Primary Cultivation and Identification of Vascular Smooth Muscle Cells from the Spiral Modiolar Artery of Guinea Pigs.
Primary Cultivation and Identification of Vascular Smooth Muscle Cells from the Spiral Modiolar Artery of Guinea Pigs.
复制标题
豚鼠蜗轴螺旋动脉血管平滑肌细胞的原代培养及鉴定
DOI：
10.12659/msm.912606
复制
发表时间：
2018-10-03
期刊：
影响因子：
--
通讯作者：
Ma K
中科院分区：
文献类型：
--
作者：
Xiao J;Zhang Z;Zhang W;Wu L;Zhang L;Wang Y;Li L;Li X;Ma K
背景本文报道了一种从豚鼠蜗轴螺旋动脉（SMA）中获取血管平滑肌细胞（SMC）的方法，为相关实验研究提供材料。材料/方法 将 SMA 从豚鼠的耳蜗中分离出来，用胰蛋白酶 (1.25 g/L) 消化，并使其粘附在 35 mm 培养皿中。对样品进行形态学研究，并通过免疫荧光分析、流式细胞术、Western blot、RT-PCR对样品进行鉴定。使用台盼蓝和流式细胞术计算细胞活力。使用全细胞膜片钳记录SMC的膜输入电阻（Rinput）、膜输入电导倒数（Ginput）、膜输入电容（Cinput）和静息膜电位（RP）。结果 镜检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记物 α-SM-actin、SM22α、calponin 和 desmin 的特异性和强阳性表达。此外，获得P3和P6细胞的以下特性：Rinput，2611±356和2477±338MΩ； G输入，0.454±0.071和0.273±0.037纳秒； C输入为17.029±0.917和18.042±1.051 pF，RP为-20.602±1.503和-22.192±1.905 mV。结论从豚鼠SMA中获得了多种高纯度的SMC。我们为体外研究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。结果可用于评价药物对血管平滑肌的作用。
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/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.
影响因子：
--
作者：
Krishnamoorthy G;Regehr K;Berge S;Scherer EQ;Wangemann P
通讯作者：
Wangemann P
影响因子：
5
作者：
Li, Xin-Zhi;Ma, Ke-Tao;Guan, Bing-Cai;Li, Li;Zhao, Lei;Zhang, Zhong-Shuang;Si, Jun-Qiang;Jiang, Zhi-Gen
通讯作者：
Jiang, Zhi-Gen