南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (07): 971-.

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血小板源性生长因子-BB对大鼠阴茎海绵体平滑肌细胞增殖、迁移及表型转化的影响

陈逢志,何书华,单海涛,张海波,梁雁冰,韦安阳   

  • 出版日期:2015-07-20 发布日期:2015-07-20

Effect of platelet-derived growth factor-BB on rat corpus cavernosum smooth muscle cell
proliferation, migration and phenotypic modulation

  • Online:2015-07-20 Published:2015-07-20

摘要: 目的了解血小板源性生长因子-BB(PDGFBBB)对大鼠阴茎海绵体平滑肌(CCSM)细胞增殖、迁移及表型转化的影响并
初步探讨其作用机制。方法采用改良的组织块培养法培养Wistar大鼠CCSM细胞,并用细胞免疫荧光法鉴定。利用CCK-8法
检测不同浓度的PDGFBB对培养的大鼠CCSM细胞增殖的影响,并筛选出最适PDGFBB作用浓度。分别用0 ng/mL PDGFBB
与最适浓度PDGFBB处理CCSM细胞,利用划痕实验检测PDGFBB对CCSM细胞迁移的影响,24 h和48 h时利用qRT-PCR检
测细胞中转录因子myocardin及收缩型表型标志物α-SMA、SMMHC mRNA表达水平;用最适浓度PDGFBB处理CCSM 细胞
0、24 和48 h 后,利用western blotting 检测细胞中myocardin 的蛋白表达水平。结果原代培养的CCSM 细胞中α-SMA 和
smoothelin 阳性率分别约为96.5%和96%;不同浓度的PDGFBB均能明显促进CCSM细胞增殖,其最适浓度为12.5 ng/mL。
PDGFBB(12.5 ng/mL)能促进CCSM细胞迁移,下调CCSM细胞中myocardin、α-SMA和SMMHC mRNA表达水平(P均<0.01);
在48 h内myocardin蛋白质的表达水平随着PDGFBB作用时间增加而降低。结论改良的组织块培养法可获得高纯的CCSM
细胞;PDGFBB可促进CCSM细胞增殖、迁移,使细胞从收缩型向合成型转化,其机制可能是通过下调myocardin。

Abstract: objective To study the effect of platelet-derived growth factor-BB (PDGFBBB) on rat corpus cavernosum smooth
muscle (CCSM) cell proliferation, migration and phenotypic modulation and explore the underlying mechanisms. Methods
Wistar rat CCSM cells were obtained through a modified tissue culture method and identified by immunofluorescence assay.
The effect of PDGFBB on the proliferation of CCSM cells was investigated using a CCK-8 kit and the optimum PDGFBB
concentration for cell treatment was determined. CCSM cells were treated with vehicle or PDGF-BB at the optimum
concentration, and the cell migration was examined using scratch assay; the mRNA expression of the transcription factor
myocardin and the contractile phenotype markers α-SMA and SMMHC in CCSM cells were determined by qRT-PCR at 24 h
and 48 h. The protein expression of myocardin in CCSM cells incubated with PDGFBB for 0, 24 and 48 h was examined by
Western blotting. Result In CCSM cell culture, 96.5% and 96% of the cells were positive for α-SMA and smoothelin,
respectively. PDGFBB at different concentrations markedly promoted the proliferation of CCSM cells; the optimum PDGFBB
concentration for enhancing cell proliferation was 12.5 ng/mL, which induced the migration of CCSM cells and significantly
reduced the mRNA expressions of myocardin, α-SMA and SMMHC (P<0.01). Exposure to PDGFBB decreased the protein
expression of myocardin as the exposure time extended (within 48 h). Conclusion CCSM cells of a high purity can be obtained
by the modified tissue culture method. PDGFBB can promote the proliferation and migration of CCSM cells and cause a
phenotypic conversion from the contractile to the synthetic type possibly by down-regulating myocardin.