维生素C对TNF-α及血清剥夺诱导的人髓核细胞凋亡的作用

EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR α AND SERUM DEPRIVATION

目的探讨维生素C(Vitamin C,Vit C)在TNF-α及血清剥夺诱导人椎间盘髓核细胞凋亡中的作用及机制。方法取脊柱矫形手术患者的髓核组织,消化法获得正常髓核细胞,取第3代细胞按不同处理因素分为Vit C作用组(A组)、TNF-α诱导组(B组)和血清剥夺组(C组),每组再分为以下亚组:A组分为A1组(基础培养液)、A2组(100μg/m L Vit C)、A3组(200μg/m L Vit C);B组分为B0组(对照组)、B1组(100 ng/m L TNF-α)、B2组(100μg/m L Vit C+100 ng/m L TNF-α)、B3组(200μg/m L Vit C+100 ng/m L TNF-α);C组分为C0组(对照组)、C1组(FBS浓度由8%降低为2%)、C2组(2%FBS+100μg/m L Vit C)、C3组(2%FBS+200μg/m L Vit C)。各组作用24 h后,采用流式细胞术检测各组膜联蛋白V、碘化丙啶双染后髓核细胞凋亡率;实时荧光定量PCR检测p53(基因编码相对分子质量为53×103的蛋白质)、脂肪酸合成酶(fatty acid synthase,FAS)、半胱氨酸天冬氨酸蛋白酶3(Caspase3)、Ⅰ型胶原、Ⅱ型胶原、Sox9和糖胺多糖(Aggrecan)m RNA表达。结果 A组:与A1组比较,A2、A3组的Vit C均能降低髓核细胞凋亡及p53、FAS、Caspase 3 m RNA表达(P<0.05),但A2、A3组间比较差异无统计学意义(P>0.05);A2、A3组的Vit C可促进髓核细胞Ⅰ型胶原、Ⅱ型胶原、Aggrecan、Sox9 m RNA表达,且A3组促进作用显著大于A2组(P<0.05)。B组:与B0组比较,B1组TNF-α促进了髓核细胞凋亡,增加了髓核细胞p53、FAS、Caspase 3 m RNA表达(P<0.05);与B1组比较,B3组的Vit C能降低髓核细胞凋亡及p53、FAS、Caspase 3 m RNA表达,而B2组的Vit C则增加其凋亡及p53、FAS、Caspase 3 m RNA表达,差异均有统计学意义(P<0.05)。C组:与C0组比较,C1组2%FBS能诱导髓核细胞凋亡,但降低了髓核细胞p53、FAS、Caspase 3 m RNA表达(P<0.05);与C1组比较,C3组的Vit C能降低髓核细胞凋亡及p53、FAS、Caspase 3 m RNA表达,而C2组的Vit C则增加其凋亡及p53、FAS m RNA表达,差异有统计学意义(P<0.05)。结论 Vit C能延缓或降低髓核细胞凋亡、促进细胞外基质合成与分泌,200μg/m L Vit C可延缓或降低100 ng/m L TNF-α和2%FBS诱导的凋亡,而100μg/m L Vit C则促进其诱导的凋亡。

Objective To explore the effect of Vitamin C（Vit C） on the apoptosis of human nucleus pulposus（NP） cells induced by tumor necrosis factor α（TNF-α） and serum deprivation. Methods The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups： Vit C group（group A）, TNF-α group（group B）, and serum deprivation group（group C）. Group A was reassigned to A1 subgroup（basic medium）, A2 subgroup（100 μg/m L Vit C）, and A3 subgroup（200 μg/m L Vit C）. Group B was reassigned to B0 subgroup（control group）, B1 subgroup（100 ng/m L TNF-α）, B2 subgroup（100 μg/m L Vit C＋100 ng/ m L TNF-α）, and B3 subgroup（200 μg/m L Vit C＋100 ng/m L TNF-α）. Group C was reassigned to C0 subgroup（Control group）, C1 subgroup（2% FBS）, C2 subgroup（2%FBS＋100 μg/m L Vit C）, and C3 subgroup（2% FBS＋200 μg/m L Vit C）. After application of 100 μg/m L or 200 μg/m L Vit C for 24 hours, NP cells were stimulated by TNF-α and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells（collagen type I, collagen type II, aggrecan, and Sox9） and apoptosis related genes（p53, FAS, and Caspase 3） were detected by real-time fluoroscent quantitative PCR. Results Group A： Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with A1 subgroup（P〈0.05）, but there was no significant difference between A2 subgroup and A3 subgroup（P〉0.05）; Vit C could promote the expressions of the extracellular matrix（collagen type I, collagen type II, aggrecan, and Sox9） of NP cells in a concentration dependent manner（P〈0.05）. Group B： TNF-α significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup（P〈0.05）; however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup（P〈0.05）. Group C： 2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup（P〈0.05）; Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup（P〈0.05）. Conclusion Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 μg/m L Vit C may delay the apoptosis induced by TNF-α and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.