目的:探索纯度较高、相对经济简便的大鼠脑微血管内皮细胞分离和原代培养的方法。方法:取1～10 d SD大鼠脑皮质经胰蛋白酶消化后,通过不同孔径的筛网过滤,再用胶原酶2次消化获得的微血管内皮细胞进行培养,采用相差显微镜形态学观察及Ⅷ...目的:探索纯度较高、相对经济简便的大鼠脑微血管内皮细胞分离和原代培养的方法。方法:取1～10 d SD大鼠脑皮质经胰蛋白酶消化后,通过不同孔径的筛网过滤,再用胶原酶2次消化获得的微血管内皮细胞进行培养,采用相差显微镜形态学观察及Ⅷ因子相关抗原免疫组化鉴定。结果:1～2 d细胞呈区域性单层贴壁生长,7～9 d呈典型的铺路卵石样征象,Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,可见细胞的胞浆及核周呈棕黄色,胞核呈阴性,阳性细胞占绝大部分。结论:采用2次消化、2次过滤技术可以成功分离培养出较理想的大鼠脑微血管内皮细胞。展开更多
Objective：To study the effects of the Weinaokang（维脑康,WNK）,the active compounds extracted from Ginkgo,Ginseng,and saffron,on ischemia/reperfusion（I/R）-induced vascular injury to cerebral microvessels after glob...Objective：To study the effects of the Weinaokang（维脑康,WNK）,the active compounds extracted from Ginkgo,Ginseng,and saffron,on ischemia/reperfusion（I/R）-induced vascular injury to cerebral microvessels after global cerebral ischemia.Methods：Male C57BL/6J mice were randomly divided into 5 groups（10 animals/group）：the sham group（0.5%CMC-Na,20 mL/kg）,the I/R model group（0.5%CMC-Na, 20 mL/kg）,the I/R＋Crocin control group（20 mg/kg）,the I/R＋high dose WNK group（20 mg/kg）,and the I/R＋low dose WNK group（10 mg/kg）.Bilateral common carotid artery occlusion（BCCAO,20 min） in mice, followed by 24 h reperfusion,was built.The generation of nitric oxide（NO）,the activity of nitric oxide synthase （NOS）,the phosphorylation of extracellular signal-regulated kinase 1/2（ERK1/2）,and the expression of matrix metalloproteinases-9（MMP-9） and G protein-coupled receptor kinase 2（GRK2） in cortical microvascular homogenates were evaluated.The ultrastructural morphology of cortical microvascular endothelial cells （CMEC） was observed.Results：The transient global cerebral ischemia（20 min）,followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS.The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC,as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression.Furthermore,enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical micovessels after l/R（20 min/24 h）.As well as the positive control Crocin（20 mg/kg, 21 days）,pre-treatment with WNK（20,10 mg/kg,21 days） markedly inhibited nitrative injury and modulated the ultrastructure of CMEC.Furthermore,WNK inhibited GRK2 translocation from cytosol to the membrane（at 20 mg/kg） and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels.Conclusion：WNK and its active compounds（Crocin） are effective to suppress l/R-induced vascular injury to cerebral mi展开更多
非创伤性股骨头坏死(nontraumatic osteonecrosis of femoral head,NONFH)是由多种原因引起的骨组织血供中断,骨细胞坏死。因早期症状不典型、诊断困难,而患者晚期会面临因股骨头塌陷所引起的髋关节疼痛、活动受限,只能行髋关节置换术...非创伤性股骨头坏死(nontraumatic osteonecrosis of femoral head,NONFH)是由多种原因引起的骨组织血供中断,骨细胞坏死。因早期症状不典型、诊断困难,而患者晚期会面临因股骨头塌陷所引起的髋关节疼痛、活动受限,只能行髋关节置换术。故研究非创伤性股骨头坏死的发病机制对疾病早期诊断和治疗有重要意义。微小核糖核酸(microRNA,miRNA)是一种非编码的单链RNA分子,参与调控基因转录后的表达。随着miRNA研究技术的发展及对非创伤性股骨头坏死这一疾病研究的深入,越来越多的miRNA被发现在非创伤性股骨头坏死中异常表达。本文通过整理非创伤性股骨头坏死不同组织和细胞中的差异表达miRNA,发现了多次报道表达异常且趋势一致的miRNA,并对这些miRNA的功能和可能的发病机制进行综述。展开更多
文摘目的:探索纯度较高、相对经济简便的大鼠脑微血管内皮细胞分离和原代培养的方法。方法:取1～10 d SD大鼠脑皮质经胰蛋白酶消化后,通过不同孔径的筛网过滤,再用胶原酶2次消化获得的微血管内皮细胞进行培养,采用相差显微镜形态学观察及Ⅷ因子相关抗原免疫组化鉴定。结果:1～2 d细胞呈区域性单层贴壁生长,7～9 d呈典型的铺路卵石样征象,Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,可见细胞的胞浆及核周呈棕黄色,胞核呈阴性,阳性细胞占绝大部分。结论:采用2次消化、2次过滤技术可以成功分离培养出较理想的大鼠脑微血管内皮细胞。
基金Supported by the Project of National Natural Science Foundation of China(No.30830118)the National Key New Drug Project(No.2009ZX09102-137,2009ZX09502-014)
文摘Objective：To study the effects of the Weinaokang（维脑康,WNK）,the active compounds extracted from Ginkgo,Ginseng,and saffron,on ischemia/reperfusion（I/R）-induced vascular injury to cerebral microvessels after global cerebral ischemia.Methods：Male C57BL/6J mice were randomly divided into 5 groups（10 animals/group）：the sham group（0.5%CMC-Na,20 mL/kg）,the I/R model group（0.5%CMC-Na, 20 mL/kg）,the I/R＋Crocin control group（20 mg/kg）,the I/R＋high dose WNK group（20 mg/kg）,and the I/R＋low dose WNK group（10 mg/kg）.Bilateral common carotid artery occlusion（BCCAO,20 min） in mice, followed by 24 h reperfusion,was built.The generation of nitric oxide（NO）,the activity of nitric oxide synthase （NOS）,the phosphorylation of extracellular signal-regulated kinase 1/2（ERK1/2）,and the expression of matrix metalloproteinases-9（MMP-9） and G protein-coupled receptor kinase 2（GRK2） in cortical microvascular homogenates were evaluated.The ultrastructural morphology of cortical microvascular endothelial cells （CMEC） was observed.Results：The transient global cerebral ischemia（20 min）,followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS.The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC,as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression.Furthermore,enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical micovessels after l/R（20 min/24 h）.As well as the positive control Crocin（20 mg/kg, 21 days）,pre-treatment with WNK（20,10 mg/kg,21 days） markedly inhibited nitrative injury and modulated the ultrastructure of CMEC.Furthermore,WNK inhibited GRK2 translocation from cytosol to the membrane（at 20 mg/kg） and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels.Conclusion：WNK and its active compounds（Crocin） are effective to suppress l/R-induced vascular injury to cerebral mi
文摘非创伤性股骨头坏死(nontraumatic osteonecrosis of femoral head,NONFH)是由多种原因引起的骨组织血供中断,骨细胞坏死。因早期症状不典型、诊断困难,而患者晚期会面临因股骨头塌陷所引起的髋关节疼痛、活动受限,只能行髋关节置换术。故研究非创伤性股骨头坏死的发病机制对疾病早期诊断和治疗有重要意义。微小核糖核酸(microRNA,miRNA)是一种非编码的单链RNA分子,参与调控基因转录后的表达。随着miRNA研究技术的发展及对非创伤性股骨头坏死这一疾病研究的深入,越来越多的miRNA被发现在非创伤性股骨头坏死中异常表达。本文通过整理非创伤性股骨头坏死不同组织和细胞中的差异表达miRNA,发现了多次报道表达异常且趋势一致的miRNA,并对这些miRNA的功能和可能的发病机制进行综述。
