| Study background:Nasopharyngeal carcinoma (NPC)is common cancer in head and neck, that is high prevalent in East South Asia, specially in South China, such in Guangdong, Guangxi, Hunan, Jiangxi, Fujian province et al, and that is a major cause of cancer deaths. Owing to anatomic restriction, radiotherapy is currently the primary choice for nasopharyngeal carcinoma. At present,5 year survival rate is about 50%,recently,lots of effects has been made for the treatment of nasopharyngeal carcinoma. both in order to increase radiation dosage for tumours and to reduce it for critical organs by using new techniques such as intensity modulated radiotherapy (IMRT) and three dimensional conformal radiotherapy (3D-CRT). With the advent of intensity modulated radiotherapy (IMRT) and three dimensional conformal radiotherapy (3D-CRT),the technical development of radiotherapy has significantly increased the previous 5-year survival rate of the nasopharyngeal carcinoma patients to more than 50%. Nasopharyngeal carcinoma is moderate sensitive to radiotherapy, partial nasopharyngeal carcinoma is resistant to radiotherapy.It has been reported that nasopharyngeal carcinoma has higher incidence of local residual and recurrence, sometimes the dose of radiation must be increased,with the increase of radiation dose,the incidence of radiation-related complication will be more than that in usual condition. Improvement of the patients'quality of life and the reduction of radiation-related complications have become pressing concerns. The researches of radiosensitization to nasopharyngeal carcinoma are emphasis of the radiotherapy oncologist in the world.Hyperbaric oxygen therapy(HBO) is a medical treatment involving the administration of pure oxygen under pressure of>1 ATM,HBO increases oxygen tension and oxygen delivery to tissues independently of the hemoglobin level.Substantially increases the amount of oxygen dissolved in the plasma,the short term of effects of hyperoxia include enhanced oxygen delivery to ischemic tissue,vosaconstriction,a reduction of edema and immuno-modulatory properties.The long term effects include neovascularizations and fibroblast proliferation,therefore, hyperoxygen therapy(HBO)has been used to treat different clinical disease,such as necroticing,soft tissue infectious,compromised skin grafts and flaps,acute traumatic ischemias and exceptional blood loss.There is some evidence that HBO in combination with radiotherapy can enhance radiotherapeutic killing of hypoxic cancer cells,reduce local recurrence and mortality,improve local tumor control as well as decrease the chance of radiation-related complications in cancers.Study objects:The mechanism of hyperbaric oxygen strengthening radiosensitivity of nasopharyngeal carcinoma is researched in vitro and in vivo tests,the study will privode guidance of hyperbaric oxygen to assist the radiotherapy for nasopharyngeal carcinoma from theory and practice.Study methods:We divided our research in two parts.The first part,our objects are to observe the radiosensitization in CNE-2 cell after HBO exposure and the changes of cell cycle and apoptosis in CNE-2 cell after HBO exposure and radiation. CNE-2 cell was cultivated and divided into control group, HBO group,radiation group and HBO+radiation group.(1) MTT was used to assess the efficacy on growth suppression of NPC cell line CNE-2 treated by HBO and radiation;(2) Clonogenic survival assay was performed to assess the change of radiosensitivity in CNE-2 cell after HBO exposure and radiation; Linear quadratic model was adopted to form the survival curve of CNE-2 cell and single hit multi-target model was used to calculate radiobiologic parameter(3) FCM mothed was carried out for analysis of cell cycle and apoptosis of CNE-2 under HBO and radiation.Study result:(1) MTT indicated that from the 2Gy to10Gy in radiation group,the cell proliferation inhinition rates increased by degrees,which were 0.1038±0.0151,0.1533±0.0135,0.2097±0.1076,0.2227±0.0353 and 0.2102±0.0112,respectively,the cell proliferation inhibition rates in the 2.0ATA HBO+2Gy,4Gy,6Gy,8Gy,10Gy were 0.1532±0.0155,0.2088±0.0188, 0.2538±0.0128,0.2518±0.0220,0.2485±0.0184.The cell proliferation inhibition of 2.5ATAHBO+2Gy,4Gy,6Gy,8Gy,10Gy radiation group were 0.2062±0.0124,0.2502±0.0199,0.3093±0.0187,0.3030±0.0257,0.3127±0. 0228. The cell proliferation inhibition rates of 2.5ATA HBO+radiation group were higher than those of 2.0ATA HBO+radiation group in the related dose points,the differences were significant (P<0.05). The cell proliferation inhibition of 2.0ATA HBO+radiation group were higher than those of the radiation group in related dose points,the difference were significant (P<0.05),(2) Clonogenic survival assay showed that D0 and SF2 of radiation group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 2.217,0.736;1.678,0.376;1.635,0.357; respectively. D0 and SF2 of 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were lower than those of radiation group.Sensitive enhancement ratio SER(D0) and SER(Dq) in 2.0ATA HBO group were 1.321 and 6.033, sensitive enhancement ratio SER(D0) and SER(Dq) in 2.5ATA HBO group were 1.341 and 6.908.The experimental data indicates HBO enhanced the radiosensitivity of CNE-2 cell.SF in the 4,6,8Gy radiation group were 0.350±0.055,0.174±0.011, 0.040±0.001.SF of 2.0ATA HBO+4,6,8Gy radiation group were 0.107±0.012,0.038±0.008,0.010±0.000.SF of 2.5ATA HBO+4,6,8Gy radiation group were 0.101±0.009,0.027±0.000,0.008±0.000. In 4,6,8Gy points,the SF of 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were lower than those in the radiation group,the difference were significant (P<0.05).(3) FCM mothed revealed that the percent rate of G2/M stage in CNE-2 in 8,10Gy radiation group and 2.0ATA HBO+6,8,10Gy radiation group and 2.5ATA HBO+4,6,8,10Gy radiation group were higher than that in the control group (P<0.05).The percent rates of S stage cell in CNE-2 of 2.0ATA HBO group and 2.5 ATA HBO group were 25.43±1.40,27.73±1.04,respectively. They were higher than that of control group,which was 16.40±1.41,the difference was significant (P<0.05). the percent rates of S stage cell in CNE-2 of 2.0ATA HBO+8,10Gy radiation group and 2.5ATA HBO+radiation group were higher than that in control group (P<0.05).Apoptotic rate of radiation group,2.0ATA HBO group and 2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were higher than that of control group (P<0.05). The apoptotic rate in 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were higher than those in radiation group in the related dose points (P<0.05).As the second part of our researches, We will detect the radiosensitization of HBO on transplanted tumor of CNE-2 cell in nude mice.Study methods:the nude mice xenografted nasopharyngeal carcinoma model with CNE-2 cell was constructed and divided into control group, radiation group, HBO group and HBO+radiation group.(1) To observe the effect on transplated tumour of NPC after HBO exposure or/and radiation.The tumor growth curve of transplanted tumor was generated and tumor growth suppression rate was calculated by measuring the volume of transplanted tumor per three days.(2) To detect the changes of protein expression of p53,Bcl-2,Bax, Ki67 and CD31(PECAM-1) in transplated tomour of NPC by immunohistochemistry after HBO exposure or/and radiation.,(3) To determine the apoptotic index(AI) in the transplanted tumor of NPC by TUNEL after HBO exposure or/and radiation.,(4) To evaluate the protein and mRNA expressions of p53,Bcl-2 and Bax in transplated tomour of NPC by Western blot and RT-PCR after HBO exposure or/and radiation.Study results:(1) The nude mice xenografted nasopharyngeal carcinoma model with CNE-2 cell trial show that the tumor growth suppression rate(%) of radiation group,2.0ATA HBO group,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 94.39±1.40,7.09±2.23,10.91±3.38,98.00±0.26 and 99.34±0.28,the tumour growth suppression rate of radiation was bigger than those in 2.0ATA HBO group and 2.5ATA HBO group,the difference was significant (P<0.05).The tumour growth suppression rate of 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were bigger than that in radiation group,the difference was significant (P<0.05).(2) Immunohistochemistry indicated that the positive rate of p53 in control group,radiation group,2.0ATA HBO group,2.5ATA HBOgroup, 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 16.67%,83.33%,83.33%,100.00%,100.00%and 100.00%.The expression of p53 protein in the radiation group,2.0ATA HBOgroup,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group was stronger than that in the control group,the difference was significant (P<0.05).It showed the radiation and HBO can induce the expression of p53 protein.The result showed significant synergistic effect of radiation combination with HBO on expression of p53 protein;the positive rate of Bcl-2 in control group,radiation group,2.0ATA HBO group,2.5ATA HBOgroup,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 83.33%,100.00%,100.00%,83.33%,83.33%and100.00%.The difference in the expression of Bcl-2 protein between control group and radiation group 2.0ATA HBOgroup,2.5ATA HBOgroup,2.0ATA HBO+radiation group,2.5ATA HBO+radiation group was not significant (P>0.05). It showed there is not effect on the radiation and HBO on the Bcl-2 protein expression;the positive expression rate of Bax protein in control group,radiation group,2.0ATA HBO group,2.5ATA HBOgroup, 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 16.67%,100.00%,83.33%,100.00%,100.00%and100.00%.The difference of expression Bax protein between control group and radiation group,2.0ATA HBO group,2.5ATA HBOgroup,2.0ATA HBO+radiation group,2.5ATA HBO+radiation group were significant (P<0.05). The result showed that HBO and radiation can induce the expression of Bax protein;the positive rate of Ki67 in control group,radiation group,2.0ATA HBO group,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 83.33%,100.00%,100.00%, 83.33%,100.00%and100.00%.The difference of the Ki67 protein expression between control group and radiation group,2.0ATA HBOgroup,2.5ATA HBOgroup,2.0ATA HBO+radiation group, 2.5ATA HBO+radiation group were not significant (P> 0.05); the expression CD31 protein in the NPC xenografted tumor were negative or weakly positive. The positive rate of CD31 in control group,radiation group,2.0ATA HBO group,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 0.00%,16.67%,0.00%,16.67%,0.00%,16.67%.The difference between control group and radiation group,2.0ATA HBOgroup,2.5ATA HBOgroup,2.0ATA HBO+radiation group,2.5ATA HBO+radiation group was not significant (P＞0.05). The radiation and HBO have not effected the expression of CD31 protein in the NPC xenografted tumor.(3)TUNEL indicated that AI(%) (apoptotic index)of control group,radiation group,2.0ATA HBO group,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were 2.21±0.97,5.54±1.24,4.43±1.09,4.89±0.94,10.67±2.62 and 15.69±3.78. The difference of AI(%) between control group and radiation group,2.0ATA HBO group,2.5ATA HBO group,2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were significant (P<0.05). AI(%) of 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group was higher than that of 2.0ATA HBO group,2.5ATA HBO group and radiation group,the difference was significant (P<0.05). It suggested that HBO induced apoptosis of transplanted tumor of NPC and showed the synergistic effect of HBO and radiation.(4)Western blot and RT-PCR showed that the expression of p53,Bax protein and mRNA detected by Western blot and RT-PCR were complete consistent with those detected by immunohistochemistry. Western blot and RT-PCR indicated the protein and mRNA expression of Bcl-2 in the HBO+radiation group were weaker than those in the HBO or radiation group,the expression of Bcl-2 protein and mRNA in the HBO or radiation group were weaker than that in the control group,it showed that HBO down-regulated the exptession of Bcl-2 in the transplanted tumor of NPC and there was synergistic effect between HBO and radiation.At last,We come to some conclusions:(1) In vitro test showed that radiation obviously inhibits the proliferation of CNE-2 cell of NPC, HBO can enhance the radiosensitivity of CNE-2 cell of NPC.(2) Radiation can result in detention on G2/M phase of CNE-2 cell,HBO can induce arrest on S phase of CNE-2 cell,HBO+radiation can mediate the blockage in the G2/M and S phase in CNE-2 cell.(3) HBO and radiation all can induce the apoptosis of CNE-2 cell,the AI(apoptotic index) of CNE-2 cell after HBO exposure and radiation is greater than that after HBO or radiation exposure.(4) In vivo test showed that tumour growth suppression rate of 2.0ATA HBO+radiation group and 2.5ATA HBO+radiation group were bigger than that in radiation group. HBO can enhance the radiosensitivity of transplanted tumor of NPC. (5) Immunohistochemistry indicated that HBO and radiation all can up-regulate the expression of p53,Bax proteins in transplated tumour of NPC and it has synergistic effect,HBO and radiation have no effect on the expression of Bcl-2,Ki67 and CD31(PECAM-1) proteins in transplated tumour of NPC,Western blot and RT-PCR indicated the HBO and radiation all can up-regulate the expression p53, Bax gene and down-regulate Bcl-2 gene in transplated tumour of NPC,it has synergistic effect. HBO or radiation can induce the apoptosis of nasopharyngeal carcinoma, HBO can enhance the radiosensitivity of nasopharyngeal carcinoma,the radiosensitization of HBO is related to the apoptosis which is associated with the expression of p53 and Bax.HBO can obviously enhance the radiosensitivity of nasopharyngeal carcinoma, promote the the p53 and Bax expression,which may be one of the mechanisms of radiosensitization by HBO in nasopharyngeal carcinoma. |
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