大肠杆菌脂多糖体外诱导小鼠血小板凋亡的研究

A study of apoptosis of murine platelet induced by lipopolysaccharide derived fromEscherichia coli in vitro

目的：观察大肠杆菌脂多糖（LPS）是否可在体外诱导小鼠血小板凋亡的发生。方法制备洗涤血小板悬液，调整血小板终浓度至3x108/mL。根据刺激剂不同分为对照组〔无钙台式缓冲液（TB）〕、凝血酶处理组（终浓度1 U/mL，无钙TB制备）和不同浓度LPS处理组（终浓度1、10、100μg/mL，无钙TB制备）。各组加入相应刺激剂后室温孵育30 min。应用化学发光法检测三磷酸腺苷（ATP）含量及天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）活性；用流式细胞仪检测血小板膜联蛋白Ⅴ（AnnexinⅤ）阳性率以反映磷酯酰丝氨酸（PS）暴露水平；用流式细胞仪测定血小板平均通道荧光强度（MCF）以反映线粒体内膜电位（ΔΨm）去极化。结果与对照组比较，凝血酶处理组血小板ATP水平明显降低〔相对荧光强度（RLU）：（5.46±0.14）x105比（6.25±0.26）x105，P＜0.05〕， AnnexinⅤ阳性率〔（50.43±2.45）%比（1.58±0.25）%，P＜0.05〕和caspase-3活性〔RLU：（26.92±1.60）x103比（1.30±0.10）x103，P＜0.05〕均明显升高，血小板MCF明显降低〔（8.32±0.58）x104比（13.05±1.10）x104，P＜0.05〕，说明ΔΨm去极化增加。给予不同浓度LPS处理后，血小板ATP水平、AnnexinⅤ阳性率和caspase-3活性均明显升高，血小板MCF明显降低，说明ΔΨm去极化增加，且呈浓度依赖性。与对照组比较，1μg/mL LPS即可使AnnexinⅤ阳性率增加〔（10.45±1.08）%比（1.58±0.25）%， P＜0.05〕，caspase-3活性升高〔RLU：（14.06±0.61）x103比（1.30±0.10）x103，P＜0.05〕，MCF明显降低〔（9.48±0.50）x104比（13.05±1.10）x104，P＜0.05〕。经100μg/mL LPS处理后血小板ATP水平、AnnexinⅤ阳性率和caspase-3活性最高，且均明显高于对照组〔ATP（RLU）：（7.00±0.03）x105比（6.25±0.26）x105， AnnexinⅤ阳性率：（55.35±2.42）%比（1.58±0.25）%，caspase-3（RLU）：（32.00±3.75）x103比（1.30±0.10）x103，均P＜0.05〕；血小板MCF最低，且明显低于对照组〔（4.69±0.55）x104比（13.05±1.10）x104，P＜0.05〕。结论大肠杆菌LPS可体外诱导小鼠血小板ATP升高、PS暴露、ΔΨm去极化及caspase-3活性增加，说明LPS可诱导血小板凋亡，且呈浓度依赖性。

ObjectiveTo observe whether lipopolysaccharide （LPS） derived fromEscherichia coli （E.coli） can induce apoptosis of murine platelets in vitro.Methods Washed platelet suspension was prepared and adjusted to the final concentration of 3x108/mL. According to the difference in stimulants, samples were divided into control group （non-calcium Tyrode buffer）, thrombin-treated group （1 U/mL final concentration and non-calcium TB） and LPS in different concentrations treated groups （1, 10 and 100μg/mL final concentration respectively and non-calcium TB）. To each specimental group corresponding stimulus was added and incubated 30 minutes at room temperature. Chemiluminescence was adopted to determine the concentration of adenosine triphosphate （ATP） and the activity of cysteinyl aspartate specific proteinase-3 （caspase-3）. The percentage of Annexin V positive platelets was determined by flow cytometry to reflect the level of phosphatidylserine （PS） exposure. Mean channel fluorescence （MCF） of platelets was determined by flow cytometry for reflecting the level of mitochondrial inner transmembrane potential （ΔΨm） depolarization.Results Compared with control group, the ATP concentration in thrombin-treated group was decreased obviously [relative light unit （RLU）： （5.46±0.14）x105 vs. （6.25±0.26）x105,P〈 0.05], Annexin V positive ratio [（50.43±2.45）% vs. （1.58±0.25）%,P〈 0.05] and caspase-3 activity [RLU： （26.92±1.60）x103 vs. （1.30±0.10） x103,P〈 0.05] were increased obviously, and platelets MCF was lowered significantly [（8.32±0.58）x104 vs. （13.05±1.10）x104,P〈 0.05], suggesting an increase inΔΨm depolarization. After being treated with different concentrations of LPS, ATP concentration, Annexin V positive ratio and caspase-3 activity were increased obviously, platelet MCF was decreased obviously, suggestingΔΨm depolarization was increased in a concentration-dependent manner. Compared with control group, 1μg/mL LPS could increase Annexin V positive ratio [（10.45±1.08）% vs. （1.58±0.25）%,P〈 0.05], elevate caspase-3 activity [RLU： （14.06±0.61）x103 vs. （1.30±0.10）x103,P〈 0.05], and decrease MCF significantly [（9.48±0.50）x104 vs. （13.05±1.10）x104,P〈 0.05]. The ATP concentration, Annexin V positive ratio and caspase-3 activity reached maximum levels after the treatment with 100μg/mL LPS, and they were higher obviously than those of the control group [ATP （RLU）： （7.00±0.03）x105 vs. （6.25±0.26）x105, Annexin V positive ratio： （55.35±2.42）% vs. （1.58±0.25）%, casepase-3 （RLU）： （32.00±3.75）x103 vs. （1.30± 0.10）x103, allP〈 0.05], and platelets MCF reached trough levels, and they were obviously lower than those of the control group [（4.69±0.55）x104 vs. （13.05±1.10）x104,P〈 0.05].ConclusionE.coli LPS can induce an increase in ATP, PS exposure,ΔΨm depolarization and activity increase of caspase-3 on mouse platelet in vitro, which indicate that LPS can induce apoptosis of platelets in a concentration-dependent manner.