亚甲蓝对大鼠椎间盘纤维环细胞增殖及凋亡的影响

Influence of methylene blue on the proliferation and apoptosis of intervertebral disc annulus fibrosus cells

中文关键词:  亚甲蓝  纤维环细胞  转化生长因子β1  金属蛋白酶组织抑制因子1  凋亡调节蛋白bax/bcl-xl  半胱氨酸天冬氨酸蛋白酶-3  增殖  凋亡

英文关键词:Methylene blue  Annulus fibrosus cells  Caspase-3  TGF-β1  TIMP-1  Bcl-xl/bax  Proliferation  Apoptosis

中文摘要:

  【摘要】 目的：探讨不同浓度亚甲蓝对大鼠椎间盘纤维环细胞增殖及凋亡的影响。方法：取3月龄清洁级SD大鼠尾椎椎间盘纤维环，采用胶原酶及胰酶序贯消化法获得纤维环细胞并进行体外扩增培养，镜下观察细胞生长情况。将第3代纤维环细胞分为对照组（不施加亚甲蓝干预）及不同浓度亚甲蓝干预组，分别为低浓度组（2μg/ml）、中浓度组（20μg/ml）及高浓度组（200μg/ml），干预后2h、4h、6h、12h、24h及72h通过CCK-8法检测细胞活力，流式细胞仪检测细胞凋亡情况，半胱氨酸天冬氨酸蛋白酶-3（caspase-3）试剂盒检测caspase-3活性，RT-PCR及Western Blot检测Ⅰ型胶原、转化生长因子β1（TGF-β1）、碱性成纤维细胞生长因子（bFGF）、金属蛋白酶组织抑制因子1（TIMP-1）、caspase-3、凋亡调节蛋白bcl-xl及bax的mRNA及蛋白表达。结果：原代纤维环细胞早期可形成细胞集落并向四周克隆生长，速度较慢，14d后达到80%~90%融合。传代后细胞生长速度明显加快，细胞多呈梭形，并有伪足伸出。亚甲蓝干预后可以显著抑制纤维环细胞增殖，促进纤维环细胞凋亡，中、高浓度组细胞凋亡率分别为74.95%及90.09%，与对照组比较，差异有统计学意义（P<0.05）；且随亚甲蓝浓度增加，抑制细胞增殖及促进细胞凋亡作用越明显，不同浓度组间比较差异均有统计学意义（P<0.05）；RT-PCR及Western Blot结果显示，亚甲蓝干预后Ⅰ型胶原、TGF-β1、bFGF、TIMP-1及bcl-xl表达显著降低，而caspase-3及bax表达升高，与对照组比较，差异有统计学意义（P<0.05）；且随亚甲蓝浓度增加，抑制作用越明显，不同浓度组间比较差异均有统计学意义（P<0.05）。结论：亚甲蓝以浓度依赖性方式抑制纤维环细胞增殖及促进细胞凋亡，其促进细胞凋亡的机制可能与bcl-xl/bax途径有关；同时亚甲蓝以浓度依赖性方式抑制纤维环细胞分泌细胞外基质，其机制可能与TIMP-1促进细胞外基质降解有关。

英文摘要:

  【Abstract】 Objectives: To investigate the effect of methylene blue at different concentrations on the proliferation and apoptosis of intervertebral disc annulus fibrosus cells(AFCs) in vitro. Methods: AFCs were isolated and cultured with collagenase and sequential trypsin digestion from the caudal intervertebral disc annulus fibrosus of 3-month-old clean-grade Sprague-Dawley rats. Cellular morphology was observed, and cellular proliferation was detected by CCK-8 assay. The third generation AFCs were incubated with control group and various concentrations of methylene blue groups, as low group(2μg/ml), medium group(20μg/ml) and high group(200μg/ml) for 2h, 4h, 6h, 12h, 24h and 72h. The activity of caspase-3 was detected by caspase-3 kit. The mRNA and protein expressions of collagen type I, basic transforming growth factor β1(TGF-β1), fibroblast growth factor(bFGF), tissue inhibitor of metalloproteinase-1(TIMP-1), caspase-3, bcl-xl and bax were detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and Western blot. Results: Primary AFCs could form cell colonies and exhibit clonal growth slowly. The passaged cells showed faster growth rate and spindle-shaped with pseudo-foot out. Methylene blue showed negative effect on the proliferation of AFCs and could induce apoptosis of AFCs, the apoptosis rate of medium and high group was 74.95% and 90.09% respectively, with a statistically significant differences compared with control group. There were also significant differences among the low group, medium group and high group(P<0.05). Methylene blue also had a negative effect on the mRNA and protein expressions of collagen type I, TGF-β1, bFGF, TIMP-1 and bcl-xl, but it could increase the expressions of caspase-3 and bax. There were significant differences between the control group and various methylene blue concentration groups(P<0.05). Conclusions: Methylene blue can inhibit proliferation and promote apoptosis of AFCs in a dose and time dependent manner, and the bcl-xl/bax mediated pathway may be involved in the apoptosis mechanism. Methylene blue can also inhibit the expressions of extracellular matrix of AFCs, and the TIMP-1 mediated pathway may be involved in the mechanism.