Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo.
Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo.
复制标题
逆转录病毒载体介导的基因在体外和体内转移到角膜细胞中。
DOI：
10.1016/s0002-9394(98)00205-0
复制
发表时间：
1998
影响因子：
4.2
通讯作者：
McDonnell,PJ
中科院分区：
文献类型：
--
作者：
Seitz,B;Moreira,L;Baktanian,E;Sanchez,D;Gray,B;Gordon,EM;Anderson,WF;McDonnell,PJ
目的探讨体细胞基因移植在浅表面性角膜切除术后角膜创面愈合中的应用潜力。方法用β-半乳糖苷酶和单纯疱疹病毒胸苷激酶基因转染人和兔角化细胞。在体外，用携带β-半乳糖苷酶或HStk（单纯疱疹病毒胸苷激酶）基因的逆转录病毒载体转染人和兔角化细胞。在体内，表面角膜切除术后局部应用载体上清液诱导兔角膜细胞。体外和体内用组织化学染色法检测β-半乳糖苷酶基因的表达。在体外，通过测定细胞在6孔板上的融合程度，测定更昔洛韦对HStk基因转导的角化细胞的细胞毒性和HStk（+）和HStk（−）角化细胞共培养10天后的旁观者效应。用Hstk转导家兔，然后用外用更昔洛韦治疗后，测量角膜雾度。结果体外转染β-半乳糖苷酶和HStk基因成功转染人和兔角质细胞。人细胞的转导效率（22%）高于兔细胞（16%），两种hstk转导的细胞系均表现出剂量依赖性更昔洛韦细胞毒性和显著的旁观者效应。在体内，vimentin阳性的角膜基质细胞中β-半乳糖苷酶的表达证实了兔角膜浅基质角膜切除术后角质细胞的转导，转导效率为25%至40%。术后局部应用更昔洛韦可减少家兔角膜间质混浊。结论间质角质细胞的遗传转导能力为理解影响角膜创面愈合的重要细胞和分子事件提供了一种新的策略，从而为减少或预防角膜浅切术后角膜混浊和瘢痕形成提供了一种潜在的方法。
PURPOSETo determine the potential of somatic gene transfer as a technique for modulating corneal wound healing after superficial keratectomy.METHODSThe transduction of human and rabbit keratocytes with β-galactosidase and herpes simplex virus thymidine kinase genes was performed. In vitro, human and rabbit keratocytes were transduced with retroviral vectors bearing β-galactosidase or HStk (herpes simplex virus thymidine kinase) genes. In vivo, rabbit keratocytes were transduced by topical application of vector supernatant after a superficial keratectomy. In vitro and in vivo, expression of the β-galactosidase gene was examined with histochemical staining. In vitro, ganciclovir cytotoxicity in HStk gene-transduced keratocytes and bystander effect in co-cultures of HStk(+) and HStk(−) keratocytes were measured by determining the degree of confluency of cells in 6-well plates after 10 days of incubation. Corneal haze in rabbits was measured after transduction with Hstk and subsequent treatment with topical ganciclovir.RESULTSIn vitro, both human and rabbit keratocytes were transduced successfully with both β-galactosidase and HStk genes. Transduction efficiency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cytotoxicity and a significant bystander effect. In vivo, expression of β-galactosidase within vimentin-positive corneal stromal cells confirmed transduction of keratocytes in the rabbit after superficial stromal keratectomy with an efficiency of 25% to 40%. Postoperative application of topical ganciclovir reduced corneal stromal haze in rabbits.CONCLUSIONSThe ability to genetically transduce stromal keratocytes provides a new strategy for understanding the important cellular and molecular events that influence corneal wound healing, thus offering a potential approach to decrease or prevent corneal haze and scarring after superficial keratectomy.