| Part 1 Experimental Studies on Induction of Apoptosis of Human Osteosarcoma Cells by Kappa-SelenocarrageenanObjective To observe the effects of inhibition of tumor growth and induction of apoptosis of human osteosarcoma cells by Kappa-Selenocarrageenan in vitro and in vivo. Methods 1. The human osteosarcoma cells OS-732 were inoculated into the 96-well-plates with intervention of three different concentrations of Kappa-Selenocarrageenan (30,60,120μg/ml). MTT assay were performed after 1,2,4,6d respectively, and then we drew the growth curves and calculate cell inhibition rate. The cell inhibition rate of Kappa-Selenocarrageenan was compared with that of Carrageenan.2. Morphological studies included transmission electron microscope observation and acridine orange (AO) and ethidium bromide (EB) double fluorescent staining of osteosarcoma cells affected by Kappa-Selenocarrageenan to detect early and late apoptotic cells and calculate apoptotic rate.Flow cytometer (FCM) with PI staining was applied to determine cell apoptotic rate and cell cycle of OS-732 after medical intervention.DNA of cells affected by Kappa-Selenocarrageenan of three different concentration gradients were extract for agarose electrophoresis to observe DNA ladder.Twenty-one nude mice were inoculated subcutaneously with OS-732 cells, randomized to three groups. They were administered with saline,low- selenium (4mg/kg/d)and high- selenium(40mg/kg/d)via abdominal cavity respectively for 10d. The mice were executed after fed for 6 weeks, then the tumor weigh and volume were measured and the tumor inhibition rate was calculated. The 3- end- labeling method of DNA (Tunel) techniques was used to detect apoptotic index (AI) of various groups of tumor tissue, and differences were analyses statistically. Results1. Growth of OS-732 cells were obviously inhibited after intervention of Kappa-Selenocarrageenan for 1,2,4,6d, with evidence of decent of growth curves. There were significant difference between intervention groups and control. The tumor growth inhibition rates were elevated with increase of medical concentration and affecting time. The tumor growth inhibition rates of Kappa-Selenocarrageenan were much higher than those of Carrageenan.2. The AO,EB double fluorescent staining showed early apoptotic cells when osteosarcoma cells affected by 30 μg/ml Kappa-Selenocarrageenan for 24h, and the early apoptotic cells increased, as well the late apoptotic cells apperaed with enhance of affecting concentration and time. Affected by 120μg/ml Kappa-Selenocarrageenan for 48h,both of the early and late apoptotic cells were observed through transmission electron microscope.3. FCM detection revealed the hypodiploid cusp appeared before G1 cusp when cells affected with 30μg/ml Kappa-Selenocarrageenan for 12h,and tumor cell apoptotic rate was 6.82%. Apoptotic rates of 60μg/ml and 120μg/ml groups were 13.26% and 17.86% respectively, and that was elevated with increase of medical concentration and affecting time. Cell cycle analysis showed that the proportion of S phase in cells of intervention groups increased while that of G2～M phase decreased markedly, which demonstrated cells were blocked in S phase.the DNA ladders presented through gel electrophoresis when OS-732 cells were affected with 30,60,120μg/ml Kappa-Selenocarrageenan for 24h.Two weeks after nude mice inoculated with OS-732 cells, subcutaneous tumors occurred. At the 6th week the volume and weigh of tumors among three groups differed distinctly, and the tumor inhibition rate among three groups differed statistically (P<0.01). The TUNEL staining assay revealed AI of high-selenium and low-selenium were significant higher than that of control group. Part 2 Experimental Studty on Inhibition of Metastasis of Human Osteosarcoma Cells by Kappa-Selenocarrageenan Objective Try to investigate the impact of Kappa-Selenocarrageenan on the chemotaxic migration and the invasion of human osteosarcoma cells in vitro and the inhibition effect on the...
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