中文摘要：

目的 研究α-半乳糖苷酶A（GLA）基因突变对细胞自噬水平的影响,并初步探讨其作用机制.方法 选取通过超微病理检查、GLA活性检测和GLA基因突变筛查确诊的两个法布里病家系,分别构建两个家系GLA基因突变型重组表达质粒[pcDNA3.1-GFP-ex1（EX1组）,pcDNA3.1-GFP-ex3（EX3组）]及GLA野生型重组表达质粒（pcDNA3.1-GFP-GLA,GLA组）,利用脂质体法将重组表达质粒瞬时转染至Hela细胞中培养.对照组为未进行转染的Hela细胞系.利用实时PCR及Western印迹法检测各组自噬基因及自噬途径相关蛋白（LC3-Ⅱ/LC3-Ⅰ,Beclin-1,P62/ SQSTM1）的表达.结果 EX1组、EX3组、GLA组和对照组LC3半定量值分别为1.495±0.064、1.490±0.020、1.285 ±0.021、1.260±0.042;P62/ SQSTM1半定量值分别为0.555±0.086、0.480±0.084、0.785±0.439、0.980±0.278;Beclin-1 mRNA 2-ΔCt值分别为0.011±0.003、0.008±0.002、0.005±0.001、0.003 ±0.001;Beclin-1蛋白半定量值分别为1.178±0.098、1.209±0.092、0.931±0.100、0.796±0.184.与野生型相比,两组转染突变型GLA组中自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ水平上调（t=5.118、4.984,P=0.007、0.008）,多聚泛素结合蛋白P62/ SQSTM1表达水平差异无统计学意义（t=1.052、1.400,P=0.323、0.199）,自噬基因Beclin-1 mRNA（t=3.800、2.445,P=0.005、0.040）及蛋白（t=2.424、2.729,P=0.042、0.026）表达水平均上调.结论 突变GLA基因可诱导细胞自噬水平上调,并可能通过Beclin-1依赖性信号通路诱导细胞自噬活化.

英文摘要：

Objective To investigate the effect of α-galactosidase A （GLA） gene mutation on cell autophagy and to elucidate its mechanism preliminarily.Methods Two families were diagnosed by ultrastructural pathological examination,GLA gene activity test and GLA gene mutation screening.Mutant type recombinant expression plasmid of two pedigrees （pcDNA3.1-GFP-ex1 （EX1 group）,pcDNA3.1-GFP-ex3 （EX3 group）） and wild type recombinant expression plasmid of GLA （pcDNA3.1-GFP-GLA,GLA group） were constructed.Hela cell line （control group） was transiently transfected with recombinant expression plasmid according to lipofectin transfection.The relative gene expression of Beclin-1 was measured with real-time PCR,and protein expression level of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and P62/SQSTM1 was examined by Western blotting.Results The LC3 protein values of groups EX1,EX3,GLA and control were 1.495 ± 0.064,1.490 ± 0.020,1.285 ± 0.021,1.260 ± 0.042,respectively;P62/ SQSTM1 values were 0.555 ± 0.086,0.480 ± 0.084,0.785 ± 0.439,0.980 ± 0.278,respectively;Beclin-1 mRNA 2-△Ct values were 0.011 ±0.003,0.008 ±0.002,0.005 ±0.001,0.003 ±0.001,respectively;Beclin-1 protein values were 1.178 ±0.098,1.209 ±0.092,0.931 ±0.100,0.796 ±0.184,respectively.Compared with the wide type group,the level of LC3-Ⅱ/LC3-Ⅰ protein was significantly higher in the mutant type groups（t =5.118,4.984;P =0.007,0.008）,though no statistically significant difference was found in the expression levels of P62/SQSTM1 （t =1.052,1.400;P =0.323,0.199）.Besides,the expression levels of Beclin-1 mRNA （t =3.800,2.445;P =0.005,0.040） and protein （t =2.424,2.729;P =0.042,0.026） were significantly higher in the mutant type groups.Conclusions GLA gene mutation can induce cell autophagic dysfunction,and signaling pathway of autophagic activation may be Beclin-1 dependent.