RESUMO
OBJECTIVE：To study the effects and mechanism o f apigenin on cisplatin sensitivity of NSCLC A 549 cells by regulating RAD51 gene. METHODS ：Human lung cancer cisplatin-resistant cells A 549/DDP were selected and divided into control group（blank culture medium ），cisplatin group （5 g/L），apigenin low-dose and high-dose groups （10，20 μmol/L）. MTT assay was used to detect the growth of A 549/DDP cells ，while the Annexin Ⅴ/PI double staining combined with flow cytometry were used to detect the apoptosis. A 549/DDP cells were collected and divided into cisplatin alone group （1，2，4，8，16 μg/mL）， apigenin combination group （10 μmol/L，based on cisplatin ）. MTT method was used to determine and calculate inhibitory rate of cell proliferation. IC 50 values of drugs were calculated by regression model ，and reversion index of apigenin was calculated. 18 nude mice were randomly divided into control group ，cisplatin alone group and apigenin combination group ，with 6 mice in each group. After A 549/DDP cells were inoculated to form the transplanted tumor ，normal saline ，cisplatin（2 mg/kg，once every other day ）， cisplatin（the same dosage and usage ）+apigenin solution （30 mg/kg，once a day ）were injected intraperitoneally respectively. After 18 days of continuous administration ，the body weight of mice and the mass of the transplanted tumor were detected and the tumor inhibition rate was calculated. Human lung cancer cells A 549 and A 549/DDP were collected and divided into normal group （A549 cells），control group （A549/DDP cells ），cisplatin group （5 g/L，A549/DDP cells ）and apigenin low-dose and high-dose groups （10，20 μmol/L，A549/DDP cells ），respectively. mRNA and protein expression of RAD51 were detected by real-time fluorescence quantitative PCR and Western blotting assay. RESULTS ： Δ 基金项目：四川省教育厅（自筹经费）科研项目（No.12ZB227） ＊讲师，硕士。研究方向：肿瘤病理。E-mail：molin212@163.com Compared with control group ，the cell growth of apigenin # 通信作者：副教授，硕士生导师，硕士。研究方向：肿瘤病理。 low-dose and high-dose groups were decreased significantly ， E-mail：xinrongH292@163.com apoptosis rates of them w ere increased significantly and higher ·708· China Pharmacy 2020Vol. 31 No. 6 中国药房 2020年第31卷第6期 than those of cisplat in group （P＜0.05 or P＜0.01）. After combined with apigenin ，proliferation inhibition rate of A 549/DDP cells was increased significantly ，compared with cisplatin alone group with the same concentration （P＜0.05）. The IC 50 in the apigenin combination group was （5.81±0.47）μg/mL，significantly lower than （14.44±0.52）μg/mL in cisplatin alone group（P＜0.05），and reversal index of apigenin was 2.49. The results of nude mice tumor inhibition experiment showed that after combined with apigenin，tumor inhibition rate of A 549/DDP bearing nude mice was 68.72％，significantly lower than 33.82％ in cisplatin alone group. Compared with A 549 cells of normal group ，relative expression of RAD51 mRNA and protein were increased significantly in A549/DDP cells of control group （P＜0.05）. Compared with A 549/DDP cells of control group ，relative expression of RAD51 mRNA and protein in A 549/DDP cells were decreased significantly in apigenin low-dose and high-dose groups （P＜0.05）. Compared with cisplatin group ，relative expression of RAD51 mRNA and protein in A 549/DDP cells of apigenin low-dose and high-dose group were decreased significantly （P＜0.05）. CONCLUSIONS ：Apigenin can effectively reverse drug resistance of cisplatin-resistant A 549/DDP cells in human lung cancer. The mechanism may be related to the reduction of RAD51 gene transcription and protein expression.
RESUMO
A new optimized experimental designing method in biomedical engineering study is provided in this paper. The characteristic of the uniform design and orthogonal design was compared. Then, a new experimental design was proposed, which was the combined use of the two experimental designs. Discussed the theoretical basis, using method and its advantages. Furthermore, we proved the validity through our experiment. This method has the specificity of uniform design, fewer times of experiment and suit for experiment with multi-factors and multi-levels. This makes full use of the advantages of orhtogonal design which is widely used and can be analyzed by simple intuitionist analysis, avoids the disadvantages of uniform design in which data can only be processed by computer software. It can be widely used in the research and development of biomedicine engineering.