【摘要】：目的:c-Met蛋白與腫瘤細(xì)胞的生長(zhǎng)、增殖、遷移、侵襲及凋亡的保護(hù)等密切相關(guān)。本實(shí)驗(yàn)研究通過建立體外熱療殘癌細(xì)胞HepG2模型,模擬肝細(xì)胞癌(hepatocellular carcinoma,HCC)經(jīng)射頻消融(radiofrequency ablation,RFA)治療后殘留癌細(xì)胞,利用免疫細(xì)胞化學(xué)方法檢測(cè)熱療后殘留癌細(xì)胞中c-Met蛋白的表達(dá)變化,旨在探討高溫?zé)岑煂?duì)殘癌細(xì)胞c-Met蛋白表達(dá)的影響,明確c-Met蛋白對(duì)殘癌細(xì)胞的作用,為RFA后HCC復(fù)發(fā)轉(zhuǎn)移的機(jī)制和以c-Met為目標(biāo)靶點(diǎn)的藥物研究提供理論依據(jù)與實(shí)驗(yàn)基礎(chǔ)。方法:1、將HePG2細(xì)胞放入正常濃度210 mL/L氧氣、50g/L二氧化碳、飽和濕度以及37°C的培養(yǎng)箱中培養(yǎng)。培養(yǎng)液為含10%胎牛血清的DMEM細(xì)胞培養(yǎng)基,隔日更換培養(yǎng)液。人肝癌細(xì)胞HepG2呈貼壁生長(zhǎng)。實(shí)驗(yàn)所用細(xì)胞均處于對(duì)數(shù)生長(zhǎng)期。2、實(shí)驗(yàn)組體外熱療殘余癌細(xì)胞模型的建立:將對(duì)數(shù)生長(zhǎng)期的HePG2細(xì)胞置于含正常濃度210 mL/L氧氣、50g/L二氧化碳及飽和濕度的細(xì)胞培養(yǎng)箱內(nèi)。培養(yǎng)液為含10%胎牛血清的DMEM細(xì)胞培養(yǎng)基。通過調(diào)整培養(yǎng)箱的溫度使其分別達(dá)到41℃、45℃、50℃,根據(jù)培養(yǎng)溫度的不同,再依次分為A、B、C三個(gè)組,各于6 hs、12hs、24 hs、48 hs觀察HePG2細(xì)胞生長(zhǎng)情況,殘留癌細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。3、對(duì)照組殘癌細(xì)胞模型的建立:將對(duì)數(shù)生長(zhǎng)期的HePG2細(xì)胞置于含正常濃度210 mL/L氧氣、50g/L二氧化碳及飽和濕度的細(xì)胞培養(yǎng)箱內(nèi)。培養(yǎng)液為含10%胎牛血清的dmem細(xì)胞培養(yǎng)基。保持培養(yǎng)箱的溫度為37℃,于6hs、12hs、24hs、48hs觀察hepg2細(xì)胞生長(zhǎng)情況,殘留癌細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。4、免疫細(xì)胞化學(xué)方法檢測(cè)各組hepg2殘癌細(xì)胞c-met蛋白的表達(dá)。5、spss軟件統(tǒng)計(jì)學(xué)分析:(1)采用χ2檢驗(yàn)比較實(shí)驗(yàn)組a、b、c兩兩間c-met蛋白表達(dá)陽性率的差異;(2)采用χ2檢驗(yàn)比較各實(shí)驗(yàn)組與對(duì)照組間c-met蛋白表達(dá)陽性率的差異。(p0.05)結(jié)果:1、經(jīng)免疫細(xì)胞化學(xué)方法檢測(cè),在光鏡下c-met蛋白陽性細(xì)胞為hepg2胞膜呈棕黃色染色,而胞核不染色。2、c-met蛋白在體外熱療殘癌細(xì)胞模型實(shí)驗(yàn)組a、b、c中表達(dá)的陽性率依次為33.2%、35.7%、36.5%。雖然三者陽性率都升高,但a與b、b與c、a與c之間依次比較,差異在統(tǒng)計(jì)學(xué)上都沒有意義(pab=0.1440.05,pbc=0.6480.05,pac=0.0550.05)。說明當(dāng)熱療溫度高于某一值(如37℃)之后,c-met蛋白陽性表達(dá)率并不一定隨溫度升高而升高,與熱療溫度之間無依賴性。3、c-met蛋白在體外熱療殘癌細(xì)胞模型實(shí)驗(yàn)組a、b、c中表達(dá)的陽性率依次為33.2%、35.7%、36.5%,對(duì)照組中c-met蛋白表達(dá)的陽性率為10.5%。a與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(pa0.001),b與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(pb0.001),c與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(pc0.001)。與對(duì)照組相比,實(shí)驗(yàn)組中殘癌細(xì)胞c-met蛋白表達(dá)的陽性率更高,說明熱療能促進(jìn)殘癌細(xì)胞c-met蛋白的表達(dá)。結(jié)論:1、體外熱療殘癌細(xì)胞hepg2中c-met蛋白表達(dá)的陽性率更高,高溫?zé)岑熆梢源龠M(jìn)殘癌細(xì)胞c-met蛋白的表達(dá)。2、c-met蛋白可能成為提示肝細(xì)胞癌復(fù)發(fā)、轉(zhuǎn)移及療效的一個(gè)新的生物標(biāo)志物。
[Abstract]:Aim: c-Met protein is closely related to the growth, proliferation, migration, invasion and apoptosis protection of tumor cells. In this study, the residual cancer cells HepG2 model in vitro hyperthermia was established to simulate the residual cancer cells after radiofrequency ablation (radiofrequency ablation,RFA) in hepatocellular carcinoma (hepatocellular carcinoma,HCC). The expression of c-Met protein in residual cancer cells after hyperthermia was detected by immunocytochemistry. The purpose of this study was to investigate the effect of hyperthermia on the expression of c-Met protein in residual cancer cells and to clarify the effect of c-Met protein on residual cancer cells. It provides a theoretical basis and experimental basis for the mechanism of recurrence and metastasis of HCC after RFA and the study of drugs targeting c-Met. Methods: 1. HePG2 cells were cultured in a incubator with normal concentration of 210 mL/L oxygen, 50g/L carbon dioxide, saturated humidity and 37 擄C. DMEM cell culture medium containing 10% fetal bovine serum was used in the culture medium, and the culture medium was changed every other day. Human hepatocellular carcinoma cell line HepG2 grew adherently. The cells used in the experiment were in the logarithmic growth phase. 2. In vitro hyperthermia model of residual cancer cells in the experimental group was established: the logarithmic growth phase of HePG2 cells was placed in the normal concentration of 210mL/L oxygen. 50g/L carbon dioxide and saturated humidity in the cell culture box. The culture medium was DMEM cell medium containing 10% fetal bovine serum. By adjusting the temperature of the incubator to 41 鈩，

本文編號(hào)：2469765