【作者】 高峰；

【导师】 刘伟国；

【作者基本信息】 浙江大学 ， 神经外科， 2007， 硕士

【摘要】 一、目的疏血通注射剂是由水蛭、地龙合理组方经低温提取、膜分离等先进工艺精制而成的。中医认为水蛭配地龙，是通络化瘀法的最佳组合。其有效成分为水蛭素和蚓激酶样作用物质，水蛭素是特异性凝血酶抑制剂，而蚓激酶能降解纤维蛋白和激活纤溶酶原。本研究拟从细胞实验角度初步探讨，疏血通在影响血管内皮生长因子受体-2和血清应答因子表达方面的作用，及其促进血管生长的可能机制。二、材料和方法1，材料1，1实验设备和器材(1)超净台(2) OLYMPUS光学显微镜(3)倒置显微镜(4)恒温水漕(5) 37度孵箱(6) 4度冰箱(7)负80度冰箱(8)移液器(9)免疫组化湿盒(10)染色缸(11)厚盖玻片(12) 5ml培养瓶(13)六孔培养板(14)高压消毒锅1，2细胞和主要试剂(1)永生化人脐静脉内皮细胞系由浙江大学医学院附属第二医院心内科友情提供。(2)血管内皮生长因子受体-2抗体购自福建迈新生物技术有限公司。(3)抗血清应答因子抗体购自北京中杉生物技术有限公司。(4) SP检测试剂盒购自北京中杉生物技术有限公司。(5)疏血通药物(规格2ml/支)由牡丹江友博药业生产。(6)培养基RPMI1640(7)胎牛血清2，细胞培养取永生化人脐静脉内皮细胞系，以RPMI1640加入胎牛血清配成10％的全培养液为培养基，按同源相同数量，传代培养第3代，接种密度3×10~4/ml，分两组接种于6孔培养板中，6孔培养板底部各放置消毒盖玻片一块，使细胞贴壁于盖玻片上。3，分组和加药实验组：按每孔3000μl培养液加300μ1比例，加入疏血通药物1：16稀释液处理；对照组：按同一比例加入生理盐水。4，指标检测血管内皮生长因子受体-2蛋白表达检测：接种并加药后24h、2d、3d、4d、5d、6d六个时间点用SP法分别做免疫细胞化学染色。血清应答因子蛋白表达检测：接种并加药后24h、2d、3d、4d、5d、6d六个时间点分别用SP法做免疫细胞化学染色。5，观察在高倍镜(400倍)下每张切片随机选取5个视野进行阳性细胞计数，取平均值作为本例的阳性细胞数值。6，统计学处理所得结果以细胞个数/高倍镜视野表示，疏血通组和生理盐水对照组数据之间进行t检验。三、结果与对照组比较，疏血通处理组细胞接种时贴壁早，生长快，细胞大多呈多边形，细胞胞体更大；免疫细胞化学染色后阳性细胞较多(如图1、图2、图3、图4所示)。疏血通组VEGFR蛋白表达和SRF蛋白表达水平与对照组相比明显增高(P＜0.001)，其差异有统计学意义(数据如表1、图5和表2、图6所示)。而VEGFR蛋白表达和SRF蛋白表达水平的增高在时间上未发现明显的趋势(如图7、图8所示)。四、结论因此我们认为，疏血通能明显增加体外培养的血管内皮细胞VEGFR-2和SRF的表达。其促血管生长的可能机制之一是通过增加局部血管内皮生长因子受体-2和血清应答因子的表达，诱导内皮细胞增殖，毛细血管瓣生成以及血管平滑肌细胞分化，促进新生血管形成。更多还原

【Abstract】 1, Study objectivesTo determine whether the traditional Chinese drug "ShuXueTong" have effects on vascular endothelial growth factor receptor (VEGFR-2) and serum response factor (SRF) expression, and to explore what mechanism it through to promote the growth of vessel.2,Materials and Methods1,1 MaterialsImmortal human umbilical vein endothelial celllines, RPMI 1640 added 10%FBS, anti-VEGFR-2 IgG anti-SRF IgG, and traditional Chinese drug "ShuXueTong" injection.1, 2 methods1, 2, 1 cell cultureImmortal human umbilical vein endothelial cells were cultured withRPMI 1640 added 10%FBS in culture flasks. At the third passage, the endothelial cells were transfered to some coverslips and cultured in 6-well culture flasks at a density of 30000/ml.1,2, 2 groupsTreated groups: "ShuXueTong" injection(1:16 dilution) were dropped into the cutlture solution to intervene the growth of human umbilical vein endothelial cells.Control groups: Normal sodium were added into the culture solution.1, 2, 3 DetectionsThe expression of VEGFR-2 and SRF were respectively detected with immunocytochemistry stain after a certain time line of 24h, 1d, 2d, 3d, 4d, 5d, and 6d.1, 2, 4, ObervationsThe stained cells were observed under high power lens. We obvered five visual fields at random in each slip and counted the positive cells in every visual field. The general average of the positive cells in the five visual fields were taken as the result.1, 2, 5 statisticsThe data of the treated groups and control groups were analyzed with Student’s t test.3, ResultsCompairing with the control cells, the expression of VEGFR-2 and SRF in treated cells was dramatically enhanced (P<0.001) .4, ConclusionsThese results suggest that "ShuXueTong" can enhance the expression of VEGFR-2 and SRF of VECs, and it may promote the growth of vessel by inducing VECs multiplication and vascular smooth muscle cell differentiation.更多还原