摘要目的 探讨氟对体外培养人软骨细胞细胞活力和超微结构的影响.方法 采用细胞培养的方法,体外原代培养24～27周龄自然流产死亡胎儿的关节软骨细胞,取第3代细胞进行实验,按染氟剂量不同分为0(对照)、10-2、5×10-3、10-3、10-4、10-5、10-6、10-7、10-8 mol/L组.采用细胞增殖与毒性检测方法(细胞计数试剂盒-8,CCK-8),在染氟24、48、72 h测定细胞活力变化；通过透射电镜观察氟对细胞超微结构的影响.结果 人软骨细胞染氟(10-2、5×10-3、10-3、104、10-5、10-6、10-7、104 mol/L)24 h后其细胞活力分别为(15.04±0.55)％、(62.53±1.03)％、(100.34±5.19)％、(111.40±3.69)％、(121.47±6.09)％、(i29.95±4.96)％、(121.81±4.97)％、(111.00±1.63)％；48 h后分别为(10.35±0.64)％、(35.23±2.41)％、(110.30±2.07)％、(113.66±6.98)％、(120.36±6.23)％、(133.40±5.80)％、(126.06±5.40)％、(115.62±7.33)％;72 h后分别为(6.19±0.16)％、(18.44±0.21)％、(120.83±4.93)％、(123.77±4.82)％、(129.09±5.21)％、(140.44±4.18)％、(131.99±7.00)％、(124.10±3.68)％.在24、48、72 h,10-2、5×10-3 mol/L染氟组细胞活力均低于对照组[(100.00±0.00)％、(100.00±0.00)％、(100.00±0.00)％,P均＜0.05].10-2与5×10-3 mol/L组比较,其细胞活力均减小(P均＜ 0.05)；氟在10-2和5×10-3 mol/L时,抑制人软骨细胞活力,促进细胞的凋亡,且其细胞活力随着染氟剂量的增加和作用时间的延长而逐渐减小.在24、48、72 h,10-3～ 10-8 mol/L染氟组细胞活力(24 h10-3 mol/L组虽高于对照组,但差异无统计学意义)均高于对照组[(100.00±0.00)％、(100.00±0.00)％、(100.00±0.00)％,P均＜0.05].10-4组与10-3 mol/L组比较(48 h、72 h,细胞活力增加,但差异无统计学意义),10-5与10-4 mol/L组比较(72 h,细胞活力增加,但差异无统计学意义),10-6与10-5 mol/L组比较,其细胞活力增加,且差异有统计学意义(P均＜ 0.05)；10-7和10-6 mol/L组比较,10-8与10-7 mol/L组比较(72 h,细胞活力减小,但差异无统计学意义),其细胞活力减小,且差异有统计学意义(P均＜ 0.05).10-3 ～ 10-8 moL/L浓度的氟促进人软骨细胞的增殖,且随着染氟时间的延长其细胞活力逐渐增加；10-6 mol/L为促进软骨细胞增殖的最佳浓度.电镜下,可见对照组软骨细胞形态规则,表面具有微柔毛结构,细胞内胞浆丰富,内有大量的线粒体,粗面内质网发达,稍微扩张,内含低电子致密物质；10-6 mol/L染氟组,可见细胞内线粒体增多,部分线粒体肿胀,粗面内质网肥大扩张；5×10-3 mol/L染氟组,可见细胞表面微绒毛减少,胞膜内陷,染色质固缩、边集,出现凋亡细胞.结论 氟在10-3 ～ 10-8 mol/L时,可以促进体外培养人软骨细胞的增殖,剂量升高时(10-2、5×10-3mol/L),则促进细胞的凋亡.

abstractsObjective To study the cell vitality and ultra-structure of in vitro cultured fetus chondrocytes exposed to different doses of fluoride.Methods Primary chondrocytes were obtained from articular cartilage of the 24-27 weeks,aborted and dead fetuses.The third generation of primary cultured chondmcytes were exposed to concentrations of 0,10-2,5 × 10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 mol/L fluoride for 24,48 and 72 h.Cell vitality was detected with Cell Counting Kit-8 (CCK-8) and ultra-structure of chondrocytes was observed by transmission electron microscope.Results The cell vitalities of chondrocytes exposed to doses of fluoride (10-2,5 ×10-3,10-3,10-4,10-5,10-6,10-7 and 10-8 moL/L) for 24,48 and 72 h were(15.04 ± 0.55)％,(62.53 ± 1.03)％,(100.34 ± 5.19)％,(111.40 ± 3.69)％,(121.47 + 6.09)％,(129.95 ± 4.96)％,(121.81 ± 4.97)％,(111.00 ± 1.63)％;(10.35 ± 0.64)％,(35.23 ± 2.41)％,(110.30 ± 2.07)％,(113.66 ± 6.98)％,(120.36 ± 6.23)％,(133.40 ± 5.80)％,(126.06 ± 5.40)％,(115.62 ± 7.33)％; (6.19 ± 0.16)％,(18.44 ± 0.21)％,(120.83 ± 4.93)％,(123.77 ± 4.82)％,(129.09 ± 5.21)％,(140.44 + 4.18)％,(131.99 ± 7.00)％,(124.10 ± 3.68)％,respectively.The cell vitalities of 10-2,5 × 10-3 mol/L fluoride groups were significantly lower than that of the control group (all P ＜ 0.05).The cell vitality of 10-2 mol/L group was significantly lower than that of the 5 × 10-3 mol/L group (P ＜ 0.05).Doses of fluoride (10-2,5 × 10-3 mol/L) could inhibit the cell vitality and promote the apoptosis of chondrocytes in vitro with increasing doses and prolonged time.The cell vitalities of 10-3,10-4,10-5,10-6,10-7,10-8 mol/L of fluoride groups were significantly higher than that of the control group (except the 24 h 10-3 mol/L,P ＜ 0.05).Between 10-4 and 10-3 mol/L groups(the vitalities of 48 h and 72 h were higher,but not significantly); 10-5 and 10-4 mol/L groups (the vitality of 72 h was higher,but not significantly); 10-6 and 10-5 mol/L groups,the cell vitalities were significantly higher than that of the control group(all P ＜ 0.05).Between 10-7 and 10-6 mol/L groups,10-8 and 10-7 mol/L groups (the vitality of 72 h was lower,but not significantly),the cell vitalities were significantly lower than that of the control group(all P ＜ 0.05).Doses of fluoride(10-3-10-8 mol/L) could promote the cell vitality of chondrocytes in vitro with prolonged time.The optimal concentration for the promotion was 10-6 mol/L.The cells of the control group were characterized as regular morphology,the abnormal surface microvillis,abundant cytoplasm and mitochondrial,abundant and slightly expanded rough endoplasmic reticulums and low electron-dense materials.The cells of 10-6 mol/L fluoride group had the following changes,increased and swell mitochondrial,hypertrophy and expanded rough endoplasmic reticulums.The cells of 5 × 10-3 mol/L fluoride group had the following changes,decreased microvillis,invaginated cell membrane,pyknosis and apoptotic body.Conclusion Doses of fluoride (10-3-10-8 mol/L) can promote the proliferation of human chondrocytes cultured in vitro.Doses of fluoride (10-2,5 × 10-3 mol/L) can promote the apoptosis of human chondrocytes cultured in vitro.