| Object:hypoxic pulmonary hypertension (HPH) is a common disease ofrespiratory system. The pathologic bases of HPH are increased contracting activity and vascular remodeling. Recent years find that the reducing of potassium channels' activity of pulmonary artery smooth muscle cells (PASMCs) is an important reason of their contraction. The proliferation of PASMCs plays an import role in the vascular remodeling progress of HPH. But the construction of vascular wall is decided by the balance between cell proliferation and apoptosis and therefore the role of apoptosis also can't be overlooked. Some author has postulated that the activity of potassium channels might play an important role in the progress of PASMCs' proliferation on the basis that inhibiting potassium channels can reduce the membrane potential, open the voltage-gated calcium channels and increase the Ca2~ influx. At the same time of influencing the proliferation and apoptosis of PASMCs, nitric oxide (NO) can also open their potassium channels. All the apoptosis of thymocyte, cortical neurons, and malignant astrocytoma has relationship with the changing activity of potassium channels. Potassium channel openers can significantly reduce the pathologic changes of HPH. So if it can be postulated that the changing of potassium channel's activity plays an important role in the proliferation and apoptosis of PASMCs? The object of this research is to answer this question by observe the effect of potassium channel blockers, openers and NO on the activity of potassium channels, cell proliferation and apoptosis of cultured PASMCs.Methods:(1)Isolation and culture of rat PASMCs The intrapulmonary branches of rat pulmonary artery were removed under microscope and were digested in-5-4bstractCa2~ free Hank's solution containing collagenase and elastase, and than cultured in 10% FCS PRMI-1640. The purity of PASMCs was identified by immunohistochemical method.(2)The potassium channels' character of acute dispersed and cultured PASMCs and the effects of channel blockers, openers and NO on them Using the whole-cell patch clamp techniques to do the test. The potassium channel's blockers, openers and NO donor in various concentration were added by a superfusing system at the rate of 1.5--2 ml/min. Membrane currents were recorded under Voltage clamp. The outward potassium currents were elicited by depolarizing cells with a series of test potentials by l0mV step between -40 and +80 mV from a holding potential of -70 mV. The pulse last 250ms, and the interpulse interval was l0s.(3)The effects of potassium channel blockers, openers and NO on cell proliferation and apoptosis of PASMCs, and the relationship between the proliferative and apoptic effects of potassium channel blockers and openers with the calcium channels The above agents in various concentration were added to cultured PASMCs between 5th and 10th passage. By using the non-radioactive cell proliferation assay the amount of A490/630 is directly proportional to the number of living cells in culture. And by the method of 3H-TdR incorporation the amount of cpm can reflect the synthesis of DNA. The cell cycle was tested by the flow cytometer. Both potassium channel and calcium channel blockers or openers were added to cultured PASMCs between 5th and 10th passage at the same time. Cell proliferation was tested by the same methods of above.The above agents in various concentration were added to culturedththPASMCs between 5 and 10 passage. Using the HE stain method, fluorescence microscope and AnnexinV apoptotic assay with flow cytometer to test the apoptic rate of PASMCs. Both potassium channel and calcium channel openers were added to cultured PASMCs between 5th and 10th passage at the same time. Apoptosis were tested by flow cytometer.-6-AbstractResults:(1)Vigorous PASMCs can be acquired more rapidly by using the collagenase with the elastase at the same time. The purity of cultured PASMCs was 98% identified by immunohistochemical metho...
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