标题：肺动脉平滑肌细胞上TMEM16A的表达及其阻滞剂对细胞增殖的作用

      作者：张锋，庞玉生，劳金泉
    (广西医科大学第一附属医院儿科，广西 南宁 530021)

      卷次： 2016年27卷11期

      【摘要】 目的 研究TMEM16A在大鼠肺动脉平滑肌细胞(PASMCs)上的表达及其阻滞剂(T16Ainh-A01)对
PASMCs增殖的作用。方法 组织块贴壁法分离正常大鼠PASMCs，原代培养。光镜下观察细胞形态，细胞免疫化
学和细胞免疫荧光进行鉴定。细胞免疫荧光检测TMEM16A在PASMCs上表达。原代培养的3~6代PASMCs分别
在 10 μmol/L T16Ainh-A01、20 μmol/L T16Ainh-A01和 50 μmol/L T16Ainh-A01下培养 24 h，用CCK-8法检测细胞增殖
情况。结果 倒置显微镜下PASMCs以长梭形为主，细胞核卵圆形，部分细胞重叠区域呈典型的“峰-谷”状结构。
免疫学检测显示胞浆内与细胞长轴平行的肌丝被染成棕黄色，标记肌丝的红色荧光也与细胞长轴平行，标记细胞
膜上的 TMEM16A的绿色荧光可见表达，而细胞核不染色。CCK-8实验检测，相对于对照组，10 μmol/L
T16Ainh-A01 组细胞活力均数为 (96.74 ± 0.92)%，20 μmol/L T16Ainh-A01 组均数为 (92.21 ± 1.25)%，50 μmol/L
T16Ainh-A01组均数为(87.41±3.27)%，差异均具有统计学意义(P<0.01)。结论 正常大鼠PASMCs表达TMEM16A；
T16Ainh-A01可抑制PASMCs增殖，其抑制作用呈浓度依赖性；TMEM16A可能参与调控PASMCs增殖。

      【关键词】 TMEM16A；肺动脉平滑肌细胞；阻滞剂；细胞增殖；原代培养

      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2016）11—1727—04


Expression of TMEM16A in pulmonary arterial smooth muscle cells and effect on cell proliferation by a selective
TMEM16A inhibitor.
ZHANG Feng, PANG Yu-sheng, LAO Jin-quan. Department of Pediatrics, the First Affiliated
Hospital of Guangxi Medical University, Nanning 530021, Guangxi, CHINA

【Abstract】 Objective To explore the expression of TMEM16A in rat pulmonary arterial hypertension
(PASMCs) and effect of a TMEM16A inhibitor (T16Ainh-A01) on cell proliferation. Methods Tissue block anchorage
was employed for the primary culture of rat PASMCs. Under light microscope, cell morphology was observed and
identified by immunocytochemistry and immunofluorescence. The expression of TMEM16A in PASMCs was detected
by immunofluorescence. The 3~6 passages of primary cultured PASMCs were respectively exposed to 10 μmol/L
T16Ainh-A01, 20 μmol/L T16Ainh-A01 and 50 μmol/L T16Ainh-A01 for 24 hours. Cell proliferation was detected by cell
counting kit-8 (CCK-8) assay. Results PASMCs tended to be long spindle, nuclear oval, and partial overlapping re-
gions of the cells grew in a typical "peak - valley" mode under light microscope. Immunology results showed that the fil-
aments in cytoplasm paralleled to cell long axis were stained brownish yellow and the red fluorescent marked filament
paralleled to cell long axis were also showed. Meanwhile, TMEM16A marked with green fluorescence were visible, and
the nuclear were non-staining. In CCK-8 assay, compared to control group, mean cell viability of 10 μmol/L, 20 μmol/L,
50 μmol/L T16Ainh-A01 group was (96.74±0.92)%, (92.21±1.25)%, (87.41±3.27)%, respectively, and the differences were
statistically significant (P<0.01). Conclusion TMEM16A were exactly expressed in normal PASMCs. T16Ainh-A01
could inhibit the proliferation of PASMCs, and its inhibitory effect was concentration-dependent. TMEM16A may regu-
late PASMCs proliferation.

      【Key words】 TMEM16A; Pulmonary arterial smooth muscle cells; Inhibitor; Cell proliferation; Primary culture

6350.2016.11.002