Rho激酶抑制减少体外培养大鼠主动脉环的血管新生

Reduced angiogenesis of in vitro cultured aortic rings of rats via inhibition of Rho Kinase

背景:目前在血管新生的研究方法中,体外器官水平主要采用鸡胚绒毛尿囊膜实验模型,此方法的应用有其局限性。研究查明,Rho激酶参与血管新生,但尚缺少体外器官水平的深入观察。目的:建立大鼠主动脉环体外培养血管新生实验模型,以Rho激酶抑制剂HA-1077为干预因素,观察Rho激酶活性抑制对血管新生的影响,以及新生血管的生长特征和规律。设计、时间及地点:以血管环为对象,观察测量对比实验,于2008-10-21/2009-01-28在广东医学院生理学研究室进行。材料:2月龄SPF级SD大鼠,雌雄不限;HA-1077(Cat:371970)为Calbiochem公司产品。方法:无菌条件下取大鼠一段胸主动脉,剪为长约1mm的血管环,随机分为对照组、HA-1077低、中、高浓度组,对照组用HG-DMEM+体积分数15%胎牛血清的培养液培养,HA-1077组分别在培养液中加入10,30,60mmol/LHA-1077,第5天终止实验。主要观察指标:拍摄微血管生长图像,计数体外培养的主动脉环新生微血管分支数;WesternBlot检测ROCKⅠ及ROCKⅡ的表达。结果:与对照组比较,HA-1077低浓度组新生微血管数明显增加(P<0.01);中、高浓度组新生微血管数较对照组明显减少(P<0.01)。对照组和HA-1077低浓度组都有ROCKⅠ和ROCKⅡ表达,组间比较差异无显著性意义;HA-1077中、高浓度组则基本未见ROCKⅠ或ROCKⅡ表达。结论:30,60mmol/LHA-1077可以抑制血管新生,并抑制Rho激酶表达。提示Rho激酶抑制可以减少体外培养大鼠主动脉环的血管新生。

BACKGROUND： Present in vitro organ level research on angiogenesis mainly uses chick chorioallantoic membrane model, which has limitations in its application. Study shows that Rho kinase has its role Jn angJogenesJs, but further researches on in vitro organ level are still few. OBJECTIVE： To establish an experimental model of in vitro cultured aortic ring angiogenesis of rats and observe the effect of Rho kinase inhibition on angiogenesis and the growth characteristics and rules of the neogenetic vessels. DESIGN, TIME AND SETTING： An observational measurement contrast experiment using vascular ring as the object was performed at the Department of Physiology of Guangdong Medical College from October 21, 2008 to January 28, 2009. MATERIALS： Two-month-old SPF level SD rats of either gender were selected; HA-1077 （Cat： 371970） was purchased from Calbiochem Company. METHODS： A section of aorta pectoralis was cut off from rats under aseptic condition and was cut into 1 mm vascular rings which were randomly divided into a control group （cultured in HG-DMEM＋1 5% fetal bovine serum medium）, HA-1077 low concentration group （cultured in medium with 10mmol/L HA-1077 added in）, intermediate concentration group （cultured in medium with 30 mmol/L HA-1077 added in） and high concentration group （cultured in medium with 60 mmol/L HA-1077 added in）. The experiment lasted for 5 clays in total. MAIN OUTCOME MEASURES： The image of microvessel growth was shot to count neogenetic microvessel branches of the in vitro cultured aortic rings. Western Blot was used to detect the expression of ROCK-1 and ROCK-2. RESULTS： Compared with the control group, the number of neogenetic microvessels increased significantly in the low concentration group （P 〈 0.01）, but decreased significantly Jn the intermediate and high concentration group （P 〈 0.01）. ROCK Ⅰ and ROCK Ⅱ was expressed in both the control group and the HA-1077 low concentration group and there was no significant difference between the two groups. However, in the HA-1077 intermediate and high concentration group, there were few ROCK Ⅰ and ROCK Ⅱ expressed. CONCLUSION： HA-1077 of 30 and 60 mmol/L have the effect of JnhibitJng angiogenesis and Rho kinase expression, which suggests that the angiogenesis of in vitro cultured aortic ring of rats can be reduced via inhibition of Rho kinase.