1、null * 8v S/9 “(I|: 2003A3020104),S E S “(I|: 51205004)null nullYT,: 020- 38688003 %#. !*刘小勇1 null 陈剑1null null 吴静2 null 周清1 null 王彦平3 null 李红4null 徐锦堂2null 赵松滨2暨南大学1附属第一医院眼科, 2医学院眼科研究室, 3病理生理学教研室, 4附属第一医院病理科(广州 510630)null nullK1null “null 建立体外培养人羊膜上皮细胞的方法,观察体外培养的羊膜上皮细胞的生物学特性bZEnull 取足月剖宫产术后羊膜,经胶原酶
2、和胰蛋白酶消化后,获取的羊膜上皮细胞接种于含 10%胎牛血清培养基中进行原代和传代培养,探索其合适的培养条件,用倒置显微镜观察培养的人羊膜上皮细胞体外生长的特征b用苏木精 -伊红染色a扫描电镜和细胞角蛋白免疫组织化学染色的方法对培养细胞进行形态学观察和鉴定bTnull 人羊膜上皮细胞可以在体外成功的培养传代,体外可连续传 8 10代b体外培养细胞呈多角形,长满后呈上皮细胞特有的铺路石样外观b扫描电镜观察细胞表面有丰富的微绒毛b细胞角蛋白 keratin单克隆抗体染色阳性b null 人羊膜上皮细胞在体外可成功进行原代和传代培养,体外培养的人羊膜上皮细胞在一定时间内可维持增殖能力b1oMnull
3、 羊膜null 上皮细胞null 细胞培养The primary culture and passage of human amniotic epithelial cellsnull LIUX iao-yong, CHEN Jian, WU Jing, et al.TheDepartment of Ophthalmology, TheFirstAffiliatedHospital of Jinan University, Guangzhou 510630, ChinaAbstractnull Objectivenull To establish amethod of culturing hum
4、an amnioticepithelial cells in vitro and to study itsbiologic features. M ethodsnull A pieceof amnioticmembrane taken from an uncomplicated elective caesarean sectionwas d-igested by collagenase and trypsin respectively. The amniotic epithelial cells were seeded in DMEM ( dulbecconullsmodifiedeaglen
5、ullsmedium) containing 10% fetal bovine serum for primary culture and passage. The optimal culture condition andfeature of the amnioticepithelial cellswere studied in vitro. The cultured cellswere investigatedmorphologically byH ema-toxylin- eosin staining, scanning electron microscope( SEM ) and we
6、re identified by cytokeratin immunohistochemistry.Resultsnull Human amniotic epithelial cellswere successfully cultured and passaged in vitro, and could be passaged succes-sively for 8- 10 times. M ostof the cultured cellswere polygon and of typicalslabstone- likeappearance. M anym icrovilliwere obs
7、erved on cell sur faces by SEM. Cytokeratin stainingwas positive. Conclusionnull Human amnioticepithelial cellscan be successfully primary cultured and passaged in vitro, and the cells can proliferate fora certain period.Keywordsnull Amnioticmembranenull Epithelial cellsnull Cell culturenull null %
8、K =, % s8?V =?7 , P V ? - % V 1b %VrHLA- A, B, CDR F,M f Q 2bBHq/, %Vs *% 3, 5 % 4, % VsF% 1byN, !ZE # 3+ 1il, V| F$b1null ZE1. 1null 试剂null !DMEM ( SGibco ), 10% d b( SGibco ), 10 ng/mlV 3y0(EGF, Speprotech ) , 4mmol/L!( SSigma ) , 100U/m,l 100 nullg/m,l( SGibco ),O ( SAmresco )b1null2null 方法1. 2.
9、1null 人羊膜上皮细胞的原代培养null |1E i OH IVaJaY# bQA U| s b| s ,cF( 100 null/m l a100 nullg/m l )PBSQ !b| ! Sgg1mm null 1mml v,F 0. 2% 37nullQh2 h, 1 000 r/m in 5m in, hA,F 0. 25%O hA i/h15m in,c b !h,200 “ V r, l“%A,1 000 r/m in 5m in,1 null 105/m l %2 m l% ! ,5% CO2a37null !Q = !,?A1Q,B5 7 d1null3.1Qb1. 2.
10、 2null 羊膜上皮细胞的传代培养null% 3980% H1# H.b !, ! F 0null25%O hA, 37nullh5m inbMA/n ,%W#9vh, “hA,F c b !h, 8u,%null181nullDnull 2007 M228 2 A,%9 1null2 31 !, !Hq ,?Ab1null3null 培养的羊膜上皮细胞的形态学观察nullA ?4% M# 3a9 b| 3j H %.%, A( PBS) !3Q, FAAA%,S !H , ,;4b ! %4, ST:|% !j ,80% H |,2. 5%= a1% %,0 ,Z s,CO2 “2a ,P
11、hilipsESEM- 30 4b F X H F8 fF ,|3j H . !%,PBS ( pH 7null3) !,Bd%5m in, X H F8aSABC fF k4(p V ) fF , DABA , 5b2nullT2. 1null 培养细胞的生长特性null !24 hvs%LC,LC%,|; , %vZ 7,|;h b%LC3 d 3,9 A,% A9,% 3b5 7 dP,% , 3b%K ?5, b ,a x,nm1b. !6 h%LC,5 d V zb4 -%,avl (B, 6 % ? 3M,% ,%9h ,.8% =Cb R ,%9 ? Ah ,vT 3,iV C
12、b2. 2null 培养细胞的形态学特征null !H ,%,% , b ,2 3 ,%,nss Mb% , Fb2. 3null 培养细胞的超微结构null%V ,M #% W?5l M 3,nm2b2. 4null 培养细胞来源鉴别 null !% %S:keratin , 5 , ! , ,nm3, !% %b3null) null null % % t+, VsF% 1, 3 f Q,F , D p,F0%b %8 !S= ,S8 ! %vl 3T3%T)!%,)!% %3a9Tb3T3% s%, cM 8 =? fQb L3T3%Hq/ ! %“,V 3T3% % !AHq, e !
13、/ ,7 O !% “ s.l%hB,F b %8 !1o !, ! ?v | % ? z %b !sF v ! hE !,F v ! F v ? !, O 8% , !HW , y v |%, 4 F1,# L 4 hEbSO sQh,N , L?CBO 4 %, OO% v,h/ %LC 3bVQ L ahZE,?C h V B %,O h+ss % , % z,LC 3b V F % )4 |!,a 0 bsY hT,y7 V ! b hT , %WhT7 %Yv, 4 % qb8 ! %,vVC,vl B, 3 Fbnull182null GuangdongM edical Journ
14、alnull Feb. 2007, Vo.l 28, No. 2,%v,2 3 , %8 !B +b/ Vn %V d ,M #%Wl M 3b %1sS, %+sVr, fF_ F8 Vk %b L X H F8 fF ,AU !% = , L !% %b Ly 8 ! %ZE,i %8 3 3+ 4, %B# F $b ID 1 null MIKIT, LEHMANN T, CAIH, eta.l Stem cellcharacteristicsofamniotic epithelialcells J. Stem Cells, 2005, 23( 10): 1 549- 1 559. 2
15、null AKLE CA, ADINOLFIM, WELSH K I, eta.l Immunogenicityofhuman amniotic epithelialcells after transplantation into volunteers J. Lance,t 1981, 2( 8 254): 1 003- 1 005. 3 null H IDENORIOKAWA, CA OSAMU OKUDA, HAJIME ARAI, eta.l Amniotic epithelial cells transform into neuron- like cells inthe ischem
16、icbrain J. Neurorepor,t 2001, 12: 4 003- 4 007. 4 null FLINIAUX I, V IALLET J P, DHOUAILLY D, et a.l Transformation ofamnion epithelium into skin and hair follicles J. Di-fferentiation, 2004, 72 (9/10): 558- 565.( l : 2006- 10- 22nullI :f )null * 2 8 Y S/1 “(I|: 0424420026)hl CD3+, CD4+,CD8+#CD4+ /C
17、D8+Y*王鹏1null 阚全程2null 余祖江2郑州大学 1护理学院基础教研室, 2第一附属医院 (郑州 450052)null nullK1null “null 探讨头孢地嗪 ( CDZM )对正常小鼠外周血中 CD3+ , CD4+ , CD8+及 CD4+ /CD8+ 的影响bZEnull 采用完全随机实验设计,分设 6组,对小鼠分别连续 7 d注射胸腺肽( Thy) 10 mg/kga头孢地嗪 ( 500, 200,50 mg/kg)a头孢曲松 500mg/kg及生理盐水后,在第 7, 14, 21天分别采集外周血,经流式细胞仪对小鼠外周血中CD3+ , CD4+ , CD8+ ,
18、 CD4+ /CD8+进行检测,观察头孢地嗪对机体免疫功能的影响bTnull CDZM具有较好的免疫调节作用,可明显地提高外周血 CD4+ /CD8+的比值,与 Thy相比, CDZM对机体免疫系统的影响更为持久b null头孢地嗪可通过调整 CD4+和 CD8+之间的平衡,有效地提高机体的免疫功能b1oMnull 头孢地嗪null T细胞亚群null 免疫功能null nullh f h f MM1,?3 H,8 F /,* Yi9Fy % )M,? 3 1- 2b 1i,7 B1y 3bnull nullh(Cefodizmi e, CDZM) B r h ) ,?C,h A f ?, ?
19、9 %,A4 %CD4+% , PCD4+ /CD8+1 6,1 %9 , PMN )T9 4,; ? z e , ? s8 f ?, F ) fT, f Jh H,h fTVCF s, F 3 ! 5bh “5 e ) 7 14 d, YV T%NlT% _, 4h8 f ? Yb1null ZE1null1null 实验动物 nullr B l , 4 5 -,8(20null 2) g,1 2 8 L, V|:410115b1null2null 试剂nullh1 Z FD0“v ZK , |: 040317;h w (Ceftriaxone, CRO)1Z 2Y 0K , |: 0403
20、01;Ft(Thymosin, Thy)1 0K , |:20040715; S: Fl F8FITC-CD4aPE-CD3aPE-CY5-CD8#M;S:| F8FITC-IgG2baPE-IgGaPE-Cy5-IgG2a (1 SeBioscience b1null3null 仪器null FACScan T%N1Becton Dick-inson b1null4null 给药方案null|l s6F, F21bNSF: NS 0null2m;l CROF: CRO 500mg/kgF; ThyF:Thy 10mg/kg; CDZM 50mg/kgF; CDZM 200mg/kgF#CDZM 500mg/kgFb LF ( 8 null183nullDnull 2007 M228 2
