Advance in in-beam PET

Tumor radiotherapy was a promising modality and over 100 years. Beams of heavy-charged particles show high RBE advantages and become the optimum tool for tumors therapy. Newly, along with the development of accelerators, scintillators, micro-electronics and computers, the heavy ion tumor therapy has been recognized more and developed.

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of '' Tail DNA (%)'' (TD) and "Olive tail moment" (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radio sensitivity

Studies on advantages of heavy ions in radiotherapy compared with gamma-rays

Hepatoma and melanoma cells were exposed to C-12(6+) beams generated by HIRFL facility and gamma-rays and the cell response was studied by colony assays as well as the analysis of RBE of carbon ions was evolved. The survival curves of cells irradiated by heavy ions were different from those of cells irradiated by gamma-rays. And two kinds of cell showed the obvious discrepancy in response to the photon and ion irradiation. The results showed that heavy ions have special physical properties and mighty potency to kill cell in both single and fractional irradiation meanwhile it can kill tumor cells with high radioresistance more efficiently. When involved in clinical therapy, heavy ions will enhance the therapy efficiency and decrease the suffering of patients because it can impair the repair for sublethal damage of cells which can lead to fewer irradiation fractions.

Pre-irradiation with low-dose C-12(6+) beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in C-12(6+) beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in gamma-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G(0)/G(1) arrest and activated G(2)/M checkpoints. The pre-exposure to C-12(6+) beam significantly improved cell to apoptosis. RBEs for the C-12(6+)+ AdCMV-p53 infection groups were 30%-60%,20% -130% and 30%-70% more than those for the C-12(6+)_irradiated only, AdCMV-p53 infected only, and gamma-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose C-12(6+) beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

Protective effects of melatonin against low- and high-LET irradiation

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/mu m carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/ cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

Survival and initial chromatid breakage in normal and tumour cells exposed in vitro to gamma rays and carbon ions at the HIRFL

Human hepatoma and normal liver cells were irradiated with C-12(6+) ion beams (linear energy transfer (LET) = 96 keV mu m(-1)) and gamma-rays at the Heavy Ion Research Facility in Lanzhou (HIRFL). The numbers and types of chromatid breaks were detected using the premature chromosome condensation technique. Irradiation with C-12(6+) ions produced a majority of isochromatid break types, while chromatid breaks were dominant for irradiation with gamma-rays. Experimental results showed that the initial level of chromatid breaks is clearly related to the absorbed dose from C-12(6+), ions and gamma-rays. The (12)C(6+)ions are relatively more effective at inducing initial chromatid breaks when compared with the gamma-rays. A relative biological effectiveness (RBE) of about 2.5 resulted for the induction of initial chromatid breaks by C-12(6+) ions relative to gamma-rays in both cell lines.

Enhanced biological effect induced by a radioactive C-9-ion beam at the depths around its Bragg peak

To explore the potential of double irradiation source, radioactive C-9-ion beam, in tumor therapy, a comparative study oil the surviving effect of human salivary gland cells at different penetration depths between C-9 and C-12-ion beams has been carried out. The 9C-ion C beam, especially at the distal side of the beam came out more efficient in cell killing at the depths around its Bragg peak than the 12 Bragg peak. Compared to the C-12 beam, an increase in RBE by a factor of up to 2.13 has been observed at the depths distal to the Bragg peak of the 9C beam. The 9C beam showed an enhanced biological effect at the penetration depths around its Bragg peak, corresponding to the stopping region of the incident C-9-ions and where the delayed low-energy particles were emitted. Further analysis revealed that cell lethality by the emitted particles from the stopping C-9-ions is responsible for the excessive biological effect at the penetration depths around the Bragg peak of the C-9 beam.

Radiobiological response of human hepatoma and normal liver cells exposed to carbon ions generated by Heavy Ion Research Facility in Lanzhou

Human hepatoma and normal liver cells were irradiated with C-12(6+), ion beams (LET= 96.05 keV/mu m) and gamma-rays at Heavy Ion Research Facility in Lanzhou (HIRFL). The chromatid breaks and break types were detected using the premature chromosome condensation technique. Our experimental results showed that chromatid breaks seem to have a good relation with C-12(6+) absorbed dose and C-12(6+) are more effective to induce chromatid breaks as compared to they-rays. For C-12(6+) ion irradiation the major break was isochromatid break, while chromatid breaks were dominant for gamma-ray irradiation. We also observed that the Relative Biology Effectiveness (RBE) of C-12(6+) ion is about 2.5 times higher than that of gamma-rays.

Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the Xray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpatdia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

重离子束治疗皮肤癌的临床研究

为探讨重离子束(12C6+)治疗皮肤癌的临床疗效及不良反应,对12例患者(志愿者)进行了重离子治疗临床研究。采用100MeV/u重离子束能量,RBE(Relative biologic effectiveness)值2.5-3.0,放疗剂量60-70GyE,单次剂量2.5-5.41Gy,1f/d,连续6-7d的治疗模式。根据治疗后3个月肿瘤大小变化评价近期疗效,由放射治疗肿瘤组(RTOG)按照急性放射损伤分级标准判断急性损伤。随访3月后发现,10位患者局部肿瘤达到完全缓解(CR),2例部分缓解(PR),局部肿瘤靶区仅有1-2级皮肤反应外,均未见其它放疗副反应。研究结果提示,重离子束治疗皮肤癌疗效好,无明显不良反应,显著缩短治疗时间,安全性高。

重离子束辐照对植物组织培养及转基因操作的影响

目的：建立重离子束辐照结合植物组织培养技术进行植物诱变的新方法，使用该方法率先开展植物组织细胞的传能线密度（LET）生物学效应的研究，尝试重离子束辐照结合农杆菌转染及质粒微注射法转基因操作。 材料与方法：采用兰州重离子研究装置(HIRFL)加速的碳离子束辐照非洲紫罗兰、丽格海棠、新几内亚凤仙以及紫花苜蓿的外植体，测定形态学指标，计算不同外植体的相对生物学效应（RBE）。以非洲紫罗兰叶片外植体为对象研究RBE随LET的变化关系。使用氖离子束辐照烟草叶片外植体结合农杆菌转染方法进行赤霉素4（GA4）基因转染实验；使用碳离子束辐照苜蓿愈伤组织结合质粒微注射方法进行β－葡萄糖苷酸酶（GUS）基因转染实验。 结果： 1. 不同剂量的936MeV的碳离子束和8MV的X射线辐照三种花卉及一种牧草的外植体后，基于存活率的RBE值分别为2.3、1.6、2.1和4.0； 2. LET值在31~151keV/μm区间的碳离子束辐照非洲紫罗兰叶片外植体。基于鲜重增殖（FWI）的RBE值随LET的增加而增加，151keV/μm时达到最高值6.7； 3. 烟草离体叶片外植体经过5Gy的1600MeV氖离子束辐照后进行农杆菌转基因操作，最终获得转染率为3.9%，单纯农杆菌转基因的转染效率为3.2%； 4. 20Gy的936MeV的碳离子束辐照苜蓿愈伤组织后结合组织表面pBI121质粒溶液微量注射处理后，获得GUS基因瞬间表达效率高达84.6%。 结论： 1. 不同花卉植物组织培养用外植体的辐照敏感性不同，本研究发现丽格海棠的辐射敏感性最高，其次是新几内亚凤仙，非洲紫罗兰的最不敏感； 2. 不同花卉植物外植体经离子束辐照诱变处理后，得到的再生植株突变类型不尽相同，主要包括叶的突变和茎的突变； 3. RBE的随LET的增大而增加可以归因于离子在生物体中能量沉积的增加，研究发现各生物学终止点受到损伤或者抑制的程度基本上是随着LET的增大而增大； 4. 通过离子束辐照结合植物组织培养方法最终获得了非洲紫罗兰叶绿素缺失突变体，该突变体通过植物组织培养技术能够稳定遗传； 5. 中能氖离子束辐照能够略微提高烟草农杆菌转基因的转化效率，辐照能够使再生植株花期提前； 6. 中能碳离子束辐照结合苜蓿愈伤组织表面微量注射质粒溶液法进行转基因操作能获得更高的基因转染效率

低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

重离子相对生物学效应的实验测定及生物有效剂量的验证

重离子高的相对生物学效应（relative biological effectiveness ，简称RBE）是将其应用于放疗治癌的生物学优势。生物有效剂量(GyE) = RBE

重离子束治癌相关生物学效应及机理研究

重离子束治癌是当今放射治疗中最科学、最先进、最有效的方法，是有代表性的高技术。目前仅有美、日、德实现了该技术，并已取得常规疗法难以实现的疗效。我国近年来开展了“重离子束治癌技术的基础研究”，其中，放射生物学及机理研究是重要内容。本论文从细胞、DNA分子、以及动物个体的三个不同层次上分别研究了重离子束治癌相关的生物学问题。在细胞研究方面。采用HeLa、B 16两种细胞分别研究了X一射线和重离子在水介质中入射的深度与相应细胞的存活率(1一失活率)，结果表明：X一射线对细胞的损伤随深度而逐渐衰减(或细胞存活随深度逐渐增加)，而重离子对细胞的损伤则为Bragg曲线(或细胞存活为倒Bragg曲线)。研究了25MeV／u ~(40)Ar~(14+)辐照人肝癌细胞SMMC一7721的微核及存活的动态变化，结果表明： 单次照射与分次照射的微核率随时间的变化规律在96h内没有明显区别，受照(单次、分次) 肝癌细胞的存活数随时间表现出衰减趋势，微核率与细胞存活数关系的动态变化为负相关性。研究了6MV X-射线和125．5keV／μm的重离子辐照B 1 6、V79细胞的2Gy存活率(SF2)，结果表明：B16和V79细胞的存活率(P<0．01)依赖于不同的辐射性质(X-射线、~(12)C离子)，其X-射线与~(12)C离子辐射这两种细胞的存活率之比分别为5．4和1．43，即~(12)C离子辐射增强了X-射线抗性细胞系的敏感性，从而显示了重离子治疗癌症的优势。研究了125．5keV／um的碳离子辐照小鼠黑色素瘤B16、人的宫颈癌HeLa、中国仓鼠肺V79、人的肝癌SMMC-7721四种细胞的相对生物学效率(RBE)，得．到了RBE依赖于细胞种类的关系、RBE随细胞存活水平的升高而增加的关系、以及当LET≥125．5keV／μm时，RBE随着LET的增大而变小的关系。在DNA分子研究方面。研究了125．5keV／μm~(12)C~(6+)辐照小鼠黑色素瘤B 16、中国仓鼠肺V79、人的宫颈癌HeLa、人的肝癌SMMC一7721细胞的灵敏度(由D50表示)、DNA双链断裂(DSB)和DNA双链断裂片段分布，结果表明：细胞敏感性与DNA双链断裂之间没有一致的关系，提出了细胞辐射敏感性的一种可能的分子机理，即DNA序列敏感性位点协同DNA双链断裂互补性机理。由此解释了四种细胞系的不同敏感性问题。在动物个体研究方面。研究了57．28MeV／u氧离子50Gy一次性局部照射对B16黑色素瘤小鼠肿瘤生长的抑制作用，并观测了受照小鼠的死亡情况，结果表明：照射B16黑色素瘤后第10天观察，肿瘤生长延迟为6天、肿瘤抑制率为66％，耐受剂量小于50Gy。研究了50MeV／u ~(12)C~(6+)离子辐照对小鼠移植性肿瘤S180的抑制作用、控制率、治愈率和病理组织学变化，结果表明：各剂量组对S180肉瘤的抑制作用均大于90％，高剂量组抑瘤作用明显强于低、中剂量组(P

重离子辐照诱导的DNA双链断裂研究

目的：重离子辐射生物学效应机理和哺乳动物细胞对重离子的辐射敏感性机理在目前仍颇有争议，是辐射生物学研究的热点。材料与方法：采用兰州重离子研究装置(HIRFL)加速的碳、氧、氩等重离子辐照体外培养的贴壁细胞，以集落法测定细胞的存活率；辐照琼脂糖包埋的细胞样品或DNA样品，以脉冲场凝胶电泳(PFGE)分析辐照诱导的DNA双链断裂(DSB)。结果：1．DNA片段释放百分比(PR)值随着剂量的增加而增加，在超过一定剂量后趋于一个准阈值；而DNA断裂水平与剂量之间呈线性关系，DSB产额为O.19-1.55DSBs/100Mbp/Gy；以~(60)Co γ射线为参照，得到重离子辐照诱导DSB的相对生物效率(RBE)为0.73-2.72。2．剂量率是影响DSB诱导及其片段分布的因素之一，剂量率越大，DSB产额越高，DSB诱导截面越大。但剂量率低可以使片段的非随机分布更为明显。3．重离子辐照诱导的DSB可以修复，修复方式主要是小片段连接成大的片段。4．无论是~(60)Coγ射线，还是碳、氧、氩等重离子，直接辐照DNA分子和辐照完整细胞诱导DSB的比值为1.64-2.64。说明细胞组分对DNA分子有一定的保护作用。5．辐照DNA分子诱导DSB的RBE随传能线密度(LET)的变化而变化，但IBE最大值远小于细胞失活的RBE最大值。结论：1．重离子辐照DNA分子诱导的DSB初始产额与细胞失活机理之间有一定的联系，但以此来解释细胞失活还不够充分；而不可修复的DSB才是细胞失活最主要的原因。2．细胞对重离子的辐射敏感性与DSB初始产额的关系不明显，但与细胞对DSB的修复能力高低密切相关。3．重离子辐照诱导的DSB片段是非随机分布的，其产生与DNA序列有关，即DNA分子上存在对重离子辐照敏感的位点。重离子辐照沉积的能量可以直接或间接地沿DNA链迁移，从而使得DNA分子上相对较弱或亲电性较强的化学键优先断裂。敏感位点即这些相对较弱或亲电性较强的化学键，而这 种化学键的产生是与敏感位点邻近的几个核苷酸相互作用的结果，即敏感位点应该是一段DNA序列。