中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2011年
7期
629-632
,共4页
侯海峰%李金平%丁国永%叶文静%焦鹏%李群伟
侯海峰%李金平%丁國永%葉文靜%焦鵬%李群偉
후해봉%리금평%정국영%협문정%초붕%리군위
软骨细胞%毒性试验%分子作用机制%脱氧雪腐镰刀菌烯醇
軟骨細胞%毒性試驗%分子作用機製%脫氧雪腐鐮刀菌烯醇
연골세포%독성시험%분자작용궤제%탈양설부렴도균희순
Chondrocytes%Toxicity tests%Molecular mechanisms of action%Deoxynivalenol(DON)
目的 探讨脱氧雪腐镰刀菌烯醇(DON)对人胚胎膝关节软骨细胞的毒性及作用机制.方法 取原代培养人胚胎膝关节软骨,用DMEM/F12培养基进行培养和传代,在培养基中加入DON,建立以下DON染毒浓度:0.1、0.2、0.4、1.0 μg/ml组和空白对照组.在处理72 h后进行相关研究指标的检测:分别用ELISA法检测培养上清基质金属蛋白酶13(MMP-13)、前列腺素E2(PGE2)含量,硝酸还原酶法检测上清液的一氧化氮(NO)含量,流式细胞术法检测软骨细胞凋亡情况、软骨细胞内诱导型一氧化氮合酶(iNOS)和Ⅱ型胶原表达的情况,RT-PCR法分析iNOS mRNA和Ⅱ型胶原mRNA的表达.结果 DON组凋亡率为6.78%～19.05%,高于对照组的1.20%,凋亡率随DON浓度增高而增加(F=174.761,P<0.05).DON组上清液NO含量为20.8～40.7 μmol/L,高于对照组的10.2 μmol/L(F=91.966,P<0.05);MMP-13含量为0.25～0.56 μmol/L,高于对照组的0 μmol/L(F=78.420,P<0.05);PGE2含量为3.2～20.6 μmol/L,高于对照组的1.6 μmol/L(F=276.453,P<0.05).DON组软骨细胞iNOS表达强度为14.8%～56.8%,高于对照组7.1%(F=214.614,P<0.05).高剂量DON组(0.4 μg/ml和1.0 μg/ml)Ⅱ型胶原表达强度分别为56.7%和52.7%,低于对照组的62.2%(F=5.134,P<0.05).DON组软骨细胞内iNOS mRNA表达的吸光度值(A值)为1.07～1.33,高于对照组的0.62(F=8.358,P<0.05).高剂量DON组(0.4、1.0 μg/ml)软骨细胞内Ⅱ型胶原mRNA表达量分别为0.83和0.82,低于对照组的1.14(F=7.887,P<0.05).结论 DON导致人软骨细胞合成NO增加,并通过NO调节MMP-13和PGE2等促进Ⅱ型胶原等软骨基质的降解和软骨毒性损伤;DON可促进软骨细胞的凋亡.
目的 探討脫氧雪腐鐮刀菌烯醇(DON)對人胚胎膝關節軟骨細胞的毒性及作用機製.方法 取原代培養人胚胎膝關節軟骨,用DMEM/F12培養基進行培養和傳代,在培養基中加入DON,建立以下DON染毒濃度:0.1、0.2、0.4、1.0 μg/ml組和空白對照組.在處理72 h後進行相關研究指標的檢測:分彆用ELISA法檢測培養上清基質金屬蛋白酶13(MMP-13)、前列腺素E2(PGE2)含量,硝痠還原酶法檢測上清液的一氧化氮(NO)含量,流式細胞術法檢測軟骨細胞凋亡情況、軟骨細胞內誘導型一氧化氮閤酶(iNOS)和Ⅱ型膠原錶達的情況,RT-PCR法分析iNOS mRNA和Ⅱ型膠原mRNA的錶達.結果 DON組凋亡率為6.78%～19.05%,高于對照組的1.20%,凋亡率隨DON濃度增高而增加(F=174.761,P<0.05).DON組上清液NO含量為20.8～40.7 μmol/L,高于對照組的10.2 μmol/L(F=91.966,P<0.05);MMP-13含量為0.25～0.56 μmol/L,高于對照組的0 μmol/L(F=78.420,P<0.05);PGE2含量為3.2～20.6 μmol/L,高于對照組的1.6 μmol/L(F=276.453,P<0.05).DON組軟骨細胞iNOS錶達彊度為14.8%～56.8%,高于對照組7.1%(F=214.614,P<0.05).高劑量DON組(0.4 μg/ml和1.0 μg/ml)Ⅱ型膠原錶達彊度分彆為56.7%和52.7%,低于對照組的62.2%(F=5.134,P<0.05).DON組軟骨細胞內iNOS mRNA錶達的吸光度值(A值)為1.07～1.33,高于對照組的0.62(F=8.358,P<0.05).高劑量DON組(0.4、1.0 μg/ml)軟骨細胞內Ⅱ型膠原mRNA錶達量分彆為0.83和0.82,低于對照組的1.14(F=7.887,P<0.05).結論 DON導緻人軟骨細胞閤成NO增加,併通過NO調節MMP-13和PGE2等促進Ⅱ型膠原等軟骨基質的降解和軟骨毒性損傷;DON可促進軟骨細胞的凋亡.
목적 탐토탈양설부렴도균희순(DON)대인배태슬관절연골세포적독성급작용궤제.방법 취원대배양인배태슬관절연골,용DMEM/F12배양기진행배양화전대,재배양기중가입DON,건립이하DON염독농도:0.1、0.2、0.4、1.0 μg/ml조화공백대조조.재처리72 h후진행상관연구지표적검측:분별용ELISA법검측배양상청기질금속단백매13(MMP-13)、전렬선소E2(PGE2)함량,초산환원매법검측상청액적일양화담(NO)함량,류식세포술법검측연골세포조망정황、연골세포내유도형일양화담합매(iNOS)화Ⅱ형효원표체적정황,RT-PCR법분석iNOS mRNA화Ⅱ형효원mRNA적표체.결과 DON조조망솔위6.78%～19.05%,고우대조조적1.20%,조망솔수DON농도증고이증가(F=174.761,P<0.05).DON조상청액NO함량위20.8～40.7 μmol/L,고우대조조적10.2 μmol/L(F=91.966,P<0.05);MMP-13함량위0.25～0.56 μmol/L,고우대조조적0 μmol/L(F=78.420,P<0.05);PGE2함량위3.2～20.6 μmol/L,고우대조조적1.6 μmol/L(F=276.453,P<0.05).DON조연골세포iNOS표체강도위14.8%～56.8%,고우대조조7.1%(F=214.614,P<0.05).고제량DON조(0.4 μg/ml화1.0 μg/ml)Ⅱ형효원표체강도분별위56.7%화52.7%,저우대조조적62.2%(F=5.134,P<0.05).DON조연골세포내iNOS mRNA표체적흡광도치(A치)위1.07～1.33,고우대조조적0.62(F=8.358,P<0.05).고제량DON조(0.4、1.0 μg/ml)연골세포내Ⅱ형효원mRNA표체량분별위0.83화0.82,저우대조조적1.14(F=7.887,P<0.05).결론 DON도치인연골세포합성NO증가,병통과NO조절MMP-13화PGE2등촉진Ⅱ형효원등연골기질적강해화연골독성손상;DON가촉진연골세포적조망.
Objective This study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol(DON)on articular chondrocytes in human embryo.Methods Articular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium.The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1,0.2,0.4,1.0 μg/ml.The effects of DON were observed 72 hours after incubation.Cell apoptosis was assayed by flow cytometry(FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits;NO was measured by Griess assay with spectrophotometer.Inducible nitric oxide synthase(iNOS) and collagen Ⅱ in cells were detected by FCM.The expression levels of iNOS,mRNA and collagen Ⅱ mRNA were measured with RT-PCR.Results The rates of cell apoptosis in DON groups were 6.78%-19.05%,which were significantly higher than that in control(1.20%,F=174.761,P<0.05).The levels of NO in DON groups were 20.8-40.7 μmol/L,which were significantly higher than that in control(10.2 μmol/L,F=91.966,P<0.05).The levels of MMP-13 in DON groups were 0.25-0.56 μmol/L,which were significantly higher than that in control(0 μmol/L,F=78.420,P<0.05).The levels of PGE2 in DON groups were 3.2-20.6 μmol/L,which were significantly higher than that in control(11.6 μmol/L,F=276.453,P<0.05).The proportions of cells with positive iNOS in DON groups were 14.8%-56.8% which were significantly higher than that in controls (7.1%,F=214.614,P<0.05).The proportions of cells with positive collagen Ⅱ in groups with DON of 0.4 μg/ml and 1.0 μg/ml were 56.7% and 52.7%,which were significantly lower than that in control(62.2%,F=5.134,P<0.05).The relative absorbance values of iNOS mRNA in DON groups were 1.07-1.33,which were significantly higher than that in control(0.62,F=8.358,P<0.05).The levels of collagen Ⅱ mRNA in groups with DON of 0.4 μg/ml and 1.0 μg/ml were 0.83 and 0.82,which were significantly lower than that in control (1.14,F=7.887,P<0.05).Conclusion DON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13,which both promoted resolution of articular cartilage matrix such as collagen Ⅱ.DON induced apoptosis in articular cartilage cells.
