T-2毒素和硒对软骨不同分化层细胞增殖的影响

Effects of T-2 toxin and selenium on cell proliferation in murine pre-ehondrogenic cell line at different differentiation stages

目的检测T-2毒素和硒对软骨不同分化层细胞增殖的影响，为大骨节病软骨损伤研究提供依据。方法用分化培养基（常规培养液含1％ITS）分别诱导鼠软骨前体细胞系ATDC50、7、14、21d，实时荧光定量PCR（Real-Time PCR）检测胶原蛋白Ⅱ（ColⅡ）、胶原蛋白X（Col X）的mRNA水平．确定其处于软骨不同分化层；T-2毒素（含量分别为1、5、10、20、50、100μg／L）和T-2毒素＋硒（0．1mg／L）分别作用于ITS诱导0、7、14、21d的ATDC5细胞24、48h，噻唑蓝（MTr）法检测T-2毒素和硒对软骨不同分化层细胞增殖的影响。结果ITS诱导ATDC5细胞14、21d，软骨细胞C01UmRNA（10．73±4．55、3．16±0．19）均高于0d（1．00±0．00，P均〈0．05）；ITS诱导ATDC5细胞21d，软骨细胞ColXmRNA（49．21±9．54）显著高于0d（1．00±0．00，P〈0．05）。低含量T-2毒素（1、5、10μg／L）作用不同分化层细胞24h出现代谢性补偿现象刺激细胞增殖，作用48h无代谢补偿现象；中高含量T-2毒素（20、50、100μg／L）作用不同分化层细胞24、48h，T-μ2毒素对21d细胞的抑制率[24h：（13．92±2．47）％、（47．78±4．22）％、（67．24±2．48）％，48h：（15．84±2．85）％、（55．31±0．50）％、（70．89±9．88）％]均低于0d[24h：（38．46±6．14）％、（53．20±6．20）％、（73．94±1．93）％，48h：（74．83±1．80）％、（88．98±1．51）％、（92．68±0．42）％]、7d[24h：（24．15±1．27）％、（45．19±1．29）％、（71．79±0．94）％，48h：（47．91±4．71）％、（77．84±0．52）％、（89．41±0．52）％]、14d[24h：（25．87±5．41）％、（62．50±2．50）％、（75．34±1．81）％，48h：（59．53±1．13）％、（80．32±4．54）％、（95．22±1．22）％，P均〈0．05]。T-2毒素＋硒作用不同分化层细胞48h，中高含量T-2毒素（20、50、100μg／L）＋硒时，T-2毒素对21d细胞的抑制率[（13．60±0．50）％、（50．76±6．99）％、（82．54±2．52）％]均低于0d[（72．65±5．80）％、（88．92±0．70）％、（90．32±2．20）％]、7d[（38．69±1．96）％、（75．75±0．22）％、（86．90±1．91）％]、14d[（77．90±2．20）％、（94．11±0．42）％、（94．10±0．46）％，P均〈0．05]。结论ITS诱导21d，ATDC5细胞对T-2毒素的敏感性低于其他不同分化层细胞，加硒后对T-2毒素的敏感性仍低于其他不同分化层细胞；提示T-2毒素有可能通过骨髓等其他途径损伤深层软骨细胞．为大骨节病软骨层坏死的机制提供了新的线索。

Objective To investigate the effects of T-2 toxin and selenium on cell proliferation in murine pre-chondrogenic cell line ATDC5 at different differentiation stages. Methods ATDC5 was induced by ITS medium respectively for 0, 7,14,21 days, and then the mRNA levels of collagen Ⅱ （Col Ⅱ） and collagen X （Col X） were determined by real-time PCR to indentify the cells at different differentiation stages. T-2 toxin （1, 5, 10,20, 50, 100 μg/L） and T-2 toxin with selenium （0.1 mg/L） were applied to ATDC5 （0, 7, 14,21 days） for 24 and 48 h, the effects of T-2 toxin and selenium on cell viability was detected by MTT assay. Results The mRNA expression of Col II was increased both at 14 and 21 days than that of 0 day （10.73 ± 4.55, 3.16 ±0.19 vs 1.00 ± 0.00, P 〈 0.05）; the mRNA expression of Col X was increased at 21 days than that of 0 day （49.21 ± 9.54 vs 1.00 ± 0.00, P 〈 0.05）. After treatment with T-2 toxin at lower concentrations （1, 5, 10μg/L）, metabolic compensation appeared at 24 h, and no difference was found among the different layers at 48 h. At the higher concentrations （20, 50, 100 Ixg/L）, the suppression ratios of cells at 21 days [24 h： （13.92 ± 2.47）%, （47.78 ± 4.22）%, （67.24 ± 2.48）%; 48 h： （15.84 ± 2.85）%, （55.31± 0.50）%, （70.89 ± 9.88）%] were lower than those of 0 d [24 h： （38.46 ± 6.14）%, （53.20 ± 6.20）%, （73.94 ± 1.93）%; 48 h： （74.83 ± 1.80）%, （88.98 ± 1.51）%, （92.68 ±0.42）], 7 d [24 h： （24.15 ± 1.27）%, （45.19 ± 1.29）%, （71.79± 0.94）%; 48 h： （47.91 ±4,71）%, （77.84± 0.52）%, （89.41 ± 0.52 ）%] and 14 d [24 h： （25.87 ± 5.41）%, （62.50±2.50）%, （75.34 ± 1,81）%; 48 h： （59.53 ± 1.13）%, （80.32 ± 4.54）%, （95.22 ± 1.22）%, all P〈 0.05]. After addition of selenium, the suppression ratios of cells at 21 days [（13.60± 0.50）%, （50.76 ＋ 6.99）%, （82.54 ± 2.52）%] were still lower than those of 0 d [（72.65 ± 5.80）%, （88.92± 0.70）%, （90.32 ± 2.20）%], 7 d [（38.69 ±1.96）%, （75.75 ± 0.22）%, （86.90± 1.91）%] and 14 d [（77.90 ± 2.20）%, （94.11 ± 0.42）%, （94.10± 0.46）%, all P 〈 0.05] at 48 h. Conclusions The sensitivity of chondrocytes to T-2 toxin in the deep layer is lower than that of other layers of cells. And the situation is not changed after addition of selenium. These data suggest a indirect hypothesis that T-2 toxin may entry the deep layer of cartilage in some special ways, such as through blood into bone marrow, making the cartilage necrosis occurred from deep to the surface.