With the method of PCR-SSCP combined sequencing,we analysed the mutations in the RB1 gene of 10 retinoblastoma samples. All 27 exons of RB1 gene have been examined. The PCR products were digested with some restriction endonuclease to the size of about 200 bp and then denatured into single strands. Loading the single strands of DNA into the 6% polyacrilamide gel,we followed the technology named SSCP to f/nd the abnormal bands in the SSCP gel. After that, the samples found with the abnormal bands in the SSCP gel were amplified again and sequenced to confirm the types and locations of the mutations in the RB1 gene. We have fonnd some kinds of mutations in the RB1 gene. （Chin J Ocul Fundus Dis,1994,10:17-20）
The Chinese human hepatocellular carcinoma cell SMMC7721 has been analysed by flow cytometry, Southern blotting and Western blotting. The results indicated that SMMC7721 cell is a hypoploid cell with a 0.81 DNA index, and that SMMC7721 cell has internal deletion in the 5'-end of its Rb gene and has no Rb gene product (Rb protein). The normal Rb cDNA has been inserted into a retrovirus vector DOL and introduced into SMMC7721 cells by electrporation transfection technique.About 75% of transfected SMMC7721 cells have been killed by Rb gene product. The remain 25% cells are alive as exogenous Rb gene has been mutationally inactivated. （Chin J Ocul Fundus Dis,1994,10:21-24）
bjective To separate the SO-Rb50 cells antigen corresponding to the monoclonal antibody of anti-retinoblastoma. Methods The antigen corresponding to the monoclonal antibody of anti-retinoblastoma was separated elementarily by ion-exchange chromatography, and was identified by dot-blotting using the monoclonal antibody of anti-retinoblastoma. The target protein band of the antigen was separated in light of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. Results A special unmixed band of SO-Rb50 cells antigen was separated with the relative molecular weight of 83×103.Conclusion The antigen corresponding to the monoclonal antibody of anti-retinoblastoma could be separated from SO-Rb50cells.(Chin J Ocul Fundus Dis，2003，19：152-155)
ObjectiveTo evaluate the pathological features of bilateral retinoblastoma (RB) and the relationship between different treatments and high-risk histopathologic features (HHF). MethodsRetrospective series of case studies. From 1999 to 2018, 73 patients with binocular RB diagnosed by pathological examination in Department of Ophthalmology, Peking University People's Hospital were included in the study. Among them, 50 patients were male (68.5%, 50/73), 23 patients were females (31.5%, 23/73); 11 patitents had a family history of RB. The mean age at the first diagnosis was 14.8±15.6 months. The average time between first diagnosis and first intervention was 3.97±4.74 months. According to the international classification standard of intraocular RB staging, among the 73 eyes, C, D and E stages were 2 (2.7%, 2/73), 15 (20.5%, 15/73), and 56 (76.7%, 56/73) eyes, respectively. Ocular images for each patient were obtained using a wide-angle contact fundus camera during examination under general anaesthesia. The treatment protocol (globe salvaging or enucleation) depended on the result of several clinical features. Globe salvaging treatment included chemotherapy combined with local therapy such as intra-arterial chemotherapy (IAC), intravitreal chemotherapeutics injection, cryotherapy, laser, transpupillary thermotherapy and radiotherapy. If globe salvaging failed, enucleation was offered and histopathologic analysis was conducted of the enucleated eye, the ophthalmic pathologist read and evaluated the presence of HHF. Independent samples t-test was performed to compare the continuous variables. The pathological features and the relationship between different treatments and HHF were analyzed. Group difference was calculated with chi-square. Results Among the 73 eyes, the first treatment was enucleation in 21 eyes (28.8%, 21/73); 52 eyes (71.2%, 52/73) were treated with eye protection. After enucleation, 9 cases (12.3%, 9/73) had recurrence and metastasis, and 7 cases (9.6%, 7/73) died. The intervention time of patients with recurrence and metastasis and those without recurrence and metastasis were 7.4±7.3 and 3.5±4.1 months respectively; the first intervention time of patients with recurrence and metastasis was significantly later than that of patients without recurrence and metastasis, but the difference was not statistically significant (t=－1.561, P=0.154). The pathological examination results showed that there were 26 eyes (35.6%, 26/73) with HHF, 4 (26.7%, 15/26) and 22 (39.3%, 22/56) eyes were in stage D and E, respectively. Those who received other treatments before enucleation had lower HHF percentages after enucleation than those who did not receive corresponding treatments, but the difference was not statistically significant (χ2=1.852, 0.074, 0.000, 1.007, 0.007, 2.729; P＞0.05). Among the 26 eyes, 5 (83.3%, 5/6) and 21 (31.3%, 21/67) eyes were treated with systemic chemotherapy combined with and without IAC, respectively, and there was a significant difference in the percentage of HHF (χ2=4.422, P=0.035). ConclusionsIAC eye-preserving therapy before enucleation has a significant effect on HHF.
Objective To investigate inhibited effects of melatonin (MLT) on proliferative activity of retinoblastoma cell line HXORB44 and its related mechanism. Methods HXO-RB44 cells were treated by MLT of different concentration (10-10, 10-9, 10-8, 10-7 mmol/L. Cell counting and tetrazolium dyereduction assay (MTT) were used to determine the effect of MLT on the survival and proliferation of HXO-RB44 cells. Apoptotic nuclei were further analyzed by HoechstPI fluorescence staining. Flow cytometry was used to measure the fluorescent intensity of ROS, cell cycle distribution and apoptosis. Results 10 -6 mmol/L (or exceed) of MLT could inhibit the proliferation of HXO-RB44cells in vitro while 10-7 mmol/L (or below) of MLT couldn't. With the increase of MLT concentration from 10-10 mmol/L to 10-7 mmol/L, HXO-RB44 cells gradually increased the expression of ROS. Hoechst staining showed that 4, 8, 12 and 24 hours after the incubation with MLT, the nuclear pyknosis and nuclear fragmentation increased in HXORB44 cells. The extent of apoptosis was proportional to the concentrations of MLT. Flow cytometry revealed that with the increasing of MLT concentration, G0/G1 and G2/M phase cells increased, S phase cells decreased. The apoptotic rate was also increased. Conclusion 10 -6 M of MLT could inhibit the proliferation of HXO-RB44 cells. This effect may relate to the increased ROS expression, cell cycle arrest at G0/G1 phase and apoptosis of HXO-RB44 cells.
ObjectiveTo explore the clinical treatment of retinoblastoma (RB) after being treated with vitrectomy (PPV) due to misdiagnosis.MethodsA retrospective case study. From July 2015 to July 2018, 5 cases and 5 eyes of RB children diagnosed by pathological examination at the Eye Center of Beijing Tongren Hospital were included in the study. Among them, there were 3 males with 3 eyes and 2 females with 2 eyes; all of them had monocular disease. The average age was 4.8±1.7 years old. At the first visit, the diagnosis was endophthalmitis in 2 eyes (40%, 2/5); vitreous hemorrhage in 3 eyes (60%, 3/5). All were treated with PPV. All children underwent slit lamp microscopy, orbital MRI and CT, and eye color Doppler ultrasound blood flow imaging. If there was no clear extraocular spread, the eyeball removal combined with artificial orbital implantation was performed; if there was clear extraocular spread, the modified orbital content enucleation operation was performed with part of the eyelid preserved. The average follow-up time after surgery was 34.6±7.9 months.ResultsAmong the 5 eyes, 2 eyes (40%, 2/5) underwent eyeball enucleation combined with stage I artificial orbital implantation, and 3 eyes (60%, 3/5) with modified orbital content enucleation. There were 2 eyes of endogenous type (40%, 2/5), 1 eye of diffuse infiltration type (20%, 1/5), and 2 eyes of mixed type (40%, 2/5). The orbit spread in 3 eyes, the tumor invaded the optic nerve in 1 eye, and regional lymph node metastasis in 2 eyes. All children received systemic chemical therapy (chemotherapy). During the follow-up period, there were no new metastatic diseases and no deaths.ConclusionsAfter RB misdiagnosis and PPV, surgical treatment should be performed as soon as possible. If there is no clear extraocular spread, eyeball removal or combined stage I orbital implantation should be performed. If there is clear extraocular spread, the orbital contents should be enucleated; Chemotherapy should be combined after surgery.