| With the rapid development of computer technology and medical imaging technology in recent years and the clinical application of three-dimensional conformal and intensity-modulated radiotherapy,radiotherapy technology has achieved a qualitative leap,and as a result,stereotactic radiotherapy technology was born.Because of the accurate physical dose distribution of stereotactic radiotherapy and the sharp decline of target volume dose,treatments can greatly improve the tumor dose without harming the surrounding normal tissue,so as to achieve better tumor control and treatment effect.For stereotactic radiotherapy on body tumors,most of the current domestic treatments follow the low dose fractionated principles while the treatments adopted abroad in recent years are mostly hypofractionation ones.And the single dose used in our domestic treatments is far less than the doses that organs of the human body can withstand,and the treatment effect is so limited.Therefore,in our study,we treat liver cancer tumor bearing rats with both X-ray accelerators and gamma knife according to clinical principles for SBRT to acquire experiment data,in order to compare and analyze them,hoping to find more supports for hypofractionation SBRT and to investigate the differences and respective features of X-ray accelerators and gamma knife treatment.ObjectiveTo investigate the curative and biological effects of SBRT in hypofractionation dose conditions,respectively using X-ray accelerator and gamma knife stereotactic body radiotherapy for hepatocellular carcinoma in different single dose and cumulative dose condition and compare the characters of them,in order to provide scientific supports for deeper studies and generalization of hypofractionation SBRT.Methods(1)Animals and groups: 104 healthy male Wistar rats aged 6 weeks were used,weighing 160-200 G.Orthotopic liver transplantation tumor was established by operation,and the rats were randomly divided into sham irradiation group and 5,7,9,12Gy(3 times)treatment groups,12-14 rats in each group.(2)Establishment of rat liver cancer model:(1)Production of liver cancer ascites: 120-140 g weight adult male Wistar rats,2.5ml PBS W256 cell suspension(about 1×106 cells)was injected into the abdominal cavity of rats.Normal feeding after injection,after about 6 to 7 days obvious fully filling in the rat abdominal side can be observed,pump out ascites suspension with syringe,centrifuge and count the cells under the microscope and made the surgery-requiring concentration suspension to establish rat model of tumor cell suspension.(2)Tumor orthotopic implantation: a 6-week-old healthy male Wistar rats was used,weighing 160-180 g,suspension 5% chloral hydrate intraperitoneal anesthesia was performed,and then a vertical incision about 1-1.5 cm on the left upper abdominal was made,then the left hepatic lobe was pushed out of the incision,W256 tumor cells were injected slowly into the the left lobe of the liver,after that abdominal suture was performed,postoperative penicillin intraperitoneal injection was given daily for three days.Then the tumor model was established 10 days after the implantation,and radiotherapy was then performed.Abdominal CT or MRI scans were performed to affirm the establishment.(3)Radiotherapy and positioning: Positioning: Rats were anesthetized by 5% chloral hydrate,fixed using self-made rat fixation and located in CT,surface location was performed with the laser assisted line(lead point markers placed on the body surface)and the CT image were derived to formulate treatment plan.The incisors,on both sides of the midline,axillary midline surface markers were reset.Planning system: the CT scan image was loaded into the planning system for the delineation of the target area and the adjustment of the dose line range,as far as possible to avoid the important organs and central nervous system irradiation.In the X-ray accelerator treatment,treatment is divided into two fields,single doses were divided into 0,5,7,9 Gy,performed on three consecutive days,and the cumulative dose reached 0,15,21,27Gy;in gamma knife treatment part,single doses were divided into 0,7,9.12 Gy,for three consecutive days,the cumulative dose reached 0,21,27,36 Gy.(4)The evaluation indicators : After the treatment,behavior(feeding and activity)and the overall situation of rats were observed.The treatment's suppressing effect on tumor growth was evaluated by measuring tumor size,observation on the survival time of rats was performed and analysis of the effect of prolonged survival time was ran.The pathology(HE)staining observing radiation damage to the tissues,as well as the staining of autophagy(MAPLC3B,Beclin1)and apoptosis(Caspase3,AIF)in tumor cells by immunohistochemistry,was performed to observe the killing effect of radiotherapy on tumor cells and explore the related mechanism.Results(1)The general status and behaviors of rats: No peritoneal metastasis ascites tumor was found in the groups,the feeding and drinking were normal,with normal defecation,no symptoms of acute radiation syndrome,such as sickness and diarrhea,were found among the rats.A certain degree of decline of weight was found one week after SBRT treatment both in the control groups of the two treatment methods and the accelerator 5Gy group(p<0.05).(2)The inhibition on tumor growth by SBRT: In the two treatment groups,with the increase of treatment dose,the tumor volume showed a significant decrease trend.The tumor volume of 7Gy group and 9Gy group was significantly lower than that of sham irradiation group,5Gy group and sham group in the accelerator treatment groups.When the single dose was above 7Gy,the inhibition effect on the growth and development of the tumor was observed(p<0.05).The tumor volumes of Gamma knife 9Gy and 12 Gy treatment group and were significantly smaller than those of sham irradiation group and 7Gy group,and when a single dose of more than 9Gy,a significant inhibition of tumor growth was observed.(3)SBRT prolonged the survival time of tumor bearing rats: After Accelerator treatment,compared with the control group,the survival time of 7Gy and 9Gy rats in the treatment groups were prolonged.Compared with the sham group,the survival time of gamma knife 7Gy,9Gy and 12 Gy treatment group were significantly prolonged.When using the same single dose(7Gy and 9Gy),compared with the accelerator treatment groups,the rats in gamma knife treatment groups had longer survival times,and by a single dose of 9Gy,rats of two treatments showed significant difference in survival time.(4)The tumor killing effect of stereotactic radiotherapy: Due to obvious biological effects and killing effect on tumor cells and tissues,the treatment groups showed patchy necroses.Along with the increased dose,the necrotic volume and severity increased(hemorrhage and local irradiation necrosis in partial liver tumor tissue of rats in accelerator 9Gy treatment group,gamma knife 9Gy and 12 Gy treatment group is obvious).With a single dose of 9Gy,2 weeks after treatment,liquefaction necroses(or the formations of cavity)in accelerator groups are more significant than those in gamma knife treatment groups,and the distribution in accelerator groups are wider,too.(5)Stereotactic radiotherapy promote autophagy in tumor cells: In accelerator and gamma knife treatment group,the tumor cell expression of MAPLC3 B in which the autophagy level wsa reflected,was increased with the dose increasing,(P<0.05),on the accelerator in the treatment group,when the single dose increased from 5Gy to 7Gy,the rate of autophagy of tumor cells there significantly increased.Similar to the accelerator,in the gamma knife treatment group,higher dose also caused the autophagy of tumor cells,an 7Gy to 9Gy dose escalation caused more significant to enhance the rate of autophagy(P<0.05).The expression level of tumor cell accelerator and gamma knife treatment group in Beclin1 were decreased with the increase of irradiation dose(P<0.05),on the accelerator in the treatment group,when the irradiation dose increased from 5Gy to 7Gy,the tumor cell Beclin1 expression was significantly increased(p<0.05).In the gamma knife treatment group,after the first week after treatment,the expression of Beclin1 showed obvious improvement(p<0.05),with the dose increasing from 7Gy to 9Gy,accordingly,the positive expression rate of tumor cells also increased significantly(p<0.05).The different treatments of the same dose were analyzed,when with a single dose of 9Gy,the tumor cells MAPLC3 B positive expression rate of gamma knife treatment group was significantly higher than that of X-ray accelerator treatment group(p<0.05).(6)Stereotactic radiotherapy can promote tumor cell apoptosis: The expression level of tumor cell Caspase3 in accelerator and gamma knife treatment groups were increased as the dose increased,the tumor cells Caspase3 expression in X-ray accelerator treatment group showed a significant increase(P<0.05),and among different dose groups comparative analysis found no statistically significant difference;in the gamma knife treatment group,the Caspase3 expression level of irradiated cells were significantly increased(P<0.05).When the dose increased from 7Gy to 9Gy,the expression of Caspase3 was significantly increased after the second week(P<0.05).When the dose increased from 9Gy to 12 Gy,the expression rate of Caspase3 was increased in the first and second week after irradiation(P<0.05).The expression level of radiotherapy of tumor cells in the incidence of AIF increased significantly in the accelerator in treatment group,after first weeks of treatment,when the single dose increased from 5Gy to 7Gy,the expression level of AIF also increased significantly(P<0.05),but the effect disappeared after the second week,when dose continued to increase from 7Gy to 9Gy,the expression of AIF did not show significant change.In the gamma knife treatment group,when dose increased from 9Gy to 12 Gy,first weeks after irradiation,the positive expression rate of AIF is significantly increased(P<0.05),and this change disappeared after the second week,but positive AIF expression rates of tumor cells was significantly higher than that of group 7Gy in the same period(P< 0.05).Conclusions(1)In the condition of strict accordance to the clinical operation,positioning,planning,and treatment regulations of stereotactic radiotherapy,hypofractionation SBRT has a significant effect on inhibiting the growth and development of liver cancer in rats.(2)SBRT treatment with hypofractionation has a significant positive effect on prolonging the survival times of rat models with liver cancer.(3)In the premise of ensuring the high accuracy,the SBRT treatment of hypofractionation can significantly enhance the level of autophagy and apoptosis of tumor cells,achieving the effect of killing tumor cells.(4)In the same dose conditions,compared to the accelerator stereotactic radiotherapy,gamma knife stereotactic radiotherapy for hepatocellular carcinoma in rats has a better local control effect and survival rate,as well as a significantly higher level of autophagic death in rats,so it has a more significant curative effect. |
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