| Background Currently, liver cancer is the most common malignant tumor, followed by the mortality of lung cancer and stomach cancer after ranking third in the high mortality rate of cancer. According to the statistics, each year, about 100-125 million people died of liver cancer, mainly in Southeast Asia, China, Japan, Singapore, South Korea region. In the recent years, the liver cancer mortality continues to show a sharp upward trend, showing a high incidence of liver cancer in China, the situation is rather grim. And, because of its high malignant and rapid progression, it is known as the "cancer of the King" by scholars. Early liver cancer has no symptom, once the symptoms appear, most are in an advanced stage. Health and quality of life of the patients with different histological type, clinical stage of liver cancer poses a serious threat to the prognosis, whether, there is distant metastasis and other factors. End-stage liver cancer with metastasis and recurrence greatly reduces the quality of life of patients with liver cancer and the survival times. Currently the reason of high mortality to cancer worldwide, mainly because the lack of screening and early diagnosis of cancer with high sensitivity and specificity of biological indicators, the lack of target specific drugs. Up to now, it has been used drugs in clinical for the treatment of cancer, have a great effect on the body damage. Thus, probing effective drugs become a hot topic of research scientists. Biologically active preparations, without its body side effects, attracted the attention of scientists.ObjectThe effect and mechanism of liver cancer against polypeptide.Materials and methods MaterialsCollect 60 cases of liver cancer patients without any treatment since January 2011 to June 2015, the First Affiliated Hospital of Zhengzhou University, aged 45 to 65 years, mean aged 55.0 + 0.3 years. All of them received operations,and all diagnosis were confirmed by postoperative pathologic. The patients received regular radiotherapy or chemotherapy after operation, while these factors weren't considered in the research. 60 cases of liver cancer patients, 18 patients with the cell type of Hep G2, another 20 with the cell type of Bel-7402. The remaining 22 cases of other cell types. Collect 10 cases who were without liver disease as controls. The researchers had informed the patients of the content of the research, and had gained approval.Collect the pathological types of Hep G2, Bel-7402 tissues each 15 cases, 10 cases of normal cell types. Mark the information and preserved in-80 C fridge in 1 hour.Methods?By CCK-8 method, different concentrations of the peptide were acting on Hep G2 cells and Bel-7402 cells,incubated 24 H, 48 H, 72 H, observe the growth inhibition rates of Bel-7402 and Hep G2 cells because of the polypeptide;?Use Immuno fluorescence observe the site of action and different concentrations of peptides on Hep G2 cells and Bel-7402 cells with the changed cell status;? flow cytometry to observe the impact of the polypeptide on tumor cell apoptosis and cell cycle.Results 1 CCK-8 assay1.1 ? = 0.05, with the time of incubation of 24 H, 48 H,72H, accompany with increasing concentrations of polypeptides, the corresponding inhibition rate increased gradually, and with the change concentration of the polypeptide, which corresponds to the different concentration, the inhibition rates difference among them were statistically significant(P<0.05); but between the incubation times 48 H and 72 H, minor differences between the inhibition rate corresponding to the concentration of the polypeptide exist, it is considered that the inhibition rate was time-dependent and the optimal incubation time is 48 H.1.2 ? = 0.05, with the concentrations 0, 0.1mg /ml, 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0 mg/ml, with the increasing incubation times, its corresponding inhibition rates gradually increased, and with the changes of incubation time, which corresponds to the difference, the differences among inhibition rates were statistically significant(P <0.05);, but when the concentration were 1.5mg/ml and 2.0mg /ml, the inhibition rates difference between them small. It is considered that the inhibition rate was concentration-dependent, and the optimal incubation concentration is 1.5mg/ml.2 Immumofluorescence Method2.1 With the polypeptide concentration of 2.0ml / ml treating normal liver cells after 24 H, we did not see the polypeptide in these cells; With the polypeptide concentrations of 1.0mg / ml, 1.5mg / ml, 2.0mg / ml were treated on hepatoma Hep G2 and Bel-7402 cells after 24 H, it shows with increasing concentrations of the drug, the drug in the cells gradually increased, promoting the apoptosis of hepatoma cells, showing a concentration-dependent manner.2.2 1) Hep G2 The significance level ? = 0.10, P(1.0mg / ml) = 0.925, P(1.5mg / ml) = 0.720, P(2.0mg / ml) = 0.944, obey the normal distribution; with the significance level ?= 0.10, check the F-distribution value tables, the statistics of homogeneity of variance test for the F0.10(2,14) = 19.42 <701.654, P = 0.701> 0.1, meeting the requirements of homogeneity of variance. With the T test: by Image- Pro Plus 6. 0 microscopic image analysis system,calculate the mean optical density of each image(AOD), AOD(2.0mg / ml)> AOD(1.5mg / ml)> AOD(1.0 mg / ml) and P < 0.05, so the differences among the AOD to concentration of 1.0mg / ml, 1.5mg / ml, 2.0mg / ml polypeptide treatment were statistically significant. 2) Bel-7402 The significance level ?= 0.10, P(1.0mg / ml) = 0.771, P(1.5mg / ml) = 0.997, P(2.0mg / ml) = 0.159, obey the normal distribution; with the significance level ?= 0.10, check the F-distribution value tables, the statistics of homogeneity of variance test for the F0.10(2,14) = 19.42> 639.062, P = 0.892> 0.1, meeting the requirements of homogeneity of variance.With the T test : by Image Pro Plus 6. 0 microscopic image analysis system,calculate the mean optical density of each image(AOD), AOD(2.0mg / ml)> AOD(1.5mg / ml)> AOD(1.0 mg / ml) and P <0.05, so the differences among the AOD to concentration of 1.0mg / ml, 1.5mg / ml, 2.0mg / ml treat liver cancer Bel-7402 cells, its AOD difference was statistically significant.3 Flow Cytometry3.1 analysis the apoptosis results(1) after the image analysis and calculation,the Hep G2 cell apoptosis in the control group was 3.875%, with the polypeptide of 0.75 mg / ml and 1.25 mg / ml, the apoptosis rates reached 16.15% and 32.45%. Compared with the control group, the difference was statistically significant(P < 0.05). Promping that polypeptide can induce apoptosis in Hep G2 cells.(2) after the image analysis and calculation, the Hep G2 cell apoptosis in the control group was 3.275%, with the polypeptide of 0.75mg/ml and 1.25mg/ml, the apoptosis rates reached 18.75% and 35.075%. Compared with the control group, the difference was statistically significant(P < 0.05). Promping that polypeptide can induce apoptosis in Bel-7402 cells.3.2 Cell Cycle Analysis Results(1) With the polypeptide intervent HepG224 H, through detecting the changes in the cell cycle, analyse its apoptosis characteristics. The results showed that cells in S period of Hep G2 control groups was 15.132%,with the polypeptide concentration of 0.75mg/ ml and 1.25 mg /ml, /each cell cycle period distribution changed obviously, the corresponding S-period cells were increased to 18.292% and 22.144%, the difference was statistically significant(F = 647.466, P < 0.05). With the the cell number of G2 / M period decreased slightly, the difference was statistically significant(F = 235.095, P <0.05). Promping that polypeptide can affect hepatoma Hep G2 cell division cycle, inducing the tumor cell apoptosis, concerned to the polypeptide block the tumor cell S period, and the effect increased with the drug dose.(2) With the polypeptide intervent Hep G224 H, through detecting the changes in the cell cycle, analyse its apoptosis characteristics. The results showed that cells in S period of Hep G2 control groups was 7.305%, with the polypeptide concentration of 0.75 mg /ml and 1.25mg/ml, each cell cycle period distribution changed obviously, the corresponding S-period cells were increased to 16.333% and 20.06%, the difference was statistically significant(F = 932.298, P < 0.05). With the the cell number of G2 / M period decreased slightly, the difference was statistically significant(F = 552.378, P < 0.05). Promping that polypeptide can affect hepatoma Bel-7402 cell division cycle, inducing the tumor cell apoptosis, concerned to the polypeptide block the tumor cell S period, and the effect increased with the drug dose.4 Western blot4.1 HepG2 cells With the increasing concentration of peptide, the expression of protein Bcl-2 and PCNA decreased, the expression of BAX protein gradually increased, and with the change of drug concentration, the differences among groups of protein expression was statistically significant(P < 0.05).Instruct that Bcl-2 family, PCNA family together play a regulatory role in polypeptides inducing apoptosis.4.2 Bel-7402 cell With the increasing concentration of peptide,the expression of protein Bcl-2 and PCNA decreased,the expression of BAX protein gradually increased, and with the change of drug concentration, the difference among groups of protein expression were statistically significant(P < 0.05).Instruct that Bcl-2 family, PCNA family together play a regulatory role in polypeptide's inducing apoptosis.Conclusion1 The polypeptide is a cytotoxic drug, which can inhibit tumor growth, and it is dose-dependent and time-dependent. 2 CCK-8 and Annexinv a FITC / PI double staining results show that polypeptide can inhibit Hep G2 cells and Bel-7402 cell proliferation and induce their apoptosis, block S period, and the effects enhance accompany with the increasing dose and duration. 3 The polypeptide exert its intracellular cytotoxic effect by entering into cells. 4 The polypeptide reduce the expression of protein Bcl-2 and PCNA and increase the expression of protein Bax. |
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