Ma R H, Tsai C C, Shieh T Y
Department of Oral and Maxillo-Facial Surgery, School of Dentistry Kaohsiung Medical College, Taiwan, ROC.
J Oral Pathol Med. 1995 Oct;24(9):407-12. doi: 10.1111/j.1600-0714.1995.tb01210.x.
Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5 x 10(5) cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-3H]--lysine labelled purified chick--embryo aorta elastin substrate. After incubation for 10 h at 37 degrees C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84 +/- 1.15 mg/ml) and lysyl oxidase activity (3558.6 +/- 345.5 cpm/10(6) cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35 +/- 0.96 mg/ml and 2436.0 +/- 352.6 cpm/10(6) cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.
在细胞培养中比较了源自人正常黏膜（NM）和与嚼槟榔相关的口腔黏膜下纤维化（OSF）的成纤维细胞的生长特性和赖氨酰氧化酶活性。通过在35mm培养皿中接种5×10⁵个细胞（第0天）来确定培养细胞的生长速率，并且每24小时通过定量细胞数量（使用血细胞计数器）来测定细胞增殖。使用第三至第七代细胞。在亚汇合期，用不含血清但补充有5mg/ml牛血清白蛋白的培养基替代原始培养基，并再培养24小时。收集培养基并用于蛋白质含量和赖氨酰氧化酶活性的测定。用[4,5-³H] - 赖氨酸标记的纯化鸡胚主动脉弹性蛋白底物测定赖氨酰氧化酶活性。在37℃孵育10小时后，使用Amicon C-10微型浓缩器通过超滤从³HHO（氚化水）中测量酶活性。结果显示，OSF成纤维细胞的平均倍增时间为3.2天，NM成纤维细胞为3.6天。根据细胞数量测定，NM成纤维细胞在第6天汇合，而OSF成纤维细胞在第5天汇合。此外，用于掺入BrdUrd的免疫酶测定显示，在标准培养条件下，OSF成纤维细胞的增殖明显快于NM成纤维细胞。OSF成纤维细胞中的总蛋白含量（10.84±1.15mg/ml）和赖氨酰氧化酶活性（3558.6±345.5cpm/10⁶个细胞）均高于NM成纤维细胞（6.35±0.96mg/ml和2436.0±352.6cpm/10⁶个细胞）。本研究结果提供了证据，表明源自口腔黏膜下纤维化（OSF）组织和正常黏膜（NM）的成纤维细胞尽管在许多方面相似，但在增殖速率和赖氨酰氧化酶活性方面表现出特定差异。此外，OSF组织中的胶原沉积可能至少部分归因于赖氨酰氧化酶活性的增加。
