本文選題：AFT蛋白 + 食管鱗癌細胞��； 參考：《中華腫瘤防治雜志》2017年05期

【摘要】：目的食管鱗狀細胞癌(esophageal squamous cell carcinoma,ESCC)多藥耐藥嚴重影響化療效果,其分子機制仍未闡明。本研究通過構建不同ATF5蛋白表達量的食管癌Eca-109細胞模型,探究過表達ATF5及低表達ATF5對食管癌Eca-109細胞耐藥性及凋亡的影響。方法利用脂質(zhì)體載體轉染技術對食管癌細胞株進行轉染,建立高、中(轉染空質(zhì)粒的對照組)、低3組不同ATF5蛋白表達量Eca-109細胞模型。蛋白質(zhì)印跡法檢測3組細胞ATF5蛋白表達水平。MTT實驗和平板克隆實驗觀察3組細胞對紫杉醇、順鉑耐受性的變化;DAPI染料核染色后觀察3組細胞在紫杉醇、順鉑作用下凋亡反應的差異;分析ATF5蛋白對食管鱗癌Eca-109細胞耐藥的影響。結果利用轉染技術成功構建的3組不同ATF5蛋白表達量的Eca-109細胞模型,表達模型組細胞中ATF5蛋白相對表達量為85.9±2.66,對照組為64.35±2.54,低表達模型組為45.3±3.11,3組差異有統(tǒng)計學意義,F=532.323,P0.001。MTT實驗顯示,紫杉醇、順鉑作用72h后,3組細胞存活率差異有統(tǒng)計學意義,F_紫=163.382,P0.001;F_順=579.36,P0.001;同一組細胞不同濃度對存活率的影響差異有統(tǒng)計學意義,F_紫=616.32,P0.001;F_順=2 558.05,P0.001;不同濃度的紫杉醇、順鉑作用3組細胞后,過表達組細胞的半數(shù)抑制率濃度(IC50)分別為4.16±2.21和1.54±0.67;對照組分別為1.54±0.92和1.27±0.65;低表達組細胞的半數(shù)抑制率濃度分別為0.33±0.21和0.53±0.62,3組差異有統(tǒng)計學意義,F_紫=198 330.768,P0.001;F_順=64 298.048,P0.001;表明過表達ATF5的細胞對紫杉醇、順鉑的藥物耐受性均明顯升高而低表達模型組細胞對紫杉醇、順鉑的藥物耐受性均明顯降低。DAPI染色實驗顯示,紫杉醇、順鉑作用3組細胞后,過表達組細胞的凋亡率分別為(14.04±1.66)%和(11.75±2.09)%;對照組分別為(26.44±2.99)%和(34.07±3.42)%;低表達組細胞的凋亡率分別為(54.85±5.88)%和(66.66±2.81)%,3組差異有統(tǒng)計學意義,F_紫=283.976,P0.001;F_順=954.464,P0.001。提示過表達ATF5蛋白細胞在紫杉醇、順鉑作用下凋亡細胞均減少,低表達ATF5細胞在紫杉醇、順鉑作用下凋亡細胞均增加。平板克隆形成實驗顯示,紫杉醇、順鉑作用3組細胞后,過表達組細胞的克隆形成率分別為(56.09±1.37)%和(62.67±1.41)%;對照組分別為(38.7±1.37)%和(34.83±3.09)%;低表達組細胞的克隆形成率分別為(25.22±1.58)%和(18.17±3.64)%,3組差異有統(tǒng)計學意義,F_紫=1157.447,P0.001;F_順=612.595,P0.001。表明過表達ATF5蛋白細胞在紫杉醇、順鉑作用下克隆形成數(shù)均更多,低表達ATF5蛋白細胞在紫杉醇、順鉑作用下克隆形成數(shù)減少。結論上調(diào)ATF5蛋白的表達能增加食管鱗癌Eca-109細胞對紫杉醇、順鉑的耐藥性,而下調(diào)ATF5蛋白的表達則能降低Eca-109細胞對紫杉醇、順鉑的耐藥性,提示食管鱗癌細胞的耐藥性可能與ATF5蛋白的表達量相關,食管鱗癌細胞中ATF5蛋白的表達情況可能能間接干擾臨床藥物化療的效果。
[Abstract]:Objective multidrug resistance (MDR) in esophageal squamous cell carcinoma (esophageal squamous cell) has a serious effect on the effect of chemotherapy, but its molecular mechanism is still unknown. This study investigated the effects of overexpression of ATF5 and low expression of ATF5 on drug resistance and apoptosis of esophageal carcinoma Eca-109 cells. Methods Eca-109 cells were transfected with liposome vector to establish Eca-109 cell model with high, middle (blank plasmid control group) and low ATF5 protein expression. The expression of ATF5 protein in three groups was detected by Western blotting. MTT assay and plate cloning assay were used to observe the changes of paclitaxel and cisplatin tolerance in the three groups. After DAPI dye nucleus staining, the three groups of cells were observed in paclitaxel. The effect of ATF5 protein on drug resistance of esophageal squamous cell carcinoma Eca-109 cells was analyzed. Results three groups of Eca-109 cell models with different ATF5 expression levels were successfully constructed by transfection technique. The relative expression of ATF5 protein was 85.9 鹵2.66 in the model group, 64.35 鹵2.54 in the control group, and 45.3 鹵3.11 in the low-expression model group. After 72 hours of treatment with paclitaxel and cisplatin, the cell survival rates of the three groups were significantly different. There were significant differences in cell survival rate between the three groups after treatment with paclitaxel and cisplatin for 72 hours. There were significant differences in the survival rate of the same group of cells treated with different concentrations of paclitaxel, 616.32C, P0.001Fx 25558.05, and different concentrations of paclitaxel. After treated with cisplatin, IC50 was 4.16 鹵2.21 and 1.54 鹵0.67 in overexpression group, 1.54 鹵0.92 and 1.27 鹵0.65 in control group, and 0.33 鹵0.21 in low-expression group and 0.53 鹵0.62ng in low expression group, respectively. ATF5 cells against paclitaxel, The drug tolerance of cisplatin group was significantly increased, while that of low expression model group was significantly decreased. The results of DAPI staining showed that paclitaxel and cisplatin treated three groups of cells. The apoptotic rate of overexpression group was (14.04 鹵1.66)% and (11.75 鹵2.09), that of control group was (26.44 鹵2.99)% and (34.07 鹵3.42), and that of low expression group was (54.85 鹵5.88)% and (66.66 鹵2.81), respectively. The results suggested that the number of apoptotic cells with overexpression of ATF5 protein in paclitaxel and cisplatin was decreased, while the apoptotic cells with low expression of ATF5 were increased under paclitaxel and cisplatin. Plate clone formation assay showed that three groups of cells were treated with paclitaxel and cisplatin. The clone formation rates of overexpression group were (56.09 鹵1.37)% and (62.67 鹵1.41), those of control group were (38.7 鹵1.37)% and (34.83 鹵3.09), and those of low expression group were (25.22 鹵1.58)% and (18.17 鹵3.64), respectively. The results showed that the over-expressed ATF5 protein cells had more clone formation under paclitaxel and cisplatin treatment, while the low expression ATF5 protein cells were reduced in taxol and cisplatin. Conclusion upregulation of ATF5 protein can increase the resistance of Eca-109 cells to paclitaxel and cisplatin, while down-regulation of ATF5 protein expression can decrease the resistance of Eca-109 cells to paclitaxel and cisplatin. The results suggest that the drug resistance of esophageal squamous carcinoma cells may be related to the expression of ATF5 protein, and the expression of ATF5 protein in esophageal squamous cell carcinoma cells may indirectly interfere with the effect of clinical drug chemotherapy.
【作者單位】： 廣州軍區(qū)廣州總醫(yī)院消化內(nèi)科;廣州中醫(yī)藥大學第二臨床醫(yī)學院;
【基金】：國家自然科學基金青年項目(81302164) 廣東省自然科學基金(2014A030313595) 廣州市科技計劃(201607010077)
【分類號】：R735.1
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本文編號：2074247