血管段孵育和原代血管平滑肌细胞培养的比较

探讨微血管段孵育方法能否与原代平滑肌细胞培养一样检测蛋白表达,以用于初步的基础研究,解决原代培养耗时、不易存活的问题。分离大鼠肠系膜动脉三级以下分支,使用胶原酶和木瓜蛋白酶混合消化单个血管平滑肌细胞,获取的平滑肌细胞采用含20%胎牛血清的DMEM培养基进行培养。培养的平滑肌细胞经特异性的α-actin进行免疫组化鉴定;血管段孵育是在无菌条件下分离大鼠肠系膜动脉三级以下分支,培养基孵育72 h。分别使用不同浓度的尼氟灭酸(niflumic acid,NFA)孵育原代培养的平滑肌细胞和肠系膜三级分支血管段24 h,观察平滑肌细胞连接蛋白43(connexin43,Cx43)的变化。形态学和免疫组织化学鉴定表明,培养的原代细胞为血管平滑肌细胞。不同浓度NFA处理原代平滑肌细胞能够使Cx43表达量呈浓度依赖性下降,相对于对照组具有统计学意义(P0.01);不同浓度NFA处理血管段能够使Cx43的表达量也呈浓度依赖性下降,相对于对照组具有统计学意义(P0.01)。由此可知,观察平滑肌细胞特异性蛋白Cx43的表达情况,使用血管段孵育的方法检测结果与细胞培养的结果相似。血管段孵育简单易行,可以作为类似实验的初步研究。

Comparative Study on Vessel Segments Incubation and Primary Culture of Vascular Smooth Muscle Cells

To investigate the method of incubating vessel segments whether or not like the primary culture of vascular smooth muscle cells（VSMC） in fundamental research to detect the expression of protein, and to solve the dilemma of time-consuming and low survival rate in primary culture.Took rat sub-grade-3 mesenteric arteries and subjected to collagenase and papain to isolate into single smooth muscle cells,then the cells were cultured in DMEM containing 20% fetal bovine serum（FBS） and identified by（SMC）-specific α-actin protein.The vessel segments incubation was that incubated rats sub-grade-3 mesenteric arteries for 72 hours. And then, different concentration of flufenamic acid（niflumic acid, NFA） was used to intervene the incubated vessel segments and primary cultured vascular smooth muscle cells for another 24 hours respectively,then to observe the changes of connexin 43（Cx43） in VSMC. Morphologic and immunohistochemical results showed that the primary cultured cells are vascular smooth muscle cells.The results showed that different concentrations of NFA down regulated the expression of Cx43 in primary cultured muscle cells,which was concentration gradient-dependent decline（P〈0.01）;It also can down regulated the expression of Cx43 in primary cultured cells,which had statistical significance compared to the control（P〈0.01）.It wasproved that the analogical results were obtained both in incubated vessel segments and primary cultured of vascular smooth muscle cells.However,the former was much easier than the later,which can be used in the similar experiment.