| Objective: Primary liver cancer (PLC) is one of the commonest malignant diseases in our country.The mortality rate is the second of all diseases.Chinese traditional medicine(TCM) is important in treatments of PLC.According to the theory of TCM and clinical study over ten years, professor YaoShukun had developed Qinggan Huayu recipe with the therapeutic principles of clearing away heat and toxic material,removing blood stasis and resolving lumps,strengthening the spleen and replenishing qi. Researches had proved that Qinggan Huayu recipe had obvious repressive effect on the genesis of PLC.The mechanisms of oncogene,antioncogene, anti-angiogenesis,apoptosis,cell cycle and immune function on Qinggan Huayu recipe had been investigated which showed the mult-targets and integral regulation of TCM.Chinese herbs serum pharmacology is a new method of in vitro experiments.Compared with using Chinese medicine directly in vitro experiments,Chinese herbs serum pharmacology is excellent in controling in vitro experimental conditions,detecting drug effects and revealing the mechanisim of drug in depth.It is an important way for pharmacodymic study of TCM.Gene chip technology is able to detection the expression of thousands of genes and their interactions in the whole genome-wide of the sample simultaneously. It reveals the gene network interactions.Gene chip technology is fast,parallel,efficient, high-throughput in biological information analysis.It is a new,credible method in Chinese medicine research to reveals mult-targets and integral regulation.Both serapharmacology and gene chip technology were used in this resezrch.Serum was taken from PLC patients withou drug and those took Qinggan Huayu Recipe for 6 weeks . The serum was pretreated by heat or acetone indepandently.For the purpose of finding anti-tumor genes and exploring anti-tumor mechanism of Qinggan Huayu recipe in genomics,32050 chips were used to detect differental expression of genes in the whole genome-wide of SMMC-7721 cells cultured by pretreated serum for 48 hours.Methods:1 SMMC-7721 cells cultured in vitro.SMMC-7721 were cultured in DMEM medium contained 10%calf serum , penicillin G 100U/ml , streptomycin 100U/ml.All cells were cultured at 37℃in 5% CO2 cell incubator,and were passaged every 3 to 4 days.Taked cells in logarithmic growth phase for the experiments.2 Pretreatments for serum from primary liver cancer patients before drug and those took Qinggan Huayu Recipe for 6 weeks.(1)heat treatment:The serum was placed in the 56oC constant temperature water bath for 30 min.(2) The serum was joined four times acetone, well mixing, centrifuged at 3000r/min for the supernatants and then acetone was evaporated by warm bath as pretreatment.3 RNA extraction.Cells cultured in DMEM medium contained 1% calf serum,15% serum pretreatmented for 48hours.Then removed the medium, added 0.25%trypsin and centrifuged to collected cells into EP without Rnase.Added Trizol 1ml into every EP to extract RNA,well mixing, preserved in -70oC.4 Gene chip analysis.Sent cell RNA to shanghai kangcheng biological company for gene chip analysis.The operation flow were conducted by kangcheng biological company,then worked for further data processing and analysis.Result:1 Differentially expressed genes.(1) acetone vs heat treatment. Differentially expressed genes were 3250 in cells cultured by before drug serum.The number of genes up regulated two and more than two times were 1555,accounting for 4.85% of the total genes.The number of genes down regulated two and more than two times were 1695,accounting for the total number of genes 5.29%.Differentially expressed genes were 2832 in cells cultured by after drug serum.The number of genes up regulated two and more than two times were 1395,accounting for 4.35% of the total enes.The number of genes down regulated two and more than two times were1428, accounting for the total number of genes 4.46%.(2)After drug vs before drug.Differentially expressed genes were 2880 in cells cultured by teatment serum.The number of genes up regulated two and more than two times were 1487,accounting for 4.64% of the total genes, the number of genes down regulated two and more than two times were 1393, accounting for the total number of genes 4.35%.Differentially expressed genes were 2314 in cells cultured by acetone serum.The number of genes up regulated two and more than two times were 1128,accounting for 3.52% of the total genes.The number of genes down regulated two and more than two times were 1186,accounting for the total number of genes 3.70%.2 According to GO and KEGG pathway analysis,differentially expressed genes were classified by gene functions.(1) acetone pretreatment vs heat pretreatment.Up-regulated genes were mainly about cell cycle,signaling pathway and cell apoptosis in after drug cells.Down-regulated genes were mainly about substances and energy metabolism,cell proliferation and cytokine in after drug cells.(2) After drug serum vs before drug serum.Up-regulated genes were mainly relateded to cell cycle,cell apoptosis,antioncogene and immune function in heat treatment cells.Down-regulated genes were mainly involoved in oncogene,cell proliferation,material and energy metabolism and cell grouth in heat treatment cells.3 Compared with before drug serum,significantly up-regulated genes were PDCD8,TGFB1,MAP4K3, BTG1,CDKN3, OVOL1.Significantly down- regulated genes were IRAK2,RHOA,DEAF1,IGFBP2,BAD,which were related to cell proliferation,cell apoptosis,signaling pathway and metabolism in cells cultured by after drug serum.Conclusions: 1 Compared with before drug serum,serum from PLC patients who took Qinggan Huayu Recipe for 6 weeks could affect gene expression of SMMC-7721 cells.The drug serum regulated genes expression of cell proliferation,signaling pathway,cell cycle,cell apoptosis and immune function.It indicated mult-targets inhabithion to SMMC-7721 cells of Qinggan Huayu Recipe.More works are need to study genes of OVOL1,BTG1,CDNK3,etal.to make sure their roles in antitumor effects of Qinggan Huayu recipe.2 Compared with acetone pretreatment serum,heat preteatment serum affected gene expression of SMMC-7721 cell was obvious with more differentially expressed genes which related to inhibiton of cell proliferation.The probable reason was that acetone made drug less in serum. |
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