地塞米松诱导红系细胞凋亡的观察_英文_

地塞米松诱导红系细胞凋亡的观察_英文_ ISS N100426453/ CN1123569 D EVELOPM ENTAL & REP ROD UCTIVE BIOLO GY Vol . 6 No. 2 pp47252 Dec . 1997 Ζ 1997 Chin . Dev. & Reprod. Biol . Socs. Observa t ion on Apoptosis of Erythroid Cell s In duced by Dexa metha sone ZHA NG Shi2f u , S HEN Jiang2bo and ZHA NG Jing2bo Depart ment of Cell Biology , Instit ute of Basic Medical Sciences , Chinese Academia of Medical ( ) Sciences and Peking U niversit y Medical College CAM S & PU MC, Beijing 100005 , China ABSTRACT : Using splen ic cell s of BAL B/ c mice inf ected with Friend Leukemia Virus ( FVA) a s a model of erythroid cell s , we invest igated the act ion of dexa m2 etha sone ( Dex) to induce apoptosis of the cell s in the presence of erythropoiet in ( EPO) ———an a poptot ic in hibitor in developing erythroid cell s. The result indi2 cated that af ter treatment with a certa in range of Dex concentrat ions and pro2 longed incubat ion , the cell s were characterized by the occurrence of D NA la dders which a ppeared on agarose electrophoresis. Transmission electron microscopy sho wed that karyopyknosis , chromat in condensat ion , dilatat ion of the perinucle2 ar space , karyorrhexis , cytopla smic vacuol izat ion and cell f ragmentat ion a p2 peared in the cell s depending on the dose of Dex and the treatment t ime. These results suggest that Dex coul d induce apoptosis in the developing erythroid cell s a s in other cell s so far reported. Key words : a poptosis , dexa metha sone , erythroid cell , erythropoiet in , transmis2 sion electron microscopy 1 . INTROD UCTIO N During no r mal develop ment , t he growt h and deat h p rocesses of eryt hroid p ro genito r cells are 1 regulated and balanced by a range of cyto kinin and ot her f acto rs. Previo us st udieshave ( ) show n t hat eryt hropoietin EPO, a glycop rotein p ro duced in mammalian kidney and liver , co nt rols differentiatio n and p revent s apop to sis during eryt hrocyte develop ment . So EPO is co nsidered an impo rtant growt h f acto r t hat may regulate t he pop ulatio n of mat ure bloo d cells in vivo . The p urpo se of t he p resent st udy is to investigate t he effect of Dex o n apop to sis of eryt hroid cells. By using splenic eryt hroid cell isolated f ro m spleens of mice infected wit h t he anemia2inducing st rain of FVA as a mo del to examine t he role of Dex , we t ried to induce apop to sis of eryt hroid cells in t he p resence of EPO . 2 . MATERIALS A ND M ETHODS Eight2 to t welve2week old female BALB/ c mice were o btained f ro m Animal Center of CAM S , Beijing. ( The FVA was kindly p rovided by Dr . Ko ury M . J . Vanderbilt U niversit y , School of ) Medicine and Veterans , Tennessee. The viruses were maintained by passage of infected () plasma into BALB/ c mice . Pure EPO 10 ,000 IU / mL was o btained as a gif t f ro m Dr . Pan of PU MC , Beijing. 2 . 1 . Isolat ion of Splen ic Erythrobla sts and Culture Condit ions. 2 ,3 Splenic eryt hro blast s were isolated as described p revio usly wit h mo dificatio n. Briefly , (eight week2old female mice were injected via tail vein wit h 0 . 2 ml FVA plasma 1 :50 dilu2 ) tio n. At 13,15 days af ter infectio n , t he mice were slaughtered and t heir spleens were re2 moved . The lat ter were washed 3 times wit h IMDM . A single cell suspensio n of spleen cells was p repared by passing t he minced splenic co ntent s t hro ugh a 100 and a 200 mesh . The spleen cells were t hen separated by sedimentatio n at 4 ?fo r 30 min in a co ntinuo us gradient 6 separatio n medium of Ficoll2Paque . Samples of 1 x10cell/ mL splenic eryt hro blast s were cul2 t ured in 3 cm2diameter plastic dishes at 37 ?in humidified air wit h 5 % CO. The medium 2 was IMDM supplemented wit h 30 % fetal bovine serum , 1 % deio nized BA S , 100 IU / mL 24 μ penicilin , 100g/ mL st rep to mycin , 10mol/ L t hioglycerol and 0 . 4 IU / mL EPO . Af ter in2cubatio n fo r 1 h , different co ncent ratio ns of Dex were added into t he dishes. Af ter different perio ds of Dex t reat ment , cells were harvested fo r mo rp hological and biological st udies. 2 . 2 . Determinat ion of Cell Number and Via bil ity. (The eryt hroid cells were t reated wit h different co ncent ratio ns of Dex fo r vario us perio ds 4 , ) 8 , 12 , 24 h respectively. The cell number and viability were estimated by t rypan blue stain2 ing. The rate of dead cells is calculated as follow s : No . of dead cells ( ) The rate of dead cells %= ×100 %No . of survival cells + No . of dead cells 2 . 3 . D NA Extract ion and Electrophoresis. 4 DNA was ext racted as described in detail p revio usly. Af ter Dex t reat ment , t he cells were harvested by cent rif ugatio n at 1000 rp m fo r 15 min and washed wit h PB S 3 times and re2 cent rif uged o nce again . The pellet was t hen resuspended in p roteinase K digestio n solutio n μco ntaining 10 mM Tris2HC I , p H 8 . 0 , 0 . 1M ED TA , 50g/ mL p roteinase K , 0 . 5 % SDS and incubated at 37 ?fo r 6 h . DNA was ext racted using equal volumes of p henol/ chlo rof ro m and chlo rof ro m2isoamyl alco hol . The aqueo us p hase of t he ext ract was collected and t he DNA was p recipitated in t wo volumes of ice2cold et hanol and 0 . 15 mM NaCl . Af ter re2suspensio n in T E buffer , t he DNA co ncent ratio n was deter mined by U V at 260 nm and t hen was ana2 μlyzed by elect rop ho resis o n 1 . 2 % agaro se gel co ntaining 0 . 1g/ mL et hidium bro mide and o bserved under U V light . Observatio n o n Apop to sis 2 . 4 . Transmission Electron Microscopy. 25 Af ter 5 ×10M Dex t reat ment , t he cells were washed wit h 0 . 2M PB S fo r 3 times and fixed wit h 2 . 5 % glutaraldehyde in 0 . 2M PB S buffer fo r 30 min at 4 ?. The cells were also po st2 fixed in 1 % o smium tet ro xide in 0 . 2M PB S. The samples were dehydrated in graded series of buffers , embedded in resin , and ult ra2t hin sectio ns were cut and viewed under t ransmis2 sio n elect ro n micro scope . 3 . RES UL TS 3 . 1 . The eff ect of Dex at D iff erent Concentrat ions on the Gro wth of Erythroid Cell s. Cell number and viability were estimated by t rypan blue ext rusio n test . Eryt hrio d cells t reat2 ed wit h different co ncent ratio ns of Dex fo r vario us perio ds in t he p resence of 0 . 4 IU / mL EPO were used to deter mine t he effect . As a co nt rol , t he eryt hroid cells were incubated o nly wit h 0 . 4 IU / mL EPO . As seen in Fig. l , t he growt h curve show s t hat t he rate of dead cells increases wit h increasing co ncent ratio ns of Dex and cult ure time . It is wo rt h to mentio n t hat t he rate of dead cells increased mar kedly af ter t reat ment fo r 12 o r 24 h , co mpared to un2 t reated co nt rol cells. During t his perio d , t he cells were at t he early eryt hto blast o r inter me2 diate2eryt hro blast stage . Co mpariso n of t he ef2 Fig. 1 fect of Dex at different co ncen2 t ratio ns o n t he growt h of Ery2 t hroid cells. Af ter incubated ( 12 h in t he p resence of Dex 5 25 24 ) ×10M , 1 ×10M, t he per2 centage of dead cell increased sharply. 3 . 2 . D NA la dder Pattern Induced by Dex necro sis o r to apop to sis , t he cells were In o rder to assess w het her t he cell deat h refers to 24 t reated wit h 1 ×10M Dex , wit h EPO2t reated cells as t he co nt rol , harvested af ter incuba2tio n fo r 4 , 8 , 12 , o r 24 h , and DNA was ext racted and separated t hro ugh agaro se gel elec2 t rop ho resis. Fig. 2 show s t hat Dex p ro duces clear DNA cleavage of early2and inter mediate2 24 eryt hro blast s , but not in ot her stages at 1 ×10M Dex . When t he cells were t reated wit h 1 26 25 25 24 ×10, 1 ×10, 5 ×10and 1 ×10M Dex , and 0 . 4 IU / mL EPO , fo r 12 h , t he result is show n in Fig. 3 . A t ypical DNA ladder of early2eryt hro blast s was also seen as t he Dex co n2 25 24 cent ratio ns were 5 ×10, and 1 ×10M . These result s suggest t hat EPO does not induceDNA breakdow n and can p revent p ro grammed cell deat h of eryt hroid cells. Thus Dex can induce apop to sis of eryt hroid cells no mat ter w het her t here is EPO p resent o r not . Gel2elect rop ho resis of t he DNAs ext racted Fig. 2 24 f ro m t he cells w hich were t reated wit h 1 ×10mol/ L Dex fo r dif2ferent time . The result indicates t hat ba2 sop hilic eryt hro blast and polychro ma2top hilic ery2 t hro blast were characterized by t he occurrence of - 4 DNA ladders at 10 mol/ L Dex co ncent ratio n . λ Lane M : DNA/ Hi n d I I I mar ker ; Lane 0 , 4 , 8 , 12 , - 4 24 : cells expo sed to 1 ×10 mol/ L Dex and 400 U / L EPO fo r 0 , 4 , 8 , 12 and 24 h ; Lane 12c , 24c : 400 U / L EPO2t reated fo r 12 , 24 h as co nt rol . Gel2elect rop hresis of DNAs ext racted Fig. 3 f ro m t he cells t reated wit h different Dex co n2 cent ratio ns and 400 U / L EPO fo r 12 h . The re2 sult indicates t hat basop hilic eryt hro blast dis2 25 played a t ypical DNA ladder at 5 ×10o r 1 × 24 10mol/ L Dex co ncent ratio n . Lane 1 , 2 , 3 and 4 : cells were incubated wit h - 4 - 5 25 1 ×10 mol/ L , 5 ×10 mol/ L , 10mol/ L , 26 10mol/ L Dex and 400 U / L EPO fo r 12 h ; Lane 5 : t reated wit h 400 U / L EPO fo r 12 h co nt rol ; Lane M : pB R322/ Hi nf I mar ker . 3 . 3 . Ultra structural Morphological Changes The ult rast ruct ural o bservatio n indicated t hat t he no r mal and integrated st ruct ure of p roery2 t hro blast was fo und wit h a large , sp herical nucleus and 1, 2 p ro minent nucleoli located att he center of t he cell . N uclear membrane was intact and hetero2chro matin was unifo r mly2 dist ributed in small pieces ( Fig. 4 ) . Af ter t reat ment wit h Dex , o bvio us ult rast ruct ural changes were o bserved , including karyop ykno sis , perip heral co ndensatio n of chro matin , di2 ) latatio n of t he perinuclear space and so me vacuoles appeared in t he cytoplasm ( Fig. 5. Fol2 lowing p rolo nged Dex t reat ment , several st ruct ural damages in t he cells , such as chro matin disintegratio n , were seen wit h t he lo ss of cell viability ; cells rapidly f ragmented into mem2 brane2bo und apop totic bo dies t hat co ntain intact o rganelles and co ndensed chro matin were al2 so noted ( Fig. 6) . Observatio n o n Apop to sis Elect ro n micrograp h of no r mal p roeryt hro blast in co nt rol gro up , showing in2 Fig. 4 tegrated cell st ruct ure and unifo r mly2dist ributed chro matin . ×10 ,000 Elect ro n micrograp h of cells Dex2t reated fo r sho rt time , showing karyop ykno2 Fig. 5 sis perip heral co ndensatio n of chro matin and dilatatio n of t he perinuclear space . × 10 ,000 Elect ro n micrograp h of cells Dex2t reated fo r lo nger time , showing severe Fig. 6 st ruct ural damages in t he cells such as chro matin disintegratio n and cell f ragmentatio n . ×8 ,000 4 . D ISCUSSIO N Apop to sis o r p ro grammed cell deat h rep resent s an impo rtant mechanism in no r mal cell t ur nover of adult tissues as well as in embryo . This p rocess is characterized by shrinkage of t he cells wit h nuclear co ndensatio n , w hile t he biochemical hallmar k of apop to sis is extensive cleavage of t he cell DNA into 180,200 bp f ragment s by endo nuclease , resulting in t ypical 5 ladder pat ter ns o n DNA gel elect rop ho resis. In 1990 , Ko ury and his colleague repo rted t hat w hen EPO levels were below no r mal , eryt hro blast underwent apop to sis rapidly. While in t he p resence of sufficient EPO , t he p rocess of apop to sis was retarded o r even blocked. Thus , t he EPO seems to be t he agent w hich t he differentiatio n of eryt hro blast is needed dur2 ing certain stage of eryt hrocytes develop ment . When EPO level increases , t he EPO2depen2 dent p ro genito rs w hich sho uld die o riginally in t he p resence of EPO at no r mal level will sur2 vive , differentiate and give rise to reticulocytes , and t hereby increases eryt hrocyte p ro duc2 tio n. So f ar , t here are several papers demo nst rating t hat several f acto rs have t he effect o n apop to sis , fo r example , glucoco rticoid induces apop to sis of no r mal cells and t umo r cells as well . In t his st udy , we used splenic cells o btained f ro m FVA infected BALB/ C mice to in2 vestigate t he actio n of Dex to induce apop to sis of t he cells in t he p resence of EPO . The result indicated t hat w hen Dex was absent , t he cells did not undergo apop to sis , and even w hen EPO existed , Dex co uld also induce significant apop to sis of early eryt hro blast o r inter medi2 - 4 ate2eryt hro blast at co ncent ratio n of 10 M and 0 . 4 IU / mL EPO . The evidence p resented here demo nst rates t hat t his p rocess is time and do se2dependent . If it is t rue , it wo uld be helpf ul to st udy t he po ssibilit y and t he mechanism by w hich it t riggers t he ent ry of malig2 nant cells of t he bloo d system into apop to sis , especially eryt hrocyto sis , in t he clinic. REFERENCE : 1 . Koury M . J . and Bo ndurant M . C. : Eryt hropoietin retards DNA breakdown and p revent s p rogrammed () deat h in eryt hroid p rogenitor cells. Science , 248 4953: 378 , 1990 . Koury M . J . , Sawyer S. T. and Bo ndurant M . C. : Splenic eryt hroblast in anemia inducing f riend dis2 2 . ( ) ease : A source of cells for st udies of eryt hropoietin2mediated differentiatio n. J Cell Physiol , 121 3 : 526 , 1984 . Bo ndurant M . C. : Co nt rol of globin gene t ransrip tio n by eryt hropoietin in eryt hroblast f ro m f riend virus2 3 . () infected mice. Mol Cell Biol , 5 4: 675 , 1985 . Sambroo k J . , Frisch E. F. and Maniatis T. : In : Molecular Clo ning , ed 2 , Cold Sp ring Harbor L abora2 4 . tory Press , 9 :16219 , 1989 . Elis R. E. , Yuan J . , Horvitz H. R. , et al : Mechanisms and f unctio ns of cell deat h. Annu Rev Cell Bi2 5 . ol , 7 : 663 , 1991 . Wal ker P. R. , Smit h C. , Youdale T. et al : Topoiso merase I I2reactive chemot herapeutic drugs induce 6 . () apop tosis in t hymocytes , Cancer Res , 51 4: 1078 , 1991 . Alnemri E. S. , Fernandes T. F. , Haldar S. et al : Involvement of BCL22 in glucocorticoid2induced 7 . () apop tosis of human Pre2B2L eukemias. Cancer Res , 52 2: 491 , 1992 . 摘要 : 地塞米松诱导红系细胞凋亡的观察 张世馥沈江波章静波 中国中国医学科学院 中国协和医科大学基础医学研究所北京 100005 ( ) 正常机体内 ,红细胞生成素 EPO诱导红系细胞分化 ,并阻止其凋亡 ,使其维持机体 ( ) 所需足够的数量 。本文采用诱发贫血病毒 FVA诱导 BALB/ c 小鼠脾细胞所形成的红 () 细胞为实验系统 ,研究了外界因子 ? ? 地塞米松 Dex对 EPO 作用的影响 。 取 628 周雌性 BALB/ c 小鼠 , 尾部静脉注射含 FVA 的小鼠血清 。15 天后 , 断颈处 死 。取脾于 IMDM 中漂洗 、剪碎 、过滤 、离心 、制成单细胞悬液 ,在 EPO 存在下 ,于平皿中 ( ) 培养至不同的时期后 ,进行不同浓度 、不同时间的 Dex 处理 。取细胞进行 : 1台盼蓝染 () () 色计数死细胞数 ; 2观察 DNA 电泳图谱 ; 3电镜检察细胞学变化 。 Fig. 1 明 : 随着 Dex 处理浓度和时间的增加 ,细胞死亡率也增加 。- 4 () () Fig. 2 表明 : 10 浓度 Dex 处理 ,即可使早 12 h、中 24 h幼期红细胞凋亡 ,DNA 断 裂成多聚核小体 ,产生明显 DNA 梯形带 。Fig. 3 表明 : 高浓度 Dex 可以使早幼期红细胞 凋亡 。 ( ) 电镜观察表明 :与对照 Fig. 4比较 ,Dex 处理后 ,细胞核固缩 ,染色质沿核膜内面周 ( ) ( 边凝聚 ,核周间隙扩张 ,胞质内出现空泡 Fig. 5;随后细胞破碎 ,出现大量凋亡小体 Fig. ) 6。 Dex 拮抗 EPO ,诱导红系细胞凋亡的现象此前未见报导 。这对深入认识 Dex 作用机 理 ,指导临床应用具有重要意义 。 ( )Received 30 J anuary 1997