先天性热休克转录因子4基因突变lop11小鼠白内障形成的分子机制

摘要

背景 研究证实热休克转录因子4(HSF4)基因突变是造成先天性常染色体隐性遗传白内障小鼠——晶状体混浊11 (lop 11)小鼠白内障的主要原因,HSF4基因突变导致lop11小鼠白内障形成的分子机制是眼科学界关注的问题.目的 探讨HSF4基因突变导致lop11小鼠白内障形成的分子机制.方法 本研究选取lop11小鼠及野生型C57BL/6小鼠各24只,分别于出生后1、7、14d两种小鼠各取8只用CO2吸入法处死,制备晶状体切片,用常规组织病理学法观察lop11小鼠出生后不同时间晶状体的病理改变,计数晶状体上皮细胞(LECs)的数量；采用Western blot法检测1日龄lop11小鼠及野生型C57BL/6小鼠晶状体中αB-晶状体蛋白(CRYAB)的含量；用定量PCR(q-PCR)法分析两种小鼠晶状体中成纤维细胞生长因子(FGF)的表达.两种小鼠上述指标检测结果的差异比较采用独立样本t检验.实验动物的喂养和使用遵循ARVO声明.结果 出生后1d的野生型C57BL/6小鼠LECs和晶状体纤维形态及排列规则,但lop11小鼠LECs增生明显,形态不规则；出生后7d的lop11小鼠晶状体纤维排列紊乱,可见空泡样变性.出生后1、7、14d的lop11小鼠每张切片上LECs数分别为(417±19)、(467±16)、(489±21)个,均明显多于同时间点野生型G57BL/6小鼠的(378±13)、(391±9)、(395±7)个,差异均有统计学意义(1 d:t=6.696,P=0.000;7 d:t=6.578,P=0.000;14 d:t=7.240,P=0.000).出生后1d的lop11小鼠晶状体中CRYAB蛋白表达较野生型G57BL/6小鼠明显减弱,而晶状体中FGF-1、FGF-4、FGF-7 mRNA的相对表达量(表达倍数)分别为2.04±0.13、2.03±0.08和4.59±0.12,野生型C57BL/6小鼠相对表达量(表达倍数)分别为1.04±0.06、0.97±0.08和1.00±0.04,差异均有统计学意义(FGF-1 mRNA:t=14.000,P＜0.01；FGF-4 mRNA:t=15.510,P＜0.01；FGF-7 mRNA:t 29.410,P＜0.01).结论 lop11基因突变影响小鼠晶状体的正常发育,其机制可能与影响晶状体蛋白的合成以及FGF的基因表达有关.%Background Mutation of the heat shock transcription factor 4 (HSF4) gene causes autosomal recessive hereditary cataract in lens opacity locus 11 (lop1 1) mouse,but the molecular mechanism of the pathogenesis has not been determined.Objective This study was to investigate the molecular mechanism of congenital cataract induced by HSF4 gene mutation in lop1 1 mouse.Methods Twenty-four lop1 1 mice and 24 wild type C57BL/6 mice were used in this study.The animals were sacrificed and the lenses were obtained on postnatal days 1,7 and 12.Regular pathological examination was carried out to evaluate the morphological changes of the lens and count the number of lens epithelial cells (LECs) in the mice.The αB-Crystallin (CRYAB) content in the lenses was detected in postnatal day 1 mice by Western blot.The expression of the fibroblast growth factor (FGF) mRNA in the lenses was assayed by quantitative PCR (q-PCR).The data were compared between the lop1 1 mice and wild type C57BL/6 mice with independent sample t test.The use and care of the experimental animals complied with the ARVO statement.Results The morphology and array of LECs were uniform and regular in the wild type C57BL/6 mice,while proliferation of LECs and disorder of lens fibers were seen in the lop11 mice on postnatal day 1.On postnatal day 7,vacuolar degeneration of the lens appeared in 7-day-old lop11 mice.The numbers of LECs were (417±19),(467±16) and (489±21) in lop11 mice on the postnatal day 1,7 and 12,respectively,and those of wild type C57BL/6 mice were (378 ± 13),(391 ±9) and (395 ±7),respectively,showing statistically significant differences between them (1 day:t=6.696,P=0.000;7 days:t=6.578,P=0.000;14 days:t=7.240,P=0.000).The expression of CRYAB in the lenses was evidently weaker in the 1-day-old lop11 mice compared with wild type C57BL/6 mice.The relative folds of expression of FGF-1,FGF-4,FGF-7 mRNA in the lenses were significantly higher in the lop11 mice than those in the wild type C57BL/6 mice (2.04±0.13 vs.1.037±0.06;2.03±0.08 vs.0.97± 0.08;4.59±0.12 vs.1.0±0.04) (FGF-1 mRNA:t=14.000,P＜0.001;FGF-4 mRNA:t=15.510,P＜0.01;FGF-7 mRNA:t =29.41,P＜0.01).Conclusions HSF4 mutation leads to the abnormal development of the lens in lop1 1 mice by arresting the expression of αB-Crystallin protein and increasing the expression of the FGF gene.