膀胱癌组织特异性溶瘤腺病毒的安全性评估

发布时间：2018-08-21 14:16

【摘要】：目的:对于膀胱癌的治疗目前仍以手术、放疗、化疗为主;此外,还包括辅助性化疗以提高肿瘤的治疗效果。尽管如此,但是膀胱癌仍有较高的的复发率。新型膀胱癌特异性溶瘤腺病毒的研究已成为当代膀胱癌生物治疗研究的热点,它可以在膀胱肿瘤细胞内特异性的增殖并且杀灭肿瘤肿瘤细胞,是肿瘤靶向治疗的重要方法。目前对溶瘤腺病毒的疗效已经进行了相应的研究,但是安全性和毒性仍存在较大的争议,并且已报道的溶瘤腺病毒的毒性主要集中于急性毒性实验和长期毒性实验,对于溶瘤腺病毒的生殖毒性的研究仍未见报道。因此本课题将我们实验室构建的膀胱癌特异性溶瘤腺病毒Ad-PSCAE-UPⅡ-E1A(APU-E1A)进行一般生殖毒性实验和致畸毒性实验;评价胎鼠的骨骼结构,并且观察F1代的实质内脏组织的发育情况。为溶瘤腺病毒的安全性提供基本的数据支持。方法:我们委托北京五加和公司将我们实验室已经构建完成的膀胱癌特异性溶瘤腺病毒Ad-PSCAE-UPⅡ-E1A(APU-E1A)进行扩增、纯化和鉴定。小鼠购买与甘肃中医药大学动物实验室。整个安全性评价体系分为三部分:急性毒性实验,一般生殖毒和致畸毒性实验。急性毒性是将扩增的溶瘤腺病毒以腹腔给药的方式一次性注射进入小鼠的腹腔,观察小鼠的行为改变和体重的变化,14天后处死小鼠检测小鼠血液中淋巴细胞和血小板以及肝脏谷丙转氨酶(ALT)和谷草转氨酶(AST)水平的测定;病理组织的切片进行HE染色后,观察溶瘤腺病毒对于各组织脏器的镜下病理改变。一般生殖毒性实验是指将小鼠给药之后,进行合笼交配,观察胎鼠和子代小鼠的发育情况;首先处理雄性小鼠:先将雄性小鼠连续给药28天,给药方式为肌肉注射,给药的时间为隔日给药;雌性小鼠,在雄性小鼠给药两周后开始给药,连续给药14天,给药方式为肌肉注射,给药时间为隔日给药;完成给药后将雌雄小鼠合笼交配。交配成功后将受孕小鼠分为两部分,一部分在受孕16天后进行安乐死,然后取出胎鼠,去除内脏和皮肤进行骨骼染色,观察骨骼改变。另一部分自然分娩,将出生后的小鼠饲养30天后处死,测定小鼠的体重变化,并且对各组织进行病理切片,观察组织在在镜下改变。致畸毒性实给药方案:将交配后的雌性小鼠在第六天开始给药,隔日给药一次,直到受孕后第14天;一部分在第16天后安乐死,取出胎鼠,观察胚胎骨骼结构的变化,另一部分自然分娩后,自然生长30天,观察生长发育过程中的改变,然后将小鼠处死,将各组织制作成病理切片,观察进行HE染色后的镜下改变。结果在小鼠的急性毒性实验中,小鼠出现扭体等腹痛的体征,实验组小鼠的体重增加快于对照组,具有统计学的差异;另外在小鼠的解剖发现实验组小鼠的的腹腔出现血性腹水,肝脏呈现出亚急性重型肝炎的表现,镜下观察小鼠的肝脏组织呈现出严重的肝组织坏死,正常的肝小叶结构遭到破坏;其他组织脏器均无明显的变化。在一般生殖毒实验的研究中发现,对照组与低剂量组、中剂量组、高剂量组之间比较,各组之间胎鼠的窝重和胎盘重均无明显的改变,胎鼠的体长和尾长之间的差异也无统计学的意义,小鼠的骨骼染色均显示正常,在子一代的发育过程之中,各组之间小鼠的体重改变有统计学的意义,但是这种差异通过小鼠的体重增长变化的曲线发现差异不大,这可能是由于个体之间的差异和受孕时雌鼠的体重不同造成的。在小鼠的致畸毒性实验的过程之中,低中高三个剂量组与对照组之间进行比较,胎鼠的胎盘和窝重各组之间的差异无统计学意义,在胎鼠的体长和尾长之间的差异也无统计学的意义,在子一代的发育过程之中,小鼠的体重增加有统计学的意义,但是从小鼠的生长曲线上看,这种差异不大,由于小鼠的各组之间生长曲线显示各组之间的差异不大,所以不认为这是由于溶瘤腺病毒所引起。结论:我们所构建的膀胱组织特异性溶瘤腺病毒((Ad-PSCAE-UPII-E1AAR(APU-E1A-AR)在急性毒性试验中,可能具有较为严重的肝脏毒性,肝脏可能为溶瘤腺病毒的靶器官;另外在给予治疗剂量的100倍时,无小鼠死亡,说明此种腺病毒的安全性还是值得肯定的。在小鼠的一般生殖毒性试验和致畸毒性实验中,各组织间的差异不是很大,无明显的改变,因此,认为我们所构建的溶瘤腺病毒对于小鼠的生殖系统的影响不大,是一种相对较为安全的肿瘤的基因治疗生物制剂,对于怀孕的病人和负责肿瘤治疗的医护人员的生育能力和致畸性影响较小
[Abstract]:Objective: Currently, the treatment of bladder cancer is still mainly surgery, radiotherapy, chemotherapy; in addition, it also includes adjuvant chemotherapy to improve the therapeutic effect of tumor. Nevertheless, bladder cancer still has a high recurrence rate. Specific proliferation and killing of tumor cells in bladder cancer cells is an important method for tumor targeted therapy. At present, the efficacy of oncolytic adenovirus has been studied, but the safety and toxicity of oncolytic adenovirus are still controversial. The reported toxicity of oncolytic adenovirus mainly focuses on acute toxicity test. The reproductive toxicity of oncolytic adenovirus (oncolytic adenovirus Ad-PSCAE-UP II-E1A), which was constructed in our laboratory, has not been reported for a long time. Methods: Beijing Wujiahe Company was commissioned to amplify, purify and identify the adenovirus Ad-PSCAE-UP II-E1A (APU-E1A), which has been constructed in our laboratory. The mice were purchased from the animal laboratory of Gansu University of Traditional Chinese Medicine. The whole safety evaluation system is divided into three parts: acute toxicity test, general reproductive toxicity test and teratogenic toxicity test. Acute toxicity is to inject the amplified oncolytic adenovirus into the abdominal cavity of mice by intraperitoneal administration, observe the changes of behavior and weight of mice, and sacrifice the mice 14 days later to detect the lymphatic fineness in the blood of mice. Cell and platelet levels of alanine aminotransferase (ALT) and glutamic oxaloacetic aminotransferase (AST) in the liver were measured; histological sections were stained with HE to observe the microscopic pathological changes of oncolytic adenovirus in various tissues and organs. Firstly, male mice were administered by intramuscular injection for 28 days in succession, and the time of administration was every other day. Female mice were administered by intramuscular injection for 14 days in succession after two weeks in male mice. After successful mating, the pregnant mice were divided into two parts. One part was euthanized 16 days after conception, then the fetus was removed, the viscera and skin were removed and stained to observe the bone changes. The teratogenic dosage regimen: female mice after mating were administered on the sixth day, once every other day, until the 14th day after conception; one was euthanized on the 16th day, fetal mice were taken out to observe the changes of embryonic skeletal structure; the other was born naturally for 30 days, and the growth and development were observed. Results In the acute toxicity test of mice, abdominal pain such as writhing occurred in mice, and the weight of mice in the experimental group increased faster than that of the control group, with statistical differences; in addition, in the experiment of mice anatomical discovery. The abdominal cavity of mice in group A showed blood ascites, and the liver showed subacute severe hepatitis. The liver tissue of mice in group A showed severe necrosis of liver tissue, and the normal structure of liver lobules was destroyed. There were no significant changes in the nest weight and placental weight of fetal mice between the middle and high dose groups. There was no significant difference in the length and tail length of fetal mice. The difference was found in the weight growth curve of mice, probably due to differences between individuals and in the weight of pregnant females. During the teratogenic test in mice, the differences in placenta and nest weight between the low, medium and high dose groups and the control group were compared. There was no statistically significant difference in fetal and tail lengths. During the development of the offspring, weight gain was statistically significant. However, from the growth curve of the mice, the difference was not significant, because the growth curves of the mice showed little difference between the groups, so Conclusion: The adenovirus-specific adenovirus of bladder tissue (ADPSCAE-UPII-E1AAR) (APU-E1A-AR) may have more severe hepatotoxicity in acute toxicity test, and the liver may be the target organ of oncolytic adenovirus. In the general reproductive toxicity test and teratogenic toxicity test in mice, there was no significant difference between the tissues. Therefore, it was considered that the oncolytic adenovirus we constructed had little effect on the reproductive system of mice and was a relatively safe swelling. Gene therapy biologics for tumors have less impact on fertility and teratogenesis in pregnant patients and health care workers responsible for cancer treatment
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14

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