Yusof, Siti Fairuz Mohd
(2018)
The role of ranibizumab as an adjunctive agent in mitomycin-C-treated glaucoma tube implantation on conjunctival fibroblast tissue culture.
Masters thesis, Universiti Sains Malaysia.
Abstract
Elevated expression of vascular endothelial growth factor (VEGF) usually
occurs after the glaucoma surgery. The elevated expression of VEGF is unnecessary
as it contributes to excessive scarring which will fail the surgery. Therefore, this
study investigated the potential role of ranibizumab, an anti – vascular endothelial
growth factor (anti – VEGF), as anti – adjunctive agent in preventing the excessive
fibrosis. The aim of this study is to determine the effect of adjunctive use of
ranibizumab with mitomycin – C on proliferation, cytotoxicity and migration of
human conjunctival fibroblasts (HConFs) cell line. In proliferation and cytotoxicity
study, HConFs were cultured in fibroblast medium and treated with 0.40 mg/ml
mitomycin – C (MMC). The cultures were then administered with ranibizumab at
different concentration (0 mg/ml, 0.30 mg/ml, 0.45 mg/ml and 0.60mg/ml). Viability
and proliferation of HConFs were assessed at 24, 48 and 72 hours by alamarBlue
assay. For cytotoxicity, the apoptosis rate of treated HConFs was evaluated via flow
cytometry using Annexin-V FITC and propidium iodide staining after 72 hours
treatment. Meanwhile, for morphology and migration study, in vitro model of
glaucoma tube implantation was used. HConFs were treated according to 4 groups;
control, MMC 0.40 mg/ml, ranibizumab 0.45 mg/ml and MMC 0.40 mg/ml
+ranibizumab 0.45 mg/ml. Migration of HConFs onto the tube was observed at day
7,14 and 21 through alamarBlue assay and scanning electron microscope (SEM).
Application of ranibizumab following MMC caused a dose – dependent inhibition of
HConF’s viability and proliferation which significant after 48 and 72 hours. It was
shown that HConF’s proliferation was significantly reduced by ranibizumab at
concentration 0.45 mg/ml, (p<0.05). In fact, surprising higher number of HConF was
noted in 0.60 mg/ml ranibizumab group in contrast to other treatment groups.
Concurrently, treatment of ranibizumab also found to induce apoptosis and reduced
necrosis among HConFs. The highest level of apoptotic HConFs and the least
necrotic HConFs were observed at concentration 0.45 mg/ml. While in tube
migration study, 7 days treatment resulted in lower percentages of HConFs migrated
on to the tube observed in MMC and MMC+ranibizumab treated group compared to
control, (p<0.05). Treatment of MMC showed lower percentage of migrated
fibroblasts than MMC+ranibizumab group, (p=0.792). In contrast, higher percentage
of migrated fibroblasts was observed in ranibizumab group with respect to control,
(p=0.985). Whereas, at day 14 and 21, percentage of migrated fibroblasts is almost
the same in all groups, (p<0.05) and (p=0.248), respectively. In conclusion,
application of ranibizumab could further inhibit the proliferation of MMC treated
HConFs by increasing the apoptosis and reducing the necrosis. It was also discovered
that ranibizumab alone was not effective in suppressing the migration of HConFs
onto the glaucoma tube. Instead, the adjunctive treatment of MMC and ranibizumab
could significantly affect the migration of fibroblasts on to the glaucoma tube.
Actions (login required)
 |
View Item |
