The Role Of CLC3 In Proliferation And Apoptosis Of Human Umbilical Vein Endothelial Cell Induced By Iodide Ion

[Objective]To research the role of CLC3 on the proliferation and apoptosis of human umbilical vein endothelial cell induced by different concentration of iodine, some kinds of methods including flow cytometry,RT-PCR,western blot were used. This study can observe the pathway which the iodine get in the human umbilical vein endothelial cell and enrich the study on trace element.[Material and Methods]1. Cell culture:The HUVEC (umbilical vein endothelial cell) were routinely cultured in RPML1640 cultivation medium containing 15% fetal bovine serum. Then the following experiment could proceed when the cells are stabilized.2. Flow cytometry analysis:Cells were cultured in different concentration iodine for 24-hours and 48-hour. Then collected the single cell suspension. And then centrifuged it and discarded the supernatant. After that, fixed the cells by 70% alcohol. Once more, centrifuged it and discarded the supernatant. Then washed the cells twice with PBS and stained with synthetic staining solution. The cells were analyzed by Flow cytometer. And data analysis was performed by ModFit software.3. Reverse Transcription and polymerase chain reaction of sodium/iodide symporter (NIS) mRNA:Cells were cultured in different concentration of iodine for 24h and 48h. Then extracted the total RNA, and detected the NIS mRNA expression by reverse transcription and polymerase chain reaction and electrophoresis. Carried it out at least three times for each sample.4. Western blot of sodium/iodide symporter (NIS) protein:Collected the cells which wered cultured in different concentrations of iodine for 24h and 48h. Extracted the total protein, then separated it on SDS-PAGE and transferred onto PVDF membranes. After that, blocked the membranes with 5% nonfat dry milk in TBS for 2h at room temperature. Then incubated it overnight at 4℃with antibodies against NIS (1:1000; Abcam) and b-actin (1:1000 Santa Cruz Biotechnology). Immunoblotting was done by incubating the membranes at 4℃overnight followed by secondary antibody (goat anti-rabbit IgG) conjugated with peroxidase for 1h at room temperature. After washing it, signals were visualized by SuperSignal. Western blot was carried out at least three times for each sample.5. Reverse Transcription and polymerase chain reaction of chloride channel (CLC3) mRNA:Cells were cultured in different concentration of iodine for 24h and 48h. Then the total RNA was extracted, after that, the CLC3 mRNA expression was detected by RT-PCR. Carried it out at least three times for each sample.6. Western blot of chloride channel (CLCN3) protein:The cells wered cultured in different concentration iodine for 24h and 48h. Collected the single cell suspension. Then visualized the signals by SuperSigna after the follow processes: Extracted the total protein, then separated it and transferred onto PVDF membranes. After that, blocked it with 5% nonfat dry milk in TBS for 2h at room temperature. Then incubated it overnight at 4℃with antibodies against CLCN3 (1:300; Abcam) and b-actin(1:1000 Santa Cruz Biotechnology). After that, incubated it with secondary antibody (goat anti-rabbit IgG; goat anti-mouse IgG) conjugated with peroxidase for 1h at room temperature and washed it with TBST.[Results]1. Flow cytometry analysis:After 24h incubation in different iodine concentration, the proliferation index in the groups of 100～5000μg/L were higher than the control group but the apoptosis rates were lower. The proliferation index in the group of 7000μg/L wasn't significantly different from control group. When the concentration was higher than 9000μg/L, the proliferation index was lower than control group and the apoptosis rate was higher. After 48h incubation, the proliferation index in the groups of 100-3000μg/L were higher than control group and the apoptosis rate were lower. The proliferation index in the group of 5000μg/L has no significant difference with control group. When the concentration was higher than 7000μg/L, the proliferation index was lower than control group while the apoptosis rate was higher.2. Reverse Transcription and polymerase chain reaction of sodium/iodide symporter(NIS) mRNA:The expression of NIS mRNA was lost in the human umbilical veins endothelial cell.3. western blot of sodium/iodide symporter(NIS) protein:The expression of NIS protein was lost in the human umbilical veins endothelial cell4. Reverse Transcription and polymerase chain reaction of chloride channel (CLC3) mRNA:After 24h incubation, the mRNA of CLC3 expression was up regulated in the groups of 1000-5000μg/L,P<0.05, the difference has statistical significance. There was no difference between the group of 7000μg/L. The mRNA of CLC3 expression was down regulated in the group of 9000μg/L, P<0.05. After 48h incubation, the mRNA of CLC3 expression was up regulated in the groups of 1000-3000μg/L, P<0.05, the difference has statistical significance. The mRNA of CLC3 expression was down regulated in the group of 7000μg/L, P<0.05. There was no difference between the group of 5000μg/L.5. Western blot of chloride channel (CLCN3) protein:After 24h incubation, the protein of CLCN3 expression increased in the groups of 1000-5000μg/L, P<0.05. In the group of 9000μg/L, the CLCN3 protein decreased, P<0.05. There was no difference between the group of 7000μg/L. After 48h incubation, the protein of CLCN3 expression increased in the groups of 1000-3000μg/L, P<0.05. In the group of 7000ug/L, the CLCN3 protein decreased, P<0.05.But there was no difference between the group of 5000μg/L.[Conclusions]1. The iodine can induce the proliferation and apoptosis of human umbilical veins endothelial cell. The effect is related to the action time and concentration.2. The sodium/iodide symporter (NIS) dosen't express on the human umbilical veins endothelial cell, so the iodine can not get in the cells by the NIS. 3. The chloride channel (CLC3) express on the human umbilical veins endothelial cell, iodine can get in the cells by CLC3 to promote the proliferation and apoptosis of the cells.