Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
Objective To investigate biological markers that differentiate states during various seizure periods of childhood absence epilepsy (CAE) by examining the spatiotemporal dynamics of magnetoencephalographic (MEG) signals from Default Mode Network (DMN) nodes, revealing the neurophysiological mechanisms underlying changes in consciousness during CAE seizures. MethodsThirty-six drug-native children diagnosed with CAE were recruited. The interictal data, ictal data of CAE children were collected using a CTF-225 channel MEG system. Conduct temporal homogeneity partitioning for all seizure period data, co-registering 14 distinct seizure states. Identify 12 brain regions associated with the default mode network (DMN) as regions of interest (ROI); employ minimum norm estimation in conjunction with the Welch method to compute the power spectral density (PSD) of the ROI; conduct differential analysis on the relative PSD values; and use a random forest model to identify significant PSD features that differentiate between states of epilepsy. ResultsPower changes in DMN-related brain regions across various frequency bands show significant synchrony. During a seizure, the power in the δ band rapidly increases at the onset and quickly decreases at the end. Meanwhile, the power in the θ-γ2 bands decreases at the beginning and gradually recovers after the seizure. During the O+2 phase following seizure onset, the power in the β band peaks briefly before rapidly declining. The medial frontal cortex has lower power in the δ frequency band during seizures compared to other DMN brain regions, but higher power in the α frequency band. The random forest model's feature importance analysis reveals that the precuneus, lateral temporal lobe and medial temporal lobe play important roles in identifying seizure states. Power changes in the precuneus in the α and δ frequency bands improve the model's classification accuracy. ConclusionsThis study investigated the dynamic spatiotemporal characteristics of the DMN during CAE seizures, revealing the typical manifestations of power changes in specific brain regions and frequency bands at the onset, maintenance, and termination of seizures. It was discovered that power of the precuneus can act as an important feature to distinguish between different stages of CAE seizures. These findings shed new light on the pathophysiological mechanisms underlying changes in consciousness states in CAE.