose was 4.0～10.0Gy. Conclusion 1,Primer was designed in each sides of the mtDNA4977bp deletion fragment and the mtDNA4977bp deletion fragment that was induced by ionizing radiation was amplified by nested-PCR methods. 2,The half-quantification PCR method was established to detect the mtDNA4977bp deletion fragment. The mtDNA 4977bp deletion quantity and ionizing radiation dose showed favourable dose-effect relation in lower radiation dose. The dose-effect relation became instability when radiation dose exceeded 4.0Gy.