低剂量X射线在优化pEgr-IL18-B7.1基因-放疗方案中的作用

目的观察低剂量X射线全身照射在接种Lewis肺癌小鼠的pEgr-IL18-B7.1基因-放疗中的抑瘤作用。方法基因-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2GyX射线局部照射和0.075GyX射线全身照射组合的不同治疗方案,观察各种治疗方案在荷瘤小鼠中的抑瘤作用。结果与单纯多次大剂量X射线局部照射相比,单次大剂量X射线局部照射后加多次低剂量全身照射在联合pEgr-IL18-B7.1基因治疗中能体现出更为有效的抑制肿瘤生长作用。结论低剂量X射线全身照射作为一种优化手段,可有效增强pEgr-IL18-B7.1基因-放疗的抑瘤效果。     

IFN-γ和内皮抑素双基因联合X射线对小鼠乳腺癌及肺转移的抑瘤作用

目的 评价IFN-γ和内皮抑素(endostatin)双基因-放射治疗在小鼠转移性乳腺癌中的抑瘤效应,并探讨其可能的作用机制。方法 用脂质体包裹pEgr-IFN-γ和 pEgr-endostatin质粒转染小鼠乳腺腺癌4T1 细胞,并用X射线照射,吸收剂量为2~20 Gy。用ELISA检测细胞培养液上清中IFN-γ和内皮抑素的浓度。小鼠下肢注4T1肿瘤细胞1×10⁵个,荷瘤小鼠随机分组为对照组、空质粒组,基因治疗组、放射治疗组及基因-放射治疗组,观察小鼠肿瘤生长及肺转移情况,计算肿瘤生长率、肿瘤/体重比及荷瘤小鼠生存率, 并用流式细胞仪检测脾脏 CTL 和 NK细胞的细胞毒活性,用免疫组织化学法检测肿瘤内部的微血管密度。结果 辐射显著增强了4T1 细胞分泌IFN-γ和内皮抑素的浓度。小鼠接受基因-放射治疗与单独接受基因治疗或者接受放射治疗相比,肿瘤生长率明显降低,同时生存率明显提高(t＝8.724,P<0.05)。双基因联合放射治疗组小鼠脾中CTL 和 NK 细胞的细胞毒活性及腹腔巨噬细胞的TNF-α水平较对照组明显升高(t=2.120、22.140和5.289,P<0.05),微血管密度明显降低(t=13.294,P<0.05)。结论 IFN-γ和内皮抑素的基因-放射治疗增强了小鼠转移性乳腺癌的抑瘤效应,其机制可能与IFN-γ激活 CTL 和 NK 细胞活性及内皮抑素引起肿瘤血管生成抑制有关。     

低剂量辐射适应性反应对辐射诱发小鼠胸腺淋巴瘤的影响

目的 探讨低剂量辐射对致癌剂量辐射诱发小鼠胸腺淋巴瘤的影响及其免疫学机理。方法 采用4次1.75GyX射线全身照射C57BL/6J小鼠诱发胸腺淋巴瘤模型, 观察不同剂量照后6个月小鼠胸腺淋巴瘤发生率, 照后1个月脾脏NK细胞毒活性、IL-2和γIFN分泌活性、腹腔巨噬细胞吞噬功能及其TNFα分泌活性以及胸腺细胞分化的变化。结果 每次1.75Gy照射前6h或12h接受25mGy或75mGy全身照射均可降低胸腺淋巴瘤发生率, 且预先接受75mGy全身照射的作用效果更为明显; 每次1.75Gy照射前12h接受75mGy照射小鼠, 上述免疫指标均比单纯1.75Gy照射组增强, 且多数指标接近假照射组; 其胸腺CD4^-CD8^-和CD4^-CD8⁺细胞较单纯1.75Gy照射组减少、CD4⁺CD8⁺细胞增多。结论 低剂量辐射可诱导辐射诱发胸腺淋巴瘤适应性反应, 对致癌剂量辐射诱发小鼠胸腺淋巴瘤有抑制作用, 其抑制作用的免疫学机理可能与低剂量辐射的免疫增强效应及诱导的免疫学适应性反应, 减轻致癌剂量辐射对机体免疫功能的损伤, 使胸腺淋巴瘤前体细胞在形成胸腺淋巴瘤之前被免疫系统清除有关。     

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。     

辐射诱导双基因质粒pEgr-1-endostatin-TNF-α的抗肿瘤作用研究

目的 探讨放射线诱导重组质粒pEgr-1-endostatin-TNF-α在小鼠体内的表达及其与射线协同抗肿瘤效应。方法 Lewis肺癌荷瘤鼠240只,随机分为4组:对照组、照射组、质粒组、质粒+照射组。将重组质粒pEgr-1-endostatin-TNF-α注射到荷瘤鼠肿瘤内,24 h后对质粒+照射组采用γ射线肿瘤局部照射10 Gy,诱导目的基因表达,ELISA法测定小鼠不同时间血清中的内皮抑素及TNF-α浓度,免疫组织化学染色检测肿瘤组织中血管内皮细胞CD31的表达,与HE染色分别计算肿瘤血管密度,测量肿瘤大小,观察肿瘤生长情况。结果 质粒+照射组内皮抑素及TNF-α表达量明显增加,第2周表达量达到高峰,分别为(52.64±4.19)和(12.01±0.87)ng/ml,持续到第4周仍有较高浓度表达,与其他3组比较差异有统计学意义(F=29.726,P<0.05)。质粒+对照组HE染色肿瘤组织血管密度较对照组明显减少[(4.7±0.8)与(10.0±1.2)个/视野, t=14.063, P<0.05],肿瘤生长较对照组和照射组受到明显抑制[(5907.2±78.6)、(4653.4±32.8)和(763.5±12.3) mm³, F=16.415,P <0.05)]。结论 重组质粒pEgr-1-endostatin-TNF-α可以通过射线的诱导在小鼠体内表达,与单纯放疗相比较表现出明显的抑制肿瘤血管形成和控制肿瘤生长的作用。     

X射线照射对小鼠辅助性T细胞相关细胞因子的影响

目的 观察不同剂量X射线照射对小鼠辅助性T细胞相关细胞因子的影响。方法 100只BALB/c小鼠用随机数字表法分为4组,包括健康对照组和X射线照射组(分别予2、4和6 Gy 照射),每组25只。于照射前、照射后7、14、21和28 d,对小鼠一般情况进行观察,对外周血进行白细胞计数,用蛋白芯片检测血清中辅助性T细胞相关细胞因子含量,并用ELISA确证细胞因子的浓度变化。结果 照射后,小鼠外周血白细胞14 d降至最低, 同一时间点的下降程度随照射剂量增加而增加 (F=86.267,P<0.05)。芯片法初筛提示,细胞因子IFN-γ、TGF-β、IL-6、IL-17上调,IL-5、IL-23、TNF-α下调。ELISA检测可见,IL-17浓度随辐射剂量增高而明显增高,6 Gy组在7、14和21 d均明显高于对照(t=23.743、21.759和17.662,P<0.05);IFN-γ/IL-4浓度在2和4 Gy照射后无明显变化,6 Gy照射后在7、14、21和28 d均比健康对照组明显增高(t=8.335、9.982、6.990和3.074,P<0.05),呈先升高后降低的变化趋势,14 d达高峰。结论 低于致死剂量的X射线照射可使小鼠血清IFN-γ、TGF-β、IL-6、IL-17细胞因子浓度升高,呈Th1细胞偏移。     

禁食对137Cs γ射线照射诱导小鼠肠道辐射损伤的代谢组学研究

目的 研究禁食对¹³⁷Cs γ射线照射诱导小鼠肠道辐射损伤的干预作用,通过非靶向代谢组学探究小鼠粪便代谢物的变化。方法 将小鼠分为健康对照组、γ射线照射（全身9 Gy或腹部15 Gy）组、禁食（24、48、72 h）+照射（全身9 Gy或腹部15 Gy）组。照射后,计算小鼠的生存率、脾脏指数和胸腺指数。非靶代谢实验测序分为4组,分别为健康对照组、禁食24 h组、腹部局部照射15 Gy组、禁食24 h组+腹部局部照射15 Gy组、每组6只。于照后3.5 d收集各组小鼠的粪便进行非靶向代谢组学检测。结果 9 Gy γ射线全身照射小鼠照射前禁食48和24 h,受照后的中位生存期提高了1和4 d;15 Gy腹部受照小鼠照射前禁食48和24 h的小鼠的存活率分别为16.67%和25%,照射前禁食24 h能够提高受照后3.5 d小鼠的体重（t=2.338,P=0.042）和脾脏指数（t=2.289,P=0.045）。非靶向代谢组学结果显示,禁食24 h和未禁食的受腹部局部照射小鼠粪便样本中有30个差异表达代谢物;代谢通路富集分析表明,类固醇激素生物合成的代谢途径存在着不平衡状态。结论 照射前禁食可以提高肠道辐射损伤小鼠的生存率,改变其肠道代谢产物,提示照射前禁食或短期内饮食营养变化参与调节肠道辐射损。     

辐射损伤后小鼠T细胞功能亚群及其IL-4和IFN-γ的变化

目的 观察不同剂量辐射损伤后小鼠T细胞功能亚群、细胞因子IL-4和IFN-γ的变化。方法 C57BL/6j小鼠分为假照射组和辐射损伤模型组。采用⁶⁰Coγ线照射诱导辐射损伤模型。吸收剂量分别为0.7、1.4、2.8及5.6 Gy。应用细胞表面标志和细胞内因子标记结合流式细胞仪分析急性照射损伤和损伤恢复期小鼠脾CD3+、CD4+、CD8+T细胞功能亚群,以及细胞因子IL-4和IFN-γ的变化。结果 1 辐射损伤后CD3+、 CD4+及 CD8+有明显的减低,其改变幅度与受照剂量有关。2 照射后1 d, IFN-γ降低幅度明显高于IL-4,受照剂量大于2.8 Gy组,IL-4/IFN-γ比值较空白对照组明显升高。3 辐射对淋巴细胞功能亚群、细胞因子IL-4和IFN-γ的损伤在照射后25 d有不同程度的恢复,但与假照射组比较仍有明显差别。 结论 电离辐射可使淋巴细胞亚群CD3+,CD4+,CD8+、 CD4+/CD8+、细胞因子IL-4和IFN-γ发生改变,并诱发受照小鼠免疫功能低下,特别是使细胞因子IL-4和IFN-γ发生失衡,再次证实辐射损伤后小鼠脾Th1/Th2模式向Th2免疫反应漂移的现象。     

纳米氧化铈对γ射线诱导小鼠免疫系统损伤的影响

目的 研究纳米氧化铈在γ射线诱导小鼠免疫系统损伤中的影响。方法 BALB/c小鼠120只按随机号法分为健康对照、照射、阳性药物与纳米氧化铈100、300和900 mg/kg组,共6组,每组20只。小鼠经3.5 Gy ⁶⁰Co γ射线一次性全身照射,照射前两周至照射后处死前连续口服灌胃给药。流式细胞术检测胸腺淋巴细胞凋亡率、外周血白细胞介素-2（IL-2）和干扰素-γ（IFN-γ）阳性率,二苯基四氮唑溴盐（MTT）法检测自然杀伤细胞（NK）活性。结果 与照射组相比,受照后3 d,纳米氧化铈各剂量组的细胞死亡率均降低,纳米氧化铈300 mg/kg组差异有统计学意义（F=4.50,P<0.05）,纳米氧化铈300 mg/kg组的IFN-γ表达显著增多（t=2.26,P<0.05）;受照后8 d,纳米氧化铈900 mg/kg组的细胞死亡率明显降低（F=4.07,P<0.05）,纳米氧化铈100 mg/kg组细胞早期凋亡率显著下降（F=3.47,P<0.05）,纳米氧化铈300 mg/kg组IFN-γ表达显著增多（t=2.47,P<0.05）,各剂量组IL-2表达均呈降低趋势,但差异无统计学意义（P>0.05）;纳米氧化铈各剂量组和阳性药物组的NK细胞活性均显著升高,差异有统计学意义（t=3.40、5.40、3.40、5.20,P<0.05）。结论 纳米氧化铈在一定程度上降低受照小鼠胸腺淋巴细胞凋亡率和死亡率,促进IFN-γ表达量增高,增强NK细胞活性,可提高机体免疫力,对辐射损伤有一定防护作用。     

电离辐射对小鼠胸腺Th17细胞相关细胞因子的影响

目的 通过研究电离辐射对小鼠胸腺Th17细胞相关细胞因子的影响,探讨高、低剂量辐射诱导不同的免疫效应中Th17细胞功能。方法 将健康ICR小鼠按随机数字表法分为健康对照组、低剂量照射组(0.05、0.075 Gy)和高剂量照射组(0.5、1.0、2.0、4.0 Gy)探讨剂量-效应关系,用X射线深部治疗机进行不同剂量的全身照射,于照射后24 h处死。同时,探讨时间-效应关系,即分为健康对照组、低剂量照射组(0.075 Gy)和高剂量照射组(2.0 Gy),于照射后12、24、48 h处死,取胸腺组织制备成组织匀浆,ELISA法检测小鼠胸腺细胞中白介素-17a(IL-17a)与白介素-21(IL-21)的浓度。结果 在时间-效应结果中,与健康对照组相比,0.075 Gy照射组胸腺细胞IL-17a和IL-21分泌量呈下降趋势,其分泌量于48 h降到最低(t=3.85、4.73,P<0.05);而2.0 Gy照射组均呈大幅度上升趋势,其分泌量在48 h达到最高(t=-6.74、-6.19,P<0.05);在剂量-效应结果中,与健康对照组相比,较低剂量照射组胸腺细胞IL-17a与IL-21分泌量下降,在0.05 Gy最低(t=8.39、16.45,P<0.05);较高剂量照射组胸腺细胞的分泌量上升,在4.0 Gy时升至最高(t=-15.60、-18.62,P<0.05)。结论 高剂量辐射可以诱导小鼠胸腺细胞IL-17a与IL-21分泌量的增加,而低剂量辐射使其下降,表明Th17细胞相关的细胞因子在低剂量辐射诱导的免疫功能增强效应和高剂量辐射诱导免疫抑制效应中起着重要的作用。     

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.     

Essays in Biochemistry,Vol. 2

Purpose:?The exploration of immune mechanisms of the tumour-inhibitory effect of exposures to low-level fractions of X-rays.

Materials and methods:?BALB/c mice were exposed to whole-body daily irradiations with 0.01, 0.02, or 0.1 Gy X-rays per day for 5 days/week for two weeks. Then, mice were intravenously injected with L1 tumour cells, killed 14 days later, and neoplastic colonies were counted in the lungs. Natural killer (NK) cell-enriched splenocytes and activated peritoneal macrophages (M?) were collected and cytotoxic activities of these cells against susceptible tumour targets were assayed. Concanamycin A (CMA) and antibody against the ligand for the Fas receptor (FasL) were used to inhibit the NK cell-mediated cytotoxicity. Production of nitric oxide (NO) was quantified using the Griess reagent. Secretion of interferon-γ (IFN-γ), interleukin-1β (IL-1β), interleukin-12 (IL-12), and tumour necrosis factor-α (TNF-α) was measured using the enzyme-linked immunosorbent assays.

Results:?All the exposures to X-rays significantly reduced the number of the induced tumour colonies and enhanced cytotoxic properties of the NK cell-enriched splenocytes and activated M?.

Conclusion:?Suppression of the growth of pulmonary tumour colonies by irradiations of mice with low-dose fractions of X-rays may result from stimulation of anti-tumour reactions mediated by NK cells and/or cytotoxic macrophages.     

Egr-IFNγ基因治疗联合125I-脱氧尿嘧啶核苷治疗抑瘤效应的实验研究

目的 探讨Egr-IFNγ基因治疗联合放射性核素 ¹²⁵I -脱氧尿嘧啶核苷治疗方案在荷H22肝癌细胞小鼠体内抑瘤效应及机制。方法 小鼠肿瘤局部注射脂质体包裹的质粒,注射后48 h,肿瘤局部注射370kBq ¹²⁵I -UdR。观察各组小鼠治疗后不同时间肿瘤生长率;治疗后第3 天,检测肿瘤胞浆蛋白中IFNγ的表达和脾脏CTL细胞毒活性。结果 基因-放射核素治疗后第6～15天,pcDNAEgr-IFNγ+ ¹²⁵I -UdR组肿瘤生长率明显低于对照组、 ¹²⁵I -UdR组及pcDNAEgr-1+ ¹²⁵I -UdR 组;基因-放射性核素治疗后第3天,pcDNAEgr-IFNγ+ ¹²⁵I -UdR组肿瘤胞浆蛋白中可检测到IFNγ的表达,其余组肿瘤胞浆蛋白中未检测到IFNγ的表达;pcDNAEgr-IFNγ+ ¹²⁵I -UdR组小鼠脾脏CTL细胞毒活性明显高于其余组 (P<0.01)。结论 pcDNAEgr-IFNγ基因治疗联合放射性核素 ¹²⁵I -UdR治疗抑瘤效应明显优于单纯 ¹²⁵I -UdR放射性核素治疗。     

Alterations of immune functions induced by 12C6+ ion irradiation in mice

Purpose:?To estimate the biological risks to the immune system of the type of space radiation, 12C6+, encountered by cosmonauts during long-term travel in space.

Materials and methods:?The Kun-Ming strain mice were whole-body irradiated by 12C6+ ion with 0, 0.01, 0.05, 0.075, 0.2, 0.3, 0.5, 0.75, 1 or 2 Gy, at a dose rate of 1 Gy/min. At 35 days after irradiation, the thymus and spleen weights were measured, the natural killer (NK) cells activity of spleen was determined by 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT), and the interferon-γ (IFN-γ) levels in serum and thymus were detected with enzyme-linked immunosorbent assays (ELISA).

Results:?The results showed that the thymus weight, IFN-γ levels in serum and the activity of splenic NK-cells had significantly increased at a dose of 0.05 Gy. With further dose increase, the weight of spleen continued to increase but the weight of thymus, IFN-γ level and NK-cells activity declined.

Conclusions:?These results suggest that the dose of 0.05 Gy irradiation has a stimulatory effect on mouse immunity; this effect declined with increasing dose.     

常规军训对新兵血清细胞因子变化的影响研究

目的研究常规军训对新兵血清细胞因子浓度的影响,判断机体损伤及适应程度。方法选择2010年底新入伍新兵共80人,在常规军训前及军训1个月后分别采静脉血,进行血清IL-2、IL-6、IL-18、TNF-α、IFN-γ等细胞因子检测,并进行前后对比。结果血清IL-6、TNF-α、IFN-γ浓度在军训前后显示良好相关性,证明其变化在所有研究对象中呈现一致性;IL-2、IL-6、IFN-γ浓度值在军训前后显著降低。结论常规军训能够促进新入伍新兵免疫功能,提升机体对训练引起细胞损伤的抵抗力。