小鼠附睾小体特异性mRNA传递入N2a和TM4细胞的研究

Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells

目的:研究小鼠附睾小体特异性mRNA能否递入Neuro-2a(N2a)及TM4细胞中,为探讨附睾小体mRNA功能提供实验依据。方法:通过RT-PCR检测附睾特异性基因(Adam7、Crisp1、Defb22、Wfdc2、Wfdc9)在成年雄性BALB/c小鼠睾丸、附睾、附睾小体和精子中,以及在人睾丸、精浆小体和精子中的存在情况。通过低速离心、过滤、超速离心方法分离BALB/c小鼠附睾小体,通过PKH26进行荧光标记,与饥饿培养24 h的N2a和TM4细胞共孵育1 h,分别以未标记PKH26的附睾小体组、无附睾小体的加PKH26染料组、未加附睾小体和PKH26染料阴性组作为对照,观察附睾小体是否与N2a和TM4细胞融合并被摄取。通过RT-PCR对共孵育1 h后N2a和TM4细胞进行附睾特异性基因的检测。结果:Adam7和Crisp1在小鼠附睾、附睾小体和精子中存在,睾丸中无表达,在人精浆小体和精子中存在,睾丸中无表达。PKH26和hoechst33258荧光双标结果显示小鼠附睾小体与N2a和TM4细胞融合并被摄取,RT-PCR结果表明共孵育1 h后N2a和TM4细胞可以检测到Adam7和Crisp1基因的mRNA。Western印迹结果显示与附睾小体孵育后的N2a和TM4细胞可以检测到CRISP1蛋白。结论:附睾小体可传递附睾特异性mRNA Adam7和Crisp1进入到细胞中,其中Crisp1可能被翻译成蛋白,其功能和意义有待进一步研究。

Objective:To investigate whether mouse epididymis-specific mRNAs Adam7 and Crispl can be delivered into N2a and TM4 cells,and to provide an experimental basis for exploring the function of epididymal mRNAs.Methods:Using RT-PCR,we detected the presence of epididymis-specific genes(Adam7,Crispl,Defb22,Wfdc2,and Wfdc9)in the testis,epididymis,epididymosome and sperm of adult male BALB/c mice as well as in the human testis,seminal vesicles and sperm.We isolated epididymosomes of BALB/c mice by low-speed centrifugation,filtration and ultracentrifugation,fluorescently labeled them by PKH26,co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture,and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling,PKH26 dye without epididymosomes,and non-epididymosome or-PKH26 dye as controls.Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR.Results:Adam7 and Crispl were present in the mouse epididymis,epididymosomes and sperm,and in the human seminal vesicles and sperm as well,but not in the testes of either the mice or men.PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested;RT-PCR revealed the mRNAs of Adam7 and Crispl in the N2a and TM4 cells after 1-hour co-incubation;and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes.Conclusion:Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crispl into N2a and TM4 cells,where Crispl may be translated into proteins,though their function and significance need to be further studied.