D NA , ) SCGE DNA.
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1 14 5 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy O cṫ 1998 SCGE D NA (, (, ) ) (SCGE) ; SCGE Χ DNA, Χ DNA, :,DNA,, Pr inc iple and M ethod of the Single Cell M icrogel E lectrophores is(scge) Techn ique to Study D NA Damage in Human Blood L ym phocytes M eng Zi2Q iang (D iv ision of E nv ironm ental B iolog ical T ox icology and D ep artm ent of L if e sciences, S hanx i U niversity, T aiy uan ) Zhang L ian2zhen (China Institute f or R ad iation P rotection, T aiy uan ) Abstract T he single cell m icrogel electropho resis (SCGE) techn ique, its p rincip le and no tice2 ab le details in the techn ique w ere described and disscu ssed Effects of DNA dam age in hum an b lood lym phocytes induced by Χ2rays radiation w ere studied u sing th is techn ique It w as show n that Χ2rays cau sed the increase of DNA m igration length in the lym phocytes in the do se2dependen t m anneṙ Key words: SCGE,DNA dam age,l ym phocyte, Ion izing radiation DNA Singh (single cell m icrogel electropho resis, SCGE), (com et assay) DNA, [ 1 3 ], SCGE, SCGE Χ DNA 1 SCGE, DNA : , :
2 , DNA, DNA SCGE, DNA,, : DNA,DNA ;, RNA, DNA, DNA, DNA DNA ; DNA DNA, DNA, DNA,, DNA DNA, DNA DNA SCGE,, DNA, DNA Betti (1994) [ 4 7 ] DNA DNA DNA DNA 5 30% [ 8 10 ] DNA, 2 SCGE Singh, SCGE (SCGE) [ 3, 4 ] m l, rgm in 2 m in 10 m l, (PBS) 5 m l, rgm in 8 m in, 2, PBS, Χ, PBS gm l, 60 Co2Χ, SCGE (, N o 380) ( ), , 1 (NM A ), 2 (LM A ), 3 LM A 1 :,, 40, Λl 015% (NM A ) Ca 2+ M g 2+ (PBS), 1, 4 10 m in NM A 2 : 10 Λl 1000 PBS 75 Λl 015% LM A Ca 2+ M g 2+ PBS 37,, LM A 605
3 1, 4 10 m in 2 LM A 3 : 2 LM A, Λl 015% LM A Ca 2+ M g 2+ PBS, (215 m o lgl N ac l, 100 mm o lgl N a2ed TA, 10 mm o lgl T ris2hc l ph 10, 1%, 1% T riton X2100, 10% ) 2 h DNA, (1 mm o lgl N a2 ED TA, 300 mm o lgl N aoh ), 0125 cm, 40 m in, DNA, DNA DNA V 300 ma, 40 m in DNA 21216, 014 mm o lg L T ris2hc l(ph 715), 15 m in, T ris2hc l, 1 h,, 50 Λl 13 Λggm l (EB ), 20 m in DNA EB EB, (U ) DNA, DNA DNA ( ) 25 DNA DNA DNA, 10 40, (A SA 400) SCGE, Χ DNA DNA DNA,, DNA, (, F ig 1) F ig 1 Χ DNA 312 D NA T ab le 1 Χ DNA, DNA, ( r = 0199, P < 01001) 606
4 F ig 1 Ionizing irradiation and typ ical pho tom icrograph s of single cell m icrogel electropho resis of hum an blood lymphocytes (com et im ages) A : untreated hum an lymphocytes; B: hum an lymphocytes expo sed to 015 Gy of Χ2rays; C: hum ann lymphocytes ex2 po sed to 110 Gy of Χ2rays; D: hum an lymphocytes expo sed to 310 Gy of Χ2rays Table 1 Effects of ionizing irradiation on DNA m igration in hum an blood lymphocytes Do seggy Percentage of cells w ith m igrated DNA (% ) L ength of DNA m igration 3 gλm (x θ S E ) V alues are the m ean the standard erro r of the m ean data based on 25 lymphocytes (including the cells w ithout DNA m igration) D iam eter of nucleus is no t included in length of DNA m igration 3 3 P < 0101, P < vs contro ls by t2test SCGE DNA DNA DNA,, Singh SCGE,, DNA, SCGE [ 1, 2 ] 607
5 SCGE DNA, DNA,DNA, SCGE ; SCGE DNA, DNA, ( ), SCGE SCGE, SCGE, DNA (apop to sis) 412 D NA, Χ DNA, [ 3 7, ],,, (S E ), SCGE V ijayalaxm i,,, DNA, DNA DNA, DNA,, DNA, SCGE, 413 SCGE, 40,,,, ;, 70%, ; DNA, ( EB 13 Λggm l),,, DNA, DNA ; DNA EB, DNA,, DNA 260 nm, DNA EB DNA, DNA,, References 1 M ckelvey2m artin V J, Green M H L, Schm ezer P, Poo l2zobel B L T he single cell gel electropho resis assay (com et assay) : A European review M u tat R es, 1993, 288: T ice R R, A ndrew s P W, H iraio, Sing N P T he single cell gel (SCG) assay: A n electropho retic technique fo r the detec2 tion of DNA dam age in individual cells In:W itm er C M ed B iolog ically R eactive Interm ed iateṡ IV M olecu lar and Cellu2 lar Ef f ects and their Imp act on H um an H ealth N ew Yo rk: P lenum P ress 1990; Singh N P T echnical repo rt: modification of alkaline m icrogel electropho resis fo r sensitive detection of DNA dam age, Int R ad iat B iol, 1994, 66: R ibas G, F renzilli G, Barale R,M arco s R H erbicide2induced DNA dam age in hum an lymphocytes evaluated by the single cell gel electropho resis (SCGE) assay M u tat R es, 1995, 344:
6 5 Singh N P,M ccoym T, T ice R R, Schneider E L A simp le technique fo r quantitation of low levels of DNA dam age in in2 dividual cells E xp Cell R es, 1988, 175: V ijayalaxm i, T ice R R, Strauss G H S A ssessnent of radiation 2induced DNA dam age in hum an blood lymphocytes using the single cell gel electropho resis technique M u tat R es, 1992, 271: Betti C, D avini R T, GiannessiL, L op rieno N, Barale R M icrogel electoopho resis assay (com et test) and SCE analysis in hum an lymphocytes from 100 no rm al subjects M u tat R es, 1994, 307: , DNA DNA (M eng Zi2Q ing, Zhang L ian2zhen A m ethod fo r determ ination of strand separation rate of DNA in alkali and its use in DNA dam age study A cta S ci C ir2 cum st), 1988, 8: Χ DNA (M eng Zi2Q iang Χ2Radia2 tion induced DNA single strand break s and their repair in m amm alian cellṡ Ch in J R ad iol M ed P rotect), 1987, 7: DNA (M eng Zi2Q iang Study on single strand break s of DNA and its rejo int repair in m amm alian cells J R ad iat R es R ad iat P roces), 1987, 5:
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