Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Medicine School of Shihezi University, Shihezi, Xinjiang, China (mainland).
Department of Physiology, Medicine School of Shihezi University, Shihezi, Xinjiang, China (mainland).
Med Sci Monit. 2018 Oct 3;24:7023-7034. doi: 10.12659/MSM.912606.
BACKGROUND This article reports a method to obtain vascular smooth muscle cells (SMCs) from the spiral modiolar artery (SMA) of guinea pigs and provides materials for related experimental studies. MATERIAL AND METHODS SMA was separated from the cochlea of guinea pigs, digested with trypsin (1.25 g/L) and allowed to adhere in a 35-mm culture dish. The morphology of the sample was investigated, and the sample was identified by immunofluorescence analysis, flow cytometry, Western blot, and RT-PCR. Cell viability was calculated using trypan blue and flow cytometry. Whole-cell patch clamp was used to record the membrane input resistance (Rinput), reciprocal membrane input conductance (Ginput), membrane input capacitance (Cinput), and resting membrane potential (RP) of the SMCs. RESULTS Microscopy results showed that the cells had typical peak-valley growth pattern. The cell growth curve was similar to an S curve, and flow cytometry results showed that the cell apoptosis rate was less than 10%. Moreover, flow cytometry, immunofluorescent staining, Western blot and RT-PCR detected the specific and intensely positive expression of cell type-specific markers α-SM-actin, SM22α, calponin and desmin. Furthermore, following properties of the P3 and P6 cells were obtained: Rinput, 2611±356 and 2477±338 MΩ; Ginput, 0.454±0.071 and 0.273±0.037 ns; Cinput, 17.029±0.917 and 18.042±1.051 pF, and RP -20.602±1.503 and -22.192±1.905 mV. CONCLUSIONS Various highly purified SMCs were obtained from the SMA of guinea pigs. We provide an ideal experimental material for the study of the pathogenesis of diseases related to the circulation disturbances in the inner ear in vitro. The results can be used to evaluate the effects of drugs on vascular smooth muscle.
本文报道了一种从豚鼠蜗旋动脉（SMA）获取血管平滑肌细胞（SMC）的方法，为相关实验研究提供了材料。
将 SMA 从豚鼠耳蜗中分离出来，用 1.25g/L 的胰蛋白酶进行消化，然后在 35mm 培养皿中进行贴壁培养。观察样本的形态，通过免疫荧光分析、流式细胞术、Western blot 和 RT-PCR 对样本进行鉴定。使用台盼蓝和流式细胞术计算细胞活力。全细胞膜片钳记录平滑肌细胞的膜输入电阻（Rinput）、膜输入电导（Ginput）、膜输入电容（Cinput）和静息膜电位（RP）。
显微镜结果显示，细胞具有典型的峰谷生长模式。细胞生长曲线类似于 S 曲线，流式细胞术结果显示细胞凋亡率小于 10%。此外，流式细胞术、免疫荧光染色、Western blot 和 RT-PCR 检测到细胞类型特异性标志物α-SM-actin、SM22α、钙调蛋白和结蛋白的特异性和强阳性表达。此外，获得了 P3 和 P6 细胞的以下特性：Rinput，2611±356 和 2477±338 MΩ；Ginput，0.454±0.071 和 0.273±0.037 ns；Cinput，17.029±0.917 和 18.042±1.051 pF，RP-20.602±1.503 和-22.192±1.905 mV。
从豚鼠 SMA 中获得了各种高度纯化的 SMC。我们为体外研究与内耳循环障碍相关疾病的发病机制提供了理想的实验材料。该结果可用于评估药物对血管平滑肌的影响。
