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目的 探讨一氧化氮 (NO)能否诱导弓形虫速殖子凋亡以及 Ca2 +信号传导途径是否参与 NO诱导的虫体凋亡。 方法 采用脱氧核苷酸末端转移酶 (Td T)介导的缺口末端标记法 (TU NEL)、透射电镜和琼脂糖凝胶电泳检测凋亡以及应用 Fura- 2荧光负载技术测定胞浆游离钙浓度 [Ca2 + ]i。 结果 (1) TU NEL 标记法检测表明 NO供体亚硝基铁氰化钠 [Na2 Fe(CN) 5 NO,SNP]可诱导弓形虫速殖子凋亡 ,并呈剂量和时间依赖性。 (2 ) NO清除剂 ,N-乙酰半胱氨酸能明显抑制 SNP诱导的速殖子凋亡 ,而不含 NO的 SNP类似物 ,K3Fe(CN ) 6 不能诱导速殖子凋亡。 (3)透射电镜下见SNP处理的速殖子具有凋亡的典型形态学特征 :核染色质凝集 ,核固缩 ,凋亡小体形成。 (4 )琼脂糖凝胶电泳显示 SNP处理的速殖子 DNA片段呈现凋亡特征性的梯形条带。 (5 )静息状态下弓形虫速殖子胞浆游离钙 [Ca2 + ]i为 10 8.49±4.87nmol/ L (n=15 )。 (6 ) SMP诱导速殖子凋亡过程胞浆[Ca2 + ]i明显升高并呈剂量和时间依赖性。 (7) N-乙酰半胱氨酸能明显抑制 SNP诱导的速殖子胞浆 [Ca2 + ]i升高 ,而K3Fe(CN) 6 不能诱导速殖子胞浆 [Ca2 + ]i升高。 (8)胞外钙螯合剂 EGTA,和 L 型电压依赖性钙通道阻滞剂 ,维拉帕米完全或部分抑制 SNP引起的速殖子胞浆 [Ca
Objective To investigate whether nitric oxide (NO) can induce apoptosis of tachyzoites in Toxoplasma gondii and whether Ca2 + signaling pathway is involved in NO-induced apoptosis. Methods Td T-mediated nick end labeling (TU NEL), transmission electron microscopy and agarose gel electrophoresis were used to detect apoptosis. Fura-2 fluorescence loading technique was used to determine the cytosolic free calcium concentration [Ca2 +] i. Results (1) TU NEL labeling assay showed that NO donor Na2F (CN) 5 NO, SNP could induce Toxoplasma gondii apoptosis in a dose-and time-dependent manner. (2) NO scavenger and N-acetylcysteine significantly inhibited SNP-induced tachyzoites apoptosis, while NO-containing SNP analogs, K3Fe (CN) 6 could not induce apoptosis of tachyzoites. (3) Under the transmission electron microscope, the SNP-treated tachyzoites have typical morphological features of apoptosis: nuclear chromatin condensation, nuclear pyknosis and apoptotic body formation. (4) agarose gel electrophoresis showed that the SNP-treated tachyzoites DNA showed apoptotic trapezoidal bands. (5) The cytosolic free calcium [Ca2 +] i of Toxoplasma gondii at resting state was 10 8.49 ± 4.87 nmol / L (n = 15). (6) The cytosolic [Ca2 +] i in TMP-induced tachyzoites was significantly increased in a dose-and time-dependent manner. (7) N-acetylcysteine significantly inhibited SNP-induced [Ca2 +] i increase in tachyzoite cytoplasm, while K3Fe (CN) 6 did not induce [Ca2 +] i increase in tachyzoite cytoplasm. (8) Extracellular calcium chelator EGTA, and L-type voltage-dependent calcium channel blocker, verapamil completely or partially inhibit SNP-induced tachyzoite cytoplasm [Ca