离体人真皮成纤维细胞热损伤变性后转归及HSP90的保护作用

发布时间：2018-09-17 11:55

【摘要】： 目的探讨体外培养的成纤维细胞(Fibroblast FB)热损伤变性后形态,功能的变化规律及HSP90的表达。方法采用体外组织细胞培养技术培养离体人真皮成纤维细胞,选择52℃浴水加热30S作为实验组,37℃30S为对照组,于3h,6h,12h,24h,36h,48h,72h,96h,120h,168h,240h等不同时相点分别采用MTT法,倒置显微镜,透射电镜,流式细胞仪,反向高效液相仪,乳酸脱氢酶(Lactic Acid Dehydrogenase,LDH)试剂盒,Na~+-K~+-ATP酶试剂盒,Ⅰ型胶原和碱性成纤维细胞生长因子(FGF_2)ELASA Kit,RT-PCR,免疫荧光及Western blot等技术对上述两组细胞的存活,形态,细胞膜通透性,Na~+-K~+-ATP酶,FGF_2的分泌,Ⅰ型胶原的转录,分泌及HSP90的合成等进行检测。结果实验组成纤维细胞生存率为50%,显著低于对照组(P＜0.05),电镜显示实验组细胞3h细胞膜,线粒体,内质网有明显损伤,透明样物,髓鞘样物增多,24h达到顶峰,细胞核形态始终未见明显异常,72h(3天)后成纤维细胞开始逐渐恢复,168h(7天)细胞形态基本恢复正常。流式细胞仪提示细胞滞留S期(S为21.13%),明显高于对照组(S为14.29%,P＜0.01);实验组细胞凋亡率(3.59%)也明显高于对照组(0.12%)(P＜0.01)。实验组细胞3h细胞膜LDH漏出率为1057.90±243.61U/L,显著高于对照组646.08±248.98 U/L(P＜0.05),24hLDH漏出率达到高峰(实验组:1493.88±80.723 U/L,对照组:62150±228.82 U/L,P＜0.01),72h后基本恢复正常。实验组胞膜中3h Na~+—K~+—ATP酶的活性为1.40±0.04 U/L,低于对照组1.49±0.04U/L(P＜0.05,为正常活性的95.3‰),24h实验组Na~+-K~+-ATP酶的活性为0.30±0.02 U/L,低于对照组0.49±0.06 U/L(P＜0.05,为正常活性的61.2‰),48h后基本恢复正常。实验组3h细胞线粒体ATP量为345439.4±33954.24μg/ml,低于对照组435416.4±23613.27μg/ml(实验组ATP量为对照组的79‰,P＜0.05),24h实验组细胞线粒体ATP量进一步降低为94731.00±1082.12μg/ml,明显低于对照组215604.2±20610.17μg/ml(实验组ATP量为对照组的43.94‰,P＜0.05)。实验组24h,168h,240h的细胞外FGF_2量与对照组无差异(P＞0.05),48h,72h,96h,120h比对照组低(P＜0.05)且它们之间相对稳定(P＞0.05).24h,72h,120h,168h,240h实验组细胞Ⅰ型胶原的转录,分泌与对照组无差别(P＞0.05)。用免疫荧光观察到FB热损伤后24h胞浆,胞核中HSP90高表达。Western blot显示3h即有HSP90表达量升高,24h达到高峰,维持到96h以上,120h恢复到正常。结论FB热损伤变性后形态改变,代谢障碍,功能降低,其功能于48h-120h(2-5天)逐渐恢复,7天基本恢复正常的形态,功能,HSP90对FB热损伤变性具有保护作用。
[Abstract]:Objective to investigate the changes of morphology and function and the expression of HSP90 in cultured fibroblasts after thermal injury and denaturation of (Fibroblast FB). Methods Human dermal fibroblasts were cultured by tissue cell culture technique in vitro. 30s were heated with water at 52 鈩，

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