作者：李新宇,李贵刚,刘磊,栗静
作者单位：430030 中国湖北省武汉市, 华中科技大学同济医学院附属同济医院眼科
【摘要】目的：观察脂质体 LipofectamineTM2000对人角膜基质细胞的影响,探索其应用于人角膜基质细胞的可行性及安全范围。方法：体外培养人角膜基质细胞,取其第35代细胞鉴定后用于实验。采用MTT法检测不同浓度和时间脂质体对人角膜基质细胞增殖率的影响;采用台盼蓝染色法检测对存活率的影响。结果：脂质体对人角膜基质细胞的影响与浓度和作用时间有关。浓度高于一定水平时可引起细胞增殖率和存活率的下降,浓度相同时作用时间越长下降越明显。浓度低于40mg/L作用24h不会对细胞增殖率和存活率产生明显影响。结论：LipofectamineTM2000在一定范围内不引起细胞毒性,有望在角膜基质细胞的基因治疗中发挥重要作用。
【关键词】 角膜基质细胞；基因转染；脂质体
Influence on cultured human keratocytes by liposome
XinYu Li, GuiGang Li, Lei Liu, Jing Li
Foundation item: Supported by the Science&Technology Foundation of Hubei Province (No.2007ABA144)
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Correspondence to:GuiGang Li.Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. [email protected]
AbstractAIM: To observe the effects on human keratocytes by cationic liposome LipofectamineTM2000 (LF2000), to investigate the efficiency and safe range applied in human keratocytes, and establish basis for gene therapy of human keratocytes.METHODS: Human keratocytes cultured in vivo within 3 to 5 passages were used in experiment after being identified. The effects on proliferation of cultured human keratocytes by LF2000 with different concentrations and time were evaluated By MTT; the effects of LF2000 on the survival rate and its relation with 5,10,20,40,80mg/L concentration and time were detected by trypan blue staining.RESULTS: LF2000s effects on human keratocytes were related with concentration and time. The cellular proliferation and survival rate declined when concentration of LF2000 was above certain level, and this effect increased as time became longer. LF2000 had no effect with concentration under 40mg/L for 24 hours.CONCLUSION：LF2000 did not cause cytotoxicity during a concentration range “tested”, and it is hoped to play an important role in gene therapy of human keratocytes.
KEYWORDS: keratocytes; gene transfection; liposome
INTRODUCTION
Changes of keratocytes in repair following trauma related closely with visual function. Clinicians have been looking for medicines which could modulate the repair of cornea trauma. Regulation on molecule and cell levels is hot at present. In this experiment, LF2000 of different concentration and time was used in cultured human keratocytes in order to obtain its safe range of concentration and time, also to provide experimental basis for gene prophylaxis and therapy of corneal diseases.
MATERIALS AND METHODS
Materials Equipments Refrigerated centrifuge; attemperator; cellular incubator; inverted phase contrast microscope; brachytely homeothermia centrifuger (Hereus Company).Reagent LipofectamineTM2000（Gibco/BRL Company）; DMEM nutritive medium（Gibco/BRL Company）.
Methods Primary culture and subculture of human keratocytes Cultured the corneal stroma tissues from donated healthy eyeball, and cells within 3~5 passages were used in the following experiment.
The effects on human keratocytes by LipofectamineTM2000 Effects of LF2000 on cell proliferation rate measured by MTT: the cells within 4 passages were seeded onto 96well plate after digestion by 2.5g/L trypsin and adjusted to a density of 4×104/mL,100μL/well. Each group including control had 5 pair wells. After culture for 24 hours, the adherent cells were incubated for 12 hours or 24 hours with LP2000 0(negative control),5,10,20,40,80mg/L. Then 5g/L MTT was added in each holes. After cultur for 4 hours, removed culture medium and added DMSO 100μL for 5minutes ordinary temperature, then the absorbance (Avalue) at 550nm wavelength of each hole was detected by ELISA Reader. Cell proliferation rate= (Avalue of LP2000 group/Avalue of negative control group) 100%.
Effects of LF2000 on cell survival rate measured by Trypan blue staining: methods of inoculation and grouping were the same with MTT. 12 hours and 24 hours after LF2000 was added, carefully removed the supernatant and added 4g/L trypan blue a drop in 5 wells of each group, and then observed cells under the inverted phase contrast microscope immediately. Dead cells with broken membrane were stained blue with trypan blue, while living cells were negative. The negative and positive cell numbers of 5 lowpower fields of each hole were counted, and mean value were used in statistical analysis. Cell survival rate=(unstained cells number/ total cells number) 100%.
Statistical method Statistical comparisons were made by ttest and ANONA with SPSS software.
RESULTS
Culture and Identification of Human Keratocytes Three days after seeding, adherent and monolayer cells migrated from verge of tissue mass, and presented with fusiform shape and round centered nuclus. Seven days after subculturing, cells reached confluence. After cells were seeded on coverslips, vimentin and keratin were immunostained to identify corneal stroma cells.
Effects of LF2000 on Human Keratocytes Effects of LF2000 on proliferation and survival rate of human keratocytes LF2000 had no obvious effects on proliferation and survival rate of human keratocytes when its concentration was below 20μg/L, which had no statistical significance compared with negative control group(P＞0.05); while proliferation and survival rate of human keratocytes would decrease significantly when concentration of LP2000 was above 40mg/L for longer than 24 hours (P＜0.05).
Proliferation and survival rate were higher in 24 hour group than that in 12 hour group when the concentration of LF2000 was 40 or 8040mg/L (P＜0.05).
Proliferation and survival rate of human keratocytes decreased as LF2000’s concentration increased (Table 1, 2).Table 1Effects of different concentrations of LF2000 on proliferation rate of cultured keratocytes (略﹚Table 2Effects of different concentrations of LF2000 on survival rate of cultured keratocytes (略﹚
DISCUSSION
Corneal stroma is composed of about 250 intertwining lamellae of collagen fibrils which made up of collagen fibrils parallel arranged into equal diameter. Keratocytes account for the major cell component of stroma, synthesize and secret fibers and also play roles for their alignment and balance. Keratocytes changes in trauma and repair have important influence on the structure and function of cornea.
Recently, most researches have carried out investigation on corneal repair in trauma, however, some mechanismsabout repair still need to be explored. With rapid development of molecular biotechnology, gene therapy obtained increasing considerations as a completely new therapeutic tool. Gene transfection technology has extensive prospect in diagnosis and therapy of ophthalmology as the basis of gene therapy [13]. The key point is the carrier which ensures exogenous gene delivered into target cell. An ideal carrier should deliver objective gene to target cell or tissue under the regulation of proper promoter without inflammatory or immune reaction; could adjust its expression time in host body according to different therapeutic demands; easy to construct and purify; high titer and without initiation of insertion mutation or wildtype viral pollution[1,2]. Carriers applied in gene therapy include naked DNA (plasmid),cationic liposome, virus, et al [4,5]. Plasmids have low transfection efficiency. Although high transfection efficiency, there are still some risks exist in the clinical application because of virus immunogenicity. As abiology method, cationicliposomes are referred to gene therapy in vivo as their safety, weaker immunogenicity, larger capacity and simplicity to operate, especially the polyvalent cationic liposomes with higher transfection efficiency [6,7]. Based on this experiment, cationic liposome LF2000 did not exert obvious influence on proliferation and survival rate of keratocytes in certain concentration and time, therefore, LF2000 could be used to deliver target genes into keratocytes because of its intervention in corneal repair in trauma at molecule level, which provides a new efficient therapeutic method for corneal diseases.
【参考文献】
1 Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science1995;270(5235):404410
2 Singh VK, Tripathi P. Gene therapy in ocular diseases. Indian J Ophthalmol2002;50(3):173181
3 Klebe S, Sykes PJ, Coster DJ, Bloom DC, Williams KA. Gene transfer to ovine corneal endothelium. Clin Experiment Ophthalmol2001;29(5):316322
4 Murata T, Kimura H, Sakamoto T, Osusky R, Spee C, Stout TJ, Hinton DR, Ryan SJ. Ocular gene therapy: experimental studies and clinical possibilities. Ophthalmic Res1997;29(5):242251
5 Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wen zM, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipidmediated DNAtransfection procedure. Proc Natl Acad Sci USA 1987;84(21):74137417
6 Felgner PL, Ringold GM. Cationic liposomemediated transfection. Nature1989;337(6205):387388
7 Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felqner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem1994; 269(4):25502561 |
