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目的观察内毒素/脂多糖(LPS)及烧伤血清对豚鼠结肠带平滑肌细胞钙激活钾通道(KCa)的影响,探讨烧伤后发生胃肠动力障碍的分子电生理机制。方法用急性酶分离法获取单个健康豚鼠结肠带平滑肌细胞,在对称性高钾溶液中采用细胞膜片钳单通道记录技术,分别记录细胞贴附式膜片(电极位于细胞膜外面)和内面向外式膜片(电极位于细胞膜内面)上的电流。引出的电流信号经转换器转换,输入计算机,用离子通道计算机分析系统进行数据处理,并测定以下指标：(1)电流幅值(CA)；(2)开放概率(PO)；(3)开放时间(OT)；(4)关闭时间(CT),以检测其特性并鉴定是否为KCa。确定为KCa后,向浴液中分别加入20、40、60、80、100 mg/L的LPS,观察LPS对两种膜片方式上KCa的影响。在浴液中分别加入正常血清和烧伤血清,观察血清对KCa的影响。结果在对称性高钾溶液中,豚鼠结肠带平滑肌细胞内面向外式膜片上的KCa电导值为(271±7)ps,是高电导的离子通道。随着膜去极化及细胞内Ca~(2+)增加,通道PO增加,该通道可被细胞外低浓度的钾通道阻断剂四乙胺(TEA,1 mmol/L)所阻断,而膜内面较高浓度的TEA(40 mmol/L)对该通道无阻断作用,证实为KCa。当Ca~(2+)为0 mol/L时,向浴液中分别加入20、40、60、80、100 mg/L的LPS,两种膜片方式KCa活性随LPS浓度的增加而增加。40 mg/L以上的LPS对KCa具有明显激活作用,通道PO显著增加(P＜0．05或0．01),用不含LPS的浴液灌流后亦不能回转。两种膜片方式下烧伤血清对KCa有激活作用,而正常血清无此作用。结论LPS和烧伤血清可通过激活肠道平滑肌细胞KCa抑制肠道蠕动。
Objective To observe the effect of LPS and burn serum on calcium-activated potassium channel (KCa) in guinea pig colonic smooth muscle cells and to explore the molecular electrophysiological mechanism of gastrointestinal motility disorder after burn injury. Methods Acute healthy guinea pig colon smooth muscle cells were obtained by acute enzymatic dissociation method. Cell patch clamp single channel recording technique was used in symmetrical high potassium solution. Cell attachment patch (the electrode was located outside the cell membrane) Current on the membrane (the electrode is on the inside of the cell membrane). The extracted current signal is converted by a converter, input into a computer, processed by an ion channel computer analysis system, and the following indexes are determined: (1) current amplitude (CA); (2) open probability (PO); Time (OT); (4) Turn off time (CT) to detect its characteristics and identify if it is KCa. After confirming as KCa, 20, 40, 60, 80 and 100 mg / L LPS were respectively added to the bath, and the effect of LPS on the KCa in the two membrane modes was observed. The normal serum and burn serum were respectively added to the bath liquid to observe the effect of serum on KCa. Results The KCa conductance of the inward-facing membrane in guinea pig colon smooth muscle cells was (271 ± 7) ps in symmetrical high potassium solution, which was a highly conductive ion channel. With the increase of membrane depolarization and intracellular Ca2 +, channel PO increased, which was blocked by tetraethylammonium (TEA, 1 mmol / L), an extracellular extracellular potassium channel blocker. The TEA (40 mmol / L) higher concentration inside the membrane on the channel without blocking, confirmed as KCa. When Ca 2+ was 0 mol / L, 20, 40, 60, 80 and 100 mg / L LPS were added into the bath, respectively. The KCa activity of the two membranes increased with the increase of LPS concentration. LPS at concentrations above 40 mg / L markedly activated KCa, while PO increased significantly (P <0.05 or 0.01). After LPS infusion, LPS also failed to revolve. Burn sera both activated the KCa in the two membrane modes, while the normal serum did not have this effect. Conclusion LPS and burn serum can inhibit intestinal motility by activating KCa of intestinal smooth muscle cells.