The Effects of Chloride Channel ClC-2 on Human Retinal Pigment Epithelial Cell’s Proliferation and Migration and on PVR’s Formation
|Keywords||Chloride channel ClC-2 Retinal pigment epithelial cell Proliferative vitreoretinopathy Proliferation Migration|
objective:The chloridion is the anion that the human body contains most . The transport of chloridion is through transmembrane transport and anion channel. This kind of conveyance is beneficial to the accomplishment of various physiology process, such as the volume of cells’regulation and variety in PH. The voltage- gated CL channel (ClC type CL channel)is a kind of CL channel. Human’s genes ClC-2s locate at chromosome3q27, the ClC-2 protein that codes 898 amino acidses, expresses in human body very extensively. Its function may possibly have some connection with the cell capacity. At present, it is indicated in some researches that the function of ClC-2 has some closed relationship with proliferation, cell cycle and migration of some cells. The cell proliferation of cultured lung epithelial cell and T leukomonocyte was significantly be inhibited after using chloride channel blockers .High activity of chloride channel has been observed in the cell of cervix cancer . Prominent ClC-2 channel expression was detected upwards in malignant gliomas. The function of cells’proliferation was decrease if expression of ClC-2 gene has been inhibited. The migration of gliomas cells and proliferation of nasopharyngeal、hepatoma,leukemic and malignant melanoma’cells was restrained if ClC-2 was blocked. Besides, Someone has observed the function of chloride channel was different in each phase of cell cycle in gliomas cell: it is the highest in G1 phase, and the lowest in S phase .The blocker of chloride channel not only can make cells not able to pass through the restrict point in G1/S and stay in G0/G1 phase ,but also has strong inhibited function in process of G0 to G1 phase.Proliferative vitreoretinopathy (PVR) is a common ophthalmopathy causing of blindness. Along with gradually thorough investigation, the mechanism of its occurrence and development has been already announced to the public gradually. At present, it is thought that Retinal pigment epithelial (RPE)cell is a main cell composition of PVR’s membrane. Its abnormality proliferation is a main reason of PVR’occurrence and a main reason for reattachment of retina during an operation, and also the remodeling stage is a key stage of PVR formation. This stage is depending on RPE cell’s phagocytize to extracellular matrix(ECM).Therefore, RPE cell’s function of proliferation、migration and phagocytosis have direct relationship with PVR.This research focuses on the cultured human RPE cell, observing the kind of binding protein of RPE cell. We intended to find out whether the function of proliferation、migration and phagocytosis were inhibited after the function of ClC-2 are inhibited by the blocker of chloride channel and siRNA technique; and we also observed whether it influence the formation and development of PVR by using chloride channel blocker in animal model experiment .contents:We found out binding protein of ClC-2 protein by utilizing Yeast-two—hybrid Technique, detected the expression of ClC-2 mRNA and protein in RPE cell by RT-PCR and Western-Blot methods,The cellular proliferation rate was determined by MTT assay, the cell cycle was measured by flow cytometry after inhibited ClC-2’function by chloride channel blocker and SiRNA. We observed the changing of RPE cell’s phagotrophic function by established phagotrophic model of emulsion strain coated with FN. We also find out whether it influence formation and development of PVR by animal model experiment .Results:we found out binding protein of ClC-2 protein by utilizing Yeast-two—hybrid. Techniques, which are known now fall in three categories : M-phase specific protein(Magoh)、protein phophatase 1α、( PPase 1α)and Smad 6. All of them are related to cell cycle and cellular proliferation. Magoh is one ingredient of exon–junction complex (EJC)’s core and has important function on transformation from pre-mRNA to mRNA; PPase 1αi?s a kind of protein phophatasen. It can catalyze protein dephophorylation that regulated cellular sinal transport. TGF—βplays a key role in cell’s growth, cellular differentiation and apoptosis. Smad’s family is downstream factor of TGF-β’s signal transportation, Smad 6 is a rejective number of Smad’s family,and play a role of regulation to TGF-βsignal transportation. These facts indicated that ClC-2 may be related to cellular proliferation and cell cycle.We find out the expression of ClC-2 mRNA and protein in RPE cell. This finding has the same expression as reports abroad.We inhibits ClC-2’s function by chloride channel blockers: NPPB and tamocifen , and finds out RPE cell’s proliferation rate was inhibited by MTT assay, 100μM’s NPPB and 50μM’s tamocifen inhibit cellular proliferation rate at about 50%-60%,and finds out there is clearly changes of RPE cell’s cycle by flow cytometry, 100μM’s NPPB and 50μM’s tamocifen can increase the number of cells in G1 phase and decrease the number of cells in G2/M phase clearly , while the number of cells in S phase has no marked change. We observe the changing of RPE cell’s phagotrophic function by established phagotrophic model of emulsion strain coated with FN, and find out that 100μM’s NPPB and 50μM’s tamocifen inhibits cellular phagotrophic rate at about 70%-75%. All these findings have marked significance in statistics.The inhibited expression of ClC-2 by SiRNA and RT-PCR and Western-Blot’s results all show that expression of ClC-2 mRNA and protein are inhibited markedly. Cellular proliferation rate of recombinant plasmids ClC-2’s group is 43% after been inhibited for 48 hours .That indicated Cellular proliferation is inhibited obviously. Cellular phagocytize index of recombinant plasmids ClC-2’s group is 8% after being inhibited for 3 hours .That indicated the function of Cellular phagocytosis is inhibited obviously.We injected chloride channel blockers to vitreous cavity of established rabbit’s PVR model , and observed the formation and development of PVR were inhibited obviously by Fastenberg’s score、fundus photograph、B model ultrasonograph and pathologic examine .Conclusions:First: Both ClC-2 mRNA and its protein expressed in RPE cell; Binding protein of ClC-2 protein are Magoh、PPase 1αand Smad 6. Second : There is markedly positived relationship between activity of ClC-2 and the function of proliferation、migration and phagocytosis of RPE cellThird: the formation and development of PVR can be inhibited obviously by chloride channel blockers depending on its dose.Achievements and its value:There are some achievements about the relationship between chloride channel and function of proliferation、migration and phagocytosis of cell. But there are still no reports about the relationship between ClC-2 and function of proliferation、migration and phagocytosis of RPE cell .In this research ,we firstly in vitro investigated the relationship between ClC-2 and function of proliferation、migration and phagocytosis of RPE cell by cultured cell and investigated the relationship between ClC-2 and formation and development of PVR, This provided a new thought about PVR’s pathomechanism, and about PVR’s drug treatment and gene treatment.