The Association Of Voltage-gated K~+ Channels And Proliferation Of Human Lung Squamous Carcinoma Cells

The lung cancer is the most common lung primary stage malignant tumor,most lung cancer origins from the bronchial tube mucous membrane epidermis,therefore it is also called the bronchial tube lung cancer.In many developed countries,the lung cancer occupies the first place in the masculine common tumor,and takes the second, third place in the feminine common tumor.For almost half a century,the lung cancer disease incidence rate and the mortality rate in every country all has sharply risen,our country is of no exception.According to investigation,lung cancer disease incidence rate in cities is higher than that in villages.In the Beijing,Shanghai,and Tianjin it holds the first place of all the malignant tumors.As one of the most important tumors in world scope,it has surpassed 500,000 new case of illness every year,the domestic lung cancer mortality rate rises also quite quick;it reaches 20.41/100,000 in the 90's. This disease incidence rate grows day by day,although massive manpower and physical resource has been put in to the research,the outcome regrettably still is unsatisfactory.In recent years,with the development of diaphragm pliers technology and the member biological technology progress,as well as in the tumor cell membrane ion channel research application,the tumor cell membrane ion channel research has become the new tidal current.The ion channel is in the cell membrane crossing membrane protein member,is one that has the selective permission,is a hydrophilic micro passageway suitable for electric charge ion passing through,and it connects the cell interior and the external environment.The research indicates that the ion channel and the tumor have close relation,the potassium ion channel is the one that lies broadly in the cell membrane and has many types.It has many important biologic functions,and thus becomes the target spot of anti-tumor medicine.Since 1976 Erwin Nether and Bert Sakmann has established diaphragm pliers technology,the ion channel,especially researches on the cardiac muscle smooth muscle cell ion channel has developed fast,further study on the cell membrane ion channel has attacked more and more attention.Researches indicate that the lung cancer correlation gene the expression,the flaw or the sudden change effects by the cell membrane signal conduction system and the cell membrane ion channel. The recent researches discover that the Cell membrane ion channel plays a very significant role in cell canceration,signal conduction of attack and shift coherent, gene expression.In 1999,Yao reported that the potassium channel expression on breast cancer cell membrane was abnormal.Other reports also discovered that there was one kind of new introverted rectification potassium electric current in nervous fibroma and pituitary tumor cell membrane potential.Up to now,there has no report about research on human lung squamous carcinoma cell and cell characteristic by suppression of K⁺ channel activity both in and abroad.To study whether the voltage-gated K⁺ channels are required for the proliferation of squamous carcinoma cell.The results could be based for the theropy of human lung adenocarcinoma cancer in clinics.Methods:prepare lung squamous carcinoma cells A₂ raised for 24-72h,choose the quiescent,rod-shaped cells with clear cross stiation to studied.1,Cell electricity physiology experiment:Ionic currents were recorded using the whole cell configuration of the voltage clamp technique.Put the micro propeller to tip of capillary electrode forming the negative pressure suction high-resistance seals meets,and attracts the broken cell membrane to form the entire cell diaphragm;Uses the depolarization to induce the lung squamous carcinoma cells A₂ gradually the extroverted potassium ion electric current,then records the effect of inhibition of K⁺ channels by TEA to the cells was studied by applying TEA 5mmol/L.2,The proliferation activity of the cells experiment:the proliferation activity of the lung squamous carcinoma cells A₂ was measured by MTT assay.The A value under the 570nm wave length of the cells treated with the complex of different concentrations of TEA for 12,24,36and48 hours.3,Morphology change perishes weakly which with the Hoechst33258 chromosome examination cells.Results:1,Under the record of whole cell configuration of the voltage clamp technique., selects the voltage immobilization method,causes the cells A₂ in the testing voltage scope for -40mV—+80mV,maintenance voltage for -70mv,the stimulation time is 500ms,along with testing voltage enlargement,the potassium ion electric current increased range also increases,explained it has the voltage dependence.2,After records the K⁺ currents of cells A₂,we add the K⁺ channel blocker—TEA to the solution with the medicine installment,then observe the K⁺ currents has changed under the test plan in the same maintenance voltage.Exposure of tumor cells to 5 mmol/L TEA reduced the peak outward K⁺ current.3,The MTT experiment result showed that,TEA has the time and the dosage correlation characteristic to the inhibition of cells proliferation,when the TEA density is bigger than was equal to when 2mmol/L,is obvious to the cells A₂ multiplication inhibitory action.4,Dyes after Hoechst33258 observes under the fluorescence microscope,after 5mmol/L TEA affects 48h,obviously has perishes weakly the shape characteristic cells A₂.Conclusions:1,On the human lung squamous carcinoma cell has the potassium ion channel. That is the charactor:(1) It is said that it has the voltage dependence,.(2)The biggest currents of K⁺ channels on the human lung squamous carcinoma cells A₂ is the 112.35±7.53 pA/pF(3) The capacitance of human lung squamous carcinoma cells A₂ is 14.13±0.45pF.2,Voltage dependent potassium channel in lung squamous carcinoma cells play the important role in the cells proliferation,moreover the potassium ion electric current is sensitive to TEA,potassium channel blocking agent TEA can suppress the proliferation of lung squamous carcinoma cells.The inhibition of cells proliferation has related to the dosage and the time of TEA.3,We investigated the electrophysiological properties of human lung squamous carcinoma cell and cell proliferation by suppression of K⁺ channel activity.To study whether the voltage-gated K⁺ channels are required for the proliferation of squamous carcinoma cell.The results could be based for the theropy of human lung adenocarcinoma cancer in clinics.4,K⁺ channel blocker—TEA can induce apoptosising of Lung squamous carcinoma cells by blocking the K⁺ channel.