The Changes Of BKca In Type2Diabetes Rat Vascular Smooth Muscle Cells

bjective: This experiment by using whole cell patch clamptechnique of perforation observed the BKca channels current changes in aortaand mesenteric artery vascular smooth muscle cells of type2diabetic rat,discuss the relationship between BKca and vascular complications in type2diabetic. Methods:1.Type2diabetes rat model: weight the adaptive fed ofmale SD rat after a week, and randomly divided into two groups, the controlgroup fed with ordinary feed, high lipid group fed with high-fat diet. Thehigh-fat group SD rats intraperitoneally injection of STZ25mg/kg (pH4.20.1mol/L citric acid buffer solution with a concentration of0.25%) after fourweeks; the control group by intraperitoneally injection volume of0.1mol/Lcitric acid buffer solution.2.Type2diabetes rat model identifition:identify the tail vein blood FBG, lipid, blood insulin levels of injected into STZa week later, model rats feeding on high diet, using3%sodium pentobarbitalintraperitoneal injection of30mg/kg after anesthesia at the end of the eight ortwelve week, and thoracic aorta and mesenteric arterial supply test.3.The patchclamp experiment:(1)cell suspension of production: the separation of thearterial cut into pieces, a cell separating medium constant temperature of37℃,oscillating water bath,the enzyme solution two step digestive cells interstitialand the surrounding connective tissue by enzymatic hydrolysis, and then will bedigested arterial tissue on the calcium free desktop liquid, the selection has strong stereo feeling, smooth muscle cells, using whole-cell patch clamptechnique to record the current perforation.(2)whole cell perforationpatch-clamp steps: experimented at room temperature, the microtubule tipelectrode and surrounding cell form electrochemical impedance insulation,about10～100GΩ, to form the whole cell configuration. With the softwareClampex10.1collect current, Clampfit10.1software system for data analysis,statistics of current amplitude and the production of I-V curve.(3)single channelpatch-clamp steps: In the formation of the whole cell configuration of theelectrode tip surface, back in the bath to form inside-out patch, usingpClamp9.0software to record the channel current.Results:1.Channel openprobability with voltage and the Ca2+concentration in the single channel bathincreases.2.In acute enzyme isolated from rat thoracic aorta smooth muscle cellBKca current basic characteristics:(1) BKca macroscopic current has obviousvoltage dependent and outwardly rectifying properties;(2) compared the BKcacurrent in the control group, high lipid group at eight weeks and twelve weeks:control group(n=8), high lipid eight weeks group(n=6), lipid twelve weeksgroup(n=6) current respectively352.31±82.56pA（+60mV）,719.25±45.03pA（+60mV）,911.58±77.82pA（+60mV）. Inspection by t test, control group andhigh lipid eight weeks group, t=9.785, P<0.05; control group and high lipidtwelve weeks group, t=12.845, P<0.05; high lipid for eight weeks and twelveweeks group, t=5.240, P<0.05.3. In acute enzyme isolated from rat mesentericartery smooth muscle cell BKca current basic characteristics:(1) BKcamacroscopic current has obvious voltage dependent and outwardly rectifying properties;(2) compared the BKca current in the control group, high lipidgroup at eight weeks and twelve weeks: control group (n=5), high lipid eightweeks group(n=5), lipid twelve weeks group(n=5)current respectively169.88±67.13pA（+60mV）,330.41±55.04pA（+60mV）,568.77±62.97pA（+60mV）. Inspection by t test, control group and high lipid eight weeksgroup, t=4.135, P<0.05; control group and high lipid twelve weeks group,t=9.690, P<0.05; high lipid for eight weeks and twelve weeks group, t=6.373,P<0.05. Conclusion:1.This experiment successfully reproduced the T2DMmodel.2.acute enzyme isolated rat thoracic aorta and mesenteric artery smoothmuscle cell BKca with voltage-dependent and Ca2+-dependent, the channelcurrent with outwardly rectifying characteristics.3. high lipid group BKcacurrent amplitudes were higher than the control group, while the high lipid fortwelve weeks in the BKca group current amplitude than the high lipid eightweek group.