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目的建立体外培养人肺内微小动脉的方法 ,并观察其对收缩剂反应的基本特性,为研究药物及各种活性因子对人肺血管的长期作用提供有效的实验模型。方法取癌旁5 cm以上的肺组织,于冰浴和无菌条件下,利用显微操作分离出肺内微小动脉,制备成1.8~2.0 mm长的血管条,分两组,一组急性分离组(新鲜组)马上进行血管张力测定;另一组(培养组)置于含DMEM-F12培养基(内含10%胎牛血清、1%青链霉素)的培养皿内,通以95%O2、5%CO2混合气,在37℃的条件下培养48 h,再进行张力测试。进行血管张力测定时,用两根直径40μm的不锈钢丝平行穿过血管腔,钢丝的四个端分别固定于微血管张力测定仪浴槽内钳夹的四个螺丝上。采用累积给药法,分别给予血管受体依赖性收缩剂血栓素A2类似物(U46619)和内皮素-1(endothelin-1,ET-1),血管非受体依赖性收缩剂60 mmol·L-1 KCL,然后测定血管的张力变化。结果培养组对血管收缩剂U46619和ET-1能产生浓度依赖性收缩,U46619的p D2为7.60±0.10,E_(max)为136.40%±6.17%(n=11);ET-1的p D2为7.17±0.22,E_(max)为137.14%±5.52%(n=9),结果类似于新鲜组:U46619的p D2为7.78±0.11,E_(max)为131.29%±3.79%(n=13);ET-1的p D2为7.53±0.15,E_(max)为139.11%±6.66%(n=10),两组比较,差异均无统计学意义(P>0.05)。培养组对血管非受体依赖性收缩剂60mmol·L-1 KCL产生的收缩力E_(max)为(7.67±0.85)m N(n=12);新鲜组的E_(max)为(7.73±0.97)m N(n=8),两组比较,差异无统计学意义。结论采用血管器官培养的方法培养人肺内微小动脉48 h,能维持血管平滑肌的基本收缩特性,可作为研究各种物质对血管长效作用的有效模型。
Objective To establish a method for culturing human pulmonary arterioles in vitro and to observe its basic characteristics of response to contractile agents and to provide an effective experimental model for studying the long-term effects of drugs and various active factors on human pulmonary blood vessels. Methods Pulmonary tissues of more than 5 cm were taken from the lungs. Micro-arteries in the lungs were isolated by micromanipulation under ice bath and aseptic conditions. Blood vessels of 1.8-2.0 mm in length were prepared and divided into two groups. One group was acutely isolated Group (fresh group) immediately vascular tension test; the other group (culture group) containing DMEM-F12 medium (containing 10% fetal bovine serum, 1% penicillin) petri dish, through 95 % O2, 5% CO2 mixture, cultured at 37 ℃ for 48 h, and then tension test. For the measurement of vascular tension, two stainless steel wires with a diameter of 40 μm were passed through the blood vessel cavity in parallel, and the four ends of the steel wire were respectively fixed on the four screws of the clamp in the microvascular tensity meter bath. Cumulative drug administration was used to induce angiotensin II receptor antagonist thromboxane A2 analogue (U46619) and endothelin-1 (ET-1), vascular non-receptor-dependent contractile agent 60 mmol·L -1 KCL, and then measure the changes in vascular tension. Results The culture group had a concentration-dependent contraction of vasoconstrictors U46619 and ET-1. The p D2 of U46619 was 7.60 ± 0.10 and the E max was 136.40% ± 6.17% (n = 11) Was 7.17 ± 0.22 and the E max was 137.14% ± 5.52% (n = 9). The results were similar to those of the fresh group: the p D2 of U46619 was 7.78 ± 0.11 and the E max was 131.29% ± 3.79% (n = 13 ). The p D2 of ET-1 was 7.53 ± 0.15 and the E max was 139.11% ± 6.66% (n = 10). There was no significant difference between the two groups (P> 0.05). The contractile force E_ (max) of 60mmol·L-1 KCL in non-receptor-dependent contraction of cultured group was (7.67 ± 0.85) m N (n = 12) 0.97) m N (n = 8). There was no significant difference between the two groups. Conclusion The culture of human pulmonary arterioles 48 h with vascular organ culture can maintain the basic contractility of vascular smooth muscle and can be used as an effective model to study the long-term effects of various substances on blood vessels.