中华风湿病学杂志
中華風濕病學雜誌
중화풍습병학잡지
CHINESE JOURNAL OF RHEUMATOLOGY
2013年
5期
327-331,361
,共6页
崔文岗%雷鸣%石岩%肖德明
崔文崗%雷鳴%石巖%肖德明
최문강%뢰명%석암%초덕명
软骨%组织工程%间质干细胞%NF-κB%抗原,CD29%白藜芦醇
軟骨%組織工程%間質榦細胞%NF-κB%抗原,CD29%白藜蘆醇
연골%조직공정%간질간세포%NF-κB%항원,CD29%백려호순
Cartilage%Tissue engineering%Mesenchymal stem cells%NF kappa B%Antigen,CD29%Resveratrol
目的 研究白藜芦醇对骨髓间充质干细胞组织工程化软骨的保护作用并探讨其机制.方法 首先将体外单层扩增的大鼠骨髓间充质干细胞在藻酸钠微球中行三维立体软骨细胞定向培养,其定向分化程度通过甲苯胺蓝染色、免疫组织化学等方法鉴定.然后将从微球释放的分化软骨细胞接种于壳聚糖-明胶复合支架,培养3周后制备组织工程化软骨,其内部软骨细胞以及支架形态用扫描电镜和激光共聚焦显微镜观察.将白细胞介素(IL)-1β作用于该细胞-支架材料上,并用白藜芦醇和(或)特异性的抗整合素β1亚单位抗体进一步干预,蛋白印迹法检测Ⅱ型胶原、基质金属蛋白酶(MMP)-13以及核因子-κB的表达,扫描蛋白印迹条带灰度并进行半定量计算,采用方差分析进行统计学处理.结果 在定向培养过程中,藻酸盐微球中的软骨细胞在细胞周围可形成明显的细胞外基质,促进分化,软骨细胞特异性Ⅱ型胶原和蛋白聚糖表达显著增高.定向分化后的软骨细胞在壳聚糖-明胶复合支架材料上能良好地贴壁、增殖和迁徙,形成组织工程化的软骨.蛋白印迹半定量分析:软骨Ⅱ型胶原对照组表达量0.484±0.006;白藜芦醇干预后表达量0.474±0.014,两者差异无统计学意义(P＞0.05)；IL-1β干预后,表达量下降至0.155±0.009,差异有统计学意义(P＜0.05)；而白藜芦醇能阻断IL-1β的负面效应,使Ⅱ型胶原表达量恢复至0.468±0.014,差异有统计学意义(P＜0.05),同对照组比较差异无统计学意义(P＞0.05)；而抗β1整合素能阻断白藜芦醇对IL-1β的拮抗作用,使胶原表达量下降至0.169±0.011,差异有统计学意义(P＜0.05),与IL-1β单独作用比较差异无统计学意义(P＞0.05).蛋白聚糖半定量统计和Ⅱ型胶原的表达趋势相同,同时伴随MMP-13、核因子-κB的反向表达.结论 白藜芦醇可能通过调节细胞膜黏附分子——整合素的活性,抑制核因子-κB在胞内的核转位,起到保护组织工程化软骨的作用.
目的 研究白藜蘆醇對骨髓間充質榦細胞組織工程化軟骨的保護作用併探討其機製.方法 首先將體外單層擴增的大鼠骨髓間充質榦細胞在藻痠鈉微毬中行三維立體軟骨細胞定嚮培養,其定嚮分化程度通過甲苯胺藍染色、免疫組織化學等方法鑒定.然後將從微毬釋放的分化軟骨細胞接種于殼聚糖-明膠複閤支架,培養3週後製備組織工程化軟骨,其內部軟骨細胞以及支架形態用掃描電鏡和激光共聚焦顯微鏡觀察.將白細胞介素(IL)-1β作用于該細胞-支架材料上,併用白藜蘆醇和(或)特異性的抗整閤素β1亞單位抗體進一步榦預,蛋白印跡法檢測Ⅱ型膠原、基質金屬蛋白酶(MMP)-13以及覈因子-κB的錶達,掃描蛋白印跡條帶灰度併進行半定量計算,採用方差分析進行統計學處理.結果 在定嚮培養過程中,藻痠鹽微毬中的軟骨細胞在細胞週圍可形成明顯的細胞外基質,促進分化,軟骨細胞特異性Ⅱ型膠原和蛋白聚糖錶達顯著增高.定嚮分化後的軟骨細胞在殼聚糖-明膠複閤支架材料上能良好地貼壁、增殖和遷徙,形成組織工程化的軟骨.蛋白印跡半定量分析:軟骨Ⅱ型膠原對照組錶達量0.484±0.006;白藜蘆醇榦預後錶達量0.474±0.014,兩者差異無統計學意義(P＞0.05)；IL-1β榦預後,錶達量下降至0.155±0.009,差異有統計學意義(P＜0.05)；而白藜蘆醇能阻斷IL-1β的負麵效應,使Ⅱ型膠原錶達量恢複至0.468±0.014,差異有統計學意義(P＜0.05),同對照組比較差異無統計學意義(P＞0.05)；而抗β1整閤素能阻斷白藜蘆醇對IL-1β的拮抗作用,使膠原錶達量下降至0.169±0.011,差異有統計學意義(P＜0.05),與IL-1β單獨作用比較差異無統計學意義(P＞0.05).蛋白聚糖半定量統計和Ⅱ型膠原的錶達趨勢相同,同時伴隨MMP-13、覈因子-κB的反嚮錶達.結論 白藜蘆醇可能通過調節細胞膜黏附分子——整閤素的活性,抑製覈因子-κB在胞內的覈轉位,起到保護組織工程化軟骨的作用.
목적 연구백려호순대골수간충질간세포조직공정화연골적보호작용병탐토기궤제.방법 수선장체외단층확증적대서골수간충질간세포재조산납미구중행삼유입체연골세포정향배양,기정향분화정도통과갑분알람염색、면역조직화학등방법감정.연후장종미구석방적분화연골세포접충우각취당-명효복합지가,배양3주후제비조직공정화연골,기내부연골세포이급지가형태용소묘전경화격광공취초현미경관찰.장백세포개소(IL)-1β작용우해세포-지가재료상,병용백려호순화(혹)특이성적항정합소β1아단위항체진일보간예,단백인적법검측Ⅱ형효원、기질금속단백매(MMP)-13이급핵인자-κB적표체,소묘단백인적조대회도병진행반정량계산,채용방차분석진행통계학처리.결과 재정향배양과정중,조산염미구중적연골세포재세포주위가형성명현적세포외기질,촉진분화,연골세포특이성Ⅱ형효원화단백취당표체현저증고.정향분화후적연골세포재각취당-명효복합지가재료상능량호지첩벽、증식화천사,형성조직공정화적연골.단백인적반정량분석:연골Ⅱ형효원대조조표체량0.484±0.006;백려호순간예후표체량0.474±0.014,량자차이무통계학의의(P＞0.05)；IL-1β간예후,표체량하강지0.155±0.009,차이유통계학의의(P＜0.05)；이백려호순능조단IL-1β적부면효응,사Ⅱ형효원표체량회복지0.468±0.014,차이유통계학의의(P＜0.05),동대조조비교차이무통계학의의(P＞0.05)；이항β1정합소능조단백려호순대IL-1β적길항작용,사효원표체량하강지0.169±0.011,차이유통계학의의(P＜0.05),여IL-1β단독작용비교차이무통계학의의(P＞0.05).단백취당반정량통계화Ⅱ형효원적표체추세상동,동시반수MMP-13、핵인자-κB적반향표체.결론 백려호순가능통과조절세포막점부분자——정합소적활성,억제핵인자-κB재포내적핵전위,기도보호조직공정화연골적작용.
Objective To investigate the mechanism of protective effects of resveratrol on tissueengineered cartilage.Methods The chondrogenesis of alginate-encapsulated bone marrow mesenchymal stem cells (BMSCs) were evaluated by toluidine blue staining and immunostain.The morphology of BMSCs-derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS) was evaluated by scanning electron microscope and laser confocal microscope.When these cells on CGS were pre-stimulated with interleukin-1β (IL-1β) or cotreated with IL-1β and resveratrol in the absence and presence of specific β1-integrin blocking antibody,collagen type Ⅱ,aggrecan,matrix metalloproteinase-13 (MMP-13) expression,and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blotting.ANOVA was used for statistical analysis.Results Alginate bead culture plus conditional medium together could induce the cartilage-specific collagen type Ⅱ,aggrecan expression and extracellular matrix accumulation in differentiated chondrocytes.CGS supported differentiated cell attachment,proliferation,and migration.When those cells cultured on CGS were stimulated with IL-1β alone,collagen type Ⅱ and aggrecan expression was inhibited.However,MMP-13 expression increased.By Western blotting semi-quantitative analysis,the expression level of cartilage-specific collagen type Ⅱ of the control group was 0.484±0.006; the expression level of resveratrol intervention group was 0.474±0.014.The difference between these two groups was not statistically significant (P＞0.05).The expression level of the IL-1β intervention group reduced to 0.155±0.009,which was statistically significant different from the above two groups(P＜0.05).Resveratrol could antagonist the negative effect of IL-1β,and increase collagen type Ⅱ to 0.468±0.014,the difference between these two was statistically significant (P＜0.05),and no significant difference when compared to the control group (P＞0.05).Specific β1-integrin blocking antibody could abrogate these effects of resveratrol,decrease collagen Ⅱ expression to 0.169±0.011,the difference was significant (P＜0.05),but there was no difference when compared to the IL-1β group (P＞0.05).Aggrecan semi-quantitative expression has the same trend in the expression of type Ⅱ collagen while the expression of MMP-13,NF-κB had the reversal trend.These indicated that the resveratrol reversed the catabolic effects by reducing the nuclear translocation of NF-κB.Specific β1-integrin blocking antibody abrogated these effects of resveratrol.Conclusion Resveratrol,by regulating β1-integrin,acts as a NF-κB nuclear trans-location inhibitor to protect tissue-engineered cartilage.
