摘要
目的在建立整合素连接激酶(ILK)基因RNAi慢病毒表达载体的基础上,研究ILK基因沉默后对人肺腺癌细胞A549细胞生物学活性的影响及中药肺岩宁方对细胞活性的影响。方法构建载体转染人肺腺癌细胞A549后,分为阴性对照病毒感染细胞组(NC组)、RNAi靶点病毒感染细胞组(KD组)、10%肺岩宁血清干预的NC组(NC+10%中药血清组)、10%肺岩宁血清干预的KD组(KD+10%中药血清组)、20%肺岩宁血清干预的NC组(NC+20%中药血清组)、20%肺岩宁血清干预的KD组(KD+20%中药血清组)及10%(10 mg/L)DDP干预的KD组(KD+10%DDP组)。MTT检测不同浓度的药物敏感性,流式细胞仪检测细胞凋亡和细胞周期变化,Transwell实验和克隆形成检测细胞的迁移和成瘤能力。结果①KD组与NC组相比,KD组细胞凋亡率上升(P<0.05),G2/M期细胞总数的百分比明显降低(P<0.01),转移率明显降低(P<0.05),克隆数目明显减少(P<0.05)。②KD+10%中药含药血清组与NC+10%中药血清组相比,KD+10%中药血清组细胞凋亡率明显上升(P<0.05),G2/M期细胞总数的百分比明显降低(P<0.01),转移率无显著性差异(P>0.05),克隆数目明显减少(P<0.05)。③KD+20%中药血清组与NC+20%中药血清相比,KD+20%中药血清细胞凋亡率下降(P<0.01),G2/M期细胞总数的百分比降低(P<0.01),转移率无显著性差异(P>0.05),克隆数目明显减少(P<0.05)。④KD+10%DDP组与NC+20%中药血清组相比,KD+10%DDP组细胞凋亡率上升(P<0.01),G2/M期细胞总数的百分比明显降低(P<0.01),转移率明显降低(P<0.01),克隆数目明显减少(P<0.01)。结论 ILK基因沉默后可以明显促进人肺腺癌细胞A549的凋亡,降低细胞迁移和成瘤能力,对细胞周期无调控作用;10%中药肺岩宁方含药血清有促进细胞凋亡及抑制细胞克隆形成的能力;20%中药含药血清可以抑制细胞克隆形成能力;10%DDP有促进细胞凋亡、抑制细胞迁移和克隆形成的能力。
Objective Based on the construction of lentiviral vector of RNA interference of ILK gene, the biological activity of human lung cancer cell line A549 and the effects of "Feiyanning Formula" (FYN) was observed after the expression of ILK was silenced. Methods Transfected with Antisense ILK on the A549, the groups were divided into the negative control (NC) , knock down (KD) , NC + 10% FYN drug serum, KD + 10% FYN drug serum, NC +20% FYN drug serum, KD + 20% FYN drug serum, and KD + 10% DDP. MTT method was used to test the drug sensitivity, the cell cycle and cell apoptosis was tested by Flow Cytometer. The cell transfer and Tumorigenic potential was tested by transwell and clone formation. Results ① Compared with the NC group, the cell apoptosis rate was increased significantly ( P 〈 0.05 ), the G2/M phase cells was decreased ( P 〈 0.01 ) ; the rate of metastasis and the number of cell clone formation were obviously decreased (P〈0.05) in KD group. ② Compared with the NC group treated with 10% FYN drug serum, the cell apoptosis rate was increased significantly(P 〈0.05) , the G2/M phase cells was decreased (P 〈0.01 ) ; No significance was found in the rate of metastasis (P 〉 0.05 ) ; the number of eel1 clone formation was obviously decreased (P 〈 0.05 ) in the KD group treated with 10% FYN drug serum. ③ Compared with the NC group treated with 20% FYN drug serum, the cell apoptosis rate and G2/M phase ceils were decreased (P 〈0.01 ) ; No significance was found in the rate of metastasis ( P 〉 0.05 ) and the number of cell clone formation was obviously decreased ( P 〈 0.05 ) in the KD group treated with 20% FYN drug serum. ④ Compared with the NC group treated with 20% FYN drug serum, the cell apoptosis rate was increased significantly( P 〈 O. 01 ), the G2/M phase cells, the rate of metastasis, the number of cell clone formation were decreased ( P 〈 0.01 ) in the KD group treated with 10% DDP drug serum. Conclusion The cell apoptosis rate can be promoted after ILK gene expression silenced and inhibit cell migration and tumorigenic potential of A549, while has no active effect on the cell cycle. 10% FYN drug serum can promote the cell apoptosis and inhibit tumorigenic potential in A549; 20% FYN drug serum can inhibit the ability of clone formation; 10% DDP can promote the cell apoptosis and inhibit the abilities of cell migration and clone formation.
出处
《上海中医药杂志》
2014年第6期86-91,共6页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金项目(81072739)
上海中医药大学杏林学者资助项目(2209.13.03)