| Objective:Primary hepatocellular carcinoma is one of the malignant tumors common in our country and the second leading malignant tumor accounting for death.It leads to the death of over 20 million people every year,which accounts for about more than half of the death toll caused by liver carcinoma in the world.Although the treatment of the tumor in China has made great progress,the main therapeutic methods remains operation, chemotherapy,radiotherapy and biological therapy etc,and the tumor thermotherapy(Hyperthermia) is appearing as the fifth kind of tumor therapeutic method following above-mentioned methods.With the further investigation and improvement of thermotherapy technology,thermotherapy has already played an important role in the synthesis therapy and become an important adjuvant therapy method of radiotherapy and chemotherapy,but the lagging theoretical research has seriously affected the further clinical practice of tumor hyperthermia.Thermal sensitivity of different types of tumor cells and its possible molecular mechanisms are worthy of study and explore.The occurrence and development of malignant tumors is practically a process of excessive cell proliferation and inhibited cell apoptosis.Inducing the cell apoptosis has become a new hot spot of tumor treatment that many scholars pay close attention to.In this experiment,human hepatocellular carcinoma Hep-G2 cells as the research object,research further the molecular biological mechanism of heating inducing cell apoptosis,in order to provide more theoretical evidence and reference for formulate the program of clinical treatment with hyperthermia.Methods: 1 Human hepatocellular carcinoma cells line Hep-G2 was chosen as the trail material.The heating temperature was 43℃,heating time for 0 h,0.5 h,1 h,1.5 h,the heating container was incubator.2 Experimental sub-groups:use of randomized principle,the control group that is heating 0 h group(normal cultured HepG2 cells) and hyperthermia groups(time by hyperthermia were divided into 3 groups:heating 0.5 h, heating 1 h,heating 1.5 h)3 After cells heating,control group and hyperthermia groups continued to cultivate them for 48 h,72 h,96 h,120 h,144 h,counted the number of survival cells under the light microscope to draw cell growth curve.4 After cells heating,control group and hyperthermia groups continued to cultivate them for 0 h,3 h,6 h,12 h,24 h,used AnnexinV-EGFP to detect the percentage of apoptotic cells by flow cytometry(FCM).Before FCM, observed hyperthermic cell growth conditions and the morphological changes under inverted microscope and photograph.5 After cells heating,control group and hyperthermia groups continued to cultivate them for 0 h,3 h,6 h,12 h,24 h,the protein expression of cell apoptosis associated genes Bax,Bcl-2 and p53 was examined by S-P immunohistochemistry technique.6 After heating 1.5 h cultivated 6 h,collected heating cells and control group cells to observe changes in cell ultrastructure under transmission electron microscope.7 With SPSS11.5 statistical software package,using factorial design analysis of variance statistics,compared with the control group,P＜0.05 were considered statistically significant.Results:1 The effect of hyperthermia on cell growth curveAfter 43℃water-bath heating 0.5 h,1 h,1.5 h at different culture time, the average cell number decreased,compared with control group differences were significant(P＜0.05,P＜0.01).It showed that thermotherapy could restrain cell growth.With the extension of heating time,the number of survival cells decreased the most obviously in heating 1.5 h group.2 The effect of hyperthermia on the percentage of apoptotic cellsAfter the Hep-G2 cells 43℃water-bath heating different times at different culture time,the flow cytometry showed that,with the extension of heating time,the percentage of apoptotic cells had an increasing trend, At the same culture time,heating 1 h group and heating 1.5 h group were compared with the control group separately,the differences were statistically significant(P＜0.01).The control group at different culture time,the percentage of apoptotic cells had no significant difference(P＞0.05).With the culture time extended,the percentage of apoptotic cells of hyperthermia groups had an increasing trend,it reached a peak at continuing to culture 6 h,and then gradually decreased.At the same heating time,culture 6 h group compared with other culture groups,there were significant differences(P＜0.05,P＜0.01).The percentage of apoptotic cells reached maximum 32.83%at heating 1.5 h cultivate 6 h,the percentage of apoptotic cells had the interaction between the heating time and culture time(Fheating*culture=5.20,P=0.00).3 The effect of hyperthermia on the expression of the 3 proteinThe immunohistochemistry results indicated that the levels of Bax and p53 proteins increased in hyperthermia groups compared with the control group,the differences had the statistical significance(P＜0.05,P＜0.01), The control group at different culture time,the expression rates of Bax and p53 proteins had no significant difference(P＞0.05).with the culture time extended,expression rates of Bax and p53 proteins of hyperthermia groups had increasing trend,while continuing to culture 6 h,it reached a peak,and then gradually reduced.The expression of Bax and p53 proteins had the interaction between the heating time and culture time(Fheating*culture= 12.55,P=0.00;Fheating*culture=3.642,P=0.00).After Heating 1.5 h cultivate 6 h,expression rates of Bax and p53 proteins reached respectively maximum 70.00%and 84.00%.The expressions of Bcl-2 proteins in heating and control groups were negative.4 The effect of hyperthermia on cell morphologyUnder inverted microscope observation,in the control group,Hep-G2 cells adherented well,showing fusiform,polygonal,sizes,cell morphology integrity,large nucleus,nucleolus obvious.43℃water bath in each group, growth status changed,cell shrinkage,round,showing float-like,nuclear lectin,cytoplasm was concentrated bubbly,nucleoli disappeared,and some membrane integrity,some cell swelling,membrane damaged,continuity interrupted,organelles ruptured.Under transmission electron microscope observation,in the control group,the cells showed that the normal morphology,boundary clear, nuclear large and round,nucleolus and nuclear membrane were clearly visibled,rich euchromatin,less heterochromatin.In heating group,cells surface microvilli disappeared,nuclear chromatin pyknosis and margination,showing crescent-shaped,wrinkled nuclear membrane,dense cytoplasm,organelles concentration,irregular or nucleolar fragmentation, there was much vacuolated cytoplasm.From the electron microscope observation,we could see the changes of cells apoptotic morphological.Conclusion:Heating Hep-G2 cells with 43℃water-bath can induce them into apoptosis and the mechanism may be realized by the promoting the expression of Bax and p53 proteins. |
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