| In our country, oral and maxillofacial malignant tumor cancer as the most common, with squamous cell carcinoma is the most rare, and among them generally accounted for more than 80%. Squamous cell carcinoma occurs in adults aged 40 to 60and more men than women. The most common with tongue cancer among them. Tongue cancer with fast growth, high malignant degree, the characteristics of invasive. For tongue cancer treatment is mainly surgery, radiotherapy, chemical therapy integrated sequence of treatment, but the long-term observation of patients with 5 years of survival rate is still low, so explore ways to positive and effective prevention and treatment to improve the curative effect of treatment of tongue cancer, reduce the local recurrence and distant metastasis, and optimize the patients quality of life is very urgent and necessary.In recent years, from the natural plant to find has antitumor effect of bioactive substances prevention and treatment of malignant tumor has become a hot spot of research at home and abroad at present. Studies have shown that the widespread in the plant flavonoids have good antitumor effect, inhibit tumor cell proliferation, invasion and metastasis and prevent new blood vessels to form is the main way of antitumor effect.This research topic with liquorice (Liquiritigenin, LQ) as the research object. Research has shown that licorice has spasmolysis, antibacterial, anti-inflammatory, anti-tumor, antiulcer effect. A study shows licorice root element can effectively inhibit the proliferation and apoptosis of cervical cancer Hela and licorice can inhibit the activity of N-acetyl transferase, thus inhibiting the growth of human colon cancer cells SW620. An animal experiment showed that Licorice root element can not only inhibit the growth of liver cancer H22 cells transplanted tumor in mice and the ability to induce apoptosis of transplanted tumor of cervical cancer Hela also can inhibit the nude mice transplantation tumor growth and angiogenesis.Based on the above research, this subject adopts the culture Cal-27 people tongue cancer cells in vitro with different concentrations of licorice role at different times to observe the inhibition of Cal-27 people tongue cancer cell proliferation and apoptosis. This experiment introduced in clinical broad-spectrum antitumor drugs Cisplatin (Cisplatin, CDDP) as positive control group. Through the observation of the licorice essence and cisplatin antitumor effect, expounds the natural plant licorice flavonoids in the antitumor function of the licorice root element, and licorice root element in the treatment of tongue cancer can provide scientific theoretical basis for clinical application.Materials and Methods1 The MaterialCal-27 cell:Ninth People’s Hospital of Shanghai Jiaotong University Oral and Maxillofacial Surgery laboratories.Liquiritigenin:Shanghai Biological Technology Co.he original leaf, purity> 98%(HPLC)Cisplatin:Qilu Pharmaceutical Co., Ltd.2 The Method2.1 Configuration of liquidGlycyrrhizin cultured with DMEM dissolved in dimethyl sulfoxide was configured to 25mmol/L stock solutions diluted before use to the required concentration with DMEM medium. Configuring liquid at 4°C.Cisplatin before use with saline arranged 10μmol/L solution dark place, and diluted to the desired concentration required. Configuring liquid at 4°C.2.2 Cell culture:the human tongue Cal-27 cells were plated using 10% fetal calf serum and penicillin (100U/ml), streptomycin (100μg/ml) in DMEM medium at 37° C, within 5% CO2 humidified incubator culture, according to the adherent cells in 24 to 48 hours passaged 1 to 0.04EDTA and 0.25% trypsin (1:1) mixture of digestion and passage.2.3 MTT colorimetric test:take the logarithm of the growth of human tongue cancer cells into the Cal-27 1.0 × 105/L cell suspension was seeded in 96-well plates, each well 100 μ L medium and cultured 24h. Were divided into three groups:1. glycyrrhizin group (100、200、300、 400、500μmol/L); 2. cisplatin (1、2、4、6、8μmol/L); 3. the control group, an equal DMEM medium amount of processing human tongue Cal-27 cells were treated 24h,48h,72h, the MTT solution was added to each well 20μL 5g/L and then 4h after withdrawing the supernatant and then added to each well 150 μL II dimethyl sulfoxide shaking shake the crystal fully dissolved, placed in each well of a microplate reader to detect the 572nm absorbance value and calculate the rate of cell growth inhibition.2.4 Inverted microscope morphological changes in each group:the human tongue Cal-27 cells were seeded in petri dishes, cells observed in each experimental group (glycyrrhizin group, cisplatin group) morphological changes under inverted microscope, including culture Oh, 24h,48h,72h cell morphology.2.5 FCM:Detect glycyrrhizin group, cisplatin group and the control group in the post-processing 24h,48,72h hours using AnnexinV-FITC and PI double staining marker in living cells by flow cytometry (FCM) detection of apoptosis.3 Statistical AnalysisUse GRAPHPAD PRISM software analysis of variance, correlation analysis and test on human tongue cancer growth inhibition and apoptosis rate Cal-27 cells were analyzed statistically.4 Results:4.1 Glycyrrhizin group and cisplatin group effect occurred after both cell morphology. Blark control cells adherent growth rapid growth was flat polygonal cytoplasm full visible parts near the cell nucleus round. Liquiritigenin Group:nuclei appeared concentrated loose contact between cells cells became circular polygonal structure disappeared. With the increase of drug concentration and dense concentration and increase intracellular free status. Cisplatin group: nuclei appeared concentrated disconnection between cells consistent size and shape round with the increase of drug concentration level cell concentrate increase the free state.4.2 MTT colorimetric assay:glycyrrhizin human tongue Cal-27 cells significantly inhibited the growth of 24h maximum growth inhibition rate of up to 50% glycyrrhizin group (100,200,300,400,500 μmol/L) human tongue Cal-27 cell proliferation inhibition rate increases with increasing concentration with the same trend with positive control cisplatin group. Statistical analysis different concentrations of drug treatment groups at different times in human tongue Cal-27 cell growth inhibition rate different (P<0.05), with increasing doses of glycyrrhizin, cell viability in a dose-response relationship.4.3 Flow Cytometry:control group human tongue Cal-27 cell apoptosis in the presence of a lower rate of apoptosis; glycyrrhizin group (200,400μmol/L) and cisplatin (2,4 μ mol/L) on the human tongue Cal-27 cell apoptosis rate were obvious the two groups with increasing the rate of apoptosis in drug concentration and treatment time increases; liquiritigenin 200μmol/L group apoptosis rate in 2μmol/L cisplatin apoptosis rate of the positive control group. After statistical analysis, the statistical significance between the concentration and time (p<0.05).Conclusion:Glycyrrhizin can significantly inhibit the proliferation of human tongue Cal-27 cells and capable of inducing apoptosis, and in a concentration and time dependent, there is a dose-response relationship. |
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