| Objective: Stomach cancer is a commom tumor. Stomach cancer's treatment is by the surgery primarily, simultaneously auxiliary by new auxiliary and the auxiliary treatment.Because diagnoses is late and the treatment result is bad, 5 year survival rate is extremely low. Malignant tumor development, not only as a result of cell's infinite multiplication,but also because the cell interior perishes weakly the (Apoptosis) machine-made barrier or the two loses the balanced result. Recently some scholars reported that the hyperthermia can induce the cell to perish weakly, but some anticancer treatment medicine (for example anthracene ring class) is also precisely perishes weakly through the induction tumour cell realizes it to suppress the tumor growth. Therefore, whether prompts us to use the tepid chemotherapy to enhance tumour cell's weak perishing rate, whether both do have the synergism, what is its mechanism? This research through in vitro experiment discussion hyperthermia killing tumour cell's mechanism and Thermochemotherapy synergism questions and so on mechanism, so as to provid a strong basis for Thermochemotherapy on the clinical application in treating cancer.Methods: 1 MGC803 cell line were maintained in vitro using MTT assay to detect the growth rate among different EPI concentration groups (0.0625,0.125,0.25,0.5,1,2μg/mL) and different time groups (12,24,48h). The working concentration of EPI was defined as its inhibiting concentration 50%(IC50)at 48 hours action against MGC803 which was determined by MTT assay. 2 The cells were divided into four groups: nomorl, chemotherapy, hyperthermia and Thermochemotherapy. The inhibition effect of cell growth were measured with MTT method. 3 Changes of apoptosis ratio after treatment with hyperthermia, chemotherapy and Thermochemotherapy groups were examined by FCM. 4 Changes of the expressions of Caspase3 and Bcl-2 after treatment with hyperthermia, chemotherapy and Thermochemotherapy groups were examined by FCM. 5 Extracting total RNA of each experimental group cell, assessing the integrality and content of RNA, the level of Caspase3 mRNA, Bcl-2 mRNA expression was examined by semiquantitative Reverse transcription polymerase chain reaction(RT-PCR) technique in the MGC803 cells treated with hyperthermia, chemotherapy and Thermochemotherapy groups.Results:1 MTT assay results: Epirubicin inhibited the proliferation of MGC803 cells significantly in dose-and time-dependent manner. 1μg/mL and 2μg/mL EPI group lethal effect was near and enter into platform phage at 37℃on 48 hour. The working concentration of Epirubicin was defined as its inhibiting concentration (IC50) at 48 hours action against MGC803 was 0.49μg/ml and subsequently used in all experiments. 2 The proliferation of MGC803 cells was inhibited in all the other groups, Compared with control group,the inhibiting rate of Thermochemotherapy groups evaluate obviously(P<0.05); Compared with hyperthermia and chemotherapy groups, the inhibiting rate of Thermochemotherapy group evaluate obviously (P<0.05). 3 The apoptosis ratio were detected by FCM: In the picture of FCM histogram apoptotic peak was detected in hyperthermia, chemotherapy and Thermochemotherapy groups. At the same level, the average apoptotic rate of Thermochemotherapy group cells was higher than chemotherapy and hyperthermia groups(P<0.05). 4 The expression of Caspase3, Bcl-2 detected by FCM: The Caspase3 gene protein expression of hyperthermia, chemotherapy and Thermochemotherapy groups was higher than normal group(P<0.05)and the Bcl-2 gene protein expression of those groups was lower than normal group(P<0.05). The effect of increasing Caspase3 protein expression and decreacing Bcl-2 protein expression of Thermochemotherapy group was stronger than hyperthermia and chemotherapy groups(P<0.05). A significant negative response correlation could be found between the expression of caspase3 and Bcl-2 protein(r=-0.990).There was positive response correlation between the apoptosis rate and the expression of caspase3 protein(r=0.891). There is negative response correlation between the apoptosis rate and the expression of Bcl-2 protein(r=-0.924). 5 The mRNA of Caspase3, Bcl-2 detected by RT-PCR:The Caspase3 mRNA expression of hyperthermia, chemotherapy and Thermochemotherapy groups was higher than normal group(P<0.05)and the Bcl-2 mRNA expression of those groups was lower than normal group(P<0.05). The effect of increasing Caspase3 mRNA expression and decreacing Bcl-2 mRNA expression of Thermochemotherapy group was stronger than hyperthermia and chemotherapy groups(P<0.05).Conclusions: 1 We comfirmed that hyperthermia(43℃, 60min) had the capability of inhibiting the proliferation and inducing apoptosis of MGC803 cells in vitro, The apoptosis rates and inhibiting rate of Thermochemotherapy are more signidficant. which proves the synergistic function of inducing apoptosis of Thermochemotherapy. 2 The decreasing expression of mRNA and protein of Bcl-2 are followed by cell apoptosis process induced by hyperthermia, chemotherapy and Thermochemotherapy. The decreasing expression of Bcl-2 of Thermochemotherapy group are more signidficant than those of hyperthermia and chemotherapy groups. It is cued that the decreasing expression of Bcl-2 maybe cause cell apoptosis. 3 The increasing expression of mRNA and protein of Caspase3 are followed by cell apoptosis process induced by hyperthermia, chemotherapy and Thermochemotherapy. The increasing expression of Caspase3 of Thermochemotherapy group are more signidficant than those of hyperthermia and chemotherapy groups. It is cued that the increasing expressions of Caspase3 maybe cause cell apoptosis. 4 A significant negative response correlation could be found between the expression of Caspase3 and Bcl-2 protein. It is cued that the apoptosis induced by the Interaction of Caspase3 and Bcl-2. |
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