体外转染荧光素酶的 4 级星形细胞瘤细胞的生物发光激活光动力治疗。

Bioluminescence-activated photodynamic therapy for luciferase transfected, grade 4 astrocytoma cells in vitro.

机构信息

UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom; National Medical Laser Centre (now Division of Surgery and Interventional Science), University College London, Charles Bell House 43-45 Foley Street, London W1W 7TS, United Kingdom.

UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom.

出版信息

Photodiagnosis Photodyn Ther. 2022 Jun;38:102856. doi: 10.1016/j.pdpdt.2022.102856. Epub 2022 Apr 6.

DOI:10.1016/j.pdpdt.2022.102856

PMID:35398261

Abstract

BACKGROUND

. Grade 4 astrocytoma is incurable due to the diffusely infiltrative nature of the disease. Photodynamic therapy (PDT) is a promising therapeutic option, but external light delivery is not feasible when cancer cells infiltrate unknown areas of normal brain. Hence the search for endogenous sources such as bioluminescence that can generate light at cancer cells. This requires a substrate (a luciferin) and an enabling enzyme (a luciferase), neither seen in mammalian cells.

METHODS

. Preliminary studies confirmed that U87 cells (derived from a human grade 4 astrocytoma) could be killed by conventional PDT using the photosensitizers hypericin or mTHPC. U87 cells were then transfected with firefly and other luciferases and light generating cell lines (U87-luc, U87-hRluc, U87-CBG68luc) identified using the appropriate substrate. Reagent doses and conditions were optimized and U87-luc cells incubated with hypericin or mTHPC with d-luciferin added to initiate bioluminescence activated PDT (bPDT). Cell survival was assessed by MTT assay, haemocytometry and growth assay. Control groups included U87-luc cells with no added active reagents, substrate only, photosensitizer only and non-transfected U87 cells. Results were expressed as a percentage of surviving cells compared with untreated U87-luc controls.

RESULTS

. There was no bPDT effect on non-transfected cells. The mean survival of treated transfected cells was 36%, (P<0.001) using hypericin and 35% (P<0.001) using mTHPC, compared with untreated U87-luc cells. bPDT effects were suppressed by the anti-oxidant, lycopene.

CONCLUSIONS

. bPDT can kill Grade 4 astrocytoma cells transfected with luciferase in vitro. This justifies progression to in vivo studies.

摘要

背景

由于疾病的弥漫浸润性质，4 级星形细胞瘤是无法治愈的。光动力疗法（PDT）是一种很有前途的治疗选择，但是当癌细胞浸润到正常大脑的未知区域时，外部光照输送是不可行的。因此，人们一直在寻找内源性的光源，如生物发光，可以在癌细胞中产生光。这需要一种底物（荧光素）和一种有效的酶（荧光素酶），而哺乳动物细胞中都没有这两种物质。

方法

初步研究证实，U87 细胞（源自人类 4 级星形细胞瘤）可以通过使用血卟啉或 mTHPC 等光敏剂进行传统 PDT 杀死。然后，将 U87 细胞转染入萤火虫和其他荧光素酶，并使用适当的底物鉴定出发光细胞系（U87-luc、U87-hRluc、U87-CBG68luc）。优化了试剂剂量和条件，并在添加 d-荧光素的情况下用 hypericin 或 mTHPC 孵育 U87-luc 细胞，以启动生物发光激活 PDT（bPDT）。通过 MTT 测定、血细胞计数和生长测定评估细胞存活率。对照组包括未添加活性试剂的 U87-luc 细胞、仅添加底物、仅添加光敏剂和未转染的 U87 细胞。结果以与未经处理的 U87-luc 对照组相比存活细胞的百分比表示。