泛素依赖的蛋白水解通路在转染脊髓小脑性共济失调3型基因的PC12细胞中的定位及作用

The role and location of ubiquitin-dependent proteolysis pathway in PC12 cells transfected with spinocerebellar ataxia type 3

目的分析泛素依赖的蛋白水解通路(UPP)在转染了脊髓小脑性共济失调3型(SCA3)基因的PC12细胞中的定位及作用。方法构建含有SCA3正常与突变基因在内的增强型绿色荧光蛋白真核表达载体(pEGFPC1-ataxin3 Q28、pEGFPC1-ataxin3 Q84)后转染大鼠肾上腺嗜铬细胞瘤(PC12)细胞,转染后24、72、120 h免疫荧光检测ataxin3、泛素(ub iqu itin)、蛋白水解酶核心成分20S及热休克蛋白40、70(Hsp40、Hsp70)在细胞内的分布情况,观察其与核内包涵体(INIs)共定位的情况;给予蛋白水解酶抑制剂lactacystin(10μmol/L)后台盼蓝染色观察细胞存活率的变化。结果正常蛋白ataxin3 Q28于胞质、胞核广泛分布,而突变蛋白ataxin3 Q84多集中于胞核内;ub iqu itin在两组的表现与目的蛋白一致。随着时间的延长20S及Hsp40、Hsp70细胞核内与INIs共定位,形成密集荧光。正常对照组与突变组120 h时INIs形成数目在Hsp40组分别为18.40±3.36、75.00±5.09;Hsp70组分别为15.43±3.05、74.75±4.99;20S组分别为4.20±1.92、92.61±3.65,统计学处理正常对照组与突变组之间P值分别为0.000、0.043、0.027,差异有统计学意义;突变组给予lactacystin后细胞死亡明显增加,前后对比120 h时细胞死亡数目分别为77.50±3.33、94.67±1.82,统计学处理前后组之间P值为0.000,差异有统计学意义。结论UPP通路中ub iqu itin、20S以及重要的辅助成分Hsp40、Hsp70与ataxin3以及由此形成的INIs紧密地共定位,抑制UPP通路中的20S活性导致细胞死亡明显增加,推测UPP在SCA3发病机制中发挥重要的作用,对UPP的干预有可能成为SCA3治疗的一个途径。

Objective To analyze the role and location of ubiquitin-dependent proteolysis pathway （UPP） in PC12 cells transfected by plasmids with spinocerebellar ataxia type 3 （ SCA3 ） gene. Methods Plasmid-enhanced green fluorescent protein （pEGFP） including normal or mutant SCA3 gene was transfected into PC12. The locations of proteasome core component 20S, heat shock protein 40 and 70 （ Hsp40, Hsp70） were observed by using immunocytochemistry after transfection of 24, 72, and 120 hours. The living cells （trypan-blue excluding） percentage was quantified after giving the protease inhibitor lactacystin （10μmol/ L）. Results Normal protein ataxin3 Q28 was distributed in the cells equally. The mutant protein ataxin3 Q84 was mainly distributed in the nucleus. Ubiquitin showed the same location. 20S and Hsp40, Hsp70 colocalized with intranuclear inclusions （INIs） respectively and gave off a dense fluorescence. INIs numbers in normal control and mutant group after 120 hours were respectively 18.40±3.36 and 75.00±5.09 for Hsp40; 15.43±3.05 and 74. 75±4.99 for Hsp70; 4. 20±1.92 and 92. 61±3.65 for 20S. P values were respectively 0. 000,0. 043,0. 027. The differences were significant. Cell death rate was improved greatly after giving lactacystin. The numbers of dead cells after 120 hours in mutant group without and with giving lactacystin were respectively 77.50±3. 33 and 94. 67±1.82. P value was 0. 000 and the difference was also significant. Conclusion UPP should play an important role in the molecular pathogenesis of SCA3. Inhibition of protease should promote the aggregation of mutant protein and cell death. It should be concluded that the interference of UPP would possibly become a target of SCA3 therapy.