| Currently,Lung cancer is one of the high morbidity and mortality malignant tumor worldwide, and the incidence and mortality of lung cancer are increasing year by year.It has become the leading cause of cancer death in urban population in China. About70%patients of primary lung cancer need to be accepted radiotherapy, but the hypoxic cell carcinoma and resistance to radiation damage, which it partly limit the use of radiotherapy.Not only radiation sensitizer improve tumor sensitivity to radiation and the level of local control greatly, but also it can reduce recurrence and metastasis of the local cancer, so studying of radiosensitizer have an important and far-reaching significance.RNA interference is a gene silencing phenomenon after specific transcription,which induced by double-strand RNA of homologous to the target gene.It has the advantages characteristics of high efficiency, high specificity and high selectivity to the target gene,could effectively inhibit expression of specific gene, and the effect is close to the gene knockout technology,which has been widely used in the study on the pathogenesis and tumor gene therapy.From the previous results,we concluded that Fuzheng Zengxiao formula has the basic conditions of radiosensitizer, the main protein of the radiation sensitization is S100A9, CyPA, However, this is only a preliminary qualitative study, rather than quantitative research,Which specific genes play a major role is unclear,which it is need validation and analysis radiation sensitization of traditional Chinese medicine to two genes in q-PCR, Western-blot technology and RNA interference.Objective:To evaluate the role and status of S100A9, and CyPA in Fuzheng Zengxiao formula increasing radiotherapy sensitivity of lung cancer.The present project was supported by National Natural Science Foundation of China (NO.81072925),Using RNAi technology to verify the radiosensitizing target-S100A9and CyPA, which it is importance to determine predictive factors of radiosensitivity and research of radiosensitizing drugs.Method:Establishment the model of transplanted human lung adenocarcinoma in nude mice, then divided into4groups, including blank group, Chinese medicine group, radiation group, Chinese medicine+radiation group, when volume of the tumor reach1cm3, we will give10Gy dose irradiation for radiation group and Chinese medicine+radiation group.Observe tumor inhibition rate;identification of protein expression by Western.Detection mRNA expression of S100A9and CyPA by real-time fluorescent quantitative RT-PCR. The silence of S100A9and CyPA in lung adenocarcinoma by gene knockdown Lentivirus-mediat ed,selecting effective interference sequence target of S100A9and CyPA by Western.Itis divided into blank group, negative control group, RNAi S100A9group, and RNAi CyPA group after Interference,then calculating the clone forming rate and survival fraction by colony forming experiment after irradiation,Evaluating cell radiation sensitivity of RNAi silencing the S100A9, CyPA in lung cancer by Click multiple target model, and cell cycle analyzed by flow cytometry.Result:Fuzheng Zengxiao formula has antitumor effects,which compared with the control group, Chinese medicine group,and radiation group,Tumor inhibition rate of traditional Chinese medicine+radiation group was striking increased.Results of Western blot showed:at6h after radiotherapy, that compared with control group, protein expression of S100A9and CyPA were decreased in each group, but no statistical difference.at12h after radiotherapy, Compared with the control group,expression of Chinese medicine+radiation group were decreased, and there were statistical significant difference(P<0.05).at24h after radiotherapy,The protein expression of S100A9,compared with the control group,radiation group was decreased,and there were statistical significant difference(P<0.05).To the other groups,expression of Chinese medicine+radiation group are decreased,there are significant.Co mpared with the control group,the protein expression of CyPA in the Chinese medicine group and radiation group was decreased,and there were statistical significant differences(P<0.05).T o the other groups,The protein expression of CyPA in Chinese medicine+radiation group were decreased,and there were statistical significant difference.Results of Real-time quantitative PCR show:at6h after radiotherapy,compared with control group,mRNA expression of S100A9and CyPA were decreased in the other group, but there were no difference. At12h after radiotherapy,Compared with the control group,mRNA expression of S100A9of radiation group were decreased,and there were statistical significant difference(P<0.05).Compared with the control group and Chinese medicine,Chinese medicine+radiation group were decreased,th ere are statistical significant difference (P<0.05).mRNA expression of CyPA,Compared with the control group,radiation group and Chinese medicine+radiation group are decreased,there are statistical significant difference(P<0.05).at24h after radiotherapy, mRNA expression of S100A9and CyPA, Compared with the control group、Chinese medicine,radiation group are decreased,there are statistical significant difference(P<0.05).To the other groups,expression of Chinese medicine+radiation group were decreased,there were statistical significant difference (P<0.05).Furthe, Construction of the lentiviral expression plasmid S100A9and CyPA RNAi gene and packing lentiviral.Select effective interference target sequence of S100A9and CyPA RNAi by Western blot,and stably transfected cells of S100A9and CyPA shRNA/PAa were prepared.It calculated the clone forming rate and survival fraction by the colony forming experiment,Using click multiple target model,it find that reduce SF2Gy value, Do value, Dq value of tumor cell after RNAi (S100A9,cyclophilin A),Increasing the radiation effect and enhance cytotoxic effects of radiation on tumour cells.The results of flow cytometry,after RNAi silencing S100A9and CyPA,without radiotherapy,Go/G1,S phase incells of every Groups had difference,but there were no statistical difference.G2/M phase in cells, Compared with the blank group, the S100A9group increased,there were statistical significant difference(P<0.05).Compared with the blank group and control group,CyPA group were increased,there were significant difference(P<0.05).After2Gy radiotherapy,Compared with the blank group, control group, G0/G1cells of S100A9and CyPA group were decreased,G2/M cells are statistical significanly increased(P<0.05).S cells of S100A9group were decreased,but no statistical difference.S cells of CyPA group were significantly decreased(P<0.05).Conclusion:The experiment demonstrated that Fuzheng Zengxiao formula had certein damps the lump rate and better radiosensitizing effect on lung adenocarcinoma.Detection the expression of S100A9and CyPA by Western blot and q-PCR,it discoved that Fuzheng Zengxiao formula played radiosensitizing effect by down-regulating expression of S100A9and CyPA in lung adenocarcinoma.24h after radiotherapy,the express was significantly decreased,and there were statistical significant difference(P<0.05).Further,Construction of the lentiviral expression plasmid S100A9and CyPA RNAi gene,select effective interference target sequence by Western blot, and stably transfected cells of S100A9and CyPA shRNA/PAa were prepared.In vitro experiment,S100A9and CyPA siRNA mediated Lentivirus can improve ratio of G2/M phase in cell,and increase radiation sensitivity of cell. |
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