Kruse F E, Tseng S C
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida.
Invest Ophthalmol Vis Sci. 1993 Jul;34(8):2501-11.
In the epidermis, proliferative basal cells can be divided into two subpopulations according to their response to phorbol ester tumor promoters. The tumor promoter-sensitive subpopulation ceases mitosis and initiates terminal differentiation and, thus, represents more differentiated transient amplifying cells. In contrast, the tumor promoter-resistant subpopulation that continues to proliferate may be the target of neoplastic transformation by chemical carcinogens and may contain stem cells. Based on this concept, we examined the differential response of stem cell-containing limbal epithelium and transient amplifying cell-containing corneal epithelium to phorbol 12-myristate 13-acetate (PMA) treatment.
A reported serum-free clonal growth assay was used. The mitogenic response was measured by colony-forming efficiency (CFE), colony size, bromodeoxyuridine (BrdU) labeling index; the differentiation was assessed by colony morphology, AE-5 monoclonal antibody staining.
The addition of PMA dose dependently inhibited the clonal proliferation of both limbal and corneal epithelial cultures with respect to CFE, colony size, and BrdU labeling index, suggesting that both cultures contain PMA-sensitive subpopulations. Nevertheless, the magnitudes of a decrease in CFE and colony size in peripheral corneal cultures were significantly greater than those in limbal cultures, indicating that the size of the PMA-resistant subpopulation is larger in the limbal epithelium. The inhibitory effect of PMA on clonal proliferation was partially reversible upon its early withdrawal, indicating that its inhibitory effect is continuous and coupled with progressive differentiation of progenitor cells in this culture system.
These results further suggest that the cell cycle length of progenitor cells correlates with the mitogenic pathway mediated via calcium- and phospholipid-dependent protein kinase C, the receptor inhibited by prolonged treatment of phorbol ester tumor promoters.
在表皮中，增殖性基底细胞可根据其对佛波酯肿瘤启动子的反应分为两个亚群。对肿瘤启动子敏感的亚群停止有丝分裂并启动终末分化，因此代表了更分化的短暂扩增细胞。相比之下，继续增殖的对肿瘤启动子耐药的亚群可能是化学致癌物致瘤转化的靶点，并且可能含有干细胞。基于这一概念，我们研究了含干细胞的角膜缘上皮和含短暂扩增细胞的角膜上皮对佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理的差异反应。
采用已报道的无血清克隆生长试验。通过集落形成效率（CFE）、集落大小、溴脱氧尿苷（BrdU）标记指数来测量有丝分裂反应；通过集落形态、AE-5单克隆抗体染色来评估分化情况。
添加PMA剂量依赖性地抑制了角膜缘和角膜上皮培养物的克隆增殖，在CFE、集落大小和BrdU标记指数方面均有体现，这表明两种培养物中都含有对PMA敏感的亚群。然而，周边角膜培养物中CFE和集落大小的降低幅度明显大于角膜缘培养物，这表明角膜缘上皮中对PMA耐药的亚群更大。PMA对克隆增殖的抑制作用在早期撤除后部分可逆，这表明其抑制作用是持续的，并且与该培养系统中祖细胞的渐进性分化相关。
这些结果进一步表明，祖细胞的细胞周期长度与通过钙和磷脂依赖性蛋白激酶C介导的有丝分裂途径相关，该受体受到佛波酯肿瘤启动子长期处理的抑制。
