微RNA-126对高糖刺激下巨噬细胞增殖的调节作用  

Regulatory effect of microRNA-126 on macrophage proliferation caused by high glucose stimulation

作　　者：穆玉竹 邓嘉胤[1] 黎家君 宋立婷 蒋少云 Mu Yuzhu;Deng Jiayin;Li Jiajun;Song Liting;Jiang Shaoyun(Department of Periodontics,Institute of Stomatology,School and Hospital of Stomatology,Tianjin Medical University,Tianjin 300070,China;Stomatological Center,Peking University Shenzhen Hospital,Shenzhen 518036,China)

机构地区：[1]天津医科大学口腔医院牙周科,300070 [2]北京大学深圳医院口腔医学中心,深圳518036

出　　处：《中华口腔医学杂志》2020年第12期969-975,共7页Chinese Journal of Stomatology

基　　金：天津市自然科学基金(17JCYBJC26300)。

摘　　要：目的研究微RNA-126(microRNA-126,miR-126)对高糖环境中人单核细胞(human myeloid leukemia mononuclear cell,THP-1)源性巨噬细胞增殖的影响,探讨miR-126在伴糖尿病牙周炎中的调节作用。方法体外培养THP-1细胞,低糖环境下(糖浓度5.5 mmol/L)以5μg/L佛波酯作用48 h诱导THP-1分化为巨噬细胞;分别用低糖、中糖(糖浓度15 mmol/L)和高糖(糖浓度25 mmol/L)培养液培养THP-1源性巨噬细胞,细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞的增殖情况,通过实时荧光定量PCR(quantitative real time-PCR,qRT-PCR)检测miR-126及增殖相关基因的表达;miR-126模拟物或抑制剂转染细胞72 h后,CCK-8法检测细胞增殖变化,qRT-PCR检测miR-126及增殖相关基因表达的变化。结果糖浓度升高可使THP-1源性巨噬细胞增殖能力显著降低(7 d时低、中、高糖组细胞A值分别为0.369±0.014、0.214±0.009和0.200±0.010,P<0.01),细胞存活率显著下降(P<0.05),并引起细胞中miR-126、B细胞淋巴瘤-2基因(B-cell lymophoma-2,Bcl-2)相关性X蛋白质(Bcl-2-associated X protein,BAX)、天冬氨酸蛋白酶3(caspase-3)表达显著升高(P<0.05),Bcl-2、醇磷脂-3激酶调节亚单位2(phosphoinositol-3 kinase regulatory subunit 2,PIK3R2)表达显著降低(P<0.05)。miR-126模拟物转染低糖培养的细胞72 h后,与阴性对照组(1.005±0.118)相比,低糖-模拟物组miR-126表达(2980.227±170.431)显著升高(P<0.05),并伴随THP-1源性巨噬细胞的增殖能力下降(阴性对照组:1.816±0.013,模拟物组:1.310±0.048,P<0.01),增殖抑制因子BAX、caspase-3的表达显著升高(P<0.01,P<0.05),增殖促进因子PIK3R2、Bcl-2的表达显著降低(P<0.05,P<0.01);miR-126抑制剂转染高糖培养的细胞72 h后,与阴性对照组相比(0.723±0.133),高糖-抑制剂组THP-1源性巨噬细胞的增殖能力显著增强(0.984±0.049)(P<0.05),增殖抑制因子BAX、caspase-3表达显著降低(P<0.01,P<0.05),增殖促进因子PIK3R2、Bcl-2表达显著升高(P<0.01,P<0.05)。结论�Objective To explore the effects of microRNA-126(miR-126)on the proliferation of human myeloid leukemia mononuclear cells(THP-1)-derived macrophages in high glucose environment and the regulatory role of miR-126 in periodontitis with diabetes.Methods THP-1 cells were cultured in vitro and 5μg/L phorbol-12-myristate-13-acetate was applied to induce THP-1 cells differentiating into macrophages for 48 h in low glucose culture medium(5.5 mmol/L).THP-1-derived macrophages were then cultured with low glucose,medium glucose(15 mmol/L)or high glucose(25 mmol/L)media respectively.The proliferation of THP-1-derived macrophages was detected by cell counting kit-8(CCK-8)method and the expressions of miR-126 and proliferation-associated factors were detected by quantitative real time PCR(qRT-PCR).The miR-126 mimic or inhibitor was transfected into THP-1-derived macrophages for 72 h.The proliferation of cells was detected by CCK-8 method and the expressions of miR-126 or proliferation-associated factors were detected by qRT-PCR.Results Increasing glucose concentration decreased the proliferation of THP-1-derived macrophages(day 7,A values in low,medium and high glucose groups were 0.369±0.014,0.214±0.009 and 0.200±0.010,respectively,P<0.01)as well as the survival rate(P<0.05),promoted the expression of miR-126,B-cell lymphoma-2(Bcl-2)-associated X protein(BAX)and caspase-3(P<0.05),and suppressed Bcl-2,phosphoinositol-3 kinase regulatory subunit 2(PIK3R2)expression(P<0.05).After the miR-126 mimic was transfected in cells in low glucose medium for 72 h,compared with negative control(1.005±0.118),the expression of miR-126 significantly increased(2980.227±170.431,P<0.05),and the proliferation of THP-1 derived macrophages decreased(negative control:1.816±0.013,mimic group:1.310±0.048,P<0.01),the level of BAX and caspase-3 significantly increased(P<0.01,P<0.05),PIK3R2 and Bcl-2 significantly decreased(P<0.05,P<0.01).After the miR-126 inhibitor was transfected in cells cultured in high glucose medium for 72 h,compared with ne

关 键 词：牙周炎 糖尿病 微RNA-126 巨噬细胞 细胞增殖 

分 类 号：R782.6[医药卫生—口腔医学] R445.2[医药卫生—临床医学]