Objective:To construct a lentiviral interference plasmid targeting collapse response regulatory protein 1(CRMP1)gene,to establish a human neuroblastoma cell line(SH-SY5Y)with stable CRMP1 knockdown,and to investigate its impact on expression of NLRP3 inflammasome protein.Methods:Double-stranded shRNA was designed and synthesized targeting h-CRMP1 mRNA sequence,and cloned into PLKO.1 vector.Recombinant shCRMP1 plasmids were constructed correctly,which was transfected into HEK-293T cells for lentiviral packaging.Obtained lentivirus supernatant was concentrated and then infected into SH-SY5Y cells.The interference effect of shCRMP1 plasmid and protein expressions of NLRP3 inflammasome components in SH-SY5Y cells were detected by Western blot.Results:DNA sequencing results showed that insertion sequences of recombinant interference plasmids pLKO.1-shCRMP1 were consistent with designed sequences,which confirmed successful construction of shCRMP1 lentivirus interfering plasmids and transfected into HEK-293T cells for lentivirus packaging,and protein level of CRMP1 in HEK-293T cells were decreased.SH-SY5Y cells were infected with lentivirus concentrate obtained from packaging and screened with puromycin.Western blot results showed that shCRMP1 recombinant lentiviral plasmids could significantly down-regulate CRMP1 protein expression in SH-SY5Y cells.It was also found that in SH-SY5Y cell line with stable CRMP1 knockdown,inhibition of CRMP1 expression could effectively inhibit NLRP3 inflammasome activation under MPP+induction.Conclusion:pLKO.1-shCRMP1 lentiviral interfering plas-mids have been successfully constructed,and interference with CRMP1 can inhibit activation of NLRP3 inflammasome in MPP+-in-duced SH-SY5Y cells.This study provides guidance for further research on mechanism of CRMP1 in neurodegenerative diseases such as Parkinson's disease.

Objective:To construct a lentiviral interference plasmid targeting collapse response regulatory protein 1(CRMP1)gene,to establish a human neuroblastoma cell line(SH-SY5Y)with stable CRMP1 knockdown,and to investigate its impact on expression of NLRP3 inflammasome protein.Methods:Double-stranded shRNA was designed and synthesized targeting h-CRMP1 mRNA sequence,and cloned into PLKO.1 vector.Recombinant shCRMP1 plasmids were constructed correctly,which was transfected into HEK-293T cells for lentiviral packaging.Obtained lentivirus supernatant was concentrated and then infected into SH-SY5Y cells.The interference effect of shCRMP1 plasmid and protein expressions of NLRP3 inflammasome components in SH-SY5Y cells were detected by Western blot.Results:DNA sequencing results showed that insertion sequences of recombinant interference plasmids pLKO.1-shCRMP1 were consistent with designed sequences,which confirmed successful construction of shCRMP1 lentivirus interfering plasmids and transfected into HEK-293T cells for lentivirus packaging,and protein level of CRMP1 in HEK-293T cells were decreased.SH-SY5Y cells were infected with lentivirus concentrate obtained from packaging and screened with puromycin.Western blot results showed that shCRMP1 recombinant lentiviral plasmids could significantly down-regulate CRMP1 protein expression in SH-SY5Y cells.It was also found that in SH-SY5Y cell line with stable CRMP1 knockdown,inhibition of CRMP1 expression could effectively inhibit NLRP3 inflammasome activation under MPP+induction.Conclusion:pLKO.1-shCRMP1 lentiviral interfering plas-mids have been successfully constructed,and interference with CRMP1 can inhibit activation of NLRP3 inflammasome in MPP+-in-duced SH-SY5Y cells.This study provides guidance for further research on mechanism of CRMP1 in neurodegenerative diseases such as Parkinson's disease.