低密度脂蛋白促进腹膜间皮细胞转分化及细胞外基质蓄积
저밀도지단백촉진복막간피세포전분화급세포외기질축적
Low-density lipoprotein promotes epithelial-mesenchymal transition and extracellular matrix accumulation in human peritoneal mesothelial cells
  • 万方数据
    中华肾脏病杂志 중화신장병잡지

    2013年 1期 44-49 ,共6页

    房艳辉%蒋兰萍%陈丽萌%李雪梅%李学旺

    방염휘%장란평%진려맹%리설매%리학왕

    脂蛋白类,LDL%细胞转分化%细胞外基质%人腹膜间皮细胞 脂蛋白類,LDL%細胞轉分化%細胞外基質%人腹膜間皮細胞
    지단백류,LDL%세포전분화%세포외기질%인복막간피세포
    Lipoproteins,LDL%Cell transdifferentiation%Extracellular matrix%Human peritoneal mesothelial cells
    目的 探讨高糖作用下低密度脂蛋白(LDL)对人腹膜间皮细胞(HPMC)转分化及细胞外基质(ECM)蓄积的影响.方法 (1)将HPMC随机分为对照组、LDL组(100 mg/L)和LDL(100 mg/L)+ LDL受体阻断剂乳铁蛋白(100 mg/L)组,共培养24 h.细胞免疫荧光法观察HPMC细胞上LDL受体的表达；油红O染色法观察HPMC对LDL的摄取情况.(2)将HPMC分别加入不同浓度LDL(0、25、50、100 mg/L)培养24 h,倒置相差显微镜下观察细胞形态的改变,细胞免疫荧光法观察细胞内α平滑肌肌动蛋白(α-SMA)表达.(3)将静止细胞分为对照组(5.6 mmol/L葡萄糖)、甘露醇组(M,2.18％甘露醇)、低糖组(LG,30 mmol/L葡萄糖)、高糖组(HG,120 mmol/L葡萄糖)和高糖+LDL组(HG+ LDL,120 mmol/L葡萄糖+100 mg/L LDL),共培养48 h.实时定量PCR法检测α-SMA、E-cadherin和1型纤溶酶原激活物抑制物(PAI-1)mRNA的表达；Western印迹法检测α-SMA蛋白表达；ELISA法检测细胞培养上清中Ⅰ型胶原(Col Ⅰ)和PAI-1蛋白含量.结果 (1)免疫荧光观察到LDL可以刺激HPMC细胞表达LDL受体.油红O染色法观察到HPMC能摄取LDL进入细胞内,加入乳铁蛋白能抑制LDL进入细胞内.(2)倒置相差显微镜下观察到,随LDL浓度增高,细胞间连接逐渐趋于松散,细胞呈现明显梭形成纤维细胞样形态；胞质内α-SMA荧光强度逐渐增强.与对照组相比,HG+ LDL组α-SMA mRNA和蛋白表达显著上调(均P＜ 0.05),E-cadherin mRNA表达显著下调(P＜0.05).上述指标在HG组与HG+ LDL组、HG组与对照组之间表达量差异无统计学意义.(3)ELISA法检测结果显示,与HG组和对照组相比,HG+LDL组细胞上清中Col Ⅰ蛋白含量[(19.27±0.17)μg/L比(14.09±0.30) μg/L、(14.81±0.91) μg/L,均P＜0.05]及PAI-1含量[(498.24±76.91) ng/L比(342.19±30.43) ng/L、(220.39±33.82) ng/L,均P＜0.05]均最著上升；同时PAI-1 mRNA水平亦升高,与对照组差异有统计学意义(P=0.022).结论 HPMC通过LDL受体摄取LDL后,能在高糖作用下诱导HPMC发生转分化,通过上调PAI-1表达抑制ECM降解,导致ECM蓄积.
    목적 탐토고당작용하저밀도지단백(LDL)대인복막간피세포(HPMC)전분화급세포외기질(ECM)축적적영향.방법 (1)장HPMC수궤분위대조조、LDL조(100 mg/L)화LDL(100 mg/L)+ LDL수체조단제유철단백(100 mg/L)조,공배양24 h.세포면역형광법관찰HPMC세포상LDL수체적표체；유홍O염색법관찰HPMC대LDL적섭취정황.(2)장HPMC분별가입불동농도LDL(0、25、50、100 mg/L)배양24 h,도치상차현미경하관찰세포형태적개변,세포면역형광법관찰세포내α평활기기동단백(α-SMA)표체.(3)장정지세포분위대조조(5.6 mmol/L포도당)、감로순조(M,2.18％감로순)、저당조(LG,30 mmol/L포도당)、고당조(HG,120 mmol/L포도당)화고당+LDL조(HG+ LDL,120 mmol/L포도당+100 mg/L LDL),공배양48 h.실시정량PCR법검측α-SMA、E-cadherin화1형섬용매원격활물억제물(PAI-1)mRNA적표체；Western인적법검측α-SMA단백표체；ELISA법검측세포배양상청중Ⅰ형효원(Col Ⅰ)화PAI-1단백함량.결과 (1)면역형광관찰도LDL가이자격HPMC세포표체LDL수체.유홍O염색법관찰도HPMC능섭취LDL진입세포내,가입유철단백능억제LDL진입세포내.(2)도치상차현미경하관찰도,수LDL농도증고,세포간련접축점추우송산,세포정현명현사형성섬유세포양형태；포질내α-SMA형광강도축점증강.여대조조상비,HG+ LDL조α-SMA mRNA화단백표체현저상조(균P＜ 0.05),E-cadherin mRNA표체현저하조(P＜0.05).상술지표재HG조여HG+ LDL조、HG조여대조조지간표체량차이무통계학의의.(3)ELISA법검측결과현시,여HG조화대조조상비,HG+LDL조세포상청중Col Ⅰ단백함량[(19.27±0.17)μg/L비(14.09±0.30) μg/L、(14.81±0.91) μg/L,균P＜0.05]급PAI-1함량[(498.24±76.91) ng/L비(342.19±30.43) ng/L、(220.39±33.82) ng/L,균P＜0.05]균최저상승；동시PAI-1 mRNA수평역승고,여대조조차이유통계학의의(P=0.022).결론 HPMC통과LDL수체섭취LDL후,능재고당작용하유도HPMC발생전분화,통과상조PAI-1표체억제ECM강해,도치ECM축적.
    Objective To investingate the effect of low-density lipoprotein (LDL) on epithelial -mesenchymal transition and extracellular matrix (ECM) accumulation in human peritoneal mesothelial cells (HPMCs).Methods (1)HPMCs were randomly divided into control group,LDL group (100 mg/L) and LDL (100 mg/L) + lactoferrin (100 mg/L,LDL receptor blocking agent) group.After co-cultured for 24 h,the expression of LDL receptor in HPMCs was examined by immunofluorescence staining,and the LDL uptake by HPMCs was observed with oil red O staining.(2)HPMCs were cultured with different concentrations of LDL (0,25,50,100 mg/L).After co-cultured for 24 h,the change of cell morphology was observed by inverted phase contrast microscope,and the expression of α-smooth muscle actin (α-SMA) was examined by immunofluorescence.(3) HPMCs were randomly divided into control group (5.6 mmol/L glucose),mannitol group (M,2.18％ mannitol),low glucose group (LG,30 mmol/L),high glucose group (HG,120 mmol/L) and HG + LDL group (120 mmol/L glucose + 100 mg/L LDL).Cocultured for 48 h,the mRNA expression of α-SMA,E-cadherin and type 1 plasminogen activator inhibitor (PAI-1) was detected by real-time quantitative PCR,the protein expression of α-SMA was detected by Western blotting,the content of type I collagen (Col I) and PAI-1 in supernatant was detected by ELISA.Results (1) After co-cultured with LDL for 24 h,the expressin of LDL receptor was found on the cell membrane of HPMCs.Oil red staining showed that LDL could be uptaken into the cells and abolished by LDL receptor blocker.(2) HPMCs tended to be loosely intercellular connected to each ofher,and prsesnted significant formation of fibroblast-like spindle morphology.The cytoplasm immunofluorescence intensity of α-SMA gradually increased with the increase of LDL concentration.Compared to the control group,the expressions of α-SMA mRNA and protein were significantly increased,and the expression of E-cadherin mRNA was decreased in HG + LDL group(all P ＜ 0.05).But the expressions of the parameters above-mentioned were not significant different between HG group and HG + LDL group or between HG group and control group.(3) Compared with HG group or control group,the concentrations of Col Ⅰ [(19.27±0.17) μg/L vs (14.09±0.30) μg/L or (14.81±0.91) μg/L,all P ＜ 0.05] and PAI-1 [(498.24±76.91) ng/L vs (342.19±30.43) ng/L or (220.39±33.82) ng/L,all P ＜ 0.05] in supernatant of HPMCs were significantly up-regulated in HG + LDL group,meanwhile the expression of PAI-1 mRNA was significantly higer than that in control group (P =0.022).Conclusions HPMCs uptake LDL into cells via LDL receptors.LDL can induce HPMCs transdifferentiation in the condition of high glucose,increase the secretion of Col Ⅰ,inhibit the degradation of ECM through up-regulating the expression of PAI-1,and lead to ECM accumulation.
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