太空环境诱变黑色素瘤细胞的实验研究

目的观察太空环境对B16细胞生物学特性的影响，寻找免疫原性增强的细胞株。方法将小鼠黑色素瘤B16细胞搭载于第20颗返回式卫星，返地后单克隆化，选择1株形态变异的单克隆细胞株（1^#），MTT法检测细胞增殖情况，接种C57BL／6J小鼠，分别观察细胞致瘤性、荷瘤小鼠免疫器官、生存期及瘤体病理变化。结果1^#太空诱变细胞株，形态呈现树突状，增殖速率与对照细胞比较差异无统计学意义（P〉0．05），接种C57BL／6J小鼠，肿瘤潜伏期延长（P〈0．01），瘤体重量减小（P〈0.01），胸腺系数增大（P〈0．01），且荷瘤小鼠生存期延长（P〈0，01），而脾系数与对照组比较差异无统计学意义（P〉0．05），病理学结果显示：1^#细胞组瘤体内血管不及对照组丰富，坏死区较少，瘤体内浸润淋巴细胞较对照组有不同程度的增多，瘤旁组织内有较多淋巴细胞。结论空间诱变1^#细胞株可能趋向分化，体内实验结果提示：1^#诱变细胞株抗原性可能被增强，激发了小鼠的免疫应答，该结果为进一步筛选免疫原性增强的细胞株，确定抗原的强化特征提供了实验基础。     

黑色素瘤细胞经太空环境诱变后致瘤性和基因表达变化

目的 筛选经太空环境诱变后B16细胞致瘤性改变的细胞株,观察其基因表达的改变,寻找与黑色素瘤发生及转移密切相关的基因.方法 将小鼠黑色素瘤B16细胞搭载于第20颗返回式卫星,返地后克隆化,随机选择5株克隆细胞株（1^#、5^#、6^#、7^#和8^#）,接种C57BL/6J小鼠,分别观察细胞致瘤性、荷瘤小鼠生存期及瘤体病理变化,用基因表达谱分析其中2株初筛致瘤性改变细胞株（1^#和8^#）的基因表达情况。结果 与对照组比较,1#细胞株接种小鼠后,肿瘤潜伏期延长（P＜0.05）,1^#和8^#细胞株移植瘤瘤体重量减小（P＜0.01）,且荷瘤小鼠生存期延长（P＜0.01）;病理诊断结果显示：1^#和8^#细胞移植瘤内血管不及对照组丰富,坏死区较少,瘤体内浸润淋巴细胞较对照组有不同程度的增多,瘤旁组织内有较多淋巴细胞;基因芯片分析结果表明：与对照B16细胞相比,2株诱变细胞（1^#和8^#）分别有145和125个基因表达水平发生改变（P＜0.05）,其中9个基因为共同变化基因,前列腺素D2合成酶基因在2株细胞中的表达均明显增高,与对照相比,差别在5倍以上。结论 1^#和8^#细胞株致瘤性减弱,且2株细胞的移植瘤肿瘤新生血管及癌旁浸润减少,癌旁组织中有较多淋巴细胞,推测可能与前列腺素D2合成酶等多种肿瘤相关基因表达的改变有关。     

太空环境对黑色素瘤B16细胞生物学特性的影响

目的初步研究太空环境对黑素瘤B16细胞生物学特性的影响.方法将B16细胞搭载于第20颗返回式卫星,返地后回收细胞,单克隆化,获得110株太空B16细胞,按顺序编号.从中选取10株的太空B16单克隆细胞株,利用光镜观察其形态变化,MTT方法检测细胞增殖曲线,FCM测定细胞DNA含量,观察太空诱变细胞周期分布.结果与对照细胞相比,太空B16细胞形态改变,有些细胞表面可见其分泌的黑色素颗粒;细胞增殖速率呈多向性变异;太空B16细胞G1期延长,S期缩短.结论太空环境的复合因素可诱导B16细胞生物学特性发生变化.     

类球红细菌空间诱变效应研究

目的利用空间诱变手段处理类球红细菌,通过诱变后菌株生理生化情况的变化,筛选辅酶Q10(CoQ10)高产菌株,为空间诱变手段筛选产业化菌株的应用提供实验和理论依据。方法利用返回式卫星搭载类球红细菌,检测搭载后菌株的抗逆性及发酵CoQ10能力。结果空间搭载后筛选到生长温度、pH值、盐浓度范围变宽,抗逆性增强的菌株,同时菌落颜色出现乳白色、粉红色等颜色变异菌株。从颜色变浅的变异菌株中筛选到一株CoQ10高产菌,发酵生产CoQ10比对照高73.13%。结论与传统单因素诱变方式相比,空间诱变是多因素综合作用,诱变效果明显,能获得高抗逆性和高CoQ10生产能力的突变株,可以作为产业化菌株筛选的诱变方式。     

太空环境对生物工程细胞CHO(dhfr-)的影响

目的 探讨空间环境对细胞的影响. 方法 利用基因工程细胞的航天搭载装置将细胞进行无源搭载,将生物工程CHO（dhfr-）细胞搭载于第18颗返回式卫星.随机选取3株诱变细胞株,MTT法测定细胞生长曲线,流式细胞仪检测细胞周期,同时观察诱变细胞染色体变化,检测细胞周期调控相关基因p21Cip1/Waf1表达情况. 结果 太空诱变CHO（dhfr-）细胞增殖速度出现快慢不一的变化;细胞周期分布改变,G1期细胞显著增多（P〈0.05）,S期细胞明显减少（P〈0.05）;细胞染色体异常核型增加;p21^Cip1/Waf1基因表达上调,mdm2基因没有明显变化. 结论 太空环境可导致细胞产生多种变异,为改造、筛选优化哺乳动物宿主细胞表达系统提供了新手段.     

IL—2基因修饰的小鼠肥大细胞瘤细胞体外增殖活性的观察

ＩＬ２基因修饰的小鼠肥大细胞瘤细胞体外增殖活性的观察李秀森郭宁刘晓丹张明伟杜德林毛宁军事医学科学院基础医学研究所北京１００８５０细胞因子基因转导可能降低肿瘤细胞的致瘤原性，提高肿瘤细胞的免疫原性〔１～３〕。动物实验结果表明，细胞因子基因修饰的肿瘤细胞...     

泰乐菌素产生菌的空间诱变育种研究

目的研究弗氏链霉菌(streptomyces fradioe)在空间条件下的变异规律,并筛选高产菌株应用于工业生产。方法将弗氏链霉菌(streptomyces fradiae)9940S^ _86连续经“神舟”1、3、4号飞船搭载进行太空诱变育种,统计筛选结果,初步探讨泰乐菌素产生菌在空间条件下的变异规律。结果复筛后得到48株效价较出发菌株提高20％以上的菌株,其中总效价最高达到14950μg／ml(摇瓶),较出发菌株提高91．5％。结论弗氏链霉菌的空间诱变受飞行时间影响较大。菌株经多次搭载后其产量变异有累积作用,并且产量变异和形态变异存在相关性。通过中试、生产实验,最终选定产量较出发生产菌株提高18％的T1—156—84—23菌株投入试生产。     

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。     

癌细胞瘤苗的小鼠抗瘤作用

高温治疗癌肿病人近年来国外学者都很重视。国内黄皎琳等利用局部高温热疗机高温治疗肿瘤以来,在这方面研究取得一定进展,并首次报道热固化后癌细胞具有一定的抗瘤作用。作者利用常规水浴方法加热处理活性小鼠肝癌H_(22)细胞株,筛选出65℃/30min处理H_(22)细胞,其抗瘤效果较佳。在此基础上,本实验用H_(22)肝癌细胞经水浴加温灭活后,制备成癌细胞固化瘤苗,免疫被接种的H_(22)荷瘤小鼠,提高小鼠被动免疫力。观察H_(22)细胞热固化瘤苗的抗癌保护作用。     

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。     

大蒜素对裸鼠体内人肺腺癌细胞SLC-89增殖的影响

目的观察大蒜素对裸鼠体内人肺腺癌细胞SLC-89增殖的影响。方法采用小鼠肉瘤S180腹水上清悬浮人肺腺癌SLC-89细胞后皮下注射SLC-89细胞给裸鼠的方法制备肿瘤模型,分别在注射细胞的同时和注射细胞后15d,连续45d腹腔注射大蒜素,对照组每天腹腔注射同等体积的生理盐水。然后处死裸鼠,测定肿块的重量,并取肿瘤组织作HE染色。结果采用小鼠肉瘤S180腹水上清悬浮人肺腺癌SLC-89细胞后每只裸鼠皮下注射5×106个SLC-89细胞的方法,可以有效复制人肺腺癌裸鼠模型,成瘤率为100％。注射细胞的同时每只裸鼠连续45d按照15mg／（kg·d）腹腔注射大蒜素溶液,所产生的肿瘤重量明显低于对照组（P〈0．05）;移植细胞后15d,再给每只裸鼠连续45d注射相同剂量的大蒜素,所产生的肿瘤重量与对照组差异不明显（P〉0．05）。与对照组比较,注射细胞的同时就给予大蒜素治疗的裸鼠肿瘤细胞减少或有肿瘤组织坏死。结论按照15mg／（kg·d）的剂量腹腔注射大蒜素,能减缓裸鼠体内人肺腺癌肿块的形成。     

空间环境对黑色素瘤细胞基因表达谱的影响

目的探讨空间环境对黑色素瘤B16细胞基因表达谱的影响。方法利用“细胞在常温条件下长期存活装置”将B16细胞进行无源搭载。返地后常规培养，以常规培养的野生型B16细胞及地面装置组作为对照，甲基噻唑基四唑法测定细胞生长曲线，基因芯片分析细胞18240个基因的差异表达。结果搭载细胞返地培养后，与对照组细胞相比，生长速率差异没有显著性意义。与常规培养的对照相比，地面装置组细胞及空间组细胞分别有61及156个基因发生明显改变；其中35个基因为地面装置组及空间组细胞二者共同与常规对照组细胞差异表达，30个基因为空间培养细胞与地面装置组细胞之间差异表达；差异表达的基因涉及Wnt及Ca^2＋等信号传导途径。结论空间环境作用后，B16肿瘤细胞基因表达改变，一些信号传导途径及能量代谢表现为对空间环境较为敏感。     

Melanoma growth and tumorigenicity in models of microgravity

INTRODUCTION: Spaceflight involves numerous biological stressors that could affect long-term cancer incidence and tumor behavior. Ground-based models of microgravity can be used to investigate in vitro and in vivo tumor growth as a preparation for later work in space. The incidence of tumor growth and carcinogenesis in microgravity is as yet unknown. Hence, we investigated the effects of modeled microgravity on tumor growth and tumorigenicity using ground-based in vitro and in vivo models. METHODS: Murine B16-F10 melanoma cells were cultured in a tissue culture flask (FL) and in a rotating-wall vessel bioreactor (BIO) designed by NASA to simulate some aspects of microgravity. We then measured cell growth, melanin production, and apoptosis. After 48 h of cultures in FL and BIO, cells were inoculated subcutaneously in C57BL/6 mice, syngeneic hosts for B16-F10 tumor cells. Tumor sizes were then measured every other day. RESULTS: BIO cultures had 50% decreases in growth when compared with FL cultures while demonstrating an inversely proportional increase in doubling time. Melanin production (a marker of differentiation) increased at 24 and 48 h in BIO. Flow cytometry analysis demonstrated that there was an increase in the percentage of apoptotic cells in the BIO when compared with that in the FL. When BIO-cultured melanoma cells were inoculated subcutaneously in mice, there was a significant increase in tumorigenicity as compared with FL-cultured cells. CONCLUSION: Our results indicate that simulated microgravity may have altered the tumor cell characteristics and enhanced the invasive property. It is possible that the microgravity analogue culture environment may have selected highly tumorigenic cells for survival despite the decreased overall growth in the microgravity analogue.     

The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity.

We have investigated the gamma-ray sensitivity of several activated c-H-ras (EJ) containing clones that have been established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to their tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p21); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p21; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p21 ras protein similar to clone I-7. Comparison of the single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2 Gy (S2) indicate that there appears to be no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance.     

新CpG寡聚脱氧核苷酸体内抗肿瘤与增强免疫活性研究

目的:探讨新设计CpG寡聚脱氧核苷酸(ODN)在小鼠体内的抗肿瘤作用及其对荷瘤小鼠免疫功能的影响.方法:建立C57BL/6小鼠腹腔、皮下荷黑素瘤肿瘤模型,腹腔注射CpG ODN,观察荷瘤鼠生存时间、肿瘤生长曲线,计算抑瘤率.用酶联免疫吸附试验(ELISA)检测小鼠血清中白细胞介素(IL)12和免疫球蛋白E(IgE)含量;3H-TDR掺入法检测脾脏B细胞、T细胞增殖活性;51Cr释放法检测NK细胞杀伤活性;中性红法检测腹腔巨噬细胞吞噬功能.结果:CpG10,CpG11能明显延长腹腔接种肿瘤小鼠的生存时间,与阳性对照CpG1826相比P＜0.01;二者平均抑瘤率(皮下荷瘤鼠)分别为(55.2±2.3)%与(40.7±1.7)%,显著高于CpG 1826 [抑瘤率(17.8±7.6) %](P＜0.05,P＜0.01).CpG10/CpG11可促使荷瘤小鼠血清IL-12含量显著升高( P＜0.01),IgE含量显著下降( P＜0.01);明显刺激B细胞、T细胞增殖能力以及NK细胞的杀伤活性( P＜0.01),对腹腔巨噬细胞的吞噬功能有显著提高( P＜0.01).CpG10免疫增强活性较CpG1826更强( P＜0.05).结论:CpG ODN能激活荷瘤鼠抗肿瘤免疫反应,从而抑制小鼠黑素瘤的生长,新结构寡核苷酸的CpG10呈现优于阳性对照的良好抗肿瘤免疫效应.     

Biodistribution of 99mTC-HMPAO in healthy rats and B16 melanoma carriers

Scintigraphic studies carried out in melanoma patients have demonstrated that the 99mTc-HMPAO complex makes it possible to locate the lesion. A biodistribution and pharmacokinetic study of the 99mTc-HMPAO complex was carried out in B16-melanoma tumor healthy and carrier mice after an intravenous injection. Radioactivity was measured in the liver, kidneys, spleen, stomach, brain, blood and tumor. It was seen that at 15 minutes of the injections, 40% of the total activity distributed in the animal body was recorded in the tumor. An interesting effect observed is an increase in the tissue distribution curves in both experimental groups at 1-2 hours post-injection. According to the seriated imaging study results with 99mTc-HMPAO in B16 melanoma bearing mice, the best image is obtained 10-30 minutes after the injection.     

Preparation and biologic evaluation of a novel radioiodinated benzylpiperazine, 123I-MEL037, for malignant melanoma.

Radiopharmaceuticals that can target the random metastatic dissemination of melanoma tumors may present opportunities for imaging and staging the disease as well as potential radiotherapeutic applications. A novel molecule, 2-(2-(4-(4-(123)I-iodobenzyl)piperazin-1-yl)-2-oxoethyl)isoindoline-1,3-dione (MEL037), was synthesized, labeled with 123I, and evaluated for application in melanoma tumor scintigraphy and radiotherapy. METHODS: The tumor imaging potential of 123I-MEL037 was studied in vivo in C57BL/6J female mice bearing the B16F0 murine melanoma tumor and in BALB/c nude mice bearing the A375 human amelanotic melanoma tumor by biodistribution, competition studies, and SPECT. RESULTS: 123I-MEL037 exhibited high and rapid uptake in the B16F0 melanoma tumor at 1 h (13 %ID/g [percentage injected dose per gram]), increasing with time to reach 25 %ID/g at 6 h. A significant uptake was also observed in the eyes (2 %ID, at 3-6 h after injection) of black mice. No uptake was observed in the tumor or in the eyes of nude mice bearing the A375 tumor. Because of high uptake and long retention in the tumor and rapid body clearance, the mean contrast ratios (MCR) of 123I-MEL037 were 30 and 60, at 24 and 48 h after injection, respectively. At 24 h after injection of mice bearing the B16 melanoma, SPECT indicated that the radioactivity was located predominately in the tumor followed by the eyes, whereas no specific localization of the radioactivity was noted in mice bearing the A375 human amelanotic tumor. In competition experiments, uptake of 123I-MEL037 in brain, lung, heart, and kidney--organs known to contain sigma-receptors--was not significantly different in haloperidol-treated animals compared with control animals. Therefore, reduction of uptake in tumor and eyes of the pigmented mice bearing the B16F0 tumor suggested that the mechanism of tumor uptake was likely due to an interaction with melanin. CONCLUSION: These findings suggested that 123I-MEL037, which displays a rapid and very high tumor uptake, appeared to be a promising imaging agent for detection of most melanoma tumors with the potential for development as a therapeutic agent in melanoma tumor proliferation.