摘要
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 investigate the influence of lipopolysaccharide （LPS） and burn sera on potassium channels （Kca） in smooth muscle cells of colon of guinea pig so as to elucidate the molecular mechanism of gastrointestinal motility dysfunction after severe burns. Methods Single smooth muscle cells were isolated from the taenia coli of guinea pig with enzyme digestion. The standard patch clamp technique was employed to record the single KCa channel currents of smooth muscle cell after challenged by LPS and burn serum. Data were recorded and analyzed by Pclamp 6.04 software, and the probability of open （PO） , mean open time （OT） , mean close time （CT） and current amplitude （CA） were determined. Subsequently, LPS in the concentration of 20,40,60,80 and 100 mg/L, respectively, and normal serum and burn serum in the concentration of 10% were respectively added into medium to examine the influence of the two clamps and sera on the KCa activity. Results In hyperkalemic solution, the KCa conductance of colonic smooth muscle cells of the guinea pig was （271 ± 7）pS, indicating it was an ionic channel with high conductivity. Subsequent to depolarization of the membrane, inner-cellular calcium level was increased, and channel PO was also increased, which could be blocked by l mmol/L tetraethylammonium （TEA） outside the membrane chaff. As 40mmol/L TEA inside the membrane chaff did not show such effect, it was proved to be KCa current. The activity of the channel as determined with two kinds of clamps was increased in a dose dependent manner with LPS challenge when the concentration of calcium was 0 mol/L. The KCa activity and PO of the channel was increased obviously when the concentration of LPS was above 40 mg/L（ P 〈 0.05 or 0.01 ） , and it could not be reversed after irrigation with non-LPS medium. By using the two kinds of patch clamps, the KCa were activated by burn sera, but not normal sera. Conclusion Both LPS and burn sera can lead to inhibition of the gastrointestinal motility by activation of KCa channels.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2006年第3期184-186,共3页
Chinese Journal of Burns
基金
四川省人事厅学术和技术带头人培养资金资助项目(4200321)
四川省教育厅科研基金(2003C011)
关键词
内毒素类
血清
离子通道
肌细胞
平滑肌
膜片钳技术
Endotoxins
Serum
Ion channels
Myocytes, smooth muscle
Patch clamp technique
