IL-1β对人颞下颌关节滑液间充质干细胞生物学特性的影响

Effect of IL-1β on human synovial fluid-derived mesenchymal stem cells in the temporomandibular joint

目的探讨白细胞介素-1β(interleukin-1β,IL-1β)对人颞下颌关节滑液间充质干细胞(human synovial fluid mesenchymal stem cells,hSFMSCs)生物学特性的影响。方法将临床收集的颞下颌关节紊乱病(temporomandibular disorder,TMD)患者颞下颌关节滑液的样本进行体外培养扩增获得hSFMSCs,将hSFMSCs分为3组:对照组用完全培养基(α-MEM细胞培养基+10%FBS+1×Gluta MAX)常规培养(命名为0ng/mL IL-1β组),IL-1β刺激组分别于完全培养基中加入rhIL-1β1ng/mL(1ng/mL IL-1β组)和rh IL-1β10 ng/m L(10ng/mL IL-1β组),根据实验不同需求分组干预。通过细胞克隆形成率比较IL-1β对h SFMSCs接种后贴壁及增殖能力的差异,CCK8法检测IL-1β对h SFMSCs细胞增殖的影响,分别使用细胞周期检测试剂盒和AnnexinV/PI凋亡检测试剂盒检测IL-1β对hSFMSCs的细胞周期和凋亡的影响。采用形态学和相关基因的定量检测评价IL-1β对h SFMSCs成骨诱导、成脂诱导、成软骨诱导等多向分化能力的影响。结果不同浓度IL-1β刺激下对h SFMSCs细胞克隆形成率(F=0.665,P=0.548)、细胞增殖(F=0.001,P=0.999)、细胞周期(FG1期=0.773,PG1期=0.503;FS期=0.636,PS期=0.562)和凋亡(F=0.196,P=0.827)的影响没有统计学意义。hSFMSCs成骨诱导中茜素红染色形成的矿化结节随IL-1β刺激浓度的增加逐渐较少,IL-1β刺激组Runt相关基因2(runt-related transcription factor 2,RUNX2)和骨钙素(osteocalcin,OCN)的m RNA表达均显著低于0 ng/m L IL-1β组(P<0.05);成脂诱导下随着IL-1β刺激浓度的增加,脂滴形成明显减少,实时荧光定量PCR检测显示成脂诱导下,与同期0ng/m L IL-1β组之间比较,IL-1β刺激组成脂分化的相关基因过氧化物酶体增殖物激活受体G2(peroxisomal proliferative receptor G2,PPARG2)和脂蛋白脂肪酶(lipoprotein lipase,LPL)m RNA表达明显降低(P<0.05)。各浓度IL-1β介导成软骨诱导后虽然均形成软骨微球,但Sry基因相关HMG box-9(sexdeter mining region Y related high-mobility group box-9,SOX9)和Ⅱ型胶原(collagenⅡ,COL-Ⅱ)基因的m RNA表达随着IL-1β的刺激而降低(P<0.05),而基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)基因在IL-1β刺激下则m RNA表达明显增加(P<0.05)。结论 IL-1β对hSFMSCs细胞生长增殖或凋亡无显著性影响,而直接影响其多向分化能力。

Objective This study investigated the effects of interleukin？1β （IL？1β） on human synovial fluid？de？rived mesenchymal stem cells （hSFMSCs） in the temporomandibular joint. Methods hSFMSCs from synovial fluid sam？ples of temporomandibular disorder （TMD） patients were cultured in vitro. hSFMSCs were divided into three groups withdifferent concentrations of rhIL？1β in complete medium （α？MEM cell culture medium ＋ 10% FBS ＋ 1× GlutaMAX）： 0ng/mL IL？1β group, 1 ng/mL IL？1β group and 10 ng/mL IL？1β group. Changes in the rate of colony formation, growthcurve, cell cycle and apoptosis of hSFMSCs under IL？1β stimulation were evaluated. The osteogenic, adipogenic and chondrogenic potential of the cells were also determined using histochemical and real？time fluorescence quantitativePCR methods. Results No significant differences in growth or proliferation capacity were observed in any IL？1β？stimu？lated group in comparisons of the colony？formation rate （F = 0.665, P=0.548）, growth curve （F=0.001, P=0.999）,cell cycle （FG1=0.773, PG1=0.503; FS =0.636, PS =0.562） or apoptosis （F=0.196, P=0.827） of the cells. However,the multidifferentiation capacity of hSFMSCs was affected in the inflammatory environment. Mineralized nodule and lip？id clusters decreased significantly, and the gene expression levels of runt？related transcription factor 2 （RUNX2）, osteo？calcin （OCN）, peroxisomal proliferative receptor G2 （PPARG2） and lipoprotein lipase （LPL） were suppressed significant？ly in IL？1β？mediated induction medium （P 〈 0.05）. In general, cartilage pellets formed in all the IL？1β？mediated chon？drogenic differentiation groups. The gene levels of sex？determining region Y？related high？mobility group box？9 （SOX9）and collagen II were decreased （P 〈 0.05）, while that of matrix metalloproteinase 13 （MMP13） was increased significant？ly in the presence of IL？1β （P 〈 0.05）. Conclusion IL？1β directly affects the multidifferentiation potential of hSFM？SCs but not their cell growth or proliferation ability.