| Malignant tumor is a major public health problem in the world,which is an incontrollable proliferation and death disorder disease,the mechanism of cancer cell apoptosis disorder plays an important role in developing of the cancer.The key point of prevention of the cancer is to suppress cancer cell proliferation and accelerat the apotosis.A number of chemotherapy agents have offered significant advantages in the treatment of cancers.However,their effects are often considered ineffective or excessively toxic.Therefore,highly effective and lowly toxic drugs are urgently required.Aspongopus chinensis Dallas has been used as a traditional Chinese medicine,which has good clinical grounding in anti-tumor in china.However,whether haemolymph from A.chinensis can induce tumor cell apoptosis and its apoptosis mechanism of tumor cells is not clear yet.How to find out its anti-tumor mechanism and apply it correctly to the treatment of malignant tumor for perfect clinical effect is what we should do.Based on the previous laboratorial results,the present study is further proved that haemolymph from A.chinensis can induce tumor cell apoptosis,and its mechanisms of antitumor activity and the molecular mechanism on apoptosis of tumor cells are also investigated,for the purpose of providing a scientific basis for the exploitation of highly effective and lowly toxic drugs.In this paper,Bradford method was used to investigate the concentrations of A.chinensis haemolymph protein.In addition,CCK-8 method was used to detect the anti-tumor activity in vitro.Apoptosis was detected by ELISA.The mechanism of haemolymph from A.chinensis was performed by RT-qPCR and western blot with extracting the total protein.The main results were as follows:1?The protein concentration of haemolymph from A.chinensis were detected by Bradford method.The standard curve of protein concentrationsY = 1.2348X+0.6240,r = 0.9827(n=7)as an indicator,calculate the content of haemolymph protein from A.chinensis: 12.2611 mg/L.2 ? The OD value was measured by CCK-8 method.Haemolymph from A.chinensis could inhibit the proliferation of SGC-7901 ?MCF-7 cells and influence their survival,and with a dose-and time-dependent manner at the concentrations of0.82?4.09?8.17?12.26 mg/L.And the ELISA experiment proved that after 24-hour treatments of different concentrations haemolymph(2.45?4.90?7.36?9.81?12.26mg/L)the contents of histone-DNA fragments in two cell lines of SGC-7901?MCF-7cells were significantly higher than that in the blank control group.It was revealed that haemolymph from A.chinensis could induce apoptosis of SGC-7901?MCF-7cells.3 ? The results of western-blot showed that the expression of Caspase-3,Caspase-9 and Bax protein in SGC-7901 and MCF-7 cells was significantly higher than that in the control group(P <0.05).The expression of Bcl-2 protein was significantly down-regulated in the two cells treated with haemolymph from A.chinensis.The results indicated that the two cells induced by haemolymph from A.chinensis may cause irreversible apoptosis of the cells by triggering the endogenous signaling pathway.4?RT-qPCR was consistent with the results of Caspase-3,Caspase-9,Bax and Bcl-2 in western-blot.MCF-7 cells and SGC-7901 cells induced by the haemolymph of A.chinensis may cause irreversible apoptosis of cancer cells by triggering endogenous signal pathway.In this study,we cleared primarily the anti-tumor mechanism of A.chinensis haemolymph,and analyzed its relevant gene and protein expression at mRNA and protein levels.The results are in favour of the development and utilization of A.chinensis,Besides,the results would provide basises and materiasl for developing safe and effective new anti-medicines,which have proprietary intellectual property rights.The project can also provide reference for the research and development of other medicinal insect resources. |
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