| Objective: Blood-brain barrier(BBB)is mainly consisted of brain microvascular endothelial cells and tight junction between neighboring endothelial cells with the selective permeability effect.Its main function is to protect the brain tissue,but also prevents therapeutic drugs transferring into the brain.The blood-tumor barrier(BTB)has the same function as the blood-brain barrier.It can prevent anti-cancer drugs into the brain and influence chemotherapy effect.Therefore,methods to selectively open the BTB are crucial for effective chemotherapy of glioma.There are two pathways of drugs being delivered through BTB into the tumor cells: paracellular pathway and transcellular pathway.Internalization induced by caveolae is one of the transcellular pathways,while caveolin-1 is necessary for the formation of caveolae;Paracellular way mainly refers to the opening of tight junction between the adjacent cells which could be caused by TJ protein down-regulation.Recent studies have reported,caveolin-1 has related with tight junction-associated protein regulation,but the results are inconsistent.e NOS activity increased in vascular endothelial cells of caveolin-1 gene knockout mice,accompanied by vascular function defects and increased vascular permeability.However,studies have shown that caveolin-1 negatively regulates the tight junction-associated protein.Zhong et al proved that in the human brain microvascular endothelial cells human immunodeficiency virus-1 Tat can cause caveolin-1 expression increased which induce tight junction-associated protein: occludin,ZO-1 and ZO-2 expression drop and increase the blood brain barrier permeability.To determine the regulatory effect of caveolin-1 on tight junction-associated protein and blood-tumor barrier permeability,we construct caveolin-1 recombinant adenovirus expression vector and silencing vector to make caveolin-1 expression increased or decreased by infecting brain microvascular endothelial cells of glioma model of rat and observe the changes of tight junction-related proteins and BBB permeability.The results of this study will provide new ideas and theoretical support for the treatment of brain tumors with open blood tumor barrier.Method: 1.Construction of the caveolin-1 recombinant adenovirus expression vectors by amplifying caveolin-1 c DNA fragment with rat brain total RNA as a template.Design of small interfering RNA fragments which silence caveolin-1,anneal and synthesis of double-stranded DNA,then construction of caveolin-1 recombinant adenoviral silencing vectors.2.C ulture of C6 glioma cells of rat and establishment of glioma model of rat.3.Wistar rats were randomly divided into 5 groups: control group(C6 glioma + saline),caveolin-1 silencing group(C6 glioma + Ad-si RNA-Cav1),silent negative control group(C6 glioma + Ad-si RNA-NC),caveolin-1 overexpression group(C6 glioma + Ad-Cav1),and negative control group(C6 glioma + Ad-EGFP).4.Up-regulation or drown-regulation of caveolin-1 expression through infecting brain microvascular endothelial cells with caveolin-1 recombinant adenoviral expression vectors or silencing vectors.5.Evans blue(EB)assay was used to determine BTB permeability after caveolin-1 recombinant adenovirus infection in glioma rats.6.The levels of tight junction-associated protein claudin-5,occludin,ZO-1 protein was determined by Western blot in brain microvascular endothelial cells after infected by caveolin-1 recombinant adenovirus.7.Ultrastructural changes of tight junctions in brain microvascular endothelial cells after infecting with caveolin-1 recombinant adenovirus was observed by transmission electron microscopy.8.Using SPSS13.0 statistical analysis,all data are presented as mean ± standard deviation((3(3 ± SD),and using single-factor analysis of variance and Bonferroni test,P <0.05 was considered statistically significant.Results: 1.C6 glioma model of Rat was established successfully.2.Caveolin-1 recombinant adenovirus expression vectors named as Ad-cav1 were successfully constructed and restriction enzyme analysis and sequencing results exactly matched.At the same time,the negative control adenovirus vectors named as Ad-EGFP were also constructed.3.Caveolin-1 recombinant adenovirus silencing vectors named as Ad-si RNA-Cav1 were successfully constructed and restriction enzyme analysis and sequencing results exactly matched.At the same time,the silencing negative control adenovirus vectors named as Ad-si RNA-NC were also constructed.4.After infection with Ad-si RNA-Cav1 for 48 h in brain microvascular endothelial cells,BTB permeability of glioma model of rat was significantly increased.On the contrary,it is decreased significantly in rats infected with Ad-cav1.BTB permeability in the control group,the silencing negative control group and over-expression negative control group was no significant difference.5.After infection with Ad-si RNA-Cav1 for 48 h in brain microvascular endothelial cells,the expression of caveolin-1 and tight junction-associated proteins: claudin-5,occludin,ZO-1 were significantly reduced;on the contrary,increased significantly in rats infected with Ad-cav1.6.Transmission electron microscopy revealed that tight junctions were opened and the gap was widened after infection with Ad-si RNA-Cav1 for 48 h in brain microvascular endothelial cells.O n the contrary,tight junctions were very dense and showed narrow black ribbon in rats infected with Ad-cav1.Conclusions: 1.Increasing or decreasing of caveolin-1 expression can the up or down the expression of tight junction proteins,showing a positive regulatory role.2.Caveolin-1 can influence the permeability of the blood tumor barrier.by regulating the expression of TJ-associated proteins and affecting the integrity of tight junctions. |
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