低剂量辐射增强小鼠肿瘤基因-放射治疗效应的免疫学机制研究

目的 探讨低剂量辐射全身照射对小鼠Lewis肺癌移植肿瘤基因-放疗方案中的免疫增强作用机制。方法 小鼠右后肢皮下接种Lewis肺癌细胞建立荷瘤模型,基因-放疗组中小鼠肿瘤局部注射由多聚乙烯亚胺包裹的pEgr-IL18-B7.1重组质粒,分别接受由2 Gy 局部照射和0.075 Gy 全身照射组合的不同治疗方案,通过³H-TdR标记方法检测小鼠CTL和NK细胞毒活性,ELISA方法检测TNF-α和IFN-γ分泌活性,观察各治疗组对荷瘤小鼠抗肿瘤免疫的作用。结果 在pEgr-IL18-B7.1基因治疗方案中,单次大剂量辐射局部照射后加多次低剂量全身照射与常规多次大剂量辐射局部照射相比,小鼠CTL和NK细胞毒活性显著增强,TNF-α和IFN-γ分泌活性有不同程度的增高。 结论 低剂量辐射可以通过促进CTL和NK细胞毒效应,上调TNF-α和IFN-γ细胞因子表达,从而增强机体抗肿瘤免疫功能,提高肿瘤基因-放疗的抑瘤效果。     

γ干扰素和内皮抑素双基因-放射治疗的体内抑瘤效应及其机制

目的探讨γ干扰素和内皮抑素双基因-放射治疗在荷Lewis肺癌小鼠的体内抑瘤效应及其机制。方法小鼠肿瘤局部注射脂质体包裹的质粒后36h，接受5Gy X射线照射，观察各组小鼠照射后不同时间肿瘤生长速率和平均存活时间；照射后第15天检测各组小鼠脾脏CTL、NK细胞毒活性和腹腔巨噬细胞TNFα分泌活性，照射后第10天用免疫组化法检测肿瘤组织微血管密度。结果基因-放射治疗后第6～18天，双基因-放射治疗组小鼠肿瘤生长速率明显低于对照组、单纯放疗组及单基因-放射治疗组，且平均生存时间明显延长。治疗后第12～18天，4次（双基因质粒＋2．5Gy）组肿瘤生长速率明显低于双基因质粒＋10Gy组，且平均生存时间明显延长。照射后第15天双基因-放射治疗组小鼠脾脏CTL、NK细胞毒活性和腹腔巨噬细胞TNFα分泌活性均明显高于对照组、单纯放疗组及内皮抑素单基因-放射治疗组，肿瘤组织微血管密度明显低于对照组、单纯放疗组及γ干扰素单基因-放射治疗组。结论双基因-放射治疗抑瘤效应明显优于单基因-放射治疗，其机理可能与辐射诱导γ干扰素和内皮抑素表达，提高机体抗肿瘤免疫功能和抗肿瘤血管生成作用有关。多次小剂量双基因-放射治疗的抑瘤效应优于单次大剂量双基因-放射治疗。     

pEgr-ssEndostatin重组质粒瘤内辐射诱导表达及其抗肿瘤作用

目的 观察pEgr-ssEndostatin基因-放射治疗对移植黑色素瘤B16小鼠的抗肿瘤作用及其在瘤内辐射诱导表达。方法 肿瘤局部注射脂质体包裹的pEgr-ssEndostatin重组质粒后42h，接受20Gyx射线照射，观察放疗后不同时间肿瘤大小，检测放疗后第2天瘤内Endostatin转录水平。结果 pEgr-ssEndostatin基因-放射治疗荷瘤小鼠能够增强放疗疗效；单纯接种pEgr-ssEndostatin质粒组和接种pEgr-ssEndostatin质粒 20Gy照射组小鼠的肿瘤组织内有Endostatin mRNA表达，且pEgr-ssEndostatin质粒照射组的EndostatinmRNA水平高于单纯pEgr-ssEndostatin质粒组。结论 pEgr-Endostatin基因-放射治疗抗肿瘤作用明显优于单纯放疗和单纯基因治疗，肿瘤局部转染重组质粒后照射瘤内Endostatin表达增强。     

pEgr-IFNγ重组质粒辐射诱导表达及其抑瘤作用的实验研究

目的 探讨pEgr-IFNγ重组质粒在接种B16黑色素瘤小鼠体内的辐射诱导表达及其抑瘤效应。方法 小鼠肿瘤局部注射脂质体包裹的重组质粒pEgr-IFNγ后36h，接受不同剂量X射线照射，观察各组小鼠照射后不同时间肿瘤生长速率和平均存活时间，照射后第3天用RT-PCR法检测瘤内IFNγ转录水平，照射后第l，3和5天用ELISA法检测小鼠血清中IFNγ含量。结果 照射后第3天重组质粒 20Gy组瘤内IFNγ转录水平明显高于重组质粒组；照射后第l，3天血清中IFNγ含量明显高于重组质粒组和对照组；照射后第9-15天，4次(质粒 5Gy)组小鼠肿瘤生长速率明显低于重组质粒 20Gy组，且平均生存时间明显延长。结论 多次基因-较小剂量放射治疗的抑瘤效应优于单次基因．较大剂量放射治疗；基因-放射治疗组可能通过诱导瘤内IFNγ表达增强，使血液中IFNγ浓度升高，从而提高荷瘤机体免疫功能和抗肿瘤能力。     

电离辐射对小鼠骨髓细胞cyclinB1、cdc2基因转录的影响

目的　研究X射线全身照射对小鼠骨髓细胞cyclinB1、cdc2基因转录的影响 ,以探讨电离辐射诱导小鼠骨髓细胞G2 期阻滞的分子机理。方法　采用流式细胞仪检测了X射线全身照射后小鼠骨髓细胞周期进程的变化。采用半定量RT PCR方法分别检测cyclinB1、cdc2基因转录水平变化。结果　 2GyX射线全身照射后 4及 1 2hG2 +M期细胞百分率升高 (P <0 0 5) ;0 5～ 6Gy照射后 1 2hG2 +M细胞百分率呈剂量依赖性增加 (P <0 0 5～P <0 0 1 )。 2GyX射线照射后 2～ 48h小鼠骨髓细胞cyclinB1转录水平在各个时间点未见明显变化 ,0 5～ 6GyX射线照射后 1 2h ,骨髓细胞cyclinB1基因转录在各剂量点均未见明显改变 ;2GyX射线照射后小鼠骨髓细胞cdc2转录水平从照后 4h即开始不同程度下降 ,1 2h达最低值 (为假照射组的 46 % ) ,照射后 48h开始恢复 ,0 5～ 6GyX射线照射后 1 2h ,骨髓细胞cdc2基因转录水平在各剂量点均明显降低 ,分别达到假照射组的40 %～ 70 %。结论　电离辐射可诱导小鼠骨髓细胞发生G2 期阻滞 ,cdc2激酶活性下降在G2 期阻滞中起重要作用。     

低剂量全身照射抑瘤作用的基础实验及临床应用

动物实验和临床研究证实,低剂量全身照射通过免疫刺激作用增强机体免疫、凋亡等生物效应产生抑瘤作用,与局部放疗联合可提高肿瘤细胞的放射治疗敏感性,减轻机体放疗的毒性反应.目前低剂量全身照射已经应用于恶性淋巴瘤等临床治疗.     

低剂量全身照射抑瘤作用的基础实验及临床应用

动物实验和临床研究证实,低剂量全身照射通过免疫刺激作用增强机体免疫、凋亡等生物效应产生抑瘤作用,与局部放疗联合可提高肿瘤细胞的放射治疗敏感性,减轻机体放疗的毒性反应.目前低剂量全身照射已经应用于恶性淋巴瘤等临床治疗.     

X射线全身照射诱导小鼠派伊氏板细胞凋亡及其机理的研究

目的 研究不同剂量X射线全身照射后小鼠派伊氏板细胞凋亡相关基因蛋白表达的变化规律。初步阐明凋亡调控的部分分子机理。方法 应用光、电镜技术观察小鼠派伊氏板细胞凋亡的形态改变,并用流式细胞术检测小鼠派伊氏板细胞Bcl-xL及Fas-L蛋白表达的变化。结果 2GyX射线全身照射后派伊氏板细胞凋亡增加,且Bcl-xL蛋白表达下降,Fas-L蛋白表达升高;75mGyX射线全身照射后,派伊氏板细胞凋亡减少,且Bcl-xL蛋白表达升高,Fas-L蛋白表达下降。结论 凋亡相关基因Bcl-xL、Fas-L在电离辐射诱导的派伊氏板细胞凋亡方面起重要的调控作用。     

肿瘤相关抗原肽与低剂量全身照射的协同抑瘤作用

目的观察低剂量X射线照射对小鼠肝癌H-22细胞膜相关抗原肽(TAP)提取物的抑瘤作用及免疫学功能的影响,为低剂量辐射(LDR)与肿瘤疫苗联合治疗肿瘤提供实验依据。方法采用微酸洗脱法制备相对分子质量≤3×106的细胞膜TAP提取物,先给予小鼠75mGyX射线全身照射,12h后以TAP提取物皮下免疫小鼠,检测脾细胞CD3、CD69和TCRαβ表面标记、脾T细胞亚组百分数的变化、脾细胞ConA反应性、IL-2和IFN-γ活性、CTL杀伤活性的变化及抑瘤效应。结果小鼠经TAP提取物免疫后,移植肿瘤的发生率降低,肿瘤平均出现时间延迟,生长速度减慢;脾细胞CD3分子和CD69分子的表达显著升高,脾细胞ConA反应性、IFNγ分泌活性和CTL杀伤活性显著增强,IL-2分泌增多。而同时给予LDR的小鼠不仅上述功能不同程度增强,脾CD8+细胞百分数显著增高。结论LDR与细胞膜TAP具有协同抑瘤作用,细胞免疫力的明显增强可能为其机制之一。     

对培养的小鼠胚胎细胞的X射线剂量分割照射和癌发生的转化

为了定量地研究低剂量照射时的剂量分割和癌发生的转化,作者采用Rezoikoff等建立的C3H10T~(1/2)c18小鼠胚胎细胞系为实验材料。研究了30～800拉德X射线照射范围内由单次剂量向多次低剂量照射的剂量分割效应。小鼠胚胎细胞系的培     

Increase in efficacy of cancer radiotherapy by combination with whole-body low dose irradiation

PURPOSE: Design of cancer radiotherapy protocol to reduce radiation dose and increase treatment efficacy in Lewis lung cancer (LLC) model. METHODS: C57BL/6J mice subcutaneously implanted with LLC were treated by conventional radiotherapy (2Gy x 6) combined with LDWBI (low dose whole-body irradiation; the second, third, fifth and sixth local doses of 2Gy each substituted by LDWBI with 0.075Gy) and/or gene therapy (intratumor injection of pEgr-IL-18-B7.1 plasmid 24 h before the first and fourth local doses). Immunologic mechanisms were explored. RESULTS: Cancer control was most significantly improved in the group receiving local radiotherapy combined with LDWBI and gene therapy as shown by prolongation of mean survival time by 60.4%, reduction in average tumor weight by 70.8%, decrease in pulmonary metastasis by 66.9% and decrease in intratumor angiogenesis by 64.8% as compared to local radiotherapy alone (p < 0.05). These changes in tumor growth and progression were accompanied with up-regulation of host immunity manifested by stimulated NK (natural killer) and CTL (cytotoxic T lymphocyte) activity, IFN (interferon)-gamma and TNF (tumor necrosis factor)-alpha secretion, PKC (protein kinase C)-theta activation and LAMP (lysosomal associated membrane protein)-1 expression. CONCLUSION: Combination of conventional radiotherapy with LDWBI and gene transfer could reduce total radiation dose by 2/3 and at the same time improve treatment efficacy of cancer accompanied with up-regulated host anticancer immunity.     

Increase in efficacy of cancer radiotherapy by combination with whole-body low dose irradiation

Purpose:Design of cancer radiotherapy protocol to reduce radiation dose and increase treatment efficacy in Lewis lung cancer (LLC) model.

Methods: C57BL/6J mice subcutaneously implanted with LLC were treated by conventional radiotherapy (2Gy × 6) combined with LDWBI (low dose whole-body irradiation; the second, third, fifth and sixth local doses of 2Gy each substituted by LDWBI with 0.075Gy) and/or gene therapy (intratumor injection of pEgr-IL-18-B7.1 plasmid 24 h before the first and fourth local doses). Immunologic mechanisms were explored.

Results: Cancer control was most significantly improved in the group receiving local radiotherapy combined with LDWBI and gene therapy as shown by prolongation of mean survival time by 60.4%, reduction in average tumor weight by 70.8%, decrease in pulmonary metastasis by 66.9% and decrease in intratumor angiogenesis by 64.8% as compared to local radiotherapy alone (p < 0.05). These changes in tumor growth and progression were accompanied with up-regulation of host immunity manifested by stimulated NK (natural killer) and CTL (cytotoxic T lymphocyte) activity, IFN (interferon)-gamma and TNF (tumor necrosis factor)-alpha secretion, PKC (protein kinase C)-theta activation and LAMP (lysosomal associated membrane protein)-1 expression.

Conclusion: Combination of conventional radiotherapy with LDWBI and gene transfer could reduce total radiation dose by 2/3 and at the same time improve treatment efficacy of cancer accompanied with up-regulated host anticancer immunity.     

Marked increase in the rate of ocular lens regeneration in the newt, Cynops pyrrhogaster, following partial body exposure to low dose X-rays

In recent years, concern over the stimulating effects of low-dose X-rays has been growing. Therefore, the effects of low-dose X-irradiation on lens regeneration in the newt were examined. Newts were subjected to sham or whole-body X-ray exposure at a dose of 0.2 Gy or 0.4 Gy, delivered at a rate of 0.43 Gy min(-1). The eyeballs were fixed in formalin solution, embedded in paraffin and assessed histologically. On day 14 after lens removal, unexposed animals showed the formation of a hollow epithelial vesicle of depigmented cells continuous with the laminae of the iris corresponding to the expected regeneration stage (Reyer's regeneration stage II). In contrast, lenses from newts exposed to a 0.2 Gy dose of X-rays showed some formation of the primary lens fibre nucleus corresponding to the fibre differentiation stage (Reyer's regeneration stage III-early). Thus, low-dose X-irradiation induced regeneration compared with the unexposed groups. An acceleration from Reyer's stage II to III-early was also found on day 14 following irradiation of only the upper belly, including the spleen. The effects of low-dose X-irradiation on lens regeneration may be mediated by changes in immune activity.     

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation

PURPOSE: Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. MATERIALS AND METHODS: The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with gamma-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). RESULTS: Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. CONCLUSIONS: Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.     

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation

Purpose : Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. Materials and methods : The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with γ-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). Results : Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.     

Residual radiation-induced damage to the kidney of the pig as assayed by retreatment.

The effect of re-irradiation on the previously irradiated kidney was studied in the Large White female pig. Both kidneys of 14-week-old pigs were initially irradiated with a single dose of 3-7 Gy of 250 kV X-rays. The individual kidney glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and the haematocrit (Hct) were serially measured up to 24 weeks after X-irradiation. Doses of 3 and 5 Gy of X-rays had little effect on the GFR. However, 7 Gy of X-rays caused a marked decline in the GFR up to 12 weeks after irradiation; the GFR then began to recover. A similar pattern of response was seen in the ERPF. In contrast, the Hct was reduced in all pigs 4 weeks after X-irradiation. The extent of the decline and subsequent recovery 24 weeks after irradiation appeared to be related to dose. Twenty-four weeks after X-irradiation both kidneys were re-irradiated with a single dose of 7.9 Gy of 60Co gamma-rays; both kidneys of four age-matched control pigs which had not previously received X-irradiation were similarly treated. Individual kidney GFR, ERPF and Hct were again serially measured up to 24 weeks after gamma-irradiation. Re-irradiation resulted in a significantly greater reduction in the GFR, ERPF and Hct compared with that seen after gamma-irradiation alone. Moreover, the severity of the reduction in the GFR and Hct observed after gamma-irradiation was related, in a dose-dependent manner, to the initial X-ray doses employed. These results indicate that the kidney fails to exhibit complete long-term recovery in function following irradiation. Re-irradiation of the kidney in patients should thus be viewed with extreme caution.     

EPR study of mouse tissues in search for adaptive responses to low level whole-body X-irradiation.

Effect of a low dose of whole-body X-irradiation (D1 = 0.075 Gy) of mice on some biochemical changes induced in the spleen and thymus by a subsequent challenge dose (D2 = 1.5 Gy) was studied by electron paramagnetic resonance (EPR). Kunming and SHK mice were used. Concentration of ribonucleotide reductase (RR) in the spleen and thymus of unirradiated Kunming mice was 0.70 and 0.46 microM respectively, and of unirradiated SKH mice--0.37 and 0.21 microM respectively (systematic error 45%). For mice exposed only to the low dose (D1 group), a stimulating effect on RR activity in spleen and thymus was found, while in mice subjected only to the D2 dose (D2 group) activity of the enzyme in the organs decreased considerably 18 h after irradiation. The group of mice irradiated with D1 and D2 doses, given at a 6-h interval (D1 plus D2 group), showed a RR activity in the organs lower than D1 and higher than D2 groups. This finding indicates that there exists an adaptation-like response to a low-dose whole-body irradiation in murine spleen and thymus. This low 'inductive' dose makes the organs' RR less susceptible to inhibition induced by a subsequent challenge dose. Preliminary results showed that the same kind of response is probably a characteristic of the RR content of splenocytes as well as of the rate of thymidine incorporation into splenocytes. 5-Thyml radicals of DNA (TH radicals) induced in whole tissues by gamma-irradiation in vitro at 77 K were also studied. Radiochemical yield of these radicals (4.0 and 5.3 nmol j-1), for spleen and thymus respectively of unirradiated Kunming mice, and 2.3 and 2.6 nmol J-1, for spleen and thymus respectively of unirradiated SHK mice (systematic error 30%), decreased significantly in both organs upon D2 irradiation. This decrease, however, was the consequence of DNA content diminishing upon D2 irradiation rather than change of DNA radiosensitivity: the beta value of TH radicals, i.e. yield of radicals per unit mass of DNA in each organ was equal for the mice from all D1, D1 plus D2, D2 and control groups. The beta values of TH radicals in mouse spleen and thymus were of the same order of magnitude compared with the yield of the single-strand breaks of DNA measured previously in rat organs just after whole-body irradiation, i.e. about 1 x 10(2) (Gy x 10(12) Da)-1.     

A radiomodifying effect of acute hypoxia on neutron-irradiated mice and dogs

Anoxia increased the survival of neutron irradiated mice with DMF = 1.66. As to haemopoietic stem cells neutron irradiated in vivo, DMF was 1.8. With X-irradiation DMF was 2.49 and 2.94, respectively. Anoxia decreased the damage of the intestinal mucous membrane after a whole-body neutron irradiation with a dose 3.0 Gy. A protective effect of acute hypoxia was demonstrated on dogs exposed to fast neutrons (4.0 Gy). Breathing of 10% gas hypoxic mixture protected more than half of the exposed animals from death and provided the development of a light form of radiation sickness instead of a serious one.     

Morphological changes in the bladder of female mice after x-ray and peak-pion irradiation (the piotron)

Early and late effects of irradiation of the urinary bladder in female NMRI mice were studied histologically. The animals were exposed either to X-rays or to negative pions in the peak (by single or fractionated local irradiation). With doses above 12.5 Gy the X-rays produced more severe late effects than the pions with the same dose.