Tissue culture of rabbit ciliary body epithelial cells on permeable supports.
Tissue culture of rabbit ciliary body epithelial cells on permeable supports.
复制标题
兔睫状体上皮细胞在渗透性支持物上的组织培养。
作者：
M. Cilluffo;M. J. Fain;G. Fain
眼房水由睫状体的上皮细胞产生，睫状体是环绕眼睛前段的复杂结构。房水的产生是通过主动转运发生的，但这一过程的机制尚不清楚。为了制备一种可以研究主动转运的制剂，我们试图制备适合于测量离子和水通量的培养物。我们在可渗透的支持物上培养了兔睫状体上皮细胞，这些细胞被几种细胞外基质蛋白包被。然后我们检查了这些蛋白质促进分化的上皮细胞层生长的能力。当在各种支持培养基上生长时，非色素细胞和色素细胞形成连续细胞片。最成功的基质是由Falcon/Becton Dickinson生产的渗透性支持物，其涂有胶原IV、层粘连蛋白和硫酸乙酰肝素的混合物。在这些条件下，培养物可维持数月，但色素细胞培养物不产生可测量的跨上皮电阻（TER），并且非色素细胞培养物的TER通常仅为20-30 Ω cm 2。在接种后3-5天，可以从非色素细胞培养物中测量到超过200 Ω cm 2的更高的TER，但这些高值是不稳定的。电子显微镜检查培养物显示细胞部分分化，形成基底膜和细胞间连接。用紧密连接蛋白（ZO-1）的特异性单克隆抗体标记表明，非色素细胞培养物显示出广泛的紧密连接形成。因此，非色素细胞培养物的低TER似乎不是由于缺乏紧密连接，而是由于细胞之间存在空间。
The aqueous humor is produced by the epithelium of the ciliary body, a complex structure encircling the anterior segment of the eye. Aqueous humor production occurs by active transport, but the mechanism of this process is not understood. To produce a preparation in which active transport can be investigated, we have attempted to prepare cultures suitable for measurements of ion and water flux. We have grown rabbit ciliary body epithelial cells on permeable supports, coated with several extracellular matrix proteins. We then examined the ability of these proteins to promote the growth of a differentiated layer of epithelial cells. Non-pigmented and pigmented cells formed sheets of contiguous cells when grown on a variety of support media. The most successful substrate was a permeable support produced by Falcon/Becton Dickinson coated with a mixture of collagen IV, laminin and heparan sulfate. Under these conditions, cultures could be maintained for several months, but pigmented cell cultures did not develop a measurable transepithelial resistance (TER), and the TER of non-pigmented cell cultures was typically only 20-30 omega cm2. Much higher TERs exceeding 200 omega cm2 could be measured from non-pigmented cell cultures 3-5 days after plating, but these high values were unstable. Examination of the cultures with electron microscopy revealed that the cells were partially differentiated with the formation of a basal lamina and intercellular junctions. Labelling with a specific monoclonal antibody marker for tight junction protein (ZO-1) suggested that non-pigmented cell cultures showed extensive tight junction formation. The low TER of the non-pigmented cell cultures appears therefore not to be due to the lack of tight junctions but rather to the presence of spaces between cells.