人突变Lumican转基因小鼠角膜组织中小分子亮氨酸蛋白聚糖成员表达改变的研究

Differential expression of small leucine-rich proteoglycans gene in Lumican transgenic mouse cornea

目的探讨人突变Lumican转基因小鼠角膜组织中小分子亮氨酸蛋白聚糖(SLRP)成员mRNA水平表达变化。方法实验研究。选取10只(雌雄各5只)人突变Lumican转基因小鼠作为实验组,选取10只(雌雄各5只)同周龄野生型C57BL/6J小鼠作为对照组。转基因小鼠Lumican基因突变位点为cDNA569T>C,使第199位氨基酸由亮氨酸突变为脯氨酸(L99P)。8周龄时脱颈法处死入组小鼠,摘取双侧眼球后获得双眼角膜组织,采用实时荧光定量聚合酶链式反应(RT-Q-PCR)技术分别检测两组小鼠角膜组织中Lumican、Decorin、Biglycan、Keratocan、Fibromodulin基因在mRNA水平的表达。采用两独立样本均数t检验对两组小鼠基因表达差异进行统计学分析,差异以倍数变化法表示。结果实验组转基因小鼠角膜组织中Lumican基因mRNA表达升高,是对照组野生型小鼠的1.497倍,差异具有统计学意义(t=4.34,P<0.05)。实验组小鼠角膜组织中Keratocan基因mRNA表达降低,是对照组的0.323倍,差异具有统计学意义(t=-95.94,P<0.05)。实验组中Decorin基因mRNA表达降低,是对照组的0.648倍,差异均具有统计学意义(t=-9.98,P<0.05)。实验组较对照组Biglycan基因mRNA表达降低,是对照组的0.522倍(t=-7.74,P<0.05),Fibromodulin基因mRNA表达升高,是对照组的1.193倍,差异无统计学意义(t=1.66,P>0.05)。结论人突变Lumican转基因小鼠与野生小鼠相比,角膜组织中SLRP成员mRNA水平表达发生改变,提示Lumican基因突变(cDNA569T>C)可能导致该基因结构破坏,调控角膜组织中其他SLRP成员基因表达的功能受损。此外,该基因位点的突变导致Lumican核心蛋白中第199位氨基酸由亮氨酸突变为脯氨酸(L199P),提示Lumican基因突变可能阻碍了Lumican与胶原蛋白分子结合,进而影响了SLRP成员的表达。

Objective To investigate the differential expression of small leucine-rich proteoglycans at mRNA level in Lumican transgenic mouse cornea with Real-time Quantitative PCR Detecting System. Methods Experimental research. Ten Lumican transgenic mice (5 male and 5 female) were chosen as experimental group and 10 wild mice (5 male and 5 female) were chosen as control group. All the mice were killed and enucleated both eyes at eight weeks of age. Gene expression levels of Lumican, Decorin, Biglycan, Keratocan, Fibromodulin in the excised corneas were analyzed by real-time quantitative polymerase chain reaction (RT-Q-PCR) using Real-time Quantitative PCR Detecting System. Differential expression within each group were analysed by fold changes and independent t-test. Results There were statistic different expression level of Lumican, Decorin, Biglycan and Keratocan mRNA between experimental and control group. The expression level of Lumican RNA was found to be 1.497-fold increased relative to the control (t=4.34, P<0.05) , while Decorin, Biglycan, Keratocan were 0.648-fold (t=-9.98, P<0.05) , 0.522-fold (t=-7.74,P<0.05), 0.323-fold (t=-95.94, P<0.05)decreased in transgenic mice. Fibromodulin mRNA up regulated 1.193-fold in transgenic mice without statistic difference (t=1.66, P>0.05). Conclusions Lumican gene mutation(cDNA 569T>C) results in abnormal SLRP expression in transgenic mouse cornea at mRNA level, which may indicate that this mutation changes the structure of Lumican and impairs the function of regulating SLRP expression. Also, Lumican gene mutation leads to amio acid exchanging(L199P), which may hinder Lumican from binding to collagens and result in abnormal expression of SLRP at mRNA level.