逆转录病毒载体携带报告基因在色素上皮细胞的转染与表达

目的 观察逆转录病毒载体携带报告基因在视网膜色素上皮(retinal pigment epithelium,RPE)细胞的表达情况,以探讨基因疗法用于抑制增生性玻璃体视网膜病变的可行性.方法 选第3～4代培养的已长满融合的RPE细胞,以1:3的分种率分种传代.随机分为对照组与实验组,每组12孔.实验组用绿色荧光蛋白基因作为报告基因,以逆转录病毒载体携带来转染RPE细胞,重组合绿色荧光蛋白基因的逆转录病毒上清液浓度为1.2×109 cfu·L-1.对照组RPE细胞以同样方法直接转染绿色荧光蛋白基因质粒DNA.观察绿色荧光蛋白基因在2组RPE细胞的表达情况.结果 基因转染后可见实验组RPE细胞的绿色荧光蛋白基因表达率可达56%～72%,对照组未见有任何表达.结论 逆转录病毒载体可以携带目的基因转染于RPE细胞,并达到较高的转染率.     

三种基因导入系统对视网膜色素上皮细胞转染效率的研究

目的：以增强型绿色荧光蛋白(enhanced green flu—oreacent protein，EGFP)为报告基因，研究lipofectamine2000、Fu—GENF6和逆转录病毒载体介导的基因导入系统对视网膜色素上皮细胞的基因转染效率，为视网膜及视网膜相关疾病的基因治疗奠定基础。方法：在体外分离培养人视网膜色素上皮细胞，构建含有EGFP基因的表达载体，分别应用阳离子脂质体lipofectamine2000、FuGENE6和逆转录病毒三种方法将EGFP基因转染到人视网膜色素上皮细胞中，转染后于48h在荧光显微镜下观察EGFP的表达情况。结果：三种方法介导EGFP基因在人视网膜色素上皮细胞中表达的阳性比例平均分别为52％、10％和72％。结论：在体外可通过逆转录病毒系统或阳离子脂质体lipofectamine2000有效地将外源基因导入到人视网膜色素上皮细胞中，其中以逆转录病毒系统转染效率为最高。     

增生性玻璃体视网膜病变模型眼内定向 转染报告基因的研究

目的观察目的基因在玻璃体增生膜的表达情况,以探讨基因治疗增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR) 的可行性。方法用β-半乳糖苷酶基因作为报告基因,以逆转录病毒载体携带,直接注入PVR模型眼玻璃体腔中,观察其在PVR 眼各组织表达情况。结果 基因转染后可见玻璃体增生膜组织有转染基因表达,表达主要位于增生膜的表面,而视网膜组织及其它眼组织未见表达。结论逆转录病毒载体用于PVR的基因治疗有靶向性作用,表明PVR基因治疗具有可行性。(中华眼底病杂志,2001,17:224-226)     

重组增强型绿色荧光蛋白腺相关病毒转染兔晶状体上皮细胞的研究

目的研究2型重组腺相关病毒(recombinant adeno-associated virus2,rAAV2)载体介导增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)对体外培养兔晶状体上皮细胞(N/N1003A)的转染和病毒载体对晶状体上皮细胞增生的影响,探讨rAAV2作为后囊膜混浊基因治疗载体的可行性。方法rAAV2-EGFP按感染复数(mul-tiplicity of infection,MOI)为104、105、2×105、106转染N/N1003A细胞,倒置荧光显微镜下观察晶状体上皮细胞中EGFP的表达,流式细胞仪检测rAAV2-EGFP对N/N1003A细胞的转染效率,MTT比色法测定rAAV2载体对N/N1003A增生的影响。结果EGFP在细胞中的表达呈绿色,弥漫于整个胞浆。rAAV2-EGFP的转染效率随着MOI的增加和时间的延长而增加,MOI=106时,转染后第7d达到71.12%.MTT法显示各转染组与未转染组A值无统计学差异。结论重组腺相关病毒载体可以介导EGFP基因高效稳定转染晶状体上皮细胞,并且对细胞增生无抑制作用,腺相关病毒作为后囊膜混浊基因治疗载体是可行的。     

腺病毒载体介导的单纯疱疹病毒胸苷激酶基因/丙氧鸟苷体系对晶状体上皮细胞的作用

目的：观察腺病毒载体介导的单纯疱疹病毒胸苷激酶（herpes simplex virus thymidine kinase,HSV-tk）基因／丙氧鸟苷（ganciclovir,GCV）体系对体外培养的牛晶状体上皮细胞的杀伤效应,并初步探讨晶状体上皮细胞死亡的机制。方法：利用携带HSV－tk基因的重组腺病毒载体adenoviral vector(ADV)/HSV-tk,感染体外培养的牛晶状体上皮细胞后用GCV治疗,并观察疗效。以切除E1区的空腺病毒载体ADV／Empty作为对照。以电镜、TUNEL法及吖啶橙－溴化乙啶染色法检测晶状体上皮细胞的凋亡及坏死情况。结果：GCV在体外对感染ADV／HSV－tk的牛晶状体上皮细胞有明显的杀伤作用,且具有旁观者效应,而对转染了ADV／Empty的细胞则无显著的毒性。GCV的杀伤效应随剂量的增大及时间延长而增强。导入HSV－th基因的晶状体上皮细胞出现明显的细胞凋亡现象,而且细胞的凋亡率（t=7.28,P＜0．01）及坏死率（t=14.72,P＜0．001）显著高于对照组。结论：腺病毒载体可将HSV-tk基因转导入牛晶状体上皮细胞中;GCV可有效杀伤表达HSV－tk的晶状体上皮细胞;HSV－tk/GCV体系引发的细胞死亡可能与亡及坏死两种机制有关;腺病毒载体介导的HSV－tk/GCV体系有可能成为后发性白内障的有效治疗方法。     

重组增强型绿色荧火蛋白腺相关病素转染兔晶状体上皮细胞的研究

目的 研究2型重组腺相关病毒(recombinant adeno-associated virus 2,Raav2)载体介导增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)对体外培养兔晶状体上皮细胞(N/N1003A)的转染和病毒载体对晶状体上皮细胞增生的影响,探讨Raav2作为后囊膜混浊基因治疗载体的可行性.方法　Raav2-EGFP按感染复数(multiplicity of infection,MOI)为104、105、2×105、106转染N/N1003A细胞,倒置荧光显微镜下观察晶状体上皮细胞中EGFP的表达,流式细胞仪检测Raav2-EGFP对N/N1003A细胞的转染效率,MTT比色法测定Raav2载体对N/N1003A增生的影响.结果 EGFP在细胞中的表达呈绿色,弥漫于整个胞浆.rAAV2-EGFP的转染效率随着MOI的增加和时间的延长而增加,MOI=106时,转染后第7 d达到71.12%.MTT法显示各转染组与未转染组A值无统计学差异.结论　重组腺相关病毒载体可以介导EGFP基因高效稳定转染晶状体上皮细胞,并且对细胞增生无抑制作用,腺相关病毒作为后囊膜混浊基因治疗载体是可行的.     

人MRG15基因荧光真核表达载体的构建及其在培养人晶状体上皮细胞的表达

目的克隆人MRG15(MORF4-related gene on chromosome15)的编码基因，构建荧光表达载体，并检测其在培养人晶状体上皮细胞中的表达，为研究MRG15与年龄相关性白内障(age related cataract，ARC)的相关性奠定基础。方法用RT—PCR的方法从人ARC的晶状体前囊膜中扩增MRG15的编码基因，克隆至pMD18-T载体并测序，结果正确后亚克隆至荧光真核表达载体pEGFP—N2中，酶切鉴定正确后采用脂质体法，瞬时转染培养人晶状体上皮细胞，荧光显微镜下检测MRG15-pEGFP-N2的定位。结果测序证实．从人ARC晶状体前囊膜中扩增出的MRG15编码基因的序列及读框全部正确。重组质粒MRG15-pEGFP-N2经酶切后产生与理论预期长度相符的片段。脂质体法转染后24h．观察到其主要在培养人晶状体上皮细胞的细胞核中表达。结论克隆了人MRG15的编码基因，成功构建了荧光真核表达载体MRG15-pEGFP-N2，并可在培养人晶状体上皮细胞中表达。     

绿色荧光蛋白逆转录病毒及其介导的视网膜色素上皮细胞基因转移研究

目的 制备携带绿色荧光蛋白（green fluorescent protein,GFP)基因的逆转录病毒,感染原代与建株的人视网膜色素上皮（retinal pigment epithelium,RPE)细胞,评价逆转录病毒对人RPE细胞的感染能力,为视网膜病变的基因治疗奠定基础。方法 分离编码GFP的cDNA片段并插入逆转录病毒载体pLNCX,借助脂质体将重组逆转录病毒载体pLNCX-GFP转入单嗜性及双嗜性包装细胞,G418筛选抗性克隆,分离GFP表达水平最高的克隆;收集含病毒颗粒的上清液感染NIH3T3及体外培养的人原代与建株的RPE细胞。结果 重组逆转录病毒载体pLNCX-GFP转染各种包装细胞后,可产生GFP逆转录病毒。由双嗜性包装细胞产生的GFP逆转病毒能够感染原代与建株的人RPE细胞,GFP基因可持续在RPE细胞内表达。结论 逆转录病毒能够简便、快速、稳定将目的基因转入RPE细胞,可作为眼底病变基因治疗介导目的基因转移的重要工具。     

腺相关病毒介导绿色荧光蛋白基因对体外培养IPE细胞的转染及毒性

目的 研究腺相关病毒(recombinant adeno-associated virus,rAAV)载体介导的绿色荧光蛋白基因(green fluorescent protein，GFP)对体外培养虹膜色素上皮细胞(iris pigment epithelial，IPE)的转染和病毒载体对IPE细胞的毒性，为rAAV携带目的基因治疗视网膜色素变性提供理论依据。方法酶辅助的显微分离方法体外培养IPE细胞并鉴定。按转染倍数(multiple of infection,MOI)为10^3,10^4,10^5,10^6转染已培养3d的传二代IPE细胞，流式细胞仪检测rAAV—GFP对IPE的转染效率。MTT法检测rAAV—GFP对IPE细胞的毒性。结果 rAAV-GFP对IPE的转染效率，MOI=10^3时为26．7％、MOI=10^4时为53．6％、MOI=10^5时为60．2％、MOI=10^6时是63．7％。AAV—GFP转染IPE细胞后，细胞生长正常。MTT法显示各转染组与未转染组的OD值无统计学差异。结论 rAAV—GFP对体外培养的IPE细胞转染效率可达60％以上，而且腺相关病毒对细胞生长无明显抑制，腺相关病毒作为视网膜色素变性基因治疗的载体是安全可行的。     

hEGF在角膜中的基因表达

目的 观察人表皮生长因子(hEGF)基因对角膜细胞的转染情况，探讨用hEGF基因对角膜损伤治疗的可行性。方法 利用基因重组技术构建了hEGF全基因序列，将该基因序列插入pcDNA3．1真核表达高效穿梭质粒，用脂质体包裹注入家兔角膜前房。从RNA、DNA和蛋白质水平分别测定了角膜组织中hEGFmRNA和DNA的表达，用免疫组化法测定了hEGF在兔角膜组织中的表达。结果 基因转染24h后在全层角膜细胞内可见hEGF mRNA、DNA和蛋白质表达，第5d达到高峰，细胞转染率高达98％以上，随后逐渐降低，可持续表达20d以上。结论 用pcDNA3．1真核表达高效穿梭质粒作为载体，携带hEGF基因在脂质体包裹下经前房注射转染角膜，可达到极高的转染率，具有良好的应用前景。     

去整合素kistrin干预兔后发性白内障形成中晶状体上皮细胞FAK及ERK基因表达的研究

目的研究去整合素kistrin对兔后发性白内障模型晶状体上皮细胞中黏着斑激酶(focal adhesion kinases,FAK)mRNA和其下游因子细胞外信号调节激酶(extracellular signal regulated kinase,ERK)mRNA表达的影响,以探讨kistrin降低兔后发性白内障形成的可能分子机制。方法 8只新西兰大白兔均行右眼透明晶状体囊外摘出术(extracapsular lens extraction,ECLE)建立后发性白内障模型。随机将8只术眼分为实验组和对照组(每组4眼),术毕实验组囊袋内注入80μg.L-1kistrin0.2mL,对照组注入等量林格氏液。随机选4眼左眼为空白组。术后14d取残留的晶状体囊袋行RT-PCR检测FAK和ERK mRNA的表达。结果正常兔晶状体上皮细胞中(空白组)FAK和ERKmRNA表达量少,分别为1.476±0.802、0.576±0.035。ECLE术后14d对照组FAK和ERK mRNA表达量分别为6.496±1.501、1.158±0.573,实验组分别为6.060±1.512、1.262±0.329,2组表达量均较空白组显著性升高,差异均有统计学意义(均为P<0.05),实验组与对照组比较差异均无统计学意义(均为P>0.05)。结论兔后发性白内障形成的早期,FAK及ERK基因表达上调。应用kistrin未改变此两基因在转录水平上的表达。     

Biocompatibility of intraocular lens materials

PURPOSE OF REVIEW: To provide an update on currently available materials used in the manufacture of intraocular lenses, as well as new materials under development, especially with regard to their uveal and capsular biocompatibility. RECENT FINDINGS: The biocompatibility of intraocular lens materials should be assessed in terms of uveal biocompatibility, related to the inflammatory foreign-body reaction of the eye against the implant, as well as in terms of capsular biocompatibility, determined by the relationship of the intraocular lens with remaining lens epithelial cells within the capsular bag. This situation may result in different entities, e.g. anterior capsule opacification, interlenticular opacification (between piggyback intraocular lenses), posterior capsule opacification and lens epithelial cell ongrowth. Reports on intraocular lens opacification suggest that the potential to calcify should also be taken into consideration when evaluating the long-term biocompatibility of a new material. SUMMARY: Intraocular lenses are being progressively implanted in much earlier stages of life (refractive lens exchange, pediatric implantation) and are expected to remain in the intraocular environment for many decades. Materials used in intraocular lens manufacture should, therefore, insure long-term uveal and capsular biocompatibility, as well as ultimate transparency after implantation.     

Intercapsular cataract surgery with lens epithelial cell removal. Part III: Long-term follow-up of posterior capsular opacification

In 144 eyes in 144 patients with senile cataract the rate of posterior capsular opacification requiring YAG capsulotomy up to 36 months following intercapsular cataract surgery with lens epithelial cell removal using ultrasound and aspiration was evaluated and compared to the rate for 471 senile cataractous eyes in patients who had had posterior chamber lens implantation following phacoemulsification and extracapsular cataract extraction without lens epithelial cell removal. Posterior capsular opacification occurred in 3.7% of patients who had lens epithelial cell removal, significantly less (P less than .01) than the 10.8% found in the control group. Lens epithelial cell removal is considered an effective method of preventing capsular opacification.     

Osteopontin: a component of matrix in capsular opacification and subcapsular cataract

PURPOSE: To examine whether tissues of human capsular opacification and subcapsular cataract contain osteopontin, an adhesive matrix protein, and whether mouse lens epithelium expresses osteopontin after injury. METHODS: An immunohistochemical examination was conducted to determine whether matrices in human postoperative capsular specimens and anterior subcapsular cataract contain osteopontin. The spatial and temporal protein expression patterns of osteopontin were then determined in epithelium of a healing mouse lens after a capsular incision. RESULTS: Human lens epithelial cells in the specimens extracted at the time of vitrectomy 10 days after cataract surgery and also after longer healing intervals were labeled with an anti-osteopontin antibody, whereas uninjured lens epithelium was not. In the later healing phase, matrix of capsular opacification was positive for osteopontin. Lens cells amid anterior subcapsular cataract tissue were also positive. Osteopontin was detected in the cell surface and membrane and the cytoplasm of lens cells, as well as in the matrix. Unlike normal uninjured specimens, anterior lens capsule of some of the healing postoperative specimens and anterior subcapsular cataract specimens also faintly or weakly stained for osteopontin. Mouse lens epithelium started to express osteopontin protein at 8 hours after injury, before the cells changed their shape from epithelial cell type to fibroblast type. Expression of osteopontin lasted during the healing interval, even after the cells transformed into fibroblast-like cells. CONCLUSIONS: Extracellular matrix in human postoperative capsular opacification and anterior subcapsular cataract contains osteopontin. Epithelial cells of a mouse lens also ectopically express osteopontin in response to capsular injury.     

Epithelial downgrowth following wound dehiscence after extracapsular cataract extraction and posterior chamber lens implantation: surgical management

Epithelial downgrowth occurred along a fistulous (nonfiltering) tract containing an incarcerated anterior capsular flap after extracapsular cataract extraction and posterior chamber lens implantation complicated by wound dehiscence. Months later, a YAG posterior capsulotomy was performed before it was realized that posterior capsule opacification was associated with epithelial downgrowth involving the posterior capsule. Surgical management of epithelial downgrowth after extracapsular cataract extraction and posterior chamber lens implantation is discussed, with emphasis on the role of combined cryotherapy, dissection of the retrocorneal membrane, and complete removal of the capsular bag. Histopathologically, we found it difficult to differentiate lens epithelial cells from corneal epithelial downgrowth within the capsular bag, but monoclonal antibody for keratin may help identify corneal epithelial cells.     

碱性成纤维细胞生长因子基因在人晶状体上皮细胞内的表达

目的:为研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)基因在人晶状体上皮细胞(human lens epithelial cell,HLEC)内表达情况。方法:采用原位杂交方法,用cDNA探针检测HLEC内bFGF mRNA的表达,并用图像分析进行相对定量。结果:HLEC内可检测到bFGF mRNA的表达。结论:HLEC内存在bFGF基因表达。     

巨噬细胞对人工晶体植入术后兔眼晶体上皮细胞增生的影响

为探讨人工晶体植入术后晶体后囊混浊的机制，采用兔眼后房型人工晶体植入术后，术眼前房内注入巨噬细胞悬液的方法，用透射电镜观察术后不同时间晶体后囊形态学变化。结果表明：人工晶体植入术后１周，晶体后囊前即覆盖一层晶体上皮细胞，排列整齐，结构正常。观察至６个月，无明显变化。前房注入巨噬细胞组，１周亦可见晶体后囊覆盖一层上皮细胞，细胞内大量微丝形成；２周可见晶体上皮细胞周围胶原纤维形成；３个月可见晶体后囊前     

Collagens XII and XIV (FACITs) in capsular opacification and in cultured lens epithelial cells

PURPOSE: We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). METHODS: Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. RESULTS: In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. CONCLUSIONS: LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.     

Modification of the surface properties of a lens material to influence posterior capsular opacification

PURPOSE: To study the effect of surface properties of materials on cellular behaviour and the formation of posterior capsular opacification (PCO). METHODS: Polymethylmethacrylate, silicone and a hydrophobic acrylic were plasma treated and used in tissue culture. The changes in surface properties were quantified by dynamic contact angle measurements. Bovine lens epithelial cells (BLECs) were seeded onto these materials and cultured for 1 month. Serial photographs were taken. The cells were then fixed and stained to facilitate counting. RESULTS: Plasma treatment significantly increased the hydrophilicity of surfaces. BLECs grew on all surfaces but significantly more cells adhered to the treated than the untreated surfaces. On the untreated surfaces the BLECs had a fibroblastic morphology whereas on the treated surfaces the cells maintained their epithelial morphology. CONCLUSIONS: Posterior capsular opacification is a form of wound healing and the behaviour of lens epithelial cells is central to its progression. Emphasis has been on the elimination of residual lens epithelial cells to combat PCO. This study demonstrated that the phenotype of BLECs was influenced by the surface properties of the intraocular lens materials. Gas plasma treatment of the materials increased their hydrophilicity and allowed the adhered BLECs to maintain their normal epithelial morphology. We believe that controlled growth of lens epithelial cells may reduce the incidence of PCO.     

Intercapsular cataract surgery with lens epithelial cell removal. Part IV: Capsular fibrosis induced by poly(methyl methacrylate)

Characteristic lens epithelial cell behavior in the pseudophakic eye was examined by comparing 30 eyes that had extracapsular cataract surgery by the intercapsular technique and posterior chamber intraocular lens (IOL) implantation with lens epithelial cell removal but without anterior capsule capsulectomy and nine aphakic eyes that had the same procedure but without posterior chamber lens implantation over a mean follow-up period of 30 and 23 months, respectively. Fibrous anterior capsule opacification was observed in 83% of the pseudophakic eyes in the area of contact with the IOL, while the region beyond the margin of the IOL remained transparent. Fibrous anterior capsular opacification was not noted in the aphakic eyes. This suggests that the IOL material, poly(methyl methacrylate), stimulates lens epithelial cells to undergo fibrous metaplasia and to produce collagen fibers. Various cytokines such as IL-1 and TGF-beta synthesized by lens epithelial cells may play a crucial role as mediators in the process. We recommend that this effect be considered as a parameter of biocompatibility in developing and evaluating new biomaterials.