RNA-seq is a transcriptome analysis method using deep sequencing technology, which uses high-throughput next-generation sequencing technology to investigate, characterize and quantify transcriptome.It can quickly and comprehensively detect almost all transcripts and gene sequences of specific cells in specific tissue of a species, and has been widely used in basic research, clinical diagnosis and drug development and other fields.Its measurement accuracy has been confirmed comparable to microarray and quantitative polymerase chain reaction by several studies.RNA-seq technology can accurately detect mRNA and non-coding RNA which play important roles in the pathogenesis of diabetic retinopathy (DR). Using this technology to investigate the gene regulation and molecular mechanism of DR is helpful to find biomarkers for early diagnosis and prognosis of DR, to comprehensively and systematically study and analyze the molecular mechanism of specific biological process, and to find new therapeutic targets.The application of RNA-seq will be of great significance for the basic research and clinical treatment of DR.In this article, RNA-seq technology was comprehensively and systematically expounded from three aspects, which were the advantages of RNA-seq technology, the selection of sequencing platform as well as library preparation and data analysis.The progress of this technology in DR research was summarized and analyzed.

Objective:To observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR).Methods:Twenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects.Results:The expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant ( tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant ( F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant ( F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant ( F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant ( P<0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group ( P=0.03) and no significant difference in FSTL1 protein expression ( P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant ( F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups. Conclusion:The expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people.

Objective:To observe the changes in refractive status of eyes with idiopathic macular hole (IMH) after vitrectomy and phacoemulsification and IOL implantation (combined surgery).Methods:A retrospective clinical study. From January 2016 to June 2019, 51patients (56 eyes) of IMH who underwent combined surgery at the Tianjin Medical University Eye Hospital. were included in the study. Among them, there were 17 males and 34 females with the average age of 66.79±4.33 years. All the affected eyes underwent BCVA, retinoscopy and axial length (AL) measurement. The IOL power was calculated according to the SRK-T formula and the refractive power (predicted value) was predicted. The average BCVA of the affected eye was 0.20±0.13. The average anterior chamber depth was 2.89±0.28 mm. The average △corneal astigmatism was 0.73±0.43 D, the average AL was 22.92±0.70 mm, the average predicted refractive power was 0.10±0.66 D. All the affected eyes underwent standard transciliary flat part three-channel 25G combined surgery. Six months after the operation, the actual value (actual value) of the diopter after the operation was measured with the same equipment and method before the operation. Paired t test was used to compare the difference between the predicted value and the actual value. Results:Six months after the operation, the actual value of the refractive power was -0.19±0.64 D. Compared with the pre-operative refractive power, the difference was not statistically significant ( t=1.665, P=0.102). The difference between the actual value and the predicted value was -0.33± 0.81 D. Conclusions:The refractive status of the IMH eye undergoes myopia drift after combined surgery. The preoperative IOL power budget can be appropriately reserved for +0.3 D hyperopia.

Objective:To observe the efficacy of dexamethasone intravitreal implant (DEX) combined with pars plana vitrectomy (PPV) in eyes with severe idiopathic epimacular membrane (IMEM).Methods:A prospective clinical case study. From December 2018 to May 2021, 24 patients with 25 eyes of severe IMEM diagnosed in Tianjin Medical University Eye Hospital were included in the study. Among them, 7 males had 7 eyes, 17 females had 18 eyes. Age was 57 to 84 years old. The IMEM stage was 3 to 4 examined by spectral domain optical coherence tomography (SD-OCT). All eyes were performed best corrected visual acuity (BCVA) and central macular thickness (CMT) by SD-OCT. The patients were randomly divided into PPV group (11 eyes) and PPV+DEX group (14 eyes). Standard PPV by three-channel 25G was performed. Phacoemulsification, membrane stripping and intraocular lens implantation were combined during the operation. Patients received vitreous injection of 0.7 mg DEX in PPV+DEX group at the end of the operation. At 1 week, 1 month, 3 months and 6 months after operation, the same equipments and methods were used to perform relevant examinations. The changes of BCVA and CMT were compared between the two groups by t test. Results:Compared with before operation, at 1, 3 and 6 months after operation, the BCVA of the eyes in the PPV+DEX group was significantly improved ( t=3.974, 4.639, 4.453), CMT was significantly decreased ( t=2.955, 3.722, 4.364), the differences were statistically significant ( P<0.05); at 3 and 6 months after surgery, the BCVA of the eyes in the PPV group was significantly improved ( t=2.983, 4.436), CMT was significantly decreased ( t=2.983, 3.461), the differences were statistically significant ( P<0.05). Conclusion:In the treatment of severe IMEM, DEX can accelerate the early postoperative visual recovery and reduce CMT.