Zheng J L, Helbig C, Gao W Q
Department of Neuroscience, Genentech, Incorporated, South San Francisco, California 94080, USA.
J Neurosci. 1997 Jan 1;17(1):216-26. doi: 10.1523/JNEUROSCI.17-01-00216.1997.
Proliferation of supporting cells in the inner ear is the early major event occurring during hair cell regeneration after acoustic trauma or aminoglycoside treatment. In the present study, we examined the possible influence of 30 growth factors on the proliferation of pure rat utricular epithelial cells in culture. Utricular epithelial sheets were separated and partially dissociated from early postnatal rats via a combined enzymatic and mechanical method. The cultured utricular epithelial cells expressed exclusively epithelial cell antigens, but not fibroblast, glial, or neuronal antigens. With tritiated thymidine incorporation assays, we found that several fibroblast growth factor (FGF) family members, insulin-like growth factor-1 (IGF-1), IGF-2, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), stimulated proliferation of the utricular epithelial cells. In contrast, neurotrophins and other growth factors did not elicit any detectable mitogenic effects. Among all of the growth factors examined, FGF-2 was the most potent mitogen. When FGF-2 was added in combination with IGF-1 or TGF-alpha to the medium, combined effects were seen. These results were confirmed with BrdU immunocytochemistry. Thus, the present culture system provides a rapid and reliable assay system to screen novel growth factors involved in proliferation of mammalian inner ear supporting cells. Furthermore, immunostainings revealed that the cultured utricular epithelial cells expressed FGF and IGF-1 receptors, and utricular hair cells produced FGF-2 in vivo. The addition of neutralizing antibodies against FGF-2 or IGF-1 to the cultures significantly inhibited the utricular epithelial cell proliferation. This work suggests that FGF-2 and IGF-1 may regulate the proliferation step during hair cell development and regeneration.
内耳支持细胞的增殖是声创伤或氨基糖苷类药物治疗后毛细胞再生过程中早期发生的主要事件。在本研究中，我们检测了30种生长因子对培养的纯大鼠椭圆囊上皮细胞增殖的可能影响。通过酶解和机械相结合的方法，从出生后早期大鼠分离并部分解离椭圆囊上皮片层。培养的椭圆囊上皮细胞仅表达上皮细胞抗原，而不表达成纤维细胞、神经胶质或神经元抗原。通过氚标记胸腺嘧啶核苷掺入试验，我们发现几种成纤维细胞生长因子（FGF）家族成员、胰岛素样生长因子-1（IGF-1）、IGF-2、转化生长因子-α（TGF-α）和表皮生长因子（EGF）可刺激椭圆囊上皮细胞的增殖。相比之下，神经营养因子和其他生长因子未引发任何可检测到的促有丝分裂作用。在所有检测的生长因子中，FGF-2是最有效的促有丝分裂原。当FGF-2与IGF-1或TGF-α联合添加到培养基中时，可观察到联合效应。这些结果通过BrdU免疫细胞化学得到证实。因此，本培养系统提供了一种快速可靠的检测系统，用于筛选参与哺乳动物内耳支持细胞增殖的新型生长因子。此外，免疫染色显示培养的椭圆囊上皮细胞表达FGF和IGF-1受体，且椭圆囊毛细胞在体内产生FGF-2。向培养物中添加针对FGF-2或IGF-1的中和抗体可显著抑制椭圆囊上皮细胞的增殖。这项工作表明FGF-2和IGF-1可能在毛细胞发育和再生过程中调节增殖步骤。
