Pre-irradiation with low-dose <Superscript>12</Superscript>C<Superscript>6+</Superscript> beam significantly enhances the efficacy of <Emphasis Type="Italic">AdCMV-p53</Emphasis> 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 MOl 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 12C6 beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6 beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6 beam significantly improved cell to apoptosis. RBEs for the 12C6 AdCMV-p53 infection groups were 30%-60%, 20% -130% and 30%-70% more than those for the 12C6 -irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6 beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.     

碳离子辐照对人肺癌细胞A549细胞周期进程的影响

选取对数生长期人肺癌细胞A549接受0—6.0 Gy 碳离子照射， 用克隆形成法检测细胞的存活率； 并于照射后12和24 h收集细胞， 用流式细胞术检测细胞周期各时相的细胞百分比， 观察不同剂量碳离子辐照对A549细胞周期进程的影响。 结果显示: 0—6.0 Gy 碳离子照射后细胞存活率显著下降； 照射后12 h细胞发生G0/G1期阻滞， 而照射后24 h， 1.0 Gy 照射组细胞在G0/G1期阻滞， 2.0—6.0 Gy 照射组细胞在G2/M期阻滞。 上述结果表明， 在A549细胞接受碳离子照射后的12 和24 h内， 1.0 Gy 照射可持续激活细胞G1期检查点， 而2.0—6.0 Gy 碳离子照射后其细胞周期进程是随时间变化的。 To investigate the effects of cell cycle progression of A549 cell induced by 12C6+ ion irradiation at different doses, the survival fractions of the A549 cells were determined by colony forming assay; cell cycles were analyzed by FACS at 12 h or 24 h after irradiation. The results showed that the percentage of survival in the A549 cells decreased with irradiation doses. Compared with control group， the percentage of the cells in G0/G1 phase significantly increased at 12 h after irradiation with different doses of 12C6+ ions. However， at 24 h after irradiation the percentage of the cells in G0/G1 phase significantly increased with 1.0 Gy 12C6+ ions， while the cells showed increasing percentage in G2/M phase with 2.0, 4.0 or 6.0 Gy 12C6+ ions. The results suggested that G1 cell cycle checkpoint was activated in 12—24 h after irradiation with 1.0 Gy 12C6+ ions， but after irradiation with 2.0—6.0 Gy 12C6+ ions， the cell cycle progression of the A549 cells changed with time.     

12C6+离子预辐射对小鼠胸腺脾脏细胞周期进程的影响

以0, 0.05, 0.1, 0.25, 0.5 Gy 12C6+ 离子全身预辐照昆明小鼠, 间隔4 h后再对小鼠进行4 Gy全身辐射。 辐照后12 h用流式细胞仪检测小鼠胸腺脾脏细胞在各细胞周期时相的百分率, 同时用单细胞电泳检测受辐射小鼠胸腺脾脏细胞DNA损伤程度。 结果显示, 相对于大剂量预照射组, 各低剂量预照射组胸腺细胞S期细胞百分率显著减少; 脾脏细胞G0/G1期细胞百分率明显减少; 同时胸腺脾脏细胞的拖尾率及拖尾长度明显减少, 以0.1 Gy预辐照效果最为明显。 这些结果表明, 低剂量预辐射处理可以减弱胸腺细胞的S期阻滞及脾脏细胞的G1期阻滞, 并明显减轻胸腺脾脏细胞的DNA损伤程度。     

重离子诱导细胞衰老研究

综述了细胞衰老诱导因素和衰老细胞的特征,提出细胞衰老在癌症发生发展过程中具有双重作用效应。同时,以X射线和12C6+离子束对黑色素瘤细胞系92-1或人正常成纤维细胞系MRC5进行辐照,观察分析DNA损伤应答效应和细胞衰老诱导。实验结果表明,相对于X射线,12C6+离子束能导致DNA团簇损伤,持续激活DNA损伤应答,更容易诱导细胞衰老;12C6+离子束能同时诱导癌细胞和正常细胞衰老。这些实验结果暗示开展12C6+离子束诱导细胞衰老研究具有紧迫性。     

重离子辐射提高腺病毒介导小鼠黑色素瘤细胞转染率的研究

探讨12C6+ 离子束辐射对用带有绿色荧光蛋白基因的缺陷性腺病毒（AdCMV GFP）转染小鼠黑色素瘤细胞(B16细胞系)的影响。 采用不同剂量的12C6+ 重离子束辐射经AdCMV GFP 转染的B16细胞， 利用流式细胞仪检测腺病毒的转染率。 结果表明， 12C6＋重离子束辐射能提高腺病毒对B16细胞的转染率， 且具有量效关系。 此外， 先转染后辐射法比起先辐射后转染法能更显著地提高转染率。The effect of 12C6+ beam irradiation on AdCMV GFP (a replication deficient recombinant adenoviral vector containing CMV promoter and green fluorescent protein) gene transfection efficiency for murine melanoma cell B16 has been investigated. B16 cells infected with AdCMV GFP were irradiated by different doses of 12C6+ beam. The transfection efficiency was assessed by flow cytometry (FCM). Results show that 12C6+ beam irradiation can improve tansfection efficiency of AdCMV GFP on murine melanoma cell B16 in a dose dependent manner. In addition， the tansfection efficiency in pre tranfection plus irradiation group is higher than that in pre irradiation plus tranfection group at the same dose irradiation dose.     

12C6+ 离子束辐照对大鼠离体主动脉环内皮依赖性舒张功能的影响

利用兰州重离子研究装置(HIRFL) 提供的高能12C6+离子束(能量为300 MeV/u,剂量率为0.5 Gy/min) 辐照大鼠离体胸主动脉环,考察了12C6+离子束辐照对主动脉环内皮依赖性舒张功能的影响,并采用NBT 还原法测定血管环生成超氧阴离子(O2􀀀) 水平,加入外源性超氧化物歧化酶(SOD) 干预探讨了O2􀀀在内皮功能损伤中的作用。研究结果表明,2.0,4.0 和6.0 Gy 的12C6+离子束辐照大鼠胸主动脉环后,可致血管内皮依赖性舒张功能的剂量依赖性明显受损(P <0.01 vs control group),并可致血管环NBT 还原能力剂量依赖性增加(4.0 Gy 时,P <0.05;6.0 Gy 时,P <0.01 vs control group)。辐照前加入外源性SOD 对6.0 Gy 12C6+离子束辐照所致血管环NBT 还原能力升高有明显抑制作用(P <0.01),对血管环内皮依赖性舒张功能也有明显的保护作用(P<0.01),但辐照后10 min 加入外源性SOD,其保护作用明显不及前者。结论显示,12C6+ 离子束辐照大鼠胸主动脉环可致血管内皮功能受损,O2 清除剂SOD 对内皮功能受损有保护作用,说明O2􀀀介导了辐照所致内皮功能损伤。Heavy ion beam has many characteristics,and it is expected to be the most suitable radiation therapy technique for malignant tumor. It is lack of depth-understanding on the potential adverse reactions caused by using this technique, because heavy ion radiotherapy is applied to clinical for a short time. Studies have shown that the vascular injury plays a pivotal role in normal tissue damage induced in the conventional radiation therapy, but there was no research report on heavy ion beam irradiation-induced vascular injury. In the present study, the isolated aortic rings of rats were irradiated by 12C6+ ion beam (300 MeV/u, 0.5 Gy/min) delivered by HIRFL(Heavy Ion Research Facility in Lanzhou), the effects of 12C6+ ion beam irradiation on aortic rings with endothelium dependent diastolic function have been investigated.NBT reduction method was used for assaying the vascular ring formation of superoxide anion (O2􀀀) level, and the involvement of superoxide anion in endothelial function injury in rats was investigated through the intervention test of exogenous superoxide dismutase (SOD) on O2. The results showed that, the vascular endothelial dependent vasodilation was impaired significantly (P < 0:01 vs control group) by irradiation with 2.0, 4.0 and 6.0 Gy 12C6+ ion beam in a dosedependent manner, and the NBT reduction of vascular rings increased dose-dependently (P <0.05 at 4.0 Gy, P <0.01 at 6.0 Gy vs control group). Adding exogenous SOD before irradiation could significantly inhibit the increasing of NBT reduction (P<0.01), and also had protective effect on vascular endothelium dependent diastolic function (P<0.01), but 10 min after irradiation with exogenous SOD, its protective function was significantly less than before. Conclusion indicated that 12C6+ ion beam irradiation could cause endothelial function impaired, O2􀀀 scavenger SOD has a protective effect on endothelial dysfunction, suggesting that O2􀀀 mediates endothelial injury induced by heavy ion irradiation.     

12C和X射线辐照人肺癌细胞H1299的生物学效应

用高传能线密度（LET）的12C离子束和低LET的X射线辐照体外培养的非小细胞肺癌H1299(p53基因缺失), 研究它们的辐照生物学效应的差异。 用克隆形成率法测定了细胞对射线的辐射敏感性; 用AnnexinV/PI试剂盒检测了细胞早期凋亡; 用流式细胞仪检测了细胞周期变化。 实验结果表明, 12C离子束辐照H1299细胞的存活率明显低于用X射线辐照的; 12C离子束引起H1299细胞的早期凋亡率明显高于X射线辐照引起的, 且持续时间更长; 12C离子束引起的H1299细胞G2/M期的抑制更明显。 说明H1299细胞对高LET的12C离子束的辐射敏感性高于对X射线的, 重离子对p53基因缺失型肿瘤的治疗可实施较低的照射剂量、 较少的照射次数和较长的时间间隔。     

辐射诱导促进AdCMV-p53 转染前列腺癌细胞

用腺病毒重组体（AdCMV p53/GFP）转染经0.5, 1.0和2.0 Gy γ射线辐射处理的前列腺癌细胞[PC 3（ nullp53）], 用克隆形成法检测细胞增殖能力, 用流式细胞分析法测定腺病毒重组体转染率和外源性p53蛋白表达。 结果提示, 辐射诱导使腺病毒重组体转染PC 3细胞提高7%—39%。 辐射联合 AdCMV p53 转染组p53表达水平提高18.5%—35.4%。 与单纯 AdCMV p53 转染组和单纯辐射组相比, 辐射联合 AdCMV-p53 转染组细胞存活率分别降低25%—64%和22%—65%。 To determine whether low dose pre irradiation could enhance adenovirus mediated p53 transfer and expression in human prostate adenocarcinoma, the PC 3 cells were pre exposed to γ rays, and then infected with replication deficient adenovirus recombinant vectors, containing human wild type p53 (AdCMV p53) or green fluorescent protein gene (AdCMV GFP) respectively (γ ray irradiation + AdCMV p53 /GFP infection). The exogenous gene transfer and expression were detected by flow cytometric analysis. The GFP transfer frequencies in γ irradiation + AdCMV GFP infection groups were 7%—39% more than those in AdCMV GFP infection groups. The p53 levels in the γ irradiation + AdCMV p53 infection groups were 18.5%—35.4% more than those in AdCMV p53 infection groups (p<0.05),suggesting that low dose (less than or equal to 1.0 Gy) irradiation could significantly promote exogenous p53 transfer and expression in the PC 3 cells. The survival fractions for the γ irradiation + AdCMV p53 infection groups were 25%—64%, 22%—65% less than those for AdCMV p53 infection, or γ irradiation groups, respectively (p<0.05).     

The4He(12C, γ)16O reaction at stellar energies

The capture reaction⁴He(¹²C, γ)¹⁶O (E _c.m.= 1.34–3.38 MeV) as well as the elastic scattering process⁴He(¹²C,¹²C)⁴He (E _c.m.=1.44–3.38 MeV) have been investigated with the use of an intense¹²C beam and a windowless and⁴He recirculating gas target system. The measurements involved two large NaI(T1) crystals in close geometry to an extended gas target, whereby angle-integrated γ-ray yields were obtained. A large area plastic detector was used for the suppression of time-independent background. A search for cascade γ-ray transitions was carried out by coincidence techniques. The measurement of absolute cross sections is also reported. Theoretical fits of the excitation function for the groundstate γ-ray transition requireE1 as well asE2 capture amplitudes, which are of equal importance at stellar energies. This result increases significantly the stellar burning rate of⁴He(¹²C, γ)¹⁶O and leads to¹⁶O as the dominant product at the end of helium burning in massive stars. The observed capture yield to the 6.92 MeV state is dominated by the direct capture mechanism and plays a small role at stellar energies.     

阿佛曼链霉菌的~(12)C离子辐照效应及高产菌株选育

选用12C6+离子辐照诱变阿维菌素B1a产生菌ZJAV-A1,研究其诱变效应。实验结果表明,12C6+离子辐照剂量50Gy时致死率97%,正突变率最高可达到34.2%。通过12C6+离子诱变处理,结合平板培养基及斜面培养基的正突变菌株筛选,最终获得一株稳定性良好,阿维菌素B1a组分产量稳定在4460—4588μg/ml之间,较出发菌株提高11.1%—14.7%的突变株ZJAV-Y1-203。     

辐照诱导人正常肝细胞系HL-7702 细胞远后的微卫星不稳定性

采用高传能线密度(LET) 的12C6+离子束和低LET 的X 射线辐照人正常肝细胞系HL-7702 细胞,利用微卫星不稳定性(MSI) 检测来分析直接受照射细胞和通过转移培养基方式旁细胞传代八代子细胞以MSI 表征的远后效应。实验结果表明,12C6+离子束诱导的远后效应较X射线的低;旁细胞的远后效应较直接受照射细胞的高;辐射引起的MSI 与杂合性丢失(LOH) 的发生率具有位点特异性。结果提示,重离子放射治疗较X 射线放射治疗对正常组织引发的辐射风险要小,可通过对MSI 高发位点的筛选来评估放疗后患者长期生存状况和二次癌症发生风险。Human normal liver cell line HL-7702 cells were irradiated with high linear energy transfer (LET) 12C6+ ions and low-LET X-rays, respectively. Delayed effect in terms of microsatellite instability (MSI) in progenies of the directly irradiated cells and bystander cells, obtained in the way of medium transfer at the 8th passage postirradiation,were examined. The delayed effect induced by the high-LET 12C6+ ions was different from that induced by the low-LET X-rays, and a higher incidence of MSI was observed in the progenies of the cells after exposure to the X-rays than to the 12C6+ ions. We also found that the delayed effect in the progenies of the bystander cells was much more severe than thoseof directly irradiated cells. Furthermore, the events of MSI and loss of heterozygosity (LOH) induced by the ionizing radiations were not randomly distributed throughout the genome and specific loci existed indeed. These results imply that the radiation risk to normal tissues is lower in heavy ion therapy than in conventional X-ray radiotherapy, and the analysis of microsatellite loci with MSI high frequency occurrence can be applied to access long-term survival condition and second cancer risk for the patients after radiotherapy.     

12C6+离子对阿维链霉菌累进辐照诱变效应研究

选用12C6+ 离子束对阿维链霉菌诱变选育高产菌株与原始菌株进行辐照诱变, 研究其累进辐照效应。实验结果表明,在辐照剂量为10 Gy时, 原始菌株比诱变高产菌株存活率高, 抗辐射能力强;辐照剂量高于30 Gy时,诱变高产菌株比原始菌株存活率高, 抗辐射能力强。原始菌株正突变率最高的辐照剂量为50 Gy, 致死率99.43%,正突变率最高, 达34.2%;对诱变高产菌株辐照剂量为30 Gy,致死率94.97%,正突变率最高, 达23.5% 。累进辐照效应降低了最佳辐照剂量。 Mutagenic effect on the mutant high producing strain ZJAV Y1 203 and the original strain ZJAV A1 irradiated by ion beam of 12C6+ have been investigated. The experimental results indicated that the original strain has higher survival rate and stronger resistance to radiation than mutant high producing Strain at dose of 10 Gy. The mutant high producing strain has higher survival rate and stronger resistance to radiation than the original strain at the dose higher than 30 Gy. The lethality was 97% and the highest rate of orthomutation was 34.2%, when ZJAV A1 was irradiated by 50 Gy 12C6+ beam. The lethality was 94.97% and the highest rate of orthomutation was 23.5% when ZJAV Y1 203 was irradiated by 30 Gy 12C6+ beam. The best radiation dose is decreased by progressivity irradiation.     

Spatial and temporal distribution of γH2AX fluorescence in human cell cultures following synchrotron‐generated X‐ray microbeams: lack of correlation between persistent γH2AX foci and apoptosis

Formation of γH2AX foci (a marker of DNA double‐strand breaks), rates of foci clearance and apoptosis were investigated in cultured normal human fibroblasts and p53 wild‐type malignant glioma cells after exposure to high‐dose synchrotron‐generated microbeams. Doses up to 283 Gy were delivered using beam geometries that included a microbeam array (50 µm wide, 400 µm spacing), single microbeams (60–570 µm wide) and a broad beam (32 mm wide). The two cell types exhibited similar trends with respect to the initial formation and time‐dependent clearance of γH2AX foci after irradiation. High levels of γH2AX foci persisted as late as 72 h post‐irradiation in the majority of cells within cultures of both cell types. Levels of persistent foci after irradiation via the 570 µm microbeam or broad beam were higher when compared with those observed after exposure to the 60 µm microbeam or microbeam array. Despite persistence of γH2AX foci, these irradiation conditions triggered apoptosis in only a small proportion (<5%) of cells within cultures of both cell types. These results contribute to the understanding of the fundamental biological consequences of high‐dose microbeam irradiations, and implicate the importance of non‐apoptotic responses such as p53‐mediated growth arrest (premature senescence).     

不同LET射线对细胞的生物学效应

以人肝癌细胞系和正常肝细胞系为材料,报道了不同传能线密度射线辐射引发细胞染色体原初断裂及24 h内的修复情况。 计算了相对生物学效应的值。 以L02染色体总断裂数量得出的RBE值96.05 keV/μm的12C6+ 为3.6, 512 keV/μm 36Ar18+ 为2.9。 而以7721染色体总断裂数量得出的RBE值: 96.05 keV/μm的12C6+ 为3.5,512keV/μm 36Ar18+也为2.9。用产生等点染色单体断裂计算,则RBE更高。对比得出,高LET对增加等点染色单体断裂量的作用要远远大于对增加染色单体断裂量的作用。等点染色单体的断裂修复难度要远远大于染色单体断裂的修复难度, 这也是高LET高致死率的一个重要原因。 Human hepatoma SMMC 7721 and normal liver L02 cells were irradiated with γ rays,12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou(HIRFL). We reported the kinetic repair of chromosome breaks of L02 and SMMC 7721 cells in 24 h of post irradiation time. The relative biological effectiveness(RBE) for inducing chromatid breaks were 3.6 for L02 and 3.5 for SMMC 7721 cell lines at the linear energy transfer(LET) peak of 96.55 keV/μm 12C6+ ions, and 2.9 (both of the two cell lines) at 512 keV/μm 36Ar18+ ions.It suggested that the RBE of isochromatid type breaks induced by 36Ar18+ was higher than those by 12C6+. We concluded that the high production of isochromatid type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high LET radiation exposure.     

内质网应激对碳离子辐照宫颈癌HeLa 细胞的影响

利用不同剂量的碳离子辐照二硫苏糖醇(2.5 mmol/L) 预处理的HeLa 细胞,探讨了内质网应激反应对碳离子辐照宫颈癌HeLa 细胞的影响。实验发现:与单独辐照组相比,二硫苏糖醇联合碳离子辐照后细胞的存活率下降,而凋亡率增加;二硫苏糖醇联合碳离子辐照加重了碳离子辐照引起的细胞周期阻滞;且联合辐照组的自噬被明显激活。结果表明,持续的内质网应激可改变宫颈癌HeLa 细胞对碳离子辐照反应,且二硫苏糖醇可能通过影响HeLa 细胞的自噬性细胞死亡通路发挥作用。To investigate the effect of endoplasmic reticulum stress on HeLa cells to 12C6+ ion irradiation,HeLa cells were pretreated with 2.5 mmol/L dithiothreitol and irradiated by 12C6+ ions with different doses.The results showed that, compared with IR alone, dithiothreitol combined with carbon ion irradiation caused HeLa cell survival decreased, and the apoptosis increased. Moreover, dithiothreitol and carbon ion radiation combination treatment led to a significant increase of G2/M phase, and autophagy was activated obviously in combination treatment group. The results imply that continuous endoplasmic reticulum stress can change the response of HeLa cells to 12C6+ irradiation, and dithiothreitol may affect HeLa cells through the autophagy cell death pathway.     

12C6+离子束辐照菘蓝干种子当代效应

利用兰州重离子研究装置(HIRFL) 提供的12C6+ 离子束辐照菘蓝干种子(辐照剂量为10,35,60,90 和140 Gy,剂量率20 Gy/min),探讨了重离子束辐照对菘蓝M1代的生物学效应。研究发现,不同剂量的12C6+ 离子束辐照后,菘蓝种子的发芽率、成苗率、株高、根长和根冠比等生物学性状以及对菘蓝中靛玉红和4(3H) 喹唑酮含量均发生了变化,其中株高和根长随辐照剂量的增加而降低;菘蓝叶和根中的4(3H) 喹唑酮和靛玉红的含量随辐照剂量增加呈马鞍形增加关系。这表明:12C6+ 重离子束辐照菘蓝种子具有明显的当代损伤效应, 并可显著提高菘蓝中靛玉红和4(3H) 喹唑酮的含量,其辐照适宜诱变剂量为35 Gy。To investigate the M1 biological effects of heavy ions on Isatis indigotica Fort, its dry seeds were irradiated by 12C6+ beam with the dose of 0, 10, 35, 60, 90 and 140 Gy respectively,at the rate of 20 Gy/min delivered by the Heavy Ion Research Facility in Lanzhou (HIRFL). The results showed that biological characters such as germinating rate, germinating potential, survival rate, plant height, root height and root-shoot ratio were changed after irradiation. Moreover, the plant height and root height decreased in a dos dependent manner. The indirubin and 4(3H) quinazolinone content of Isatis indigotica Fort was improved and exhibited obviously “saddle” trends with irradiation dose increasing.Data suggest that exposure with low-dose 12C6+ to seeds of Isatis indigotica Fort has obvious injury effects at the first generation, and the active ingredient content of Isatis indigotica Fort may be improved by carbon ion beamirradiation. It is concluded that the suitable irradiation dose of mutation breeding is 35 Gy for the seeds of Isatis indigotica Fort.     

Increased viability of odontoblast-like cells subjected to low-level laser irradiation

Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm² were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO₂ at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm² + 5% FBS; G2: 1.5 J/cm² + 10% FBS; G3: 5 J/cm² + 5% FBS; G4: 5 J/cm² + 10% FBS; G5: 19 J/cm² + 5% FBS; G6: 19 J/cm² + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm². These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.     

羧甲基-β-1,3-葡聚糖对人肝癌HepG2细胞的放射增敏作用

本研究旨在探讨羧甲基-β-1,3葡聚糖（CMG）对人肝癌HepG2细胞X射线或12C6+离子束辐射敏感性的影响。首先用CCK-8法检测CMG对HepG2细胞的生长抑制情况,得到半数抑制浓度（IC50）为120.6μg/mL。用浓度为0.1×IC50的CMG预处理HepG2细胞24 h,再给予2 Gy X射线或12C6+离子束辐照（CMG+辐照组）;CMG未处理组直接接受2 Gy X射线或12C6+离子束辐照（辐照组）。对比分析辐照组和CMG+辐照组细胞的克隆存活、DNA损伤、凋亡与周期分布、细胞内活性氧（ROS）水平。发现:与X射线辐照组相比,相同剂量的12C6+离子辐照组克隆存活率更小,DNA损伤和周期阻滞更加严重,细胞凋亡率和细胞内ROS水平也更高。与单独X射线或12C6+离子束辐照组相比,CMG+辐照组克隆存活率明显降低,细胞凋亡率随辐照后CMG作用时间的延长而明显增加,CMG使辐照后细胞内ROS维持在一个较高的水平,同时CMG明显加重了单独辐照诱导的DNA损伤和周期阻滞。结果表明,与X射线相比,HepG2细胞对相同剂量的12C6+离子辐射更敏感;CMG可增加HepG2细胞对X射线或12C6+离子辐射的敏感性;CMG可能通过增加受照HepG2细胞内的ROS水平,加剧辐照诱导的DNA损伤,促进辐射诱导细胞凋亡而起到辐射增敏作用。This study aims to investigate the effect of carboxymethy-β-1, 3-glucan (CMG) on the sensitivity of human hepatoma HepG2 cells to X-rays or 12C6+ ions irradiation. First, the inhibitory effect of CMG on the growth of HepG2 cells was detected by CCK-8 assay, and the half maximal inhibitory concentration (IC50) was 120.6 μg/mL. HepG2 cells were pretreated with CMG at a concentration of 0.1×IC50 for 24 h and then irradiated with 2 Gy X-ray or 12C6+ ion beams (CMG + irradiation group). CMG untreated group was directly irradiated by 2 Gy X-rays or 12C6+ ions beam (irradiation group). The clone survival, DNA damage, cell apoptosis, cell cycle distribution, and intracellular reactive oxygen species (ROS) levels in irradiation group and CMG + irradiation group were comparatively analyzed. The results showed that the clone survival rate was lower, DNA damage and cycle arrest were more serious, and the rate of apoptosis and intracellular ROS levels were higher in 12C6+ ions irradiation group than those in the same dose of X-rays irradiation group. Compared with X-rays or 12C6+ ions irradiation group, the clone survival rate of CMG + irradiation group was significantly decreased, and the apoptosis rate significantly increased with the prolongation of CMG treatment post-irradiation; CMG maintained intracellular ROS at a higher level after irradiation, CMG also significantly aggravated radiation-induced DNA damage and cycle arrest. These results indicated that HepG2 cells were more sensitive to 12C6+ ions radiation than those at the same dose of X-rays. CMG increased the sensitivity of HepG2 cells to X-rays or 12C6+ ions irradiation by increasing intracellular ROS level, exacerbating radiation-induced DNA damage and promoting radiation-induced apoptosis in irradiated HepG2 cells.     

γ射线对肝癌细胞G2染色体的原初损伤

用Calyculin—A诱导的早熟染色体凝集技术研究了γ射线诱导人肝癌细胞株HepG2细胞G2期染色体的原初损伤。结果表明:G2等点染色单体断裂畸变与辐射剂量呈线性平方关系,G2染色单体断裂畸变和G2期染色单体断裂畸变总数与辐射剂量呈线性正相关关系,发生各类断裂畸变的细胞率与剂量也呈线性正相关关系。γ射线诱发的断裂畸变主要是G2染色单体断裂畸变,断裂畸变的细胞主要是发生G2染色单体断裂畸变。A chemically induced premature chromosome condensation technique with Calyculin-A has been employed to estimate the initial chromosome damage in HepG2 condensed in G2 phase and the percentage of aberrant cells after exposure to γ-rays. The results show that the dose-response for iso-chromatid breaks is linear-quadratic manner, while chromatid-type breaks and total chromatid breaks show a positive linear dose-response. The percent tages of all kinds of aberrant cells are increasing linearly with increasing doses. G2 chromatid-type breaks and the percentage of G2 chromatid-type aberrant cells are predominate in G2 total chromatid breaks induced b y γ-rays.     

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm²) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.