丹参酮对大鼠睑板腺上皮细胞增生、分化及脂质合成的影响

The effect of tanshinone on meibomian gland cell proliferation,differentiation and lipid synthesis

目的观察不同剂量丹参酮的2种主要单体成分隐丹参酮和丹参酮ⅡA对体外原代培养的SD大鼠睑板腺上皮细胞增生、分化及其脂质合成的影响。方法取清洁级2月龄SD大鼠,分离取出双眼睑板腺组织并与3T3滋养细胞共培养5d。隐丹参酮和丹参酮ⅡA用二甲基亚砜(DMSO)配制成不同浓度,按照培养液中添加的隐丹参酮和丹参酮ⅡA浓度不同将混合培养的细胞分为0.125μmol/L药物组、0.250μmol/L药物组、0.500μmol/L药物组、1.250μmol/L药物组和2.500μmol/L药物组,分别加入相应浓度的隐丹参酮或丹参酮ⅡA处理细胞48h,溶剂对照组仅加入相应体积的DMSO溶剂。采用免疫荧光染色法测定睑板腺组织冰冻切片和睑板腺细胞克隆细胞中角蛋白14(K14)和p63的定位和表达;采用实时荧光定量PCR法检测睑板腺细胞克隆中K16、细胞增生相关抗原(Ki67)及CCAAT增强子结合蛋白α(C/EBPα)的基因表达情况;采用结晶紫染色和油红O染色法分别评估睑板腺上皮细胞的克隆形成率和脂质合成情况。结果睑板腺细胞体外原代培养至第4~5天可见克隆形成,至培养第7天克隆面积明显增大,克隆细胞中p63和K14蛋白表达阳性。与溶剂对照组比较,0.500μmol/L隐丹参酮组、1.250μmol/L隐丹参酮组和2.500μmol/L隐丹参酮组Ki67mRNA相对表达量明显升高,0.500μmol/L丹参酮ⅡA组和1.250μmol/L丹参酮ⅡA组细胞中Ki67mRNA相对表达量明显升高,差异均有统计学意义(均P<0.05);不同浓度隐丹参酮或丹参酮ⅡA组细胞中K16和C/EBPαmRNA相对表达量总体比较,差异均无统计学意义(均P>0.05)。睑板腺细胞克隆内未见油红O染色,克隆边缘交界处细胞团出现堆积状油红O阳性染色。1.250μmol/L隐丹参酮组睑板腺细胞平均克隆形成率为(2.55±0.20)%,高于溶剂对照组的(2.05±0.13)%,差异有统计学意义(t=4.379,P<0.05);1.250μmol/L丹参酮ⅡA组睑板腺细胞的平均克隆形成率为(2.25±0.20)%,与溶剂对照组比较差异无统计学意义(t=1.616,P>0.05)。结论隐丹参酮和丹参酮ⅡA可以促进睑板腺细胞增生,但不影响睑板腺上皮细胞的分化及脂质合成。隐丹参酮可促进睑板腺细胞体外克隆的形成。

Objective To investigate the effects of cryptotanshinone and tanshinone ⅡA, two major monomer components of tanshinone, on the proliferation, differentiation and lipid synthesis of rat meibomian gland epithelial cells in vitro. Methods The eyelid meibomian gland tissue was isolated from 2-month-old SD rats and co-cultured with 3T3 trophoblasts for 5 days.Cryptotanshinone and tanshinone ⅡA were prepared with DMSO into different concentrations.The cells were grouped to 0.125 μmol/L, 0.250 μmol/L, 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L drug groups and treated for 48 hours, respectively.Only dimethyl sulfoxide (DMSO) was added in medium in the DMSO control group.The expressions of keratin 14 (K14) and p63 in frozen sections of meibomian gland tissue and clones of meibomian gland cells were detected by immunofluorescence.Real-time fluorescence quantitative PCR was used to assay the relative expression of K16, cell proliferation related antigen 67 (Ki67) and CCAAT enhancer binding protein α(C/EBPα) in meibomian gland cell clones.Crystal violet staining and oil red staining were used to evaluate the colony formation rate and lipid synthesis of meibomian gland epithelial cells. Results Primary cultured meibomian gland cells were cloned on day 4-5 in vivo, and the cloning area was increased on day 7 after culture, p63 and K14 were positively expressed in clones.Compared with the DMSO control group, the relative expression levels of Ki67 mRNA were significantly elevated in the 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L cryptotanshinone groups (all at P<0.05). The relative expressions of Ki67 mRNA in the 0.500 μmol/Land 1.250 μmol/L tanshinone ⅡA groups were significantly higher than those in the DMSO control group (all at P<0.05). No significant difference was found in the relative expression of K16 and C/EBPα mRNA among different concentrations of cryptotanshinone or tanshinone ⅡA group (all at P>0.05). No lipid drop was found in the tarsal gland cell clones;however, the accumulation of lipid was seen in the cell clusters at the margin of the clones by oil red O staining.The average clone formation rate of tarsal gland cells in the 1.250 μmol/L cryptotanshinone group was (2.55±0.20)%, which was significantly higher than (2.05±0.13)% in the DMSO control group (t=4.379, P<0.05). The average clone formation rate of tarsal gland cells in the 1.250 μmol/L tanshinone ⅡA group was (2.25±0.20)%, there was no significant difference between 1.250 μmol/L tanshinone ⅡA group and DMSO control group (t=1.616, P>0.05). Conclusions Cryptotanshinone and tanshinone ⅡA promote the proliferation of meibomian gland epithelia cells, but play less impacts to lipid synthesis of meibomian gland epithelia cells in vitro.cryptotanshinone promote the clone tormation of meibomian gland epithelia cells.