Proliferation and regeneration effects of constructed human beta-nerve growth factor gene on transfected cat corneal endothelial cells in vitro

OBJECTIVE: To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF) on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. METHODS: It was a experimental study. The human pcDNA4-beta-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene lipofectine transfection technique. The expression of the reporter gene pcDNA4-beta-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium (MTT) value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human beta-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. RESULTS: A human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF)was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. Effectene lipofectine mediated transfection efficiency of pcDNA4-beta-NGF into cat corneal endothelial cells in vitro was 11.3%. The human beta-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTT value and G1 stage cell numbers, as well as traumatically defected endothelial cells numbers during the healing process of human beta-NGF transfected corneal endothelial cells were statistically differed from the pre-transfected cells and control groups. CONCLUSIONS: Effectene lipofectine transfection technique could be effectively used for transfecting pcDNA4-beta-NGF into cat corneal endothelial cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat corneal endothelial cells. This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human beta-NGF gene into human corneal endothelial cells. Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.     

人β神经生长因子基因转染体外培养猫角膜内皮细胞促进分裂再生的研究

目的 探讨人β神经生长因子真核表达载体(pcDNA4-13-NGF)转染体外培养猫角膜内皮细胞并促进细胞分裂再生的机制,为将该基因应用于促进人角膜内皮细胞再生的研究打下基础.方法 为实验研究.通过EffecteneTM脂质体介导将自行构建并经测序证实的人pcDNA4-B-NGF转染到体外培养的猫角膜内皮细胞中.采用分组对照的方法研究转染前后猫角膜内皮细胞分裂再生能力.转基因后48 h通过逆转录聚合酶链反应(RT-PCR)和免疫组织化学染色方法分别在mRNA和蛋白水平检测人β神经生长因子(β-NGF)的表达.转基因后96 h采用细胞四甲基偶氮唑盐染色(MTT)测量吸光度值、细胞有丝分裂指数、流式细胞仪检测G1期细胞比例及细胞损伤后愈合面积测量等方法检测目的 基因对猫角膜内皮细胞增殖活性的作用.结果 EffecteneTM脂质体可有效介导重组真核表达载体peDNA4-β-NGF转染到经改良方法体外培养的猫角膜内皮细胞中,转染效率为11.3%,并促进该细胞表达β-NGF.正常对照组β-NGF/β-actin比值结果为3.14,加脂质体组为3.23,pcDNA4质粒转染组为3.21,pcDNA4-β-NGF重组质粒转染组为4.53.培养液、正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组平均A值分别为0.178±0.007、0.482±0.033、0.488±0.017、0.520±0.021及0.623±0.041.正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组细胞有丝分裂指数分别为3.3%、3.0%、3.1%及7.7%.正常对照组、加脂质体组、pcDNA4质粒转染组及pcDNA4-β-NGF重组质粒转染组G1期细胞比例分别为68.1%、51.6%、60.4%及87.9%.结论 人β-NGF基因可通过EffecteneTM脂质体介导有效转染到体外培养的猫角膜内皮细胞中,并促进细胞分裂再生,为进一步将此基因转入人角膜内皮细胞的研究提供实验经验.     

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B(PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CECs, which promotes the viability and proliferation of cells.     

Inhibition of corneal neovascularization by vascular endothelia growth inhibitor gene

AIM: To evaluate the effect of EffecteneTM lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV). METHODS: Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to establish the animal model and divided into 4 random group, ten per each group: group A: transfected by pcDNA4-VEGI gene mediated by EffecteneTM lipofectine transfection, group B: by Plasmid pcDNA4, group C: by EffecteneTM, and group D: by normal saline. Length and area of CNV were measured under slit lamp every day after transfection, immunohistochemistry was used to detected the expression of VEGI protein in cornea at 3, 7, 14 and 21 days. RESULTS: Average occurrence of CNV in the pcDNA4-VEGI gene transfected group (group A) was 6.3 days, in plasmid pcDNA4 control group (group B) was 3.1 days, in EffecteneTM lipofectine control group (group C) was 3.2 days, in normal saline control group (group D) was 3.2 days. Differences between groups A and B, C, D were statistically significant (P<0.01), while differences in groups B, C and D were meaningless (P>0.05). Lenth and average area of CNV in each period in group A was meaningful different from that in groups B, C, and D (P<0.01), while differences in group B, C and D were meaningless (P>0.05). Immunohistochemistry result: VEGI positive cells could be seen in epithelium, stroma, endothelium and the cliff of CNV in group A at 3 days after transfection. VEGI cells changed with the decrease of CNV. None positive cells were in the control groups (groups B, C and D) all the time. CONCLUSION: EffecteneTM lipofectine transfection technique can be effectively used in transfecting pcDNA4-VEGI gene into rabbit cornea and the lenth and areas of CNV can be inhibited by VEGI gene.     

Inhibition of corneal neovascularization by vascular endothelia growth inhibitor gene

AIM: To evaluate the effect of EffecteneTM lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV). METHODS: Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to induce CNV and divided into 4 random teams, ten per each team: team A: transfected by pcDNA4-VEGI gene mediated by EffecteneTM lipofectine transfection; team B: by plasmid pcDNA4; team C: by EffecteneTM, and team D: by normal saline. Length and area of CNV were observed under slit lamp every day after tran- sfection. Immunohistochemistry was performed to detect the expression of VEGI protein in corneas at day 3, 7, 14 and 21. RESULTS: 1) Average occurrence of CNV was 6.3 days in team A, 3.1 days in team B, 3.2 days in team C, and 3.2 days in team D. Difference was significant between A and other teams (P<0.01); 2) Length and average area of CNV in each period in team A was significantly different from those in team B, C and D (P<0.01); 3) VEGI expressions were observed in epithelium, stroma, endothelium and the cliff of CNV in team A at 3 days after transfection by immunohistochemical staining. None VEGI positive cells were found in the control teams (team B, C and D) all the time. CONCLUSION: EffecteneTM lipofectine transfection technique can effectively transfect pcDNA4-VEGI gene into rabbit cornea and the length and CNV areas can be inhibited by VEGI gene.     

VEGI基因转移抑制角膜新生血管的实验研究

目的:探讨以阳离子脂质体介导重组人VEGI基因转移对角膜新生血管(corneal neovascularization,CNV)的抑制作用。方法:角膜缝线法制作兔CNV模型,用结膜下注射的方法将阳离子脂质体包裹的VEGI重组质粒(pcDNA4-VEGI)转染入兔角膜,裂隙灯显微镜下观察记录各组兔CNV长出的时间、长度和数量,并分别于基因转染后3,7,14及21d以免疫组织化学方法观察VEGI基因表达情况,观察其对CNV的抑制作用。结果:基因转染组CNV平均出现时间为6.3d,对照组分别为3.1,3.2,3.2d不等,差异有统计学意义(F=39.838,P<0.01);基因转染后3d,转染组实验兔未出现CNV,对照组已有部分兔眼出现CNV;转染后第7d,转染组实验兔的CNV纤细,累及钟点数局限于缝线周围,对照组兔的CNV最长为2.9mm,血管密集;转染后第14d,转染组兔CNV最长达4.0mm,对照组新生血管最长达6.4mm,累及钟点数为3.2个;各时间段基因转染组CNV长度、平均面积与对照组相比,差异均有统计学意义(q=17.386,P<0.01)。免疫组织化学显示角膜上皮、基质、新生血管管壁细胞的VEGI表达阳性。各实验指标与对照组比较,差异有统计学意义(均P<0.05)。结论:VEGI基因对CNV有抑制作用。     

人血小板源性生长因子B链基因的克隆表达及其对猫角膜内皮细胞体外增殖的影响

目的构建血小板源性生长因子B（PDGF-B）原核表达载体,表达足量的PDGF-BB蛋白,作用于体外培养的猫角膜内皮细胞,观察角膜内皮细胞增殖情况。方法从健康剖宫产妇胎盘组织中提取总RNA,通过逆转录聚合酶链反应（RT-PCR）,扩增出PDGF-BcDNA,将其克隆至含T7启动子的质粒pET-28a（＋）中,构建表达质粒pET-PDGF-B,转化大肠杆菌BL21（DE3）,获得表达菌株BL-PDGF-B,进行PDGF-BB蛋白的表达,以Ni^2＋-NTA树脂对表达蛋白纯化、复性;将得到的PDGF-BB蛋白作用于体外培养的猫角膜内皮细胞,用MTY法分析其对细胞增殖的影响;通过倒置相差显微镜、透射电镜观察细胞形态变化以及细胞内部超微结构。结果经基因测序证明,成功构建出pET-PDGF-B质粒;凝胶自动扫描分析表明,PDGF-BB在BL21（DE3）大肠杆菌中得到了高效表达,表达的PDGF-B单体蛋白经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）,显示了1条特异蛋白带,相对分子质量约为27000;MTY法证明所表达的PDGF-BB蛋白可促进体外培养的猫角膜内皮细胞增殖。结论pET-PDGF-B原核表达载体的成功构建和PDGF-BB蛋白制备纯化为生产活性PDGF-BB蛋白及其进一步功能研究奠定了基础。PDGF-BB蛋白具有促进猫角膜内皮细胞增殖的作用。（中华眼科杂志,2006,42：415-419）     

人血管内皮细胞生长抑制因子基因转移抑制角膜新生血管的实验研究

目的 探讨以阳离子脂质体介导重组人pcDNA4-血管内皮细胞生长抑制因子(VEGI)基因转移对角膜新生血管的抑制作用.方法 实验研究.40只健康成年新西兰大白兔(40只眼),采用随机数字表法分为4组:A组(10只兔)为阳离子脂质体-重组人pcDNA4-VEGI基因转染组;B组(10只兔)为空白载体转染组;C组(10只兔)为阳离子脂质体转染组;D组(10只兔)为空白对照组.自行构建真核表达的重组人血管内皮细胞生长抑制因子(VEGI)基因,并检测重组基因的正确性;角膜缝线法制作兔角膜新生血管模型,用结膜下注射的方法将阳离子脂质体包裹的VEGI重组质粒(pcDNA4-VEGI)转染入兔角膜,裂隙灯显微镜下观察记录各组兔角膜新生血管长出的时间、角膜新生血管的长度和数量,并分别于基因转染后1、3、7、14及21 d以免疫组织化学方法观察VEGI基因表达情况,观察其对角膜新生血管的抑制作用.采用SPSS 10.0单因素方差分析行数据统计.结果 计算机自动测序证实成功构建真核表达的重组人VEGI基因;动物实验中基因转染组角膜新生血管平均出现时间为6.3 d,对照组分别为3.1、3.2、3.2 d不等,差异有统计学意义(F=39.838,P=0.00);基因转染后第3天,转染组实验兔未出现角膜新生血管,对照组已有部分兔眼出现角膜新生血管;转染后第7天,转染组实验兔的角膜新生血管纤细,累及钟点数局限于缝线周围,对照组兔的角膜新生血管最长为2.9 mm,血管密集;转染后第14天,转染组兔角膜新生血管最长达4.0 mm,对照组新生血管最长达6.4 mm,累及钟点数为3.2个;各时间段基因转染组角膜新生血管长度、平均面积与对照组相比,差异均有统计学意义.A组角膜新生血管长度与B组相比,第7、14、21天q值分别为17.386、20.944、8.892,P值均＜0.01;与C组相比,第7、14、21天q值分别为19.488、19.795、7.483,P值均＜0.01;与D组相比,第7、14、21天q值分别为19.583、20.413、8.941,P值均＜0.01.A组角膜新生血管面积与B组相比,第7、14、21天q值分别为30.238、57.820、35.543,P值均＜0.01;与C组相比,第7、14、21天q值分别为32.607、57.843、36.653,P值均＜0.01;与D组相比,第7、14、21天q值分别为33.873、57.590、34.724,P值均＜0.01.免疫组织化学显示角膜上皮、基质、新生血管管壁细胞的VEGI表达阳性.结论 利用人工合成引物和聚合酶链反应方法可以将连接于原核表达载体的VEGI基因切下并连接于真核表达载体;阳离子脂质体能够介导重组人VEGI基因转染入角膜组织并使其分泌VEGI蛋白,对角膜新生血管有抑制作用.     

体外培养表达人β-NGF基因的猫角膜内皮细胞

目的:体外培养表达人β-神经生长因子基因(β-nerve growth factor,β-NGF)的工程化猫角膜内皮细胞,为进一步猫角膜内皮细胞的移植做准备。方法:将人β-NGF重组真核表达载体pcDNA4-β-NGF,通过EffecteneTM脂质体介导转染到体外培养的猫角膜内皮细胞中,选择性培养基筛选转基因后细胞,人NGF单克隆抗体行细胞免疫组织化学染色(immunohistochemistry)。结果:选择性培养基培养2wk后筛选出独立细胞克隆,该细胞高表达人β-NGF基因。结论:转基因方法可体外培养出表达人β-NGF基因的工程化猫角膜内皮细胞。     

Studies on the expression of mRNA for anion transport related proteins in corneal endothelial cells

PURPOSE: Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium. METHODS: RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing. RESULTS: RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium. CONCLUSIONS: CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.     

Regulation of bovine corneal endothelial cell cycle by transforming growth factor-beta

PURPOSE: The transforming growth factor-beta (TGF-beta) family includes three multifunctional proteins, TGF-beta1, TGF-beta2 and TGF-beta3, expressed in ocular tissue, which are involved in regulating cell differentiation, cell proliferation and other cell functions. TGF-beta is present in aqueous humour and regulates corneal endothelial cells. This study explores the mechanism by which TGF-beta regulates the cell cycle in cultured corneal endothelial cells. METHODS: The expression of specific receptors for the TGF-beta family was investigated at the protein level by affinity cross-linking with radio-iodinated TGF-beta1 and immunoprecipitation with specific antibodies to TGF-beta receptors. Regulation of entry into the S-phase of the cell cycle was determined by 5-bromo-2' deoxyuridine (BrdU) incorporation into the cells. The signal transduction pathways were investigated using various blocking agents for protein kinase transducers involved in intracytoplasmic signal transduction. RESULTS: Cultured bovine corneal endothelial cells were confirmed to express TGF-beta type 1 and type 2 receptors and endoglin. In the confluent state, TGF-beta1 and TGF-beta2 stimulated the cells to progress to the S-phase of the cell cycle through platelet-derived growth factor-B (PDGF-B) chain production and protein kinase C. CONCLUSIONS: TGF-beta accelerated cell cycle progression from the G0/G1 phase to the S-phase in cultured corneal endothelial cells, under our experimental conditions, through pathways involving protein kinase C. These pathways are related to the cross-talk between TGF-beta and other cytokines. The conditions employed in the present experiments may be useful for investigating the complex cross-talk between various cytokines and growth factors.     

小鼠TGFBI基因真核表达载体的构建和表达

目的构建小鼠TGFBI基因的真核表达载体,为研究角膜营养不良的发病机制奠定基础。方法提取BALB/cBy小鼠正常角膜组织总RNA,经反转录-PCR合成TGFBI cDNA,克隆入真核表达载体pcDNA3.1并测序验证。用不同剂量重组质粒pcDNA3.1-TGFBI转染NIH3T3细胞,通过SYBR荧光实时定量PCR和Western blot检测TGFBI在细胞中的表达。结果测序结果显示扩增到的TGFBI cDNA以正确序列和方式插入载体,实时定量PCR和Western blot结果显示TGFBI在NIH3T3细胞中表达增强。结论成功构建了小鼠TGFBI基因真核表达载体,并在细胞中进行表达,为进一步研究TGFBI在角膜内的生理、病理功能奠定基础。     

角膜内皮细胞高压力损伤机制的实验研究

目的探讨青光眼急性高眼压对角膜内皮细胞的损伤机制。设计实验性研究。研究对象体外培养的角膜内皮细胞。方法采用后弹力层撕除联合酶消化法获取角膜内皮细胞,免疫组化法鉴定细胞。实验分两组：A组：急性压力增高组,压力为6．67kPa;B组：压力仿生培养,压力为2．0kPa。倒置显微镜定期观察细胞形态及生长规律;HE染色观察细胞形态结构变化;台盼兰一茜素红染色观察高压对细胞的损伤作用;流式细胞术分析细胞活性;免疫荧光检测细胞胞浆中细胞色素C（CytC）的表达。主要指标角膜内皮细胞形态结构、凋亡率及胞浆中CytC的表达。结果获取的细胞经免疫法证实为角膜内皮细胞表型。两组细胞分别培养24hr后,流式细胞术分析显示,高压力组的早、晚期细胞凋亡率分别为（16．40±0．95）％和（41．37±1．29）％;而正常压力组早、晚期细胞凋亡率分别为（1．07±0．40）％和（0．70±0．00）％,差异有统计学意义（P=0．000）。免疫荧光检测到高压力组角膜内皮细胞胞浆CytC呈阳性表达。结论高压力对角膜内皮细胞损伤呈时间敏感性,细胞的凋亡启动是其角膜内皮细胞损伤的机制之一。f眼科,2011,20：155-159）     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.     

Morphometric analysis of corneal endothelial giant cells in normal and traumatized corneas

Corneal endothelial cells from normal and traumatized human, primate, cat and rabbit eyes were studied by specular microscopy. Morphometric analysis was performed on micrographs of corneal endothelium using a semi-automated image analysis system. The results showed that under normal conditions the corneal endothelium of all four species exhibit major morphological similarities (mean cell areas: human 317 ± 32 μm², primate 246 ± 22 μm², cat 357 ± 25 μm², rabbit 308 ± 35 μm²). The normal corneal endothelium in man was found to be more polymegethous than that of the other species. Trauma to cat, primate and human corneas resulted in a long-term reduction in endothelial cell density and enhanced polymegethism. In contrast, the reparative response of the rabbit ensured the reformation of an essentially normal monolayer following injury. Endothelial giant cells were a normal inclusion in the rabbit corneal endothelium but were only significant in cat, primate and man following trauma. The presence of corneal endothelial giant cells in amitotic corneas may therefore represent a compensatory response in the absence of mitotic potential.