糖皮质激素及二膦酸盐对人骨组织及骨髓间充质干细胞Hedgehog通路的影响
당피질격소급이련산염대인골조직급골수간충질간세포Hedgehog통로적영향
Effects of glucocorticoid and bisphosphonates on Hedgehog signaling pathway in human bone mesenchymal stem cells and bone tissue
  • 万方数据
    中华风湿病学杂志 중화풍습병학잡지
    CHINESE JOURNAL OF RHEUMATOLOGY
    2013年 11期 760-763,封3 ,共5页

    刘颖%张晓%张光锋%崔阳%雷云霞%李玲%董光富

    류영%장효%장광봉%최양%뢰운하%리령%동광부

    糖皮质激素类%二膦酸盐类%骨和骨组织%间质干细胞 糖皮質激素類%二膦痠鹽類%骨和骨組織%間質榦細胞
    당피질격소류%이련산염류%골화골조직%간질간세포
    Glucocorticoids%Diphosphonates%Bone and bones%Mesenchymal stem cells
    目的 探讨糖皮质激素及二膦酸盐类制剂对人骨髓间充质干细胞(hBMSC)及骨组织Hedgehog通路的影响.方法 骨活检检测:40例系统性红斑狼疮(SLE)患者分成初诊组20例(未使用糖皮质激素),激素组20例(标准剂量糖皮质激素).其中激素组又按随机数字表法分成对照组10例(标准剂量糖皮质激素治疗即泼尼松首始剂量≥1 mg·kg-1·d-,治疗过程规律减量,如有复发,随病情需要加用糖皮质激素),治疗组10例(标准剂量糖皮质激素治疗+阿仑膦酸钠).24周后行骨髓穿刺术,检测Gli1平均积分吸光度.体外细胞培养检测:将鉴定成功的P2～P4代hBMSC,在普通培养下,分成重组人SHHN(rh-SHHN)细胞因子与高、中浓度甲泼尼龙(分别为10-3 mol/L,10-5 mol/L)单独或共培养组,用荧光定量核酸检测系统(qPCR)检测不同干预组细胞Gli1-mRNA水平的表达,茜素红染色法观察其矿化结节的程度.在成骨诱导培养下,分成低浓度二膦酸盐(10-9 mol/L)与高浓度甲泼尼龙单独或共培养组,用qPCR法检测2组细胞Gli1 mRNA的表达,茜素红染色法观察二膦酸盐对细胞矿化程度的影响.2组间比较采用t检验,多组间多因素的统计学分析采用析因方差分析.结果 第24周,服激素组骨组织Gli1蛋白平均积分吸光度(47±7)与初诊组(61±12)相比显著降低(t=4.442,P＜0.01)；单用标准剂量糖皮质激素患者骨组织Gli1蛋白平均积分吸光度(42±6)与二膦酸盐治疗组(51±6)相比显著下降(t=3.701,P=0.002).普通培养下:第7天,加入高、中浓度甲泼尼龙共刺激后hBMSCs Gli1 mRNA水平(0.38±0.04,0.68±0.24)与单用细胞因子组(2.01±0.38)相比显著降低(F=8.748,P＜0.01),细胞矿化结节程度明显降低；成骨诱导培养下,第14天,相比单用高浓度激素组(0.024±0.011),加入低浓度二膦酸盐后,hBMSCs Gli1 mRNA(0.034±0.006)表达显著升高(t=7.62,P＜0.01),细胞矿化结节程度明显增加.结论 高中浓度的糖皮质激素可以通过抑制Gli1的表达降低成骨作用,低浓度的二膦酸盐可以对抗糖皮质激素对Gli1表达及成骨的抑制作用.
    목적 탐토당피질격소급이련산염류제제대인골수간충질간세포(hBMSC)급골조직Hedgehog통로적영향.방법 골활검검측:40례계통성홍반랑창(SLE)환자분성초진조20례(미사용당피질격소),격소조20례(표준제량당피질격소).기중격소조우안수궤수자표법분성대조조10례(표준제량당피질격소치료즉발니송수시제량≥1 mg·kg-1·d-,치료과정규률감량,여유복발,수병정수요가용당피질격소),치료조10례(표준제량당피질격소치료+아륜련산납).24주후행골수천자술,검측Gli1평균적분흡광도.체외세포배양검측:장감정성공적P2～P4대hBMSC,재보통배양하,분성중조인SHHN(rh-SHHN)세포인자여고、중농도갑발니룡(분별위10-3 mol/L,10-5 mol/L)단독혹공배양조,용형광정량핵산검측계통(qPCR)검측불동간예조세포Gli1-mRNA수평적표체,천소홍염색법관찰기광화결절적정도.재성골유도배양하,분성저농도이련산염(10-9 mol/L)여고농도갑발니룡단독혹공배양조,용qPCR법검측2조세포Gli1 mRNA적표체,천소홍염색법관찰이련산염대세포광화정도적영향.2조간비교채용t검험,다조간다인소적통계학분석채용석인방차분석.결과 제24주,복격소조골조직Gli1단백평균적분흡광도(47±7)여초진조(61±12)상비현저강저(t=4.442,P＜0.01)；단용표준제량당피질격소환자골조직Gli1단백평균적분흡광도(42±6)여이련산염치료조(51±6)상비현저하강(t=3.701,P=0.002).보통배양하:제7천,가입고、중농도갑발니룡공자격후hBMSCs Gli1 mRNA수평(0.38±0.04,0.68±0.24)여단용세포인자조(2.01±0.38)상비현저강저(F=8.748,P＜0.01),세포광화결절정도명현강저；성골유도배양하,제14천,상비단용고농도격소조(0.024±0.011),가입저농도이련산염후,hBMSCs Gli1 mRNA(0.034±0.006)표체현저승고(t=7.62,P＜0.01),세포광화결절정도명현증가.결론 고중농도적당피질격소가이통과억제Gli1적표체강저성골작용,저농도적이련산염가이대항당피질격소대Gli1표체급성골적억제작용.
    Objective To investigate the effect of glucocorticoid and bisphosphonate on Hedgehog signaling pathway in human bone mesenchymal stem cells (hBMSCs) and bone tissue.Methods ① Bone biopsy test:forty cases of systemic lupus erythematosus (SLE) patients were divided into 2 groups:20 cases in newly diagnosis group,20 cases in the GCs group (the dosage of glucocorticoids was higher than 1 mg·kg-1·d-1).Patients in the GCs group was randomly divided into 2 groups:10 cases in the control group were without anti-osteoporosis treatment,10 cases in treatment group received alendronate 70 mg once a week.All of the patients had bone marrow puncture after24 weeks,and the value of average optical density of Gli1 was tested with immunohistochemistry.② Cell culture:hBMSCs cultured in normal medium were intervened with rh-SHHN and methylprednisolone (10-3 mol/L,10-5 mol/L) after successfully identified.Quantitative polymerase chain reaction (PCR) was used to detectthe mRNA expression of Gli1.The final calcium nodules was detected by Alizarin red staining.hBMSCs cultured in osteogenesis medium were intervened with bisphosphonate and methylprednisolone (10-3 mol/L) after successfully identified.Quantitative PCR was used to detect the expression of Gli1 mRNA.Alizarin red staining was used to detect the final calcium nodules.Comparisons between the two groups were carried out using t-test while the difference analysis of multi-groups were tested by factorial analysis.Results The average optical density of Gli1 in the GCs group (47±7) was less than the newly diagnosed group (61±12) (t=4.442,P＜0.01),and it was less in the control group (51±6) than in the treatment group (42±6) (t=3.701,P=0.002).When normally cultured,high and moderate concentration of methylprednisolone suppressed the mRNA (0.38±0.04; 0.68±0.24) (F=8.748,P＜0.01) expression of Gli1 which was initially stimulated by rh-SHHN (2.01 ±0.38).And the final calcium nodules in groups which contained methylprednisolone were much less than rh-SHHN only group.When hBMSCs were cultured in osteogenesis medium,compared with the methylprednisolone group (0.024±0.011),the expression of Gli1mRNA(0.034-0.006) (t=7.62,P＜0.01) and the final calcium nodules were significantly improved by bisphosphonate.Conclusion High and moderate doses of glucocorticoids inhibit hBMSC osteogenesis by inhibiting Gli1.Low concentration of alendronate can not only stimulate hBMSC osteogenesis differentiation but also can remit the inhibition effects of GCs through the way of Hedgehog.
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