IGF-1调控β-catenin信号参与糖尿病角膜上皮细胞创伤愈合的机理
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摘要
第一部分：
IGF-1在高糖培养的角膜上皮细胞创伤愈合中的作用研究
目的：
研究高糖培养的人永生化角膜上皮细胞(Human telomerase-immortalized corneal epithelial cells, THCE)及糖尿病大鼠角膜上皮中,高糖对创伤刺激的IGF-1及IGF-1R表达的影响,探讨外源性IGF-1对高糖培养的THCE细胞增殖、迁移、凋亡的影响及相关分子机制,阐明IGF-1在高糖培养的角膜上皮细胞创伤愈合中的作用。
方法：
1.根据文献报道的THCE高糖干预模型,分别用5mM-45mM浓度的葡萄糖处理THCE细胞24h或48h,MTT法检测THCE细胞存活率,筛选合适的高糖浓度及作用时间。根据文献报道的外源性IGF-1干预浓度,分别用10-200ng/ml的IGF-1处理THCE细胞48h,MTT法检测THCE细胞存活率,筛选合适的IGF-1浓度。
2.分别用5mM、25mM浓度的葡萄糖预处理THCE细胞48h,经生长因子饥饿12h后,收集未划伤及划伤后不同时间点的两组细胞及细胞培养上清,利用real-time PCR法检测IGF-1及IGF-1R mRNA表达,酶联免疫吸附试验(ELISA)测定IGF-1蛋白水平,western blot分析IGF-1R蛋白表达,免疫荧光细胞化学法检测IGF-1、IGF-1R蛋白分布及表达。
3.比较正常及糖尿病大鼠角膜上皮创伤愈合情况,研究创伤后角膜上皮中IGF-1、IGF-1R mRNA及蛋白的表达。
4.高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1(50ng/ml)。于刺激后不同时间点分别对5mM葡萄糖、25mmM葡萄糖、25mmM葡萄糖+IGF-1处理的三组细胞,利用MTT法检测细胞增殖能力；细胞划痕实验检测细胞迁移能力；Annexin V-FITC/PI双荧光染色法+流式细胞仪检测细胞凋亡。
5.高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1(50ng/ml)。于刺激后不同时间点分别收集5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+IGF-1处理的三组细胞,利用real-time PCR法、western blot及免疫荧光细胞化学法检测增殖、迁移及凋亡相关因子ki-67、MMP-2、bax、bcl-2mRNA及蛋白的表达。
结果：
1.制作高糖干预模型的合适条件为25mM浓度的葡萄糖处理THCE细胞48h。外源性添加IGF-1的合适浓度为50ng/ml。
2.在正常或高糖培养的两组THCE细胞中,划伤可刺激IGF-1及IGF-1R mRNA表达上调；划伤后12及24h,高糖组IGF-1及IGF-1R mRNA表达显著低于正常组；划伤后24及48h,高糖组IGF-1及IGF-1R蛋白表达显著低于正常组；划伤后48h,高糖组IGF-1及IGF-1R荧光染色强度低于正常组。
3.与正常大鼠相比,糖尿病大鼠角膜上皮创伤愈合显著延迟。伤后1,2,3天,糖尿病大鼠角膜上皮中IGF-1、IGF-1R mRNA表达显著低于正常大鼠。伤后2,3天,糖尿病大鼠角膜上皮中IGF-1、IGF-1R荧光染色强度低于正常大鼠。
4.高糖抑制THCE细胞存活率,下调增殖相关因子ki-67mRNA及蛋白表达。高糖+IGF-1组的细胞存活率、ki-67mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
5.高糖抑制THCE细胞迁移能力,下调迁移相关因子MMP-2mRNA及蛋白表达。高糖+IGF-1组的细胞迁移能力、MMP-2mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
6.高糖诱导THCE细胞凋亡,上调促凋亡因子bax mRNA及蛋白表达,下调凋亡抑制因子bcl-2mRNA及蛋白表达。添加IGF-1可改善高糖诱导的细胞凋亡及bax、bcl-2表达变化,但与正常组相比,仍存在显著差异。
结论：
在THCE细胞及糖尿病大鼠角膜上皮中,高糖抑制创伤刺激的IGF-1及IGF-1R表达。高糖通过抑制IGF-1表达,引起ki-67、MMP-2、bcl-2表达下调、bax表达上调,从而降低THCE细胞增殖、迁移,诱导THCE细胞凋亡,这可能是导致角膜上皮细胞创伤愈合延迟的原因之一。
第二部分：
β-catenin信号在高糖培养的角膜上皮细胞创伤愈合中的作用研究
目的：
研究高糖培养的THCE划伤后β-catenin信号的表达,探讨激活β-catenin信号对高糖培养的THCE细胞增殖、迁移、凋亡的影响及相关分子机制,阐明β-catenin信号在高糖培养的角膜上皮细胞创伤愈合中的作用。
方法：
1.高糖预处理THCE细胞且生长因子饥饿后,外源性添加β-catenin信号激活剂Licl(0.5mM)。对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,进行划伤刺激,于不同时间点利用real-time PCR法检测P-catenin、 cyclinD1、c-myc mRNA表达,western blot分析β-catenin、cyclin D1、c-Myc蛋白表达。
2.高糖预处理THCE细胞且生长因子饥饿后,外源性添加0.5mM Licl。于刺激后不同时间点分别对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,利用MTT法检测细胞增殖能力；细胞划痕实验检测细胞迁移能力；Annexin V-FITC/PI双荧光染色法+流式细胞仪检测细胞凋亡。
3.高糖预处理THCE细胞且生长因子饥饿后,外源性添加0.5mM Licl。于刺激后不同时间点分别收集5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,利用real-time PCR法、western blot及免疫荧光细胞化学法检测增殖、迁移及凋亡相关因子ki-67、MMP-2、bax、bcl-2mRNA及蛋白的表达。
结果：
1.高糖抑制P-catenin cyclin D1、c-Myc mRNA及蛋白表达,外源性添加Licl可上调P-catenin、cyclin D1、c-Myc mRNA及蛋白表达,激活P-catenin信号。
2.高糖抑制THCE细胞存活率,下调增殖相关因子ki-67mRNA及蛋白表达。高糖+Licl组的细胞存活率、ki-67mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
3.高糖抑制THCE细胞迁移能力,下调迁移相关因子MMP-2mRNA及蛋白表达。高糖+Licl组的细胞迁移能力、MMP-2mRNA及蛋白表达显著高于高糖组,但与正常组相比,仍存在差异。
4.高糖诱导THCE细胞凋亡,上调促凋亡因子bax mRNA及蛋白表达,下调凋亡抑制因子bcl-2mRNA及蛋白表达。添加Lic1可改善高糖诱导的细胞凋亡及bax、bcl-2表达变化,但与正常组相比,存在显著差异。
结论：
在THCE细胞中,高糖通过抑制β-catenin及下游靶基因cyclin D1、c-Myc的表达,引起ki-67、MMP-2、bcl-2表达下调、bax表达上调,从而降低THCE细胞增殖、迁移,诱导THCE细胞凋亡,这可能是导致角膜上皮细胞创伤愈合延迟的原因之一。
第三部分：
IGF-1调控β-catenin信号参与高糖培养的角膜上皮细胞创伤愈合的研究
目的：
研究高糖培养的THCE划伤后,外源性IGF-1对P-catenin信号表达的影响,明确IGF-1是否通过调控β-catenin信号共同参与高糖损害的角膜上皮细胞创伤愈合。
方法：
高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1。对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+IGF-1处理的三组细胞,进行划伤刺激,于不同时间点利用real-time PCR法检测P-catenin cyclin D1mRNA表达,western blot分析β-catenin、cyclin D1蛋白表达。
结果：
高糖抑制β-catenin、cyclin D1mRNA及蛋白表达,外源性添加IGF-1可显著上调高糖抑制的β-catenin、cyclin D1mRNA及蛋白表达。
结论：
IGF-1可调控β-catenin信号共同参与糖尿病角膜上皮创伤愈合。
Part I:
Role of IGF-1in high-glucose impaired corneal epithelial cells wound healing
Purpose
To investigate the expression and role of insulin-like growth factor-1(IGF-1) in high glucose-impaired corneal epithelial cells wound healing.
Methods
1. THCE cells were treated with glucose solutions (5mM-45mM) for24h or48h and cell viability was measured by MTT to determine the best concentration of glucose and time. THCE cells were treated with IGF-1(10-200ng/ml) for48h and cell viability was measured by MTT to determine the best concentration of IGF-1.
2. Respectively, with a concentration of5mM or25mM D-glucose treatment THCE cells for48h, then cells were growth factors starved for12h. After wounding, cells were harvested and real-time PCR, ELISA, western blot and immunofluorescence staining were performed to detect IGF-1and IGF-1R mRNA and protein expression.
3. In wounded corneal epithelium of streptozotocin induced diabetic rats, IGF-1and IGF-1R mRNA and protein expression were measured by real-time PCR and immunofluorescence.
4. THCE cells were pretreated with25mM D-glucose. For experiments,50ng/ml IGF-1was added into high glucose medium. Then, three groups of cells were formed: cells grown in normal glucose medium, high glucose medium, or high glucose medium plus50ng/ml IGF-1. The effects of IGF-1in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining.
5. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.
Results
1. For experiments, the proper concentration was25mM glucose or50ng/ml IGF-1.
2. After wounding, both IGF-1and IGF-1R mRNA and protein expression were increased. At12and24h, the expressions were significantly lower in high glucose group than that in normal group. As for IGF-1and IGF-1R protein, the expressions were significantly lower in high glucose group than that in normal group at24and48h.
3. Delayed wound healing was observed in wounded corneal epithelium of diabetic rats which were accompanied with reduced IGF-1and IGF-1R mRNA and protein expression.
4. High glucose reduced THCE cells proliferation and migration, induced cell apoptosis via downregulating ki-67, MMP-2, bcl-2mRNA and protein expression, upregulating bax expression. Treatment with IGF-1could reverse these high glucose effects.
Conclusions
High glucose-inhibited IGF-1/IGF-1R expression caused THCE cell dysregulation including decreased cell proliferation, migration and increased cell apoptosis, which might result, at least in part, in delayed corneal epithelial cell wound healing.
Part Ⅱ:
Role of β-catenin Signaling in high-glucose impaired corneal epithelial cells wound healing
Purpose
To investigate the expression and role of P-catenin signaling in high glucose-impaired corneal epithelial cells wound healing.
Methods
1. THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, real-time PCR and western blot were performed to detect J3-catenin> cyclin D1、c-Myc mRNA and protein expression.
2. THCE cells were pretreated with25mM D-glucose and growth factors starved. For experiments,0.5mM Licl was added into high glucose medium. Then, three groups of cells were formed:cells grown in normal glucose medium, high glucose medium, or high glucose medium plus0.5mM Licl. The effects of Licl in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining.
3. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.
Results
High glucose downregulated β-catenin、cyclin D1、c-Myc mRNA and protein expression while Licl upregulated these factors expression in wounded THCE cells cultured in high glucose. Licl improved high glucose-impaired THCE cells proliferation, migration and apoptosis via upregulating ki-67, MMP-2,'bcl-2and downregulating bax mRNA and protein expression.
Conclusions
High glucose decreased THCE cell proliferation, migration and increased cell apoptosis through inhibited β-catenin signaling.
Part Ⅲ:
IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cells wound healing
Purpose
To investigate whether IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cell wound healing.
Methods
THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, IGF-1was added into high glucose medium. Real-time PCR and western blot were performed to detect β-catenin, cyclin D1mRNA and protein expression.
Results
The addition of IGF-1significantly increased β-catenin、cyclin D1mRNA and protein expression compaired with high glucose-treated control.
Conclusions
IGF-1mediated β-catenin signaling to modulate high glucose-impaired corneal epithelial cell wound healing.
IGF-1在高糖培养的角膜上皮细胞创伤愈合中的作用研究
目的：
研究高糖培养的人永生化角膜上皮细胞(Human telomerase-immortalized corneal epithelial cells, THCE)及糖尿病大鼠角膜上皮中,高糖对创伤刺激的IGF-1及IGF-1R表达的影响,探讨外源性IGF-1对高糖培养的THCE细胞增殖、迁移、凋亡的影响及相关分子机制,阐明IGF-1在高糖培养的角膜上皮细胞创伤愈合中的作用。
方法：
1.根据文献报道的THCE高糖干预模型,分别用5mM-45mM浓度的葡萄糖处理THCE细胞24h或48h,MTT法检测THCE细胞存活率,筛选合适的高糖浓度及作用时间。根据文献报道的外源性IGF-1干预浓度,分别用10-200ng/ml的IGF-1处理THCE细胞48h,MTT法检测THCE细胞存活率,筛选合适的IGF-1浓度。
2.分别用5mM、25mM浓度的葡萄糖预处理THCE细胞48h,经生长因子饥饿12h后,收集未划伤及划伤后不同时间点的两组细胞及细胞培养上清,利用real-time PCR法检测IGF-1及IGF-1R mRNA表达,酶联免疫吸附试验(ELISA)测定IGF-1蛋白水平,western blot分析IGF-1R蛋白表达,免疫荧光细胞化学法检测IGF-1、IGF-1R蛋白分布及表达。
3.比较正常及糖尿病大鼠角膜上皮创伤愈合情况,研究创伤后角膜上皮中IGF-1、IGF-1R mRNA及蛋白的表达。
4.高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1(50ng/ml)。于刺激后不同时间点分别对5mM葡萄糖、25mmM葡萄糖、25mmM葡萄糖+IGF-1处理的三组细胞,利用MTT法检测细胞增殖能力；细胞划痕实验检测细胞迁移能力；Annexin V-FITC/PI双荧光染色法+流式细胞仪检测细胞凋亡。
5.高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1(50ng/ml)。于刺激后不同时间点分别收集5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+IGF-1处理的三组细胞,利用real-time PCR法、western blot及免疫荧光细胞化学法检测增殖、迁移及凋亡相关因子ki-67、MMP-2、bax、bcl-2mRNA及蛋白的表达。
结果：
1.制作高糖干预模型的合适条件为25mM浓度的葡萄糖处理THCE细胞48h。外源性添加IGF-1的合适浓度为50ng/ml。
2.在正常或高糖培养的两组THCE细胞中,划伤可刺激IGF-1及IGF-1R mRNA表达上调；划伤后12及24h,高糖组IGF-1及IGF-1R mRNA表达显著低于正常组；划伤后24及48h,高糖组IGF-1及IGF-1R蛋白表达显著低于正常组；划伤后48h,高糖组IGF-1及IGF-1R荧光染色强度低于正常组。
3.与正常大鼠相比,糖尿病大鼠角膜上皮创伤愈合显著延迟。伤后1,2,3天,糖尿病大鼠角膜上皮中IGF-1、IGF-1R mRNA表达显著低于正常大鼠。伤后2,3天,糖尿病大鼠角膜上皮中IGF-1、IGF-1R荧光染色强度低于正常大鼠。
4.高糖抑制THCE细胞存活率,下调增殖相关因子ki-67mRNA及蛋白表达。高糖+IGF-1组的细胞存活率、ki-67mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
5.高糖抑制THCE细胞迁移能力,下调迁移相关因子MMP-2mRNA及蛋白表达。高糖+IGF-1组的细胞迁移能力、MMP-2mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
6.高糖诱导THCE细胞凋亡,上调促凋亡因子bax mRNA及蛋白表达,下调凋亡抑制因子bcl-2mRNA及蛋白表达。添加IGF-1可改善高糖诱导的细胞凋亡及bax、bcl-2表达变化,但与正常组相比,仍存在显著差异。
结论：
在THCE细胞及糖尿病大鼠角膜上皮中,高糖抑制创伤刺激的IGF-1及IGF-1R表达。高糖通过抑制IGF-1表达,引起ki-67、MMP-2、bcl-2表达下调、bax表达上调,从而降低THCE细胞增殖、迁移,诱导THCE细胞凋亡,这可能是导致角膜上皮细胞创伤愈合延迟的原因之一。
第二部分：
β-catenin信号在高糖培养的角膜上皮细胞创伤愈合中的作用研究
目的：
研究高糖培养的THCE划伤后β-catenin信号的表达,探讨激活β-catenin信号对高糖培养的THCE细胞增殖、迁移、凋亡的影响及相关分子机制,阐明β-catenin信号在高糖培养的角膜上皮细胞创伤愈合中的作用。
方法：
1.高糖预处理THCE细胞且生长因子饥饿后,外源性添加β-catenin信号激活剂Licl(0.5mM)。对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,进行划伤刺激,于不同时间点利用real-time PCR法检测P-catenin、 cyclinD1、c-myc mRNA表达,western blot分析β-catenin、cyclin D1、c-Myc蛋白表达。
2.高糖预处理THCE细胞且生长因子饥饿后,外源性添加0.5mM Licl。于刺激后不同时间点分别对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,利用MTT法检测细胞增殖能力；细胞划痕实验检测细胞迁移能力；Annexin V-FITC/PI双荧光染色法+流式细胞仪检测细胞凋亡。
3.高糖预处理THCE细胞且生长因子饥饿后,外源性添加0.5mM Licl。于刺激后不同时间点分别收集5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+Licl处理的三组细胞,利用real-time PCR法、western blot及免疫荧光细胞化学法检测增殖、迁移及凋亡相关因子ki-67、MMP-2、bax、bcl-2mRNA及蛋白的表达。
结果：
1.高糖抑制P-catenin cyclin D1、c-Myc mRNA及蛋白表达,外源性添加Licl可上调P-catenin、cyclin D1、c-Myc mRNA及蛋白表达,激活P-catenin信号。
2.高糖抑制THCE细胞存活率,下调增殖相关因子ki-67mRNA及蛋白表达。高糖+Licl组的细胞存活率、ki-67mRNA及蛋白表达显著高于高糖组,与正常组无显著差异。
3.高糖抑制THCE细胞迁移能力,下调迁移相关因子MMP-2mRNA及蛋白表达。高糖+Licl组的细胞迁移能力、MMP-2mRNA及蛋白表达显著高于高糖组,但与正常组相比,仍存在差异。
4.高糖诱导THCE细胞凋亡,上调促凋亡因子bax mRNA及蛋白表达,下调凋亡抑制因子bcl-2mRNA及蛋白表达。添加Lic1可改善高糖诱导的细胞凋亡及bax、bcl-2表达变化,但与正常组相比,存在显著差异。
结论：
在THCE细胞中,高糖通过抑制β-catenin及下游靶基因cyclin D1、c-Myc的表达,引起ki-67、MMP-2、bcl-2表达下调、bax表达上调,从而降低THCE细胞增殖、迁移,诱导THCE细胞凋亡,这可能是导致角膜上皮细胞创伤愈合延迟的原因之一。
第三部分：
IGF-1调控β-catenin信号参与高糖培养的角膜上皮细胞创伤愈合的研究
目的：
研究高糖培养的THCE划伤后,外源性IGF-1对P-catenin信号表达的影响,明确IGF-1是否通过调控β-catenin信号共同参与高糖损害的角膜上皮细胞创伤愈合。
方法：
高糖预处理THCE细胞且生长因子饥饿后,外源性添加IGF-1。对5mM葡萄糖、25mM葡萄糖、25mM葡萄糖+IGF-1处理的三组细胞,进行划伤刺激,于不同时间点利用real-time PCR法检测P-catenin cyclin D1mRNA表达,western blot分析β-catenin、cyclin D1蛋白表达。
结果：
高糖抑制β-catenin、cyclin D1mRNA及蛋白表达,外源性添加IGF-1可显著上调高糖抑制的β-catenin、cyclin D1mRNA及蛋白表达。
结论：
IGF-1可调控β-catenin信号共同参与糖尿病角膜上皮创伤愈合。
Part I:
Role of IGF-1in high-glucose impaired corneal epithelial cells wound healing
Purpose
To investigate the expression and role of insulin-like growth factor-1(IGF-1) in high glucose-impaired corneal epithelial cells wound healing.
Methods
1. THCE cells were treated with glucose solutions (5mM-45mM) for24h or48h and cell viability was measured by MTT to determine the best concentration of glucose and time. THCE cells were treated with IGF-1(10-200ng/ml) for48h and cell viability was measured by MTT to determine the best concentration of IGF-1.
2. Respectively, with a concentration of5mM or25mM D-glucose treatment THCE cells for48h, then cells were growth factors starved for12h. After wounding, cells were harvested and real-time PCR, ELISA, western blot and immunofluorescence staining were performed to detect IGF-1and IGF-1R mRNA and protein expression.
3. In wounded corneal epithelium of streptozotocin induced diabetic rats, IGF-1and IGF-1R mRNA and protein expression were measured by real-time PCR and immunofluorescence.
4. THCE cells were pretreated with25mM D-glucose. For experiments,50ng/ml IGF-1was added into high glucose medium. Then, three groups of cells were formed: cells grown in normal glucose medium, high glucose medium, or high glucose medium plus50ng/ml IGF-1. The effects of IGF-1in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining.
5. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.
Results
1. For experiments, the proper concentration was25mM glucose or50ng/ml IGF-1.
2. After wounding, both IGF-1and IGF-1R mRNA and protein expression were increased. At12and24h, the expressions were significantly lower in high glucose group than that in normal group. As for IGF-1and IGF-1R protein, the expressions were significantly lower in high glucose group than that in normal group at24and48h.
3. Delayed wound healing was observed in wounded corneal epithelium of diabetic rats which were accompanied with reduced IGF-1and IGF-1R mRNA and protein expression.
4. High glucose reduced THCE cells proliferation and migration, induced cell apoptosis via downregulating ki-67, MMP-2, bcl-2mRNA and protein expression, upregulating bax expression. Treatment with IGF-1could reverse these high glucose effects.
Conclusions
High glucose-inhibited IGF-1/IGF-1R expression caused THCE cell dysregulation including decreased cell proliferation, migration and increased cell apoptosis, which might result, at least in part, in delayed corneal epithelial cell wound healing.
Part Ⅱ:
Role of β-catenin Signaling in high-glucose impaired corneal epithelial cells wound healing
Purpose
To investigate the expression and role of P-catenin signaling in high glucose-impaired corneal epithelial cells wound healing.
Methods
1. THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, real-time PCR and western blot were performed to detect J3-catenin> cyclin D1、c-Myc mRNA and protein expression.
2. THCE cells were pretreated with25mM D-glucose and growth factors starved. For experiments,0.5mM Licl was added into high glucose medium. Then, three groups of cells were formed:cells grown in normal glucose medium, high glucose medium, or high glucose medium plus0.5mM Licl. The effects of Licl in the presence of high glucose on cell proliferation, migration and apoptosis were evaluated by MTT, cell restitution assay and flow cytometry after PI-Annexin-V staining.
3. The possible molecular mechanisms were detected by the levels of ki-67, MMP-2, bax, and bcl-2expression using real-time PCR, immunohistochemistry or western blot.
Results
High glucose downregulated β-catenin、cyclin D1、c-Myc mRNA and protein expression while Licl upregulated these factors expression in wounded THCE cells cultured in high glucose. Licl improved high glucose-impaired THCE cells proliferation, migration and apoptosis via upregulating ki-67, MMP-2,'bcl-2and downregulating bax mRNA and protein expression.
Conclusions
High glucose decreased THCE cell proliferation, migration and increased cell apoptosis through inhibited β-catenin signaling.
Part Ⅲ:
IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cells wound healing
Purpose
To investigate whether IGF-1mediated β-catenin signaling in high glucose-impaired corneal epithelial cell wound healing.
Methods
THCE cells were pretreated with25mM D-glucose and growth factors starved. After wounding, IGF-1was added into high glucose medium. Real-time PCR and western blot were performed to detect β-catenin, cyclin D1mRNA and protein expression.
Results
The addition of IGF-1significantly increased β-catenin、cyclin D1mRNA and protein expression compaired with high glucose-treated control.
Conclusions
IGF-1mediated β-catenin signaling to modulate high glucose-impaired corneal epithelial cell wound healing.
引文
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