青蒿素对人鼻咽癌CNE细胞的放射增敏作用及机制的研究

Objective To investigate the radiosensitizing effects of artemisinin on CNE human nasopharyngeal carcinoma cells in vitro.Methods CNE human nasopharyngeal carcinoma cell line was used in this study.Cell growth kinetics was determined by MTT assay.Effect of the drug on radiosensitivity of CNE cells was analyzed by clonogenic assay.The change of cell cycle was measured by flow cytometry.Results The inhibition of CNE cells growth by artemisinin was increased with concentrations.Artemisinin (1 μmol/L)could enhance the radiosensitizing effects on CNE cell line,and the sensitizing enhancement ratio(SER)was 1.26.Artemisinin abrogated radiation-induced G2/M arrest of the tested CNE cells.Compared with the radiation alone group,the proportion of G2/M phase cells increased in radiation combined with drug group.Conclusions Artemisinin could reduce radiation-induced G2/M arrest and enhance the cytotoxicity of γ-irradiation on the CNE ceils.     

青蒿素对人鼻咽癌CNE细胞的放射增敏作用及机制的研究

Objective To investigate the radiosensitizing effects of artemisinin on CNE human nasopharyngeal carcinoma cells in vitro.Methods CNE human nasopharyngeal carcinoma cell line was used in this study.Cell growth kinetics was determined by MTT assay.Effect of the drug on radiosensitivity of CNE cells was analyzed by clonogenic assay.The change of cell cycle was measured by flow cytometry.Results The inhibition of CNE cells growth by artemisinin was increased with concentrations.Artemisinin (1 μmol/L)could enhance the radiosensitizing effects on CNE cell line,and the sensitizing enhancement ratio(SER)was 1.26.Artemisinin abrogated radiation-induced G2/M arrest of the tested CNE cells.Compared with the radiation alone group,the proportion of G2/M phase cells increased in radiation combined with drug group.Conclusions Artemisinin could reduce radiation-induced G2/M arrest and enhance the cytotoxicity of γ-irradiation on the CNE ceils.     

18F-FDG PET-CT对裸鼠鼻咽癌移植瘤放疗疗效的早期评估及与Ki67表达的相关性研究

Objective To investigate the value of 18F-FDG PET-CT scans on early assessing the therapeutic effects on human nasopharyngeal carcinoma (NPC) xenograft in nude mice after radiotherapy,and preliminarily analyze the relation of 18F-FDG PET-CT scans and the positive expression rate of Ki67, and provide a basis for further clinical studies. Methods Fifteen xenograft nude mice of NPC were randomly divided into three groups (5 nude mice per group). Control group: all mice immediately executed after the 18F-FDG PET-CT scans and taken pathology; 6 Gy radiotherapy group: all mice took 18F-FDG PET-CT scans before and 1 d after receiving 6 Gy irradiation, and were killed immediately to take pathology; 12 Gy radiotherapy group: all mice received 12 Gy irradiation (6 Gy each time, totally 2 times). They took 18F-FDG PET-CT scans before radiotherapy, after receiving the first 6 Gy irradiation, 2 d and 6 d after receiving 12 Gy irradiation respectively ,and then were executed to take pathology after the last scan. The expression of Ki67 in xenografts of different groups was detected and the relation between their changes and T/NT were analyzed.Results ①For 12 Gy radiotherapy group, the average T/NT ratios of the NPC xenograft of pre-radiotherapy, after the first 6 Gy irradiation, 2 d and 6 d after 12 Gy radiotherapy were 2.17±0.31, 1.68±0.42, 1.41±0.40,and 0.70±0.12, respectively. The average T/NT ratios were not statistically difference before and after the first 6 Gy irradiation. The average T/NT ratio of 2 d after radiotherapy decreased significantly than pre-radiotherapy (t=2.80, P<0.05) and after the first 6 Gy irradiation (t=3.14, P<0.05); The average T/NT ratio of 6d after radiotherapy was decreased seriously than 2 d after radiotherapy(t=3.49, P<0.05, compared with preradiotherapy t=8.01, P<0.01). ② The positive expression rates of Ki67 of NPC xenograft markedly decreased after receiving irradiation. There were significant differences in the positive expressions of Ki67 between different group s(F=21.95, P<0.01).The positive expression of Ki67 in the 12 Gy radiotherapy group decreased significantly compared with the control group (t=7.145, P<0.01) and the 6 Gy radiotherapy group (t=2.384, P<0.05). The positive expression of Ki67 in the 6 Gy radiotherapy group was less than the control group, also have statistical significance(t=4.320, P<0.01).③There was no correlation between the T/NT ratio of NPC xenograft and the positive expression of Ki67. ④Positive correlation was found between the variance of T/NT ratio and the variance of the positive expression of Ki67 of NPC xenograft (r=0.532, P<0.05).Conclusions 18F-FDG PET-CT imaging has an important value on early assessing the therapeutic effects of NPC xenograft in nude mice after radiotherapy, and the 6th day after radiotherapy is an appropriate time point.18F-FDG PET-CT imaging can reflect the variance of the positive expression of Ki67 of NPC, and yet indirectly reflect the sensitivity of NPC to radiotheraphy.     

hNIS基因转染介导Hela细胞移植瘤放射性核素显像和治疗的实验研究

Objective To explore the feasibility of imaging and treatment of cervical cancer xenograft model using 131I mediated by hNIS gene transfection. Methods The cervical cancer xenograft models were established with Hela-NIS( +) cells and Hela cells, respectively. Five Hela-NIS( +) xenograft models and five Hela xenograft models were dynamically imaged at 0.5, 1, 2, 4, 8, 16 and 20 h postinjection of 131I(7.4 MBq). Five Hela-NIS( +) xenograft models were imaged at 0. 5,1,2,4,8,16, 20 and 25 h postinjection of 99TcmO4-(11.1 MBq). Twenty Hela-NIS( +) cervical cancer xenograft models were randomly divided into four groups: Three 131I treating groups and one control group. The therapeutic effects of 131I at threelevels (74,111,148 MBq) were investigated following intraperitoneal injection. Results Hela-NIS( +)human cervical cancer xenografts were established successfully in nude mice. The Hela-NIS( +) xenografts significantly accumulated radioactivity after intraperitoneal injection of 131I, and the radioactivity was persistently present until 20 h postinjection, but Hela xenografts had no radioactive accumulation. The T/B value of the Hela-NIS( +) xenografts reached 17.34 at 8 h postinjection. The imaging with 99TcmO4- showed that the radioactivity was persistently present in Hela-NIS( +) xenografts for almost 25 h. The Hela-NIS( +)xenografts shrinked after 131I treatment. The inhibition ratios of tumor growth in 111 MBq and 148 MBq groups were both significantly higher than that of 74 MBq group (t: 2.74-5.75, P ＜0.05). Conclusions Hela-NIS( +) cervical cancer xenografts in nude mice could persistently accumulate 131I and 99TcmO4- and could be treated successfully with 131 I. 131 I treatment mediated by hNIS gene transfection could be a promising cancer treatment method.     

双功能制剂99Tcm-Gd-DTPA-DG的制备及在荷瘤裸鼠体内的生物分布

Objective To evaluate the stability and biodistribution of a novel SPECT-MRI bi-functional agem 99Tcm-Gd-DTPA-DG in tumor-bearing nude mice. Methods DTPA-DG was synthesized and then conjugated with Gd2O3 to generate Gd-DTPA-DG. The tumor-bearing nude mice were scanned by MRI to evaluate the tumor targeting ability of Gd-DTPA-DG. The orthogonal experiment was applied to optimize pH value of reaction medium and reaction temperature. The radiolabeling efficiency was measured by thin layer chromatography. The distribution of 99Tcm-Gd-DTPA-DG in nude mice was evaluated by scintigrapy in vivo. The % ID/g was measured at different time after intravenous injection of 99Tcm-Gd-DTPA-DG. Results The tumor was significantly enhanced by Gd-DTPA-DG with MRI. The radiochemical purity of 99Tcm-Gd-DTPADG was about 98.5% and remained 96.2% at room temperature for 6 h. The tumor was well visualized by 99TcmGd-DTPA-DG SPECT at 2 h after injection. The tumor uptake was (1.48 ±0.12) %ID/g, and the rumor to muscle radioactivity ratio was 2.91. Conclusions MRI contrast of Gd-DTPA-DG may enhance tumor detection. 99Tcm-labeled Gd-DTPA-DG may be useful for tumor imaging and might have a potential role as a SPECT-MRI bi-functional agent.     

HPV16 E6/E7 Negatively Affect Radiosensitivity of Lung Cancer Cells

Objective Lung cancer cells associated with radioresistance are likely to give rise to local recurrence and distant metastatic relapse,but little is known about its underlying mechanisms.In the present paper,the effects of the HPV16 E6 and HPV16 E7 oncoprotein on the radiosensitivity of lung cancer cell lines were investigated.Methods The HPV16 E6 or HPV16 E7 oncoprotein was expressed by a transient transfection with pcDNA3-HPV16 E6 or pcDNA3-HPV16 E7 expression vector.Human lung cancer H2179 cells and mouse lung cancer Lewis cells were exposed to a γ-ray radiation source,cellular survival was evaluated by using a colony formation assay.The expression of HPV16 oncoproteins E6/E7,extracellular signal-regulated kinases 1/2(ERK1/2) and AKT signaling was determined by Western blot assay.VEGF secretion was determined by ELISA.Results Both HPV16 oncoproteins E6 and E7 significantly decreased radiosensitivity of H2179 cells,associated with a promotion of the ERK1/2 and AKT phosphorylation.A decrease of reactive oxygen species(ROS) and an increase of VEGF levels were observed in the cells expressing the HPV16 oncoproteins E6 and E7.Furthermore,a similar reduction of radiosensitivity mediated by the HPV16 oncoproteins E6 and E7 was also observed in a mouse lung cancer Lewis cells.Conclusion The findings indicate that the HPV16 oncoproteins E6 and E7 negatively affects susceptibility of lung cancer cells to radiotherapy via regulation of the ERK1/2 and Akt signaling pathway and VEGF expression.     

Artemis蛋白磷酸化对紫外线C引发的复制检测点的调控

Objective To investigate Artemis phosphorylation on S516 and S645 in response to stalled replication forks and its role in regulation of cell cycle replication checkpoint.Methods Western-blotting was used to measure the expression of phosphorylation of Artemis on S516 and S645 after UVC irradiation.The nonphosphorylatable double mutant (S516-645A) and the mimicking phosphorylation mutant (S516-645D) plasmids were constructed.HEK 293 cells with stable expression of wild type Artemis and the corresponding mutants were established by transfection.Cell cycles of the cells treated with UVC irradiation were analyzed by flow cytometry,Western-blotting was used to measure the expression of Chk1,γ-H2AX and Cdk2.IP-kinase assay was used to measure the kinase activity of Cdk2 2,6 and 12 h after UVC irradiation.Results Artemis got rapid and prolonged pbosphorylation on S516 and S645 after treatment with UVC irradiation and the major responsible kinase was ATR.The S516-645A mutant caused prolonged arrest in replication checkpoint in S phase.The Cdk2 IP kinase activity was inhibited in S516-645A mutant ceils,but the expression levels of Chk1,Cdk2 and γ-H2AX were not affected.Conclusion The ATR phosphorylation on S516 and S645 of Artemis promotes cell cycle recovery from UVC induced replication checkpoint.     

Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.