高压氧对鼻咽癌放射敏感性的影响及其作用机制的研究
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摘要
研究背景：鼻咽癌是常见的头颈部恶性肿瘤,在东南亚地区,尤其我国南方,如广东、广西、湖南、江西、福建等省高发。成为癌症死亡的主要原因,鼻咽癌首选放射治疗,5年生存率50%左右。调强适形放疗(intensity modulated radiotherapy IMRT)和三维适形放疗(3 dimensional conformal radiotherapy 3D-CRT)的出现,提高了肿瘤组织的放射剂量,降低了正常组织的放射剂量,使鼻咽癌5年生存率有进一步的提高。但鼻咽癌对放疗中度敏感,部分患者放疗后仍有肿瘤残留,为提高局控率增加放射剂量将增加放射并发症,严重影响患者生活质量。因此寻找放射增敏手段,减少放射并发症,提高患者生活质量,成为世界范围内放射肿瘤学研究的内容。
高压氧是吸入氧分压大于一个大气压的氧气的治疗方法,由于高压氧可以增加组织氧浓度、氧分压、增大氧的弥散距离,具有减轻水肿和各种缺血缺氧性损伤,促进血管生成,激活纤维母细胞,调节机体免疫力等多种作用。因此广泛应用于组织坏死感染、创伤。已有研究表明高压氧能增强放射治疗对肿瘤乏氧细胞的杀伤作用,提高肿瘤局控率,减少肿瘤复发,降低放疗并发症。
研究目的：通过体外及体内试验探索高压氧对鼻咽癌放射敏感性的影响及其作用机制,为高压氧辅助放射治疗应用于鼻咽癌的临床研究提供理论和实践依据。整个研究内容分两部分进行,第一部分是高压氧对鼻咽癌放射敏感性影响的体外研究。
研究方法：鼻咽癌细胞株CNE-2经传代培养后,分为对照组、高压氧组、放射组、高压氧+放射组。
(1)应用MTT检测高压氧和放射治疗对鼻咽癌CNE-2细胞增殖能力的影响；
(2)应用克隆形成试验通过线性二次方程拟合细胞存活曲线,单击多靶模型计算放射生物学参数,评价高压氧治疗对鼻咽癌CNE-2细胞放射敏感性的影响；
(3)应用FCM分析高压氧和放射治疗对鼻咽癌CNE-2细胞细胞周期和细胞凋亡的影响。
结果：
(1)MTT观察：放射组2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.1038±0.0151、0.1533±0.0135和0.2097±0.1076,0.2227±0.0353,0.2102±0.0112；2.0ATA高压氧±2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.1532±0.0155、0.2088±0.0188、0.2538±0.0128、0.2518±0.0220、0.2485±0.0184,各组与放射各组比较均有差异(P<0.05)；2.5ATA高压氧+2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.2062±0.0124、0.2502±0.0199、0.3093±0.0187、0.3030±0.0257、0.3127±0.0228,各组与放射各组比较均具有差异(P<0.05)。2.5ATA高压氧+不同剂量放射组CNE-2细胞增殖抑制率均高于2.0ATA高压氧+不同剂量放射组,差异均具有显著性(P<0.05)。
(2)克隆形成试验显示：放射组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组D0(一次照射杀死63%细胞的剂量)、SF2(照射2Gy时存活分数)分别为2.217,0.736；1.678,0.376；1.635,0.357。2.0ATA高压氧放射增敏比SER(D0)和SER(Dq)分别为1.321和6.033,2.5ATA高压氧放射增敏比SER(D0)和SER(Dq)分别为1.341和6.908。
放射组4、6、8Gy CNE-2细胞存活分数分别为0.350±0.055、0.174±0.011、0.040±0.001；2.0ATA高压氧+4、6、8Gy放射组CNE-2细胞存活分数分别为0.107±0.012、0.038±0.008、0.010±0.000,各组与放射各组比较均有差异(P<0.05)；2.5ATA高压氧+4、6、8Gy放射组CNE-2细胞存活分数分别为0.101±0.069、0.027±0.000、0.008±0.000,各组与放射各组比较均有差异(P<0.05)。
(3)FCM检测表明：放射组8、10Gy、2.0ATA高压氧+6Gy、8、10Gy及2.5ATA高压氧+4、6、8、10Gy组G2/M期细胞百分率均高于对照组(P<0.05)；2.0ATA高压氧组和2.5ATA高压氧组S期细胞百分率分别为25.43±1.40和27.73±1.04,均高于对照组S期细胞百分率16.40±1.41(P<0.05)；2.0ATA高压氧+8、10Gy组和2.5ATA高压氧+不同放射剂量组S期细胞百分比均高于对照组(P<0.05)。
放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组、2.5ATA高压氧+放射组细胞凋亡率均大于对照组(P<0.05)。2.0ATA高压氧+不同放射剂量组及2.5ATA高压氧+不同剂量放射组细胞凋亡率均大于不同剂量放射组(P<0.05)。
本研究的第二部分是高压氧对鼻咽癌放射敏感性影响的体内试验研究。
研究方法：试验动物分为对照组、放射组、高压氧组、高压氧+放射组。
(1)建立鼻咽癌裸鼠移植瘤模型,绘制移植瘤生长曲线,计算各组肿瘤抑制率,评价高压氧对鼻咽癌裸鼠移植瘤放射敏感性的影响；
(2)应用免疫组化法检测高压氧和放射治疗对鼻咽癌移植瘤组织中p53、Bcl-2、Bax、Ki67、CD31 (PECAM-1)表达的影响；
(3)应用TUNEL试验评价高压氧和放射治疗对鼻咽癌裸鼠移植瘤凋亡指数(apoptotic index AI)的影响；
(4)应用Western blot和RT-PCR方法检测高压氧和放射治疗对鼻咽癌移植瘤组织中p53、Bcl-2、Bax蛋白和mRNA表达的影响。
结果：
(1)放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组以及2.5ATA高压氧+放射组肿瘤抑制率(%)分别为94.39±1.40,7.09±2.23,10.91±3.38,98.00±0.26和99.34±0.28。2.0ATA高压氧+放射组和2.5ATA高压氧+放射组肿瘤抑制率均大于放射组(P＜0.05)。
(2)免疫组化显示：对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组p53蛋白表达阳性率分别为16.67%、83.33%、83.33%、100.00%、100.00%和100.00%。各治疗组p53蛋白表达阳性率均高于对照组(P<0.05)。说明高压氧和放射可不同程度诱导p53蛋白的表达；Bcl-2免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Bcl-2蛋白表达阳性表达率分别为83.33%,100.00%,100.00%,83.33%,83.33%,100.00%。各治疗组与对照组比较均无明显差异(P>0.05)；Bax免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Bax蛋白表达阳性率分别为16.67%、100.00%、83.33%、100.00%、100.00%、100.00%。各治疗组与对照组比较均有明显差异(P<0.05)。说明高压氧和放射有促进Bax蛋白表达增高的趋势；Ki67免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Ki67蛋白表达阳性率分别为83.33%、100.00%、100.00%、83.33%、100.00%和100.00%。各组比较均无差异(P>0.05)；CD31(PECAM-1)蛋白在各组移植瘤组织中有阴性至弱阳性的表达。对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组CD31蛋白表达阳性率分别为0.00%、16.67%、0.00%、16.67%、0.00%、16.67%。各治疗组和对照组表达无明显差异(P>0.05)。
(3)TUNEL试验显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组凋亡指数AI(%)分别为2.21±0.97,5.54士1.24,4.43±1.09,4.89±0.94,10.67±2.62和15.69±3.78。各治疗组与对照组比较均有显著差异(P＜0.05)。
(4) Western blot和RT-PCR观察到p53和Bax在鼻咽癌移植瘤组织中的蛋白和mRNA水平的表达与免疫组化分析的结果相似,对照组Bcl-2在蛋白和mRNA水平的表达明显弱于高压氧组和放射组,高压氧+放射组Bcl-2蛋白和mRNA水平的表达又弱于高压氧组或放射组,提示高压氧和放射均抑制Bcl-2在移植瘤组织中的表达,且表现为协同效应。
本研究最后得到以下结论：
(1)体外实验显示放射治疗对鼻咽癌CNE-2细胞的增殖有明显的抑制作用。高压氧治疗对鼻咽癌CNE-2细胞有放射增敏作用。
(2)高压氧治疗引起鼻咽癌CNE-2细胞阻滞于S期,放射治疗导致鼻咽癌CNE-2细胞阻滞于G2/M期,高压氧+放射治疗诱导鼻咽癌CNE-2细胞阻滞于S期和G2/M期。
(3)高压氧和放射治疗均可诱导鼻咽癌CNE-2细胞凋亡,高压氧+放射治疗对鼻咽癌CNE-2细胞的凋亡指数(AI)大于单纯高压氧或单纯放射。
(4)体内试验显示2.0ATA高压氧+放射组和2.5ATA高压氧+放射组肿瘤抑制率均大于放射组。高压氧对鼻咽癌裸鼠移植瘤有放射增敏作用。
(5)免疫组化显示高压氧和放射治疗均可上调鼻咽癌p53、Bax基因的表达,且具有协同作用。Western blot和RT-PCR进一步显示高压氧和放射均可上调鼻咽癌p53、Bax基因的表达,且具有协同作用。高压氧上调p53、Bax基因在鼻咽癌组织中的表达,进而促进鼻咽癌细胞的凋亡,增强鼻咽癌的放射敏感性,可能是高压氧对鼻咽癌放射增敏的机制之一。
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.
高压氧是吸入氧分压大于一个大气压的氧气的治疗方法,由于高压氧可以增加组织氧浓度、氧分压、增大氧的弥散距离,具有减轻水肿和各种缺血缺氧性损伤,促进血管生成,激活纤维母细胞,调节机体免疫力等多种作用。因此广泛应用于组织坏死感染、创伤。已有研究表明高压氧能增强放射治疗对肿瘤乏氧细胞的杀伤作用,提高肿瘤局控率,减少肿瘤复发,降低放疗并发症。
研究目的：通过体外及体内试验探索高压氧对鼻咽癌放射敏感性的影响及其作用机制,为高压氧辅助放射治疗应用于鼻咽癌的临床研究提供理论和实践依据。整个研究内容分两部分进行,第一部分是高压氧对鼻咽癌放射敏感性影响的体外研究。
研究方法：鼻咽癌细胞株CNE-2经传代培养后,分为对照组、高压氧组、放射组、高压氧+放射组。
(1)应用MTT检测高压氧和放射治疗对鼻咽癌CNE-2细胞增殖能力的影响；
(2)应用克隆形成试验通过线性二次方程拟合细胞存活曲线,单击多靶模型计算放射生物学参数,评价高压氧治疗对鼻咽癌CNE-2细胞放射敏感性的影响；
(3)应用FCM分析高压氧和放射治疗对鼻咽癌CNE-2细胞细胞周期和细胞凋亡的影响。
结果：
(1)MTT观察：放射组2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.1038±0.0151、0.1533±0.0135和0.2097±0.1076,0.2227±0.0353,0.2102±0.0112；2.0ATA高压氧±2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.1532±0.0155、0.2088±0.0188、0.2538±0.0128、0.2518±0.0220、0.2485±0.0184,各组与放射各组比较均有差异(P<0.05)；2.5ATA高压氧+2Gy、4Gy、6Gy、8Gy、10Gy治疗后CNE-2细胞增殖抑制率分别为0.2062±0.0124、0.2502±0.0199、0.3093±0.0187、0.3030±0.0257、0.3127±0.0228,各组与放射各组比较均具有差异(P<0.05)。2.5ATA高压氧+不同剂量放射组CNE-2细胞增殖抑制率均高于2.0ATA高压氧+不同剂量放射组,差异均具有显著性(P<0.05)。
(2)克隆形成试验显示：放射组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组D0(一次照射杀死63%细胞的剂量)、SF2(照射2Gy时存活分数)分别为2.217,0.736；1.678,0.376；1.635,0.357。2.0ATA高压氧放射增敏比SER(D0)和SER(Dq)分别为1.321和6.033,2.5ATA高压氧放射增敏比SER(D0)和SER(Dq)分别为1.341和6.908。
放射组4、6、8Gy CNE-2细胞存活分数分别为0.350±0.055、0.174±0.011、0.040±0.001；2.0ATA高压氧+4、6、8Gy放射组CNE-2细胞存活分数分别为0.107±0.012、0.038±0.008、0.010±0.000,各组与放射各组比较均有差异(P<0.05)；2.5ATA高压氧+4、6、8Gy放射组CNE-2细胞存活分数分别为0.101±0.069、0.027±0.000、0.008±0.000,各组与放射各组比较均有差异(P<0.05)。
(3)FCM检测表明：放射组8、10Gy、2.0ATA高压氧+6Gy、8、10Gy及2.5ATA高压氧+4、6、8、10Gy组G2/M期细胞百分率均高于对照组(P<0.05)；2.0ATA高压氧组和2.5ATA高压氧组S期细胞百分率分别为25.43±1.40和27.73±1.04,均高于对照组S期细胞百分率16.40±1.41(P<0.05)；2.0ATA高压氧+8、10Gy组和2.5ATA高压氧+不同放射剂量组S期细胞百分比均高于对照组(P<0.05)。
放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组、2.5ATA高压氧+放射组细胞凋亡率均大于对照组(P<0.05)。2.0ATA高压氧+不同放射剂量组及2.5ATA高压氧+不同剂量放射组细胞凋亡率均大于不同剂量放射组(P<0.05)。
本研究的第二部分是高压氧对鼻咽癌放射敏感性影响的体内试验研究。
研究方法：试验动物分为对照组、放射组、高压氧组、高压氧+放射组。
(1)建立鼻咽癌裸鼠移植瘤模型,绘制移植瘤生长曲线,计算各组肿瘤抑制率,评价高压氧对鼻咽癌裸鼠移植瘤放射敏感性的影响；
(2)应用免疫组化法检测高压氧和放射治疗对鼻咽癌移植瘤组织中p53、Bcl-2、Bax、Ki67、CD31 (PECAM-1)表达的影响；
(3)应用TUNEL试验评价高压氧和放射治疗对鼻咽癌裸鼠移植瘤凋亡指数(apoptotic index AI)的影响；
(4)应用Western blot和RT-PCR方法检测高压氧和放射治疗对鼻咽癌移植瘤组织中p53、Bcl-2、Bax蛋白和mRNA表达的影响。
结果：
(1)放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组以及2.5ATA高压氧+放射组肿瘤抑制率(%)分别为94.39±1.40,7.09±2.23,10.91±3.38,98.00±0.26和99.34±0.28。2.0ATA高压氧+放射组和2.5ATA高压氧+放射组肿瘤抑制率均大于放射组(P＜0.05)。
(2)免疫组化显示：对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组p53蛋白表达阳性率分别为16.67%、83.33%、83.33%、100.00%、100.00%和100.00%。各治疗组p53蛋白表达阳性率均高于对照组(P<0.05)。说明高压氧和放射可不同程度诱导p53蛋白的表达；Bcl-2免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Bcl-2蛋白表达阳性表达率分别为83.33%,100.00%,100.00%,83.33%,83.33%,100.00%。各治疗组与对照组比较均无明显差异(P>0.05)；Bax免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Bax蛋白表达阳性率分别为16.67%、100.00%、83.33%、100.00%、100.00%、100.00%。各治疗组与对照组比较均有明显差异(P<0.05)。说明高压氧和放射有促进Bax蛋白表达增高的趋势；Ki67免疫组化显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组Ki67蛋白表达阳性率分别为83.33%、100.00%、100.00%、83.33%、100.00%和100.00%。各组比较均无差异(P>0.05)；CD31(PECAM-1)蛋白在各组移植瘤组织中有阴性至弱阳性的表达。对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组CD31蛋白表达阳性率分别为0.00%、16.67%、0.00%、16.67%、0.00%、16.67%。各治疗组和对照组表达无明显差异(P>0.05)。
(3)TUNEL试验显示对照组、放射组、2.0ATA高压氧组、2.5ATA高压氧组、2.0ATA高压氧+放射组和2.5ATA高压氧+放射组凋亡指数AI(%)分别为2.21±0.97,5.54士1.24,4.43±1.09,4.89±0.94,10.67±2.62和15.69±3.78。各治疗组与对照组比较均有显著差异(P＜0.05)。
(4) Western blot和RT-PCR观察到p53和Bax在鼻咽癌移植瘤组织中的蛋白和mRNA水平的表达与免疫组化分析的结果相似,对照组Bcl-2在蛋白和mRNA水平的表达明显弱于高压氧组和放射组,高压氧+放射组Bcl-2蛋白和mRNA水平的表达又弱于高压氧组或放射组,提示高压氧和放射均抑制Bcl-2在移植瘤组织中的表达,且表现为协同效应。
本研究最后得到以下结论：
(1)体外实验显示放射治疗对鼻咽癌CNE-2细胞的增殖有明显的抑制作用。高压氧治疗对鼻咽癌CNE-2细胞有放射增敏作用。
(2)高压氧治疗引起鼻咽癌CNE-2细胞阻滞于S期,放射治疗导致鼻咽癌CNE-2细胞阻滞于G2/M期,高压氧+放射治疗诱导鼻咽癌CNE-2细胞阻滞于S期和G2/M期。
(3)高压氧和放射治疗均可诱导鼻咽癌CNE-2细胞凋亡,高压氧+放射治疗对鼻咽癌CNE-2细胞的凋亡指数(AI)大于单纯高压氧或单纯放射。
(4)体内试验显示2.0ATA高压氧+放射组和2.5ATA高压氧+放射组肿瘤抑制率均大于放射组。高压氧对鼻咽癌裸鼠移植瘤有放射增敏作用。
(5)免疫组化显示高压氧和放射治疗均可上调鼻咽癌p53、Bax基因的表达,且具有协同作用。Western blot和RT-PCR进一步显示高压氧和放射均可上调鼻咽癌p53、Bax基因的表达,且具有协同作用。高压氧上调p53、Bax基因在鼻咽癌组织中的表达,进而促进鼻咽癌细胞的凋亡,增强鼻咽癌的放射敏感性,可能是高压氧对鼻咽癌放射增敏的机制之一。
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.
引文
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