【摘要】： 目的探討體外培養(yǎng)的成纖維細胞(Fibroblast FB)熱損傷變性后形態(tài),功能的變化規(guī)律及HSP90的表達。方法采用體外組織細胞培養(yǎng)技術(shù)培養(yǎng)離體人真皮成纖維細胞,選擇52℃浴水加熱30S作為實驗組,37℃30S為對照組,于3h,6h,12h,24h,36h,48h,72h,96h,120h,168h,240h等不同時相點分別采用MTT法,倒置顯微鏡,透射電鏡,流式細胞儀,反向高效液相儀,乳酸脫氫酶(Lactic Acid Dehydrogenase,LDH)試劑盒,Na~+-K~+-ATP酶試劑盒,Ⅰ型膠原和堿性成纖維細胞生長因子(FGF_2)ELASA Kit,RT-PCR,免疫熒光及Western blot等技術(shù)對上述兩組細胞的存活,形態(tài),細胞膜通透性,Na~+-K~+-ATP酶,FGF_2的分泌,Ⅰ型膠原的轉(zhuǎn)錄,分泌及HSP90的合成等進行檢測。結(jié)果實驗組成纖維細胞生存率為50%,顯著低于對照組(P＜0.05),電鏡顯示實驗組細胞3h細胞膜,線粒體,內(nèi)質(zhì)網(wǎng)有明顯損傷,透明樣物,髓鞘樣物增多,24h達到頂峰,細胞核形態(tài)始終未見明顯異常,72h(3天)后成纖維細胞開始逐漸恢復(fù),168h(7天)細胞形態(tài)基本恢復(fù)正常。流式細胞儀提示細胞滯留S期(S為21.13%),明顯高于對照組(S為14.29%,P＜0.01);實驗組細胞凋亡率(3.59%)也明顯高于對照組(0.12%)(P＜0.01)。實驗組細胞3h細胞膜LDH漏出率為1057.90±243.61U/L,顯著高于對照組646.08±248.98 U/L(P＜0.05),24hLDH漏出率達到高峰(實驗組:1493.88±80.723 U/L,對照組:62150±228.82 U/L,P＜0.01),72h后基本恢復(fù)正常。實驗組胞膜中3h Na~+—K~+—ATP酶的活性為1.40±0.04 U/L,低于對照組1.49±0.04U/L(P＜0.05,為正�；钚缘�95.3‰),24h實驗組Na~+-K~+-ATP酶的活性為0.30±0.02 U/L,低于對照組0.49±0.06 U/L(P＜0.05,為正�；钚缘�61.2‰),48h后基本恢復(fù)正常。實驗組3h細胞線粒體ATP量為345439.4±33954.24μg/ml,低于對照組435416.4±23613.27μg/ml(實驗組ATP量為對照組的79‰,P＜0.05),24h實驗組細胞線粒體ATP量進一步降低為94731.00±1082.12μg/ml,明顯低于對照組215604.2±20610.17μg/ml(實驗組ATP量為對照組的43.94‰,P＜0.05)。實驗組24h,168h,240h的細胞外FGF_2量與對照組無差異(P＞0.05),48h,72h,96h,120h比對照組低(P＜0.05)且它們之間相對穩(wěn)定(P＞0.05).24h,72h,120h,168h,240h實驗組細胞Ⅰ型膠原的轉(zhuǎn)錄,分泌與對照組無差別(P＞0.05)。用免疫熒光觀察到FB熱損傷后24h胞漿,胞核中HSP90高表達。Western blot顯示3h即有HSP90表達量升高,24h達到高峰,維持到96h以上,120h恢復(fù)到正常。結(jié)論FB熱損傷變性后形態(tài)改變,代謝障礙,功能降低,其功能于48h-120h(2-5天)逐漸恢復(fù),7天基本恢復(fù)正常的形態(tài),功能,HSP90對FB熱損傷變性具有保護作用。
[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，

本文編號：2245831