建立猪血管内皮细胞对人血清适应的模型及其评估

Establishment and evaluation of porcine vascular endothelial cell models adaptive to human normal serum

目的:观察人免疫球蛋白G、免疫球蛋白M和正常人AB型血清诱导猪血管内皮细胞适应状态的效果。方法:实验于2002-05/2003-02在解放军第四军医大学分子生物学实验室完成。①正常人AB型血清和猪血清制备:取健康AB血型成年人血,每次50mL,置于干燥管内,垂直静置6h,3000r/min离心10min,取上清。操作4℃条件下进行,以保证补体的活性。获得血清于-20℃保存备用,用前0.22μm滤膜过滤除菌。猪血清制备同上。②猪血管内皮细胞培养、纯化及鉴定:取体质量3kg实验用乳猪,自左侧髂总动脉向心侧插入大隐静脉导管20cm,注射肝素500U后持续滴注含肝素抗生素盐水(肝素1×105U/L,青霉素4×107U/L),取腹主动脉和胸主动脉,以4℃含肝素抗生素盐水冲洗管腔和外膜后注入37℃预热的10g/LⅠ型胶原酶,消化5min后收集消化液,以1000r/min离心5min,收集细胞放于CO2孵箱内培养并传代,第3代以后不再使用抗生素。在光学显微镜下观察细胞生长形态,进行培养细胞类型的鉴定。③正常人血清对猪血管内皮细胞溶解试验:将猪血管内皮细胞制成浓度5×106L-1的细胞悬液,接种于96孔培养板,每孔200μL,培养至第4天细胞基本铺满培养板底部后加入含正常人AB型血清的培养液,终浓度分别为5%、10%、25%、40%,以相应浓度猪血清为对照,每组各取3孔。放入37℃孵箱内孵育2h后以四唑盐比色试验测各孔的吸光度值。④猪血管内皮细胞对人血清适应模型的建立及评估:培养猪血管内皮细胞,分别以免疫球蛋白G、免疫球蛋白M和低浓度正常人AB型血清诱导猪血管内皮细胞6d,以四唑盐比色法检测细胞存活率,并以免疫组化方法检测其血红素氧合酶1表达的情况。结果:①接种细胞形态呈圆形、三角形或短梭形,胞体丰满、胞浆均匀、核仁清晰。部分细胞呈团块样,大部分细胞为单细胞分布,其间混杂有少许血细胞,说明培养细胞为血管内皮细胞,无明显杂细胞污染。②在25%的正常人AB型血清作用下,猪血管内皮细胞的存活率为(71±22)%。与对照组相比较,100mg/L免疫球蛋白M和5%、10%正常人AB型血清诱导猪血管内皮细胞存活率分别为(81±31)%,(93±25)%,(90±42)%。③诱导后猪血管内皮细胞胞浆内血红素氧合酶1的表达明显增加。结论:100mg/L免疫球蛋白M和正常人AB型血清能够诱导猪血管内皮细胞的适应,后者效果更为明显。

AIM： To establish the porcine vascular endothelial cell （PVEC） model and evaluate the effect of inducing PVEC accommodation with human immunoglobulin （Ig）G, IgM and AB-type normal human serum （NHS）. METHODS： The experiment was carded out in the Laboratory of Molecular Biology, Fourth Military Medical University of Chinese PLA from May 2002 to February 2003.①Preparation of AB-type NHS and porcine blood serum： Blood serum of 50 mL ware laid vertically in drying tube for 6 hours and centrifugated for 10 minutes （3 000 r per minute）, followed by supematant desertion. The temperature was required to remain at 4℃ to keep the activity of complement. Then the serum was preserved at -20℃ and sterilized by former 0.22 μm filtration. ②Culture, purification and identification of PVEC： A great saphenous vein tube ware inserted 20 cm into the porker weighing 3 kg via left common iliac artery, with the injection of 500 U heparin and saline （containing 1×10s U/L heparin and 4×10^7 U/L benzylpenicillin） continuously. At 4℃, the bore and adventitia of both abdominal aorta and thoracic aorta ware washed with saline and injected with the pre-heated 1% type Ⅰ collagenase at 37℃. Five minutes later, the digestive liquid was collected and centrifugated for 5 minutes （3 000r per minute）, and then PVEC was cultured in CO2 incubator. Light microscope was used to observe the growth of cells so as to identify the type of cells. ③Dissolving test of PVEC to NHS： PVEC suspension with the concentration of 5 × 10^8 L^-1 was inoculated into 96-hole culture plate, each hole containing 200μL, and then was co-cultured 2 hours by AB-type NHS with the final concentration of 5%, 10%, 25% and 40% on the fourth day of culture, when the cells covered the bottom of culture plate. While porcine blood serum with the corresponding concentrations ware taken as controls, 3 holes in each group. MTT chromatometry was applied to assess the absorbance value of each hole （A value）.④Establishment and evaluation of PVEC accommodation to NHS： PVEC was induced by IgG, IgM and lowly concentrated AB-type NHS for 6 days, and the survival rate was detected through MTT chromatometry, while the expression of heme oxygenase （HO） 1 was observed with immunohistochemistry method. RESULTS： ①The inoculated cells shaped as round, triangle or short fusiform, with the great soma, equal plasm and clear nucleolus. Some cells distributed in balls, but most of the cells presented monocellular appearance, which ware mixed with few blood cell, suggesting that those cells ware vascular endothelial cells and ware not infected with heterocells obviously.②The survival rate of PVEC was （71±22）% under action of 25% AB-type NHS. Compared with the controls, the survival rates of PVEC induced by 100 mg/L IgM, 5% and 10% AB-type NHS ware increased [（81±31）%, （93±2.5）%, （90±42）%].③The expression of HO 1 ware also upgraded significantly after inducing PVEC. CONCLUSION： IgM of 100 mg/L and AB-type NHS can induce PVEC accommodation, and the later one is more effective.