摘要
目的 探讨烧伤患者血清 (以下称烧伤血清 )、痂下水肿液诱导人脐静脉血管内皮细胞株ECV30 4凋亡的分子机制。 方法 将体外培养的ECV30 4随机分为 :A组 ,用含体积分数 30 %正常人血清的培养基培养 ;B、C组 ,分别用含烧伤血清、痂下水肿液 (体积分数同A组 )的培养基培养。 6、12、2 4、36h后采用Hochest 332 5 8荧光染色观察 3组细胞核形态学改变 ,并计算细胞凋亡百分率。培养 12h后采用半胱氨酸天冬氨酸蛋白酶 (caspase)检测试剂盒测定caspase 3、8、9的活化情况。培养 12、2 4h后进行琼脂糖凝胶电泳观察细胞凋亡的特征性DNA梯状条带。 结果 A组各时相点下可见胞核呈弥散均匀荧光 ,B、C组细胞培养 2 4h后胞核呈现深染、致密的颗粒块状荧光。培养12～ 36hB、C组的凋亡百分率均高于A组 (P <0.0 1),其中 36h时B、C组的凋亡百分率分别为 (38.9± 7 3) %、(4 9.5± 6 .5 ) % ,明显高于A组 (2 .2± 0 .3) % (P <0.0 1)。B、C组培养 12h后caspase 3、8、9活性均高于A组 (P <0.0 1)。B、C组DNA琼脂糖电泳出现明显梯状条带 ,A组未见此现象。 结论 烧伤血清、痂下水肿液通过同时激活死亡受体信号通路和线粒体信号通路导致ECV30
Objective To explore the molecular mechanism of apoptosis of the human umbilical vein endothelial cells (HUVECs) induced by burn serum and subeschar tissue fluid. Methods In vitro cultured HUVECs were randomly divided into A(treated by normal serum, n=6), B(treated by burn serum, n=6) and C (treated by subeschar tissue fluid,n=6) groups. The change in cellular nuclear morphology was determined by Hochest 33258 fluorescent staining, and the apoptotic rate was calculated at 24th and 36th post treatment hours (PTHs).The DNA ladder strips were observed with agarose electrophoresis at 12th and 24th PTH. The activity of caspase-3, 8, 9 was assayed by caspase colorimetric assay kit at 12 PTH. Results It was found that nuclei appeared to be condensed and highly fluorescent granular in form in B and C groups. The nucleic apoptotic rate in B and C groups was (38.9±7.3)% and (49.5±6.5)%, respectively, which was evidently higher than that in A group (P<0.01). The DNA ladder strips could be found by agarose electrophoresis in B and C groups. The activity of caspase-3, 8, 9 in B and C groups was significantly higher than that in A group after 12 PTHs (P<0.01). Conclusion Burn serum and subeschar tissue fluid might induce apoptosis of HUVEC by means of the activation of death receptor and mitochondrial signal pathways simultaneously.
出处
《中华烧伤杂志》
CAS
CSCD
2004年第5期275-277,共3页
Chinese Journal of Burns
