R&D Laboratories, POLA PHARMA INC., Yokohama, Japan.
J Dermatol Sci. 2012 Dec;68(3):179-86. doi: 10.1016/j.jdermsci.2012.09.012. Epub 2012 Sep 28.
The inhibition of transforming growth factor β (TGF-β)-induced myofibroblast differentiation is a key objective for the treatment of hypertrophic scarring. We previously reported that knockdown of the electron transfer flavoprotein β subunit (ETFB) reduced mechanoregulated cell number in fibroblast-populated collagen gel cultures [1].
To characterize the effects of ETFB knockdown, we investigated gel contraction, TGF-β-induced collagen, α-SMA mRNA expression and stress fiber formation.
Fibroblasts were transfected with negative control or ETFB-specific siRNAs and embedded in collagen gels in an attached or detached condition. The gel contraction assay was performed in three different concentrations of collagen (0.5, 1.0 or 1.5mg/mL) and was analyzed by measuring the changes in the gel area throughout the culture period. The attached collagen gel culture was performed in the presence of rTGF-β and the mRNA levels of α-SMA and COL1A1 were measured by qRT-PCR. The effect of ETFB knockdown on proliferation and stress fiber organization in monolayer cultures was investigated by conducting AlamarBlue assays and phalloidin staining.
The transfection of ETFB siRNA did not alter gel contraction compared to the negative control in all collagen concentrations. When the cells were treated with TGF-β under mechanical stress conditions, ETFB knockdown attenuated α-SMA mRNA expression to a level comparable to that observed in the absence of TGF-β. However, no inhibitory effect on COL1A1 mRNA levels was observed. The AlamarBlue assay indicated that the knockdown had no effect on the proliferation of cells cultured on plastic. Phalloidin staining of a monolayer culture showed that ETFB knockdown weakened the stress fiber organization induced by rTGF-β.
ETFB knockdown can affect TGF-β-induced tissue remodeling and/or fibrotic processes in vitro.
抑制转化生长因子β（TGF-β）诱导的肌成纤维细胞分化是治疗增生性瘢痕的关键目标。我们之前的研究报告表明，电子传递黄素蛋白β亚基（ETFB）的敲低减少了成纤维细胞在富含胶原的凝胶培养物中的机械调节细胞数量[1]。
为了研究 ETBF 敲低的影响，我们研究了凝胶收缩、TGF-β诱导的胶原、α-SMA mRNA 表达和应激纤维形成。
将阴性对照或 ETBF 特异性 siRNA 转染到成纤维细胞中，并在附着或不附着条件下嵌入胶原凝胶中。通过测量培养过程中凝胶面积的变化，在三种不同浓度的胶原（0.5、1.0 或 1.5mg/ml）下进行凝胶收缩测定。在存在 rTGF-β的情况下进行附着胶原凝胶培养，并通过 qRT-PCR 测量α-SMA 和 COL1A1 的 mRNA 水平。通过进行阿尔玛蓝测定和鬼笔环肽染色，研究 ETBF 敲低对单层培养物中的增殖和应激纤维组织的影响。
与阴性对照相比，在所有胶原浓度下，ETFB siRNA 的转染对凝胶收缩没有影响。当细胞在机械应激条件下接受 TGF-β处理时，ETFB 敲低使α-SMA mRNA 表达减弱到与未添加 TGF-β时相当的水平。然而，COL1A1 mRNA 水平没有观察到抑制作用。阿尔玛蓝测定表明，在塑料培养的细胞中，敲低对增殖没有影响。单层培养物的鬼笔环肽染色表明，ETFB 敲低削弱了 rTGF-β诱导的应激纤维组织。
ETFB 敲低可以影响 TGF-β诱导的组织重塑和/或纤维化过程。
