Sec62/Sec63是位于内质网膜上的跨膜蛋白复合物,辅助新合成的前体蛋白或新生多肽链转运至内质网,进行后期的折叠加工。为研究Sec62/Sec63复合物对乙型脑炎病毒(JEV)复制的影响,本研究利用CRISPR/Cas9基因编辑技术分别构建SEC62、SEC63...Sec62/Sec63是位于内质网膜上的跨膜蛋白复合物,辅助新合成的前体蛋白或新生多肽链转运至内质网,进行后期的折叠加工。为研究Sec62/Sec63复合物对乙型脑炎病毒(JEV)复制的影响,本研究利用CRISPR/Cas9基因编辑技术分别构建SEC62、SEC63基因敲除的HEK-293细胞系,经测序鉴定以及western blot检测,获得两个基因敲除细胞系,分别命名为SEC62 KO HEK-293细胞和SEC63 KO HEK-293细胞。分别以MOI为1、0.05的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,利用间接免疫荧光试验检测Sec62/Sec63复合物对JEV复制的影响。结果显示,不同剂量JEV感染SEC62 KO HEK-293细胞与SEC63 KO HEK-293细胞中荧光信号阳性细胞均少于HEK-293细胞对照组;以MOI为1的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,经western blot检测,结果显示于感染后12 h、24 h和48 h,SEC62 KO HEK-293、SEC63 KO HEK-293细胞中均未检测出JEV E蛋白条带,而对照HEK-293细胞中JEV E蛋白的表达量逐渐增加。以MOI为1的JEV感染上述3种细胞后检测细胞培养液中病毒滴度,结果显示JEV感染12 h后SEC62 KO HEK-293、SEC63 KO HEK-293细胞与HEK-293细胞病毒滴度无差异(p>0.05),而感染后24 h与48 h时,SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.01)。以MOI为1、0.5、0.05的JEV感染上述3种细胞后检测48 h后细胞培养液中病毒滴度,结果显示SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.001)。以上结果表明,SEC62、SEC63基因敲除后均抑制了JEV的复制,也表明Sec62、Sec63是影响JEV在细胞内复制的重要宿主因子。本研究为进一步探究Sec62、Sec63等宿主因子对JEV复制的影响机制提供了参考依据。展开更多
Polycystic liver diseases(PCLDs) are a heterogeneous group of genetic disorders characterized by the development of multiple fluid-filled cysts in the liver,which derive from cholangiocytes,the epithelial cells lining...Polycystic liver diseases(PCLDs) are a heterogeneous group of genetic disorders characterized by the development of multiple fluid-filled cysts in the liver,which derive from cholangiocytes,the epithelial cells lining the bile ducts.When these cysts grow,symptoms such as abdominal distension,nausea,and abdominal pain may occur.PCLDs may exist isolated(i.e.,autosomal dominant polycystic liver disease,ADPLD) or in combination with renal cystogenesis(i.e.,autosomal dominant polycystic kidney disease and autosomal recessive polycystic liver disease).The exact prevalence of PCLDs is unknown,but is estimated to occur in approximately 1:1000 persons.Although the pathogenesis of each form of PCLD appears to be different,increasing evidences indicate that hepatic cystogenesis is a phenomenon that may involve somatic loss of heterozygosity(LOH) in those pathological conditions inherited in a dominant form.A recent report,using highly sophisticated methodology,demonstrated that ADPLD patients with a germline mutation in the protein kinase C substrate 80K-H(PRKCSH) gene mostly develop hepatic cystogenesis through a second somatic mutation.While hepatocystin,the PRKCSH-encoding protein,was absent in the hepatic cysts with LOH,it was still expressed in the heterozygous cysts.On the other hand,no additional trans-heterozygous mutations on the SEC63 homolog(S.cerevisiae /SEC63) gene(also involved in the development of PCLDs) were observed.These data indicate that PCLD is recessive at the cellular level,and point out the important role of hepatocystin loss in cystogenesis.In this commentary,we discuss the knowledge regarding the role of somatic second-hit mutations in the development of PCLDs,and the most relevant findings have been highlighted.展开更多
文摘Sec62/Sec63是位于内质网膜上的跨膜蛋白复合物,辅助新合成的前体蛋白或新生多肽链转运至内质网,进行后期的折叠加工。为研究Sec62/Sec63复合物对乙型脑炎病毒(JEV)复制的影响,本研究利用CRISPR/Cas9基因编辑技术分别构建SEC62、SEC63基因敲除的HEK-293细胞系,经测序鉴定以及western blot检测,获得两个基因敲除细胞系,分别命名为SEC62 KO HEK-293细胞和SEC63 KO HEK-293细胞。分别以MOI为1、0.05的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,利用间接免疫荧光试验检测Sec62/Sec63复合物对JEV复制的影响。结果显示,不同剂量JEV感染SEC62 KO HEK-293细胞与SEC63 KO HEK-293细胞中荧光信号阳性细胞均少于HEK-293细胞对照组;以MOI为1的JEV感染HEK-293、SEC62 KO HEK-293、SEC63 KO HEK-293细胞后,经western blot检测,结果显示于感染后12 h、24 h和48 h,SEC62 KO HEK-293、SEC63 KO HEK-293细胞中均未检测出JEV E蛋白条带,而对照HEK-293细胞中JEV E蛋白的表达量逐渐增加。以MOI为1的JEV感染上述3种细胞后检测细胞培养液中病毒滴度,结果显示JEV感染12 h后SEC62 KO HEK-293、SEC63 KO HEK-293细胞与HEK-293细胞病毒滴度无差异(p>0.05),而感染后24 h与48 h时,SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.01)。以MOI为1、0.5、0.05的JEV感染上述3种细胞后检测48 h后细胞培养液中病毒滴度,结果显示SEC62 KO HEK-293、SEC63 KO HEK-293细胞培养液中病毒滴度均显著低于HEK-293细胞对照组(p<0.001)。以上结果表明,SEC62、SEC63基因敲除后均抑制了JEV的复制,也表明Sec62、Sec63是影响JEV在细胞内复制的重要宿主因子。本研究为进一步探究Sec62、Sec63等宿主因子对JEV复制的影响机制提供了参考依据。
文摘Polycystic liver diseases(PCLDs) are a heterogeneous group of genetic disorders characterized by the development of multiple fluid-filled cysts in the liver,which derive from cholangiocytes,the epithelial cells lining the bile ducts.When these cysts grow,symptoms such as abdominal distension,nausea,and abdominal pain may occur.PCLDs may exist isolated(i.e.,autosomal dominant polycystic liver disease,ADPLD) or in combination with renal cystogenesis(i.e.,autosomal dominant polycystic kidney disease and autosomal recessive polycystic liver disease).The exact prevalence of PCLDs is unknown,but is estimated to occur in approximately 1:1000 persons.Although the pathogenesis of each form of PCLD appears to be different,increasing evidences indicate that hepatic cystogenesis is a phenomenon that may involve somatic loss of heterozygosity(LOH) in those pathological conditions inherited in a dominant form.A recent report,using highly sophisticated methodology,demonstrated that ADPLD patients with a germline mutation in the protein kinase C substrate 80K-H(PRKCSH) gene mostly develop hepatic cystogenesis through a second somatic mutation.While hepatocystin,the PRKCSH-encoding protein,was absent in the hepatic cysts with LOH,it was still expressed in the heterozygous cysts.On the other hand,no additional trans-heterozygous mutations on the SEC63 homolog(S.cerevisiae /SEC63) gene(also involved in the development of PCLDs) were observed.These data indicate that PCLD is recessive at the cellular level,and point out the important role of hepatocystin loss in cystogenesis.In this commentary,we discuss the knowledge regarding the role of somatic second-hit mutations in the development of PCLDs,and the most relevant findings have been highlighted.
