人晶状体上皮细胞的组织块培养

目的建立人晶状体上皮细胞体外培养的简单有效方法,观察不同年龄人晶状体上皮细胞的体外生长规律和特点。方法应用改良组织块培养法对胎儿、成人和年龄相关性白内障的晶状体上皮细胞进行体外培养,在倒置显微镜下观察其生长、分化规律。结果胎儿、成人和年龄相关性白内障晶状体上皮细胞都具有增殖能力,胎儿和成人晶状体上皮细胞可传3代,年龄相关性白内障晶状体上皮细胞传代培养基本不能增殖。结论人晶状体上皮细胞体外培养困难,不同年龄人晶状体上皮细胞体外均能增殖,但增殖能力均很有限;改良组织块培养法是晶状体上皮细胞体外培养的较好方法。     

人外伤性白内障晶状体上皮细胞的组织块培养

目的建立人外伤性白内障晶状体上皮细胞体外培养的简单有效方法，观察晶状体上皮细胞的体外生长规律和特点。方法应用改良组织块贴附培养法对儿童及成人外伤性白内障的晶状体上皮细胞进行体外培养，倒置显微镜下观察其生长规律。结果儿童和成人外伤性白内障晶状体上皮细胞都具有增生能力，晶状体上皮细胞均可传3代，晶状体上皮细胞多次传代后生长缓慢。结论儿童和成人外伤性白内障晶状体上皮细胞体外培养增生能力有限，儿童晶状体上皮细胞增生能力较强。改良组织块贴附培养法简单易行，重复性好，是晶状体上皮细胞体外培养的较好方法。     

兔晶状体培养及其上皮细胞超微结构观察

探讨兔晶状体培养及其体外生长情况，试图找到一种简单，易行的实验方法，方法对４０只兔晶状体分３组进行体外培养。对生长３，５，７天的３组兔晶状体分别观察，并将其前囊制成透射电镜标本，对其上皮细胞的超微结构进行观察。     

白内障术后后囊浑浊发生机制的研究--老年性白内障晶状体上皮细胞的体外培养

目的观察老年性白内障晶状体上皮细胞体外培养的生长特点。为研究老年性白内障及术后后囊浑浊的发生机制及防治奠定基础。方法应用改良组织块培养法对超声乳化术中老年性白内障晶状体前囊上皮细胞进行体外培养，在倒置显微镜下观察其生长和分化的规律。结果前囊接种3—5天，有新生上皮细胞自囊片的边缘长出并向四周延伸，第3-4周部分细胞内出现空泡和颗粒等结构改变，生长近于停止；传代培养细胞不能增生。结论老年性白内障晶状体前囊上皮细胞体外增生能力有限。改良组织块培养法培养晶状体上皮细胞简单有效。     

不同成分培养基对原代兔晶状体上皮细胞生长的影响

目的:研究不同培养基对兔晶状体上皮细胞(LEC)培养的影响。方法:使用组织块贴壁法原代培养兔LEC,倒置显微镜观察不同培养基(DMEM低糖、高糖、F12)下兔LEC形态、生长速度的情况。结果:DMEM低糖和高糖培养基使培养2wk后的细胞开始出现分化,并停止生长,晶状体上皮细胞明显成纤维细胞化。DMEM/F12培养基使细胞生长良好,传至第5代时细胞开始发生转化变为成纤维细胞。结论:DMEM低糖和高糖培养基造成兔LEC增殖抑制,DMEM/F12培养基适合兔LEC的生长。     

兔眼晶状体上皮细胞的体外培养

目的 建立兔晶状体上皮细胞体外培养的模型。方法 采用组织块培养法,对兔眼晶状体前囊膜进行培养,并利用形态学检查方法和免疫组化技术鉴定。结果 组织块接种２４ｈ后即可见细胞生长,且保持上皮细胞形态,１ｗｋ左右细胞融合,在体外可传至７代,５代以后细胞呈成纤维细胞状,α－晶状体蛋白间接免疫荧光呈阳性反应。结论 成功地建立晶状体上皮细胞体外培养模型,可用于后发性白内障发病机制的研究。     

巨噬细胞对培养兔晶体上皮细胞增殖率及DNA合成率的影响

采用细胞计数及Ｈ＾３ＴＤＲ液闪测定的方法，观察巨噬细胞对体外培养兔晶体上皮细胞增殖率的影响。结果表明，在巨噬细胞调理液作用２４小时后，晶体上皮细胞增殖率及ＤＮＡ合成率均较对照组明显增高（Ｐ〈０．０５），并保持至作用后第５天。由此证实，巨噬细胞及其活性因子可促进晶体上皮细胞增殖。该机制可能与白内障术后后囊混浊有关。     

人结膜上皮细胞的培养鉴定及液氮冻存

目的：探索人眼结膜上皮细胞体外培养和细胞保存的最佳方法，建立人结膜上皮细胞，进一步研究人结膜的病理、生理特点及为毒理试验提供可靠的细胞模型。方法：分别用组织块培养法、机械分离法及混合消化液培养法体外培养正常成年人结膜上皮细胞，通过观察细胞形态、生长特性并用原位免疫组化方法鉴定培养细胞；收集第３代和第４代融合的细胞液氮冻存，保存３０天后复苏，观察复苏成功率。结果：３种取材方法中，混合消化液培养法细胞     

TGF-β1在后发性白内障形成中的作用--TGF-β1对牛晶状体上皮细胞明胶酶表达的影响

目的探讨转化生长因子β1(TGF-β1)对牛晶状体上皮细胞明胶酶活性的影响及其在后发性白内障形成中的作用。方法对牛晶状体上皮细胞进行体外原代及传代培养。分别收集原代、1代、2代、3代体外培养牛晶状体上皮细胞培养液；对1代细胞加入TGF-β1(终浓度10ng/mL)，收集12h、24h、36h、48h和72h细胞培养液，酶谱法检测明胶酶活性。结果不加TGF-β1的原代、1代、2代、3代细胞培养液中未检测到明胶酶活性；加入TGF-β1后各个时间点均能检测到明胶酶活性，且条代密度随时间呈上升趋势。结论TGF-β1能够诱导牛晶状体上皮细胞明胶酶的表达。TGF-β1和明胶酶共同作用，可能加速后发性白内障的形成。     

Genistein对兔晶状体上皮细胞增殖的影响

目的　探讨染料木黄酮 (genistein)对体外培养的兔晶状体上皮细胞增殖的影响。方法　用不同浓度的 genisteinDMSO溶液处理常规培养的第 3代兔晶状体上皮细胞 ,采用细胞计数法以及MTT法检测对晶状体上皮细胞增殖的影响。结果　 12 .5mg·L-1genistein对晶状体上皮细胞增殖无显著影响 ,genistein在 2 5mg·L-1可显著抑制体外培养的兔晶状体上皮细胞的增殖 ,抑制率为 32 .86 % ,5 0mg·L-1genistein抑制率为 4 9.2 4 % ,10 0mg·L-1genistein抑制作用最强 ,抑制率达 70 .72 % ,且genistein在 2 5～ 10 0mg·L-1其抑制作用呈剂量依赖性。结论　genistein在浓度≥ 2 5mg·L-1时可显著抑制体外培养的兔晶状体上皮细胞的增殖 ,且呈剂量依赖性。提示genistein可能在临床防治后发性白内障中有所帮助。     

Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture

Human lens cells from 5-91-year old individuals were cultured in 8 different basal media containing fetal bovine, adult bovine, rabbit or human serum or human plasma or in a serum-containing medium supplemented with insulin, epidermal growth factor, fibroblast growth factor plus other hormones or trace elements. Cultures were initiated from explants of the capsule and epithelium or following enzymatic dissociation of cells from the capsule. Under all conditions the epithelial cells had a limited doubling potential. As a function of time in culture, cells enlarged, displayed numerous filaments and exhibited apparent in vitro senescence. Lens epithelia from 4-6 day old rabbits cultured under identical conditions mimicked the behavior of human lens cells. Lens epithelia from newborn rabbits may be a suitable model for investigating the basis of apparent in vitro senescence in this cell type and could help in defining the conditions required for the long-term growth of human lens cells. The limited growth of human lens epithelia suggests that these cells require tissue-specific nutrients or hormonal supplements not present in standard tissue culture media.     

p21和p27在兔晶状体上皮细胞中的表达

目的:研究细胞周期蛋白激酶抑制因子p21和p27蛋白在不同年龄段兔晶状体上皮细胞（lens epithelial cells,LECs）中的表达规律。方法:分别取10,20,30周龄兔晶状体前囊膜组织,采用RT-PCR和Western-blot检测该组织中p21和p27的mRNA水平和蛋白水平。结果:兔晶状体前囊膜组织中p21与p27的mRNA水平和蛋白水平相似,青年最高,老年次之,中年最低。p21和p27在青年、中年及老年兔LECs中的表达水平有差异（P<0.05）。结论:p21和p27在兔LECs周期调控过程中可能起着平衡作用,对防止PCO的研究具有一定参考价值。     

重组人白细胞介素-1受体拮抗剂对体外培养的胎儿晶状体上皮细胞的影响

目的:探讨重组人白介素-1受体拮抗剂对培养的胎儿晶状体上皮细胞黏附和增殖的影响。方法:培养晶状体上皮细胞,取生长良好的第2代细胞用于实验。将第2代胎儿LEC于不同浓度的rhIL-1ra孵育后,用四甲基偶氮唑盐(methylthio tetrazole,MTT)法记录各孔黏附细胞数量的吸光度(A)值,并与空白组进行对照;将第2代胎儿LEC培养24h待细胞贴壁后,与不同浓度的rhIL-1ra再共育24h后,MTT法记录各孔细胞数量的吸光度(A)值,并与空白组进行对照。结果:除1μg/L浓度组外,其余各组抑制胎儿晶状体上皮细胞与胶原的黏附呈剂量依赖性,与空白组对照具有显著性差异。100μg/L浓度组细胞收缩、变圆,但仍保持贴壁状态,与空白组对照可显著抑制已贴壁胎儿晶状体上皮细胞的增殖。结论:有>1μg/L的各组rhIL-1ra能够抑制体外培养的胎儿LEC与胶原的黏附,并且浓度越大抑制作用越明显,具有浓度依赖性。仅100μg/LrhIL-1ra浓度组作用24h可显著地抑制已贴壁胎儿晶状体上皮细胞的增殖,但仍保持贴壁状态,提示该药可能具有较轻的毒副作用。     

Donor age influences the growth of rabbit lens epithelial cells in vitro

Experiments were designed to optimize the conditions required for the establishment of cell lines from individual rabbit and human lenses. The influence of donor age and the response of the epithelia to various media, sera, substrata and growth factors were determined. Epithelial cells were obtained from 4–6-day-, 3-, 5-, 8-, 10-week- and 4-months- to 4-yr-old rabbits. Cell lines from animals aged 4 months or older were maintained in culture for over 2 yr, had a population doubling time of ca. 2.5 days, required serum for growth, and contained polypeptides that comigrated with and had an apparent molecular weight of known lens crystallins. Explants or enzymatically dissociated cells from 4–6-day-, 3-or 5-week-old donors exhibited little mitosis. This limited growth occurred in cells cultured in Eagle's MEM, BME. Dulbecco's modified MEM, medium 199, RPMI-1640, Ham's F-14, Leibovitz's L-15, KEI-4 or in a modified Ca²⁺-free MEM, supplemented with heat-inactivated rabbit or bovine sera or in these sera-containing-media supplemented with growth factors. Cells from the 4–6-day-olds migrated from the explant, synthesized DNA, enlarged, but did not show a marked increase in cell number. Analysis of video tapes indicated that the outgrowth from the explants of 4–6-day-olds was due solely to cell migration. Under similar culture conditions fibroblasts from 4–6-day-old rabbits or lens epithelia from rabbits 4 months or older were capable of generating continuous cell lines. Under like culture conditions, lens epithelia from humans aged 30–89 yr underwent a maximum of only three subcultures, enlarged and did not divide. Human lens cells have been maintained in a viable non-dividing condition for 1 yr.Cell lines possessing lens-specific proteins could be obtained from 4–6-day-old or 3-week-old rabbits if the cells were cultured in MEM containing non-heat inactivated rabbit serum. Clearly, the growth requirements of lens epithelial cells from younger rabbits can be markedly different from those of their older counterparts.     

Ex vivo canine lens capsular sac explants

Background: Lens capsular sac explants from human cadaver eyes were used to investigate posterior capsular opacification (PCO). The purpose of this study was to characterize a similar model using canine tissue and to determine whether transferrin (Tf), transforming growth factor β-2 (TGF-β2), and insulin-like growth factor-1 (IGF-1) are secreted by lens epithelial cells (LEC) of these ex vivo sacs. Methods: The lens from canine eyes was removed by extracapsular cataract extraction, the lens sac dissected free, pinned to a petri dish, and cultured in either serum-supplemented or serum-free medium. Morphologic characteristics and growth rate to confluence on the posterior capsule were studied by phase-contrast microscopy. Vimentin, alpha smooth muscle actin, and panTGF-β expression by LEC were determined by immunohistochemistry. Tf, TGF-β2, and IGF-1 levels were measured by ELISA in the supernatant of sacs cultured in serum-free medium. Results: The mean time to confluence of LEC onto the posterior capsule was 5.4±1.1 days (n=22) and 14.7±3.7 days (n=14) for sacs in serum-supplemented and serum-free medium, respectively. Following development of confluence, explants displayed opacification and light scatter from cellular proliferation and capsular contraction. Confluent LEC expressed vimentin, alpha smooth muscle actin, and TGF-β2, and both Tf and TGF-β2 were secreted into the culture supernatant. Conclusion: Canine lens sac explants have characteristics virtually identical to those of human origin, and appear to be a useful alternative tissue source for this model when human cadaver eyes are unavailable. Tf and TGFβ-2, but not IGF-1, are secreted by LEC in explanted lens sacs and may influence the proliferation and metaplasia of LEC during the development of PCO. Received: 7 December 1999 Revised: 2 March 2000 Accepted: 21 March 2000