[關(guān)鍵詞]
[摘要]
目的 研究半枝蓮總黃酮(TF-SB)對人胃腺癌細(xì)胞AGS增殖、凋亡和放療敏感性的影響和分子機(jī)制。方法 采用細(xì)胞計數(shù)試劑盒(CCK-8)法檢測TF-SB(0、25、50、100、200 μg·mL−1)作用24 h對AGS細(xì)胞存活率的影響。將AGS細(xì)胞分為對照組、溶劑對照(含相同濃度的甲醇)組、TF-SB(100 μg·mL−1)組、質(zhì)??蛰d體(pcDNA,200 nmol·L−1)組、遷移侵襲抑制蛋白(MIIP)過表達(dá)質(zhì)粒(pcDNA-MIIP,200 nmol·L−1)組、TF-SB(100 μg·mL−1)+小干擾RNA對照(si-con,200 nmol·L−1)組、TF-SB(100 μg·mL−1)+MIIP小干擾RNA(si-MIIP,200 nmol·L−1)組,轉(zhuǎn)染質(zhì)粒后TF-SB處理24 h,CCK-8法檢測細(xì)胞存活率;流式細(xì)胞術(shù)檢測細(xì)胞凋亡;Western blotting法檢測B細(xì)胞淋巴瘤-xl(Bcl-xl)、裂解的半胱氨酸天冬氨酸蛋白酶(cleaved Caspase-3)、MIIP表達(dá);不同照射量X射線(0、2、4、6、8 Gy)照射后,克隆形成實驗檢測細(xì)胞存活率,并繪制單擊多靶模型擬合曲線,Western blotting法檢測γ-H2AX蛋白表達(dá)。結(jié)果 與TF-SB 0 μg·mL−1組比較,TFSB 25、50、100、200 μg·mL−1組AGS細(xì)胞存活率顯著降低(P<0.05);與對照組及溶劑對照組比較,TF-SB 100 μg·mL−1組AGS細(xì)胞凋亡率顯著升高(P<0.05),Bcl-xl蛋白表達(dá)顯著降低(P< 0.05),cleaved Caspase-3和MIIP蛋白表達(dá)顯著升高(P<0.05);放療后,TF-SB組細(xì)胞存活率顯著降低(P<0.05),γ-H2AX蛋白表達(dá)顯著升高(P<0.05),放療敏感性增加。與pcDNA組比較,pcDNA-MIIP組AGS細(xì)胞凋亡率顯著增加,細(xì)胞存活率顯著降低,Bcl-xl蛋白表達(dá)顯著降低,cleavedCaspase-3蛋白表達(dá)增加(P<0.05);放療后,pcDNA-MIIP組細(xì)胞存活率顯著降低(P<0.05),γ-H2AX蛋白表達(dá)顯著升高(P<0.05),敏感性增加。與TF-SB+si-con組比較,TF-SB+si-MIIP組AGS細(xì)胞凋亡率顯著降低,細(xì)胞存活率顯著增加,Bcl-xl蛋白表達(dá)顯著增加,cleaved Caspase-3蛋白表達(dá)顯著降低(P<0.05);放療后,TF-SB+si-MIIP組細(xì)胞存活率顯著升高(P<0.05),γ-H2AX蛋白表達(dá)顯著降低(P<0.05),敏感性降低。結(jié)論 TF-SB通過上調(diào)MIIP可抑制AGS細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡,提高其放療敏感性。
[Key word]
[Abstract]
Objective To investigate the effect and molecular mechanism of total flavonoids of Scutellaria barbata (TF-SB) on the proliferation, apoptosis and radiosensitivity of gastric cancer cell AGS. Methods The cell counting kit (CCK-8) method was used to detect the effects of different concentrations (0, 25, 50, 100, 200 μg·mL−1) of TF-SB on the survival of gastric cancer cells to determine the experimental concentration. AGS was divided into control group, solvent control group (methanol), TF-SB (100 μg·mL−1), pcDNA (200 nmol·L−1), pcDNA-MIIP (200 nmol·L−1), TF-SB (100 μg·mL−1) + si-con (200 nmol·L−1), TF-SB (100 μg·mL−1) + siMIIP (200 nmol·L−1) group. Cell viability was detected by CCK-8 method. Flow cytometry was used to detect apoptosis. Clone formation test was used to detect the cell survival fraction after irradiation with different rays, and a one-click multi-target model was fitted to fit the curve. Western blotting was used to detect B cell lymphoma-xl (Bcl-xl), cleaved Caspase-3, migration invasion inhibitor protein (MIIP), γ -H2AX protein expression. Results Compared with TF-SB 0 μg·mL−1 group, the survival rate of AGS cells in TF-SB 25, 50, 100 and 200 μg·mL−1 groups was significantly decreased (P < 0.05). Compared with control group and solvent control group, the apoptosis rate of AGS cells in TF-SB 100 μg·mL−1 group was significantly increased (P < 0.05), the expression of Bcl-XL protein was significantly decreased (P < 0.05), and the expression of Cleaved Caspase-3 and MIIP protein was significantly increased (P < 0.05). After radiotherapy, cell survival rate of TF-SB group was significantly decreased (P < 0.05), γ-H2AX protein expression was significantly increased (P < 0.05), and radiotherapy sensitivity was increased. Compared with the pcDNA group, the apoptosis rate of AGS cells in the pcDNA-MIIP group was significantly increased, the cell survival rate was significantly decreased, the expression of Bcl-XL protein was significantly decreased, and the expression of Cleaved Caspase-3 protein was increased (P < 0.05). After radiotherapy, the survival rate of pcDNA-MIIP group was significantly decreased (P < 0.05), the expression of γ-H2AX protein was significantly increased (P < 0.05), and the sensitivity was increased. Compared with TF-SB + si-con group, apoptosis rate of AGS cells in TF-SB + si-MIIP group was significantly decreased, cell survival rate was significantly increased, Bcl-XL protein expression was significantly increased, and Cleaved Caspase-3 protein expression was significantly decreased (P < 0.05). After radiotherapy, the survival rate of TF-SB + si-MIIP group was significantly increased (P < 0.05), the expression of γ -H2AX protein was significantly decreased (P < 0.05), and the sensitivity was decreased. Conclusions TF-SB could inhibit the proliferation of gastric cancer cell line AGS, induce apoptosis, and improve its radiotherapy sensitivity through up-reglating MIIP.
[中圖分類號]
R285.5
[基金項目]