人腹膜微血管内皮细胞的原代培养

目的 分离和培养人腹膜微血管内皮细胞.方法 取非炎症且非转移性肿瘤患者施行腹部外科手术的大网膜组织,经Ⅰ型胶原酶消化,两次筛网过滤、离心等步骤获取人微血管段,接种24 h待内皮细胞从微血管段爬出贴壁后,去除微血管段获得内皮细胞进行传代培养.组织形态学观察及免疫组化法鉴定所培养的细胞.结果 运用此法进行的原代培养,血细胞在换液和传代过程中被去除,成纤维细胞污染被减至最少;组织形态学观察显示内皮细胞呈铺路石样生长,免疫组化鉴定表明Ⅷ因子阳性;证实所培养的细胞为人腹膜微血管内皮细胞,其纯度较高,生长状态良好.结论 实验成功分离和培养了人腹膜微血管内皮细胞,为体外研究腹膜透析的病理生理学改变,提供了细胞来源.     

对腹膜透析中腹膜血管生成的认识

长期腹膜透析会引起腹膜血管生成,对腹膜功能有重大的影响。本文就尿毒症腹膜透析患者腹膜血管生成的原因,血管形成的病理生理过程及腹膜血管新生对腹膜功能的影响做一综述。     

血管生成素2与腹膜透析时腹膜血管新生的关系

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [(5±3)/HP vs (1±1)/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [(10±5)/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control (P<0.01), and such expressions were significantly higher in the PD groups as compared to non-PD group (P<0.01), but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density(r=0.7756, P<0.01; r=0.5223, P<0.05). Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.     

脂肪细胞通过TNF-α-p38 MAPK信号通路促进内皮细胞ICAM-1表达

目的血管内皮细胞功能紊乱是腹膜透析患者心血管疾病(cardiovascular disease,CVD)发生的重要原因。本研究模拟腹膜透析液的高糖环境,观察高糖作用下脂肪细胞对微血管内皮细胞细胞间粘附分子-1(intercellular adhesion molecule,ICAM-1)表达的影响,探讨腹膜透析患者内皮功能紊乱导致CVD发生的可能机制。方法体外诱导3T3-L1分化成为脂肪细胞。将实验所用内皮细胞分成6组,分别是:对照组,高糖组,高渗组,对照脂肪细胞组(普通培养基培养脂肪细胞所得上清干预内皮细胞),高糖脂肪细胞组(高糖培养脂肪细胞所得上清干预内皮细胞)及高渗脂肪细胞组(高渗培养脂肪细胞所得上清作干预内皮细胞)。细胞增殖检测试剂盒检测b End.3增殖能力。酶联免疫吸附试验检测脂肪细胞高糖、高渗作用后上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达量的改变。Western blot法检测脂肪细胞条件培养基对内皮细胞ICAM-1表达量的影响,及加用丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路阻断剂SB203580作用于内皮细胞后ICAM-1表达量的变化。结果与对照组相比,高糖培养液直接干预内皮细胞或培养脂肪细胞后干预内皮细胞均可以促进内皮细胞增殖(高糖组/对照组:1.600±0.104比1.000±0.000,t=9.954,P=0.010;高糖脂肪细胞组/对照脂肪细胞组:1.563±0.181比1.213±0.097,t=2.945,P=0.042)。高糖可显著促进脂肪细胞TNF-α的表达(2515.313±277.434比788.683±167.267,t=9.232,P=0.001)。高糖培养脂肪细胞的上清显著促进内皮细胞ICAM-1表达(1.533±0.058比1.133±0.153,t=4.243,P=0.013),该作用可被p38 MAPK信号通路阻断剂SB203580部分抑制(0.850±0.111比1.124±0.108,t=-3.053,P=0.038)。结论高糖培养脂肪细胞后,可通过TNF-α-p38 MAPK信号通路促进内皮细胞ICAM-1表达,可能和腹膜透析患者内皮细胞功能紊乱及CVD发生有关。     

高糖和甲基乙二醛对人脐静脉内皮细胞Ang-Tie2信号链表达的影响

目的观察高浓度葡萄糖对体外培养的人脐静脉内皮细胞(HUVECs)血管生成素2(Ang2)和酪氨酸激酶2受体(Tie2)表达的影响,探讨腹膜透析患者腹膜血管新生的可能机制。方法分别以4.25%葡萄糖(高糖组)、4.25%甘露醇(甘露醇组)和800μmol/L葡萄糖降解产物甲基乙二醛(MGO)(MGO组)对体外培养的HUVECs进行干预,设立空白对照组。以MTT比色法测定的D(490 nm)比较各组HUVECs干预后12、24、48 h时点的细胞增殖情况;分别采用Real-Time PCR技术、ELISA法和Western blotting法检测各组HUVECs中Ang2和Tie2 mRNA表达、细胞培养上清液中Ang2分泌量及HUVECs中Tie2蛋白的表达。结果高糖组、甘露醇组和MGO组HUVECs干预后12 h时点的D(490 nm)均明显高于空白对照组(P<0.05),干预后24 h时点的D(490 nm)则显著低于空白对照组(P<0.05)。与空白对照组比较,高糖组、甘露醇组和MGO组HUVECs中Ang2mRNA表达均明显上调,高糖组和甘露醇组细胞培养上清液中Ang2分泌量均显著增加(P<0.05),...     

血管生成素2与腹膜透析时腹膜血管新生的关系

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [(5±3)/HP vs (1±1)/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [(10±5)/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control (P<0.01), and such expressions were significantly higher in the PD groups as compared to non-PD group (P<0.01), but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density(r=0.7756, P<0.01; r=0.5223, P<0.05). Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.     

血管生成素2与腹膜透析时腹膜血管新生的关系

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [(5±3)/HP vs (1±1)/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [(10±5)/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control (P<0.01), and such expressions were significantly higher in the PD groups as compared to non-PD group (P<0.01), but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density(r=0.7756, P<0.01; r=0.5223, P<0.05). Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.     

可溶性酪氨酸激酶2融合蛋白对尿毒症腹膜透析大鼠腹膜形态和功能的影响

目的 研究可溶性酪氨酸激酶2融合蛋白（sTie-2-Fc）对尿毒症腹膜透析大鼠腹膜血管新生、溶质转运和超滤功能的影响。 方法 32只雄性Wistar大鼠按数字随机法分为假手术组、尿毒症组、尿毒症腹透组和sTie-2-Fc干预组（均n=8）。尿毒症腹透组和sTie-2-Fc干预组大鼠经腹透管每天2次腹腔灌注4.25%葡萄糖透析液（3 ml/100 g体质量）共4周,sTie-2-Fc干预组大鼠每次灌注时在透析液中加入1 μg sTie-2-Fc。各组大鼠处死前行腹膜平衡试验,检测腹膜转运和超滤功能,取大网膜标本行抗CD31免疫组化染色并计血管数。 结果 与假手术组大鼠相比,尿毒症组大鼠的2 h腹透液和血肌酐比值（D/Pcr）增高（0.78±0.05比0.70±0.09,P = 0.028）,腹透液2 h与0 h葡萄糖比值（D/D0）降低（0.69±0.05比0.76±0.07,P = 0.033）,腹膜超滤量（UF,ml）减少（2.29±0.50比4.58±1.64,P = 0.005）,腹膜血管数量增加[（5.8±3.0）/HP比（1.6±0.5）/HP,P < 0.01]。尿毒症腹透组大鼠的溶质转运较尿毒症组大鼠进一步增高（D/Pcr: 0.89±0.05比0.78±0.05,P < 0.01;D/D0:0.47±0.09 比0.69±0.05, P < 0.01）,UF（ml）减少（0.40±0.59比2.29±0.50,P = 0.005）,腹膜血管数量增多[（16.7±1.2）/HP比（5.8±3.0）/HP,P < 0.01]。干预组大鼠使用sTie-2-Fc后,UF（ml）较尿毒症腹透组大鼠显著增加（1.56±0.48比0.40±0.59,P = 0.014）,腹膜血管数量显著减少[（9.2±1.2）/HP比（16.7±1.2）/HP,P ＜ 0.01],但两组大鼠的D/Pcr和D/D0差异均无统计学意义。 结论 sTie-2-Fc使尿毒症腹透大鼠腹膜血管新生减少,超滤增加,有利于保护腹膜结构和功能,可能是防治腹透后腹膜结构和功能改变的另一靶点。     

人腹膜微血管内皮细胞的原代培养

目的分离和培养人腹膜微血管内皮细胞。方法取非炎症且非转移性肿瘤患者施行腹部外科手术的大网膜组织,经Ⅰ型胶原酶消化,两次筛网过滤、离心等步骤获取人微血管段,接种24 h待内皮细胞从微血管段爬出贴壁后,去除微血管段获得内皮细胞进行传代培养。组织形态学观察及免疫组化法鉴定所培养的细胞。结果运用此法进行的原代培养,血细胞在换液和传代过程中被去除,成纤维细胞污染被减至最少;组织形态学观察显示内皮细胞呈铺路石样生长,免疫组化鉴定表明Ⅷ因子阳性;证实所培养的细胞为人腹膜微血管内皮细胞,其纯度较高,生长状态良好。结论实验成功分离和培养了人腹膜微血管内皮细胞,为体外研究腹膜透析的病理生理学改变,提供了细胞来源。     

血管生成素2与腹膜透析时腹膜血管新生的关系

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [(5±3)/HP vs (1±1)/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [(10±5)/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control (P<0.01), and such expressions were significantly higher in the PD groups as compared to non-PD group (P<0.01), but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density(r=0.7756, P<0.01; r=0.5223, P<0.05). Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.