【摘要】： 目的探討體外培養(yǎng)的成纖維細(xì)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(dá)。方法采用體外組織細(xì)胞培養(yǎng)技術(shù)培養(yǎng)離體人真皮成纖維細(xì)胞,選擇52℃浴水加熱30S作為實(shí)驗(yàn)組,37℃30S為對(duì)照組,于3h,6h,12h,24h,36h,48h,72h,96h,120h,168h,240h等不同時(shí)相點(diǎn)分別采用MTT法,倒置顯微鏡,透射電鏡,流式細(xì)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(xì)胞生長(zhǎng)因子(FGF_2)ELASA Kit,RT-PCR,免疫熒光及Western blot等技術(shù)對(duì)上述兩組細(xì)胞的存活,形態(tài),細(xì)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(jìn)行檢測(cè)。結(jié)果實(shí)驗(yàn)組成纖維細(xì)胞生存率為50%,顯著低于對(duì)照組(P＜0.05),電鏡顯示實(shí)驗(yàn)組細(xì)胞3h細(xì)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(dá)到頂峰,細(xì)胞核形態(tài)始終未見(jiàn)明顯異常,72h(3天)后成纖維細(xì)胞開(kāi)始逐漸恢復(fù),168h(7天)細(xì)胞形態(tài)基本恢復(fù)正常。流式細(xì)胞儀提示細(xì)胞滯留S期(S為21.13%),明顯高于對(duì)照組(S為14.29%,P＜0.01);實(shí)驗(yàn)組細(xì)胞凋亡率(3.59%)也明顯高于對(duì)照組(0.12%)(P＜0.01)。實(shí)驗(yàn)組細(xì)胞3h細(xì)胞膜LDH漏出率為1057.90±243.61U/L,顯著高于對(duì)照組646.08±248.98 U/L(P＜0.05),24hLDH漏出率達(dá)到高峰(實(shí)驗(yàn)組:1493.88±80.723 U/L,對(duì)照組:62150±228.82 U/L,P＜0.01),72h后基本恢復(fù)正常。實(shí)驗(yàn)組胞膜中3h Na~+—K~+—ATP酶的活性為1.40±0.04 U/L,低于對(duì)照組1.49±0.04U/L(P＜0.05,為正�；钚缘�95.3‰),24h實(shí)驗(yàn)組Na~+-K~+-ATP酶的活性為0.30±0.02 U/L,低于對(duì)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(shí)驗(yàn)組3h細(xì)胞線粒體ATP量為345439.4±33954.24μg/ml,低于對(duì)照組435416.4±23613.27μg/ml(實(shí)驗(yàn)組ATP量為對(duì)照組的79‰,P＜0.05),24h實(shí)驗(yàn)組細(xì)胞線粒體ATP量進(jìn)一步降低為94731.00±1082.12μg/ml,明顯低于對(duì)照組215604.2±20610.17μg/ml(實(shí)驗(yàn)組ATP量為對(duì)照組的43.94‰,P＜0.05)。實(shí)驗(yàn)組24h,168h,240h的細(xì)胞外FGF_2量與對(duì)照組無(wú)差異(P＞0.05),48h,72h,96h,120h比對(duì)照組低(P＜0.05)且它們之間相對(duì)穩(wěn)定(P＞0.05).24h,72h,120h,168h,240h實(shí)驗(yàn)組細(xì)胞Ⅰ型膠原的轉(zhuǎn)錄,分泌與對(duì)照組無(wú)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(dá)。Western blot顯示3h即有HSP90表達(dá)量升高,24h達(dá)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(duì)FB熱損傷變性具有保護(hù)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(hào)：2245831