| Background: According to the analysis of WHO, cancer remains one of the leading causes of death throughout the world. The rat glial tumor (C6) cell is a kind of frequent malignant cancer which morbidity has increased obviously and the survival time of cured patients is about one year. Presently lots of therapeutic agents inhibit the proliferation of tumor cells through affecting their cell cycle and inducing apoptosis, while sensitizing agents can enhance these effects and reduce its toxicity by different manner. So, this kind of research can provide some therotic basis to tumor biology and cancer chemotherapy.Objectives: To investigate the growth inhibition of C6 cell line in vitro in treatment of LPS ,CCDP or LPS+CCDP in order to get more knowledge about the mechenism of how these therapeutic agents act on C6 and provide some cell-level analysis for the therapy of the cancer.Effect of LPS+CDDP on cell proliferation: Morphological changes were observed under phase contrast microscope. Viable cells were determined by cell counting assay, using a hemocytometer.Affection of C6 apoptosis: Membrane blebbing and apoptotic bodies was observed by morphologic assay using fluorescence microscope. The nucleus Changes were detected using H33258 staining through which we could observe the typical phenomena of apoptosis. The integrality of membrane was examined by LDH assay.Determination of p53 protein in C6: The expression of p53 was determined by immunofluorescence assay using laser scanning confocal microscope. Detection of the activity of ROS in C6: After treated with different drugs, C6 was marked by ROS specificity fluorescence probe DCFH-DA, it's content of ROS was determined by colorimetric assay.Cell cycle analysis: Flow cytometry was used to detect the changes about the cell cycle of C6 after treated with different drugs. Results:1. Observed by phase microscope, the configuration of C6 had not visible changes after being treated with LPS or CDDP only. But when two drugs were used together, the number and shape of C6 was decreased obviously and apoptosis or necrosis was observed. When treated with CDDP alone or LPS+CDDP, the characteristic of apoptosis was presented such as cell prominency tailed off, particle and vacuole increased and cell necleus largened.2. The results of trypan blue dyeing assay showed that the percentage inhibition of C6 after 24h treated with different drugs is 19% (LPS), 27.8% (CDDP) and 46.5% (LPS+CDDP) respectively. The percentage inhibition of C6 after 24h treated with different drugs is 54.4% (LPS), 60.2% (CDDP) and 72% (LPS+CDDP), respectively. Combination of two agents can inhibit the growth of C6 more effectively. All groups present the time and dosage dependent3. Observed by the fluorescence microscope, the phenomenon of apoptosis is not obvious when LPS is used alone, but parts of cells take on apoptosis or necrosis when used CDDP. With the treatment of LPS+CDDP, the main form of cell death regards apoptosis as principle. The chromatin in apoptic cell nucleus assemble and turn to the edge, or break into crumb's section.The C6 cell treated with LPS after 48h ,its death form is apoptosis.4. We measurated the content of LDH in the culture liquid and found that: The content of LDH with the treatment of LPS+CDDP is low, compared with CDDP alone, but the difference is not obvious (P>0.05)5. The result of the activity test of ROS: The activity of ROS is lower in LPS and CDDP alone section than in LPS+CDDP section, but there is no obvious difference (P>0.05).6. The immune fluorescence dying test of p53 reveals: The p53 expresses the quantity to compares with the control group, LPS group was not different (P>0.05), while CDDP group has obvious difference (P<0.05). Compared to sole agent, the p53 expression quantity of two agents group increases and difference is remarkable (P>0.05).7. The cell cycle of C6 under different circumstance was examined by FACs: Compared with the control group , the cell cycle is arrested in the G2 phase with CCDP, the cell cycle was arrested in the G1 phase with LPS, combined of LPS and CDDP the cell cycle of C6 was arrested in the S phase,Conclusion:1. Compared to one agent group, the C6 of LPS+CDDP group obviously enhanced the C6 lethality.2. CDDP could cause C6 not only apoptosis, but also could kill tumor cells through other paths such as necrosis, when it was in combination with LPS, but there was no obvious difference (P>0.05).3. The result of the activity test of the ROS shows: This enzyme in all group have enhanced and the synergetic effect of LPS and CDDP has less relation.4. LPS and CDDP jointly to C6 inhibitory action is related with p53 protein expression upward.5. LPS arrest the cell cycle of C6 in the Gl phase, CDDP arrested the cell cycle of C6 in the G2 phase, the union agents hinder the cell cycle of C6 in S phase. |
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