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[Deletion of extracellular loops of occludin affects the tight junction of TM4 cells in mice].

Objective
To analyze the functions of the two extracellular loops of occludin in the tight junction of TM4 cells in mice.


METHODS
Using genetic engineering, we separately or simultaneously deleted two extracellular loops of occludin, cloned the three occludin genes without extracellular loops into the pcDNA3.1 expression vector, and transfected them into TM4 cells. Then we determined the expression of occludin by RT-PCR and Western blot, and analyze the effects of the extracellular loops of occludin on the tight junction of the TM4 cells with the in vitro cell line model.


RESULTS
The results of sequencing showed that the expression vector of pcDNA3.1 - occludin Δ OCC1, pcDNA3.1 - occludin Δ OCC2 and pcDNA3.1 - occludin Δ OCC1 + OCC2 was constructed successfully. The mRNA and protein expressions of occludin in the non-extracellular loop groups were significantly higher than in the control group. Both the extracellular loops of occludin increased the tight junction of the TM4 cells. The macromolecular permeability in the TM4 cells was significantly lower in the pcDNA3.1 - occludin Δ OCC1 than in the pcDNA3.1 - occludin Δ OCC2 group (P < 0.05), indicating a higher impact of the second than the first extracellular loop on the tight junction of the TM4 cells.


CONCLUSIONS
Both of the two extracellular loops of occludin can affect the tight junction of TM4 cells, the second even more significantly than the first one.