Construction Of ApoB-Cystatin C Fusion Protein Targeting The Blood Brain Barrier And Its Effect On The Formation Of A? In The Brain Of AD Model Mouse

Objective: In recent years,people have found many endogenous proteins have a strong neuroprotective effect,and they have broad prospects for the treatment of diseases of the central nervous system such as AD.However,this requires a delivery of drug molecules throughout the central nervous system to work.The blood brain barrier(BBB)blocks most of the drug molecules from entering the brain.Only400-500 Da of lipid-soluble small molecule proteins can cross the BBB,and large proteins entering the central nervous system require other pathways,such as receptor-mediated endocytosis.The brain endothelial cells in BBB express some receptors,such as transferrin receptor,insulin type 2 receptor,low density lipoprotein receptor(LDLR)and so on,so we design a ligand for specific receptors on BBB.As a carrier for drug delivery,it can enter the brain to play a role.At present,studies have reported that due to the characteristics of the low-density lipoprotein targeting receptor(LDLR)binding domain of apolipoprotein B(ApoB),people have used this feature to design and construct a lentiviral vector and recombine the fusion expressed ApoB.The protein is able to cross the BBB and increase the enzymatic activity of the animal's central nervous system by 50% compared to the control group.In previous experiments,we found that under normal conditions,Cystatin C activates the transcription of ADAM10 by up-regulating the expression of SIRT1 in human brain microvascular endothelial cells(HBMEC),thereby increasing the secretion of sAPP?by HBMEC and inhibiting the production of A?.Based on this,we used the ApoB as a targeting vector to construct a lentivirus-mediated recombinant plasmid expressing Cystatin C.To investigate whether ApoB can mediate the entry of Cystatin C into the blood-brain barrier and affect the deposition of A? in the brain of APP/PS1 mice.Methods:1.Molecular Cloning three lentiviral vector systems were constructed:PLV-ss-Cys C-V5-ApoB(experimental group),PLV-ss-BACE1 inhibitor-V5-ApoB(positive control group),PLV-ss-V5-ApoB(negative control group),the lentivirus particles were packaged after gene sequencing.2.The purified lentiviral particles were intraperitoneally injected into APP/PS1 mice to observe the post-infection state.3.To detect the influence of the recombinant protein ApoB-Cystatin C across the blood-brain barrier and the deposition of A? in the brain of mice.Western blot was used to detect the effect of Cystatin C on the expression of total APP protein and secreted enzyme in N2 a 695 cells under normal conditions.Western blot was used to detect the effect of Cystatin C on ?-secretase activity in N2 a 695 cells under oxidative stress.Results:1.The packaged and purified lentivirus was injected into mice.After 30 days of infection,the recombinant fusion protein was significantly expressed in the spleen and liver in the APP/PS1 mice.The recombinant protein ApoB-Cystatin C can enter the brain parenchyma and can be detected in the brain in APP/PS1 mice infected with experimental group PLV-ss-Cys C-V5-ApoB.3.In the cerebellum of APP/PS1 mice 30 days after lentivirus infection,the recombinant fusion protein ApoB-Cystatin C was significantly expressed compared to the negative control group.4.The recombinant protein ApoB-Cystatin C colocalized with neurons in brain parenchyma and had little colocalization with astrocytes.5.Detection of A? deposition in brain parenchyma of 9-month-old APP/PS1 mice 3 months after the injection of lentivirus.The positive control group was compared and the PLV-ss-Cys C-V5-ApoB in the experimental group was compared with the negative control.The group PLV-ss-V5-ApoB inhibited the deposition of A?.Under normal conditions,Cystatin C had no significant effect on the expression of total APP protein and secreted enzyme in N2 a 695 cells.7.Under oxidative stress,Cystatin C can inhibit the expression of?-secretase in N2 a 695 cells.Conclusion: 1.The APP/PS1 mice were injected intraperitoneally.The lentivirus could infect the liver and spleen and express ApoB-Cystatin C recombinant fusion protein.The recombinant fusion protein was abundantly expressed in APP/PS1 mouse cerebellum across the blood-brain barrier.2.In the brain of APP/PS1 mice,ApoB-Cystatin C recombinant fusion protein was found to colocalize with neurons and it could reduce the deposition of A? in the brain.In vitro simulation of AD oxidative stress environment,observed that Cystatin C can inhibit BACE1 expression in N2 a 695 cells.