外子宮頸 (exocervix) 的上皮細胞結構為複層鱗狀上皮。動物研究發現大鼠外子宮頸分層化 (stratification) 的程度會隨著動情週期而改變。有關細胞研究亦指出，人類外子宮頸細胞經飼養層細胞共同培養或培養在含生長因子之培養基中可以進行細胞增生及分化，但卻無法呈現完整上皮分層化的結構。曾有研究團隊利用器官培養 (organotypic culture) 的方式培養外子宮頸小組織塊，可觀察到上皮細胞的多層結構，然而，該研究使用10 % 牛血清之培養基，含有許多影響細胞增生及分化之生長因子。本研究的目的探討在無添加血清及生長因子培養條件下，外子宮頸上皮組織是否能進行細胞增生及分化。因此，我將人類外子宮頸組織塊培養於明膠海綿上以不含血清之培養液培養不同天後，使用免疫組織染色的方式偵測細胞增生及分化標誌蛋白keratin 1 (K1)、keratin 14 (K14) 之表現。正常外子宮頸上皮細胞可分成四層，包括基底層 (basal layer)、副基底層 (parabasal layer)、中間層 (midzone) 及外表皮層 (superficial layer)。基底層細胞表現K14，但不表現K1；其他層次細胞則表現K1，而不表現K14。經過7-10天無血清培養後，具空泡的細胞層消失，副基底層細胞上層處出現單獨或同時表現K1、K14之緻密扁平狀細胞層，以及一層不表現K1及K14之伊紅深染角質化細胞層。最外層之表皮層中有些細胞表現K1或K14，部份細胞則同時表現K1及K14。Bromodeoxyuridine所標誌之增生細胞則主要出現在基底層及副基底層的細胞。我們的研究結果指出，即使在無血清的培養下，外子宮頸上皮細胞依然可進行增生及分化，並出現角質化的現象。由於外子宮頸組織含上皮細胞及基質細胞，我們的研究結果顯示上皮細胞或基質細胞可能分泌生長因子去調控細胞增生及分化。

The exocervix is composed of squamous epithelium and stroma. It has been shown that rat cervical epithelium undergo cyclic proliferation, differentiation and stratification during estrous cycle. Ectocervical epithelial cells cultured with fibroblast feeder cells or supplemented with specific growth medium proliferate and differentiate to some extent. In organotypic culture, the tissue fragments are cultured on sponge in air-liquid surface. Previous studies show that the stratified structure of exocervix in organotypic culture with 10 % fetal calf serum resembled in vivo condition. The purpose of this study is to investigate whether the exocervical epithelium undergoes cell proliferation and differentiation in organotypic culture under serum-free condition. At different days after culture, the tissue fragments were collected for H&E and immunostaining of keratin 1 (K1) and keratin 14 (K14) expression, and bromodeoxyuridine (BrdU) incorporation. Before culturing, exocervical epithelium consisted of basal, parabasal, midzone and superficial layers. K14 only expressed in the cells of basal layers, and K1 expressed in other layers. After culture for 7 to 10 days, midzone cells with large vacuoles disappeared. They were replaced by flat and compact cells in the upper area of parabasal layer which expressed K1 and/or K14. In addition, on the top of these layers, an eosinphilic and K1/K14-negative layer appeared. In the superficial layers, cells expressed K1 and/or K14. BrdU staining showed that cell proliferation occurred in basal cell and parabasal cells. Taken together, our results showed cell proliferation, differentiation and stratification occurred in exocervical epithelium in organotypic culture without exogenous supply of serum and growth factors. It suggests that cell proliferation and differentiation of exocervical epithelium may be regulated by autocrine and paracrine factors secreted from the tissue itself.

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