Objective: To investigate the effect of cobalt chloride (CoCl2) on transgene expression and viral particle titers in tumor cells infected by conditionally replicating adenovirus expression vector with hypoxia response elements(HRE)-regulated E1AE1B expression (Ad-5HRE-E1AE1B-RFP) and non-HRE regulated replication-deficient adenovirus expression vector (Ad-EGFP, Ad-Luc) in vitro and in vivo. Methods: Ad-5HRE-E1AE1B-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concentrations of CoCl2. The hypoxia-inducible factor 1 alpha (HIF-1α) protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus (Ad-Luc) was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results: Western blotting results showed that CoCl2 at 0.4 and 0.08 μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 cells, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4 μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-5HRE-E1AE1B-RFP had no replication. CoCl2 0.4 μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-5HRE-E1AE1B-RFP and Ad-Luc significantly increased the expression of Ad-Luc in nude mice.Conclusion: CoCl2 markedly enhances transgene expression of recombinant adenovirus. However, the underlying mechanism is not only related to the CoCl2-induced hypoxia, but also probably related to regulation of gene transcription.

Objective:To evaluate the efficacy and safety of comprehensive treatment for retinoblastoma (RB).Methods:A retrospective clinical study. From January to December in 2019, 157 cases (203 eyes) of RB who were diagnosed by the Department of Ophthalmology of Xinhua Hospital and received comprehensive treatment were included in this study. Of cases, 76 were male, and 81 were female; 111 were unilateral, and 46 were bilateral. The medium of age at diagnosis was 20.1 months. All patients received treatment for the first time. Patients with intraocular tumors were divided into A-E stages, extraocular stage and distant metastasis according to international intraocular RB classification standard. The median follow-up time was 37.4 months. Clinical features, treatment, prognosis and ocular complications of all cases were recorded.Results:Among 157 cases (203 eyes), 137 cases (180 eyes) were in intraocular stage; 6, 14, 10, 98, and 52 of eyes were in A-E stages, respectively. Twelve cases (12 eyes) were in extraocular stage; 8 cases (11 eyes) were in distant metastasis stag; 8 cases died due to distant metastasis; 149 cases (94.9%, 149/157) survived; 48 eyes were enucleated, 34 of which underwent initial enucleation, and 14 eyes underwent enucleation after eye-preserving treatment. The overall global salvage rate was 155 eyes (76.4%,155/203), and that after eye-preserving treatment was 91.7% (155/169). Severer eye for bilateral cases was taken into account for statistic; 120 cases (120 eyes) received initial eye-preserving treatment. Among them, 36 and 84 eyes underwent initial intravenous chemotherapy (IVC) and initial intra-arterial chemotherapy (IAC), respectively. The enucleation of the two groups was 7(19.4%, 7/36), 7(8.3%, 7/84); 33 (91.7%, 33/36) and 33 (39.3%, 33/84) eyes received the second treatment, respectively. There was no significant difference in the rate of enucleation between the two treatments ( χ2=2.037, P=0.154). There was significant difference in the percentage of secondary treatment ( χ2=27.937, P<0.001). Fifty-four eyes (45.0%, 54/120) stabilized after initial treatment, and 66 eyes (55.0%, 66/120) underwent secondary treatments due to poor response or tumor recurrence. For 66 eyes receiving secondary treatments, enucleation, IAC, intravitreous chemotherapy (IVitC), IAC combined with IVitC, and laser and/or cryotherapy was performed in 6, 18, 12, 13, and 17 eyes, respectively. The number of eyes of enucleation among the IAC, IVitC, and IAC combined with IVitC group was 5 (27.8%, 5/18), 1 (9.3%, 1/12), and 2 (15.4%, 2/13) eyes, which was no significantly different ( χ2=2.001, P=0.368). Until the last follow-up, visual acuity outcomes were acquired in 148 eyes (72.9%, 148/203). Among them, 41, 53, 16 and 38 eyes had no light perception, light perception to finger counting, 20/400, and ≥20/200, respectively. In total, among 203 eyes, 121 eyes received IAC, of which 2, 4, and 1 eyes had optic disc atrophy, vitreous hemorrhage, and severe retinal-choroidal atrophy, respectively; 60 eyes received IVitC, of which one and one eye had vitreous hemorrhage and macular hemorrhagic necrosis, respectively. Conclusions:In this study, the overall survival rate was 94.9% after comprehensive treatment and the rate of global salvage after eye-preserving treatment was 91.7%. The comprehensive treatment of retinoblastoma had a relatively high efficacy and safety.

Objective To observe the clinical characteristics of rhegmatogenous retinal detachment (RRD) secondary to conservative therapy in retinoblastoma (RB) patients.Methods A retrospective study. From July 2013 to May 2017, 20 RRD patients (20 eyes) of 456 RB patients (573 eyes) treated in Xinhua Hospital of Shanghai Jiao Tong University School of Medicine were included in the study. Eleven patients (11 eyes) were boy and 9 patients (9 eyes) were girls. Thirteen patients demonstrated bilateral RB and 7 patients had unilateral RB. Average age when diagnosed with RB was 25 months. International Classification of Retinoblastoma groups were C in 1 eye, D in 17 eyes, and E in 2 eyes. These patients received intra-arterial chemotherapy (17 eyes), intravenous chemotherapy (11 eyes), intravitreal chemotherapy (8 eyes), laser (14 eyes) and/or cryotherapy (5 eyes). Twelve patients (12 eyes) received vitreoretinal surgery including vitrectomy (6 eyes) and scleral buckling (7 eyes). The mean follow-up was 39 months. Fundus examination was performed under general anesthesia during comprehensive treatment and follow-up. The time interval of fundus examination varied from 1 to 6 months depending on the stability of the tumor.Results RRD was noted in 20 eyes (3.5％) with RB. Retinal hole was found in 15 eyes (75％). The cause of RRD was atrophic hole in calcified tumor (6 eyes, 30％), cryotherapy-related hole (5 eyes, 25％) and laser-related hole (9 eyes, 45％). Multiple atrophic hole in calcified tumor was noted in 3 eyes. Size of hole smaller than 2 DD was noted in 8 eyes (53％), and larger than 2 DD was noted in 7 eyes (47％). Holes were in posterior (3 eyes), equator (2 eyes) and periphery (10 eyes). Severe proliferated was noted in 1 eye. No tear was found. No bulbar retinal detachment and choroidal detachment was noted. Among 12 eyes who underwent vitreoretinal surgery, reattachment was achieved in 9 eyes (75％). No metastasis was noted.Conclusions Calcified regression of tumor, cryotherapy and laser were main reasons of RRD. Most of the holes are small in diameter and located in the periphery.