Objective To explore transthyretin (TTR) effect on retinal vascular endothelial cells (hREC) under high glucose and hypoxia environment. Methods hREC and human retinal pigment epithelial cell (hRPEC) were cultured at low-glucose (LG), high glucose (HG) and hypoxia. The glucose concentration was increased from 5.5 mmol/L up to 25 mmol/L, and hypoxia was induced by 200 μmol/L CoCl2. The cells were divided into LG group, LG-hypoxia group, HG group, HG-hypoxia group according to the different cell culture environment. The growth index was detected at 0, 4, 8, 16, 24, 36, 48, 60, 72 hours after cultured. Furthermore, hREC and hRPEC were also cultured with additional TTR (4 μmol/L), respectively. Then transwell co-culture system was employed to reveal the effects of hRPEC on the growth of hREC. Results At 72 hours after cultured, the growth index of hREC and hRPEC in LG group were increased as compared with LG-hypoxia group and HG group (hREC: F=17.098, 22.970; P < 0.05. hRPEC: F=45.442, 9.011; P < 0.05); the growth index of hREC and hRPEC were decreased in HG group and HG-hypoxia group (hREC: F=146.184, P < 0.05；hRPEC: F=27.907, P < 0.05). Additionally, hREC could be significantly repressed by added TTR during culture with high concentration of glucose (F=161.430, 24.106; P < 0.05). hREC could be significantly increased by added TTR during culture with low concentration of glucose (F=200.486, 48.662; P < 0.05). In co-culture process, hRPEC revealed inhibition activity against hREC under both natural and abnormal environment (LG group: F=15.711, P < 0.05; LG-hypoxia group: F=45.659, P < 0.05; HG group: F=7.857, P < 0.05; HG-hypoxia group: F=6.348, P < 0.05). Conclusion Under high glucose and hypoxia environment, the growth of hREC from neovascular could be inhibited by TTR.
The mechanisms behind diabetic retinopathy (DR) can be ascribed primarily to retinal microvascular abnormalities, excessive inflammatory response and neurodegeneration. Circular RNA (circRNA) is a type of endogenous non-coding RNA with a special circular structure, which is mainly composed of precursor RNA after shearing and processing. It is widely present in the retina and participates in the occurrence and development of various fundus diseases. CircRNAs express in an abnormal way in retina, serving as “the sponge” for miRNA so as to play roles in dysfunction of retinal vascular, inflammatory response and neurodegeneration in the development of DR. Further studies for circRNAs in DR will illustrate pathophysiology of DR more deeply, shedding light on circRNAs becoming novel biomarkers and molecular targets for diagnosis and treatment, thus achieving the goal of early diagnosis and precise therapy of DR.
ObjectiveTo explore the effects of transthyretin (TTR) on biological behavior of retinal microvascular epithelial cell (RMVEC). MethodsRMVEC was cultured in medium with 0 μmol/L and 4 μmol/L TTR. The proliferation, migration and healing abilities (0, 24, 48 hours) of RMVEC with different concentrations of TTR were measured by methyl thiazol tetrazolium (MTT) assay, transwell assay and scarification test. ResultsMTT assay shows that RMVEC with the concentrations of 4 μmol/L TTR [absorbance (A) value=0.17±0.02] glows faster than with the concentrations of 0 μmol/L TTR (A value=0.40±0.03), the difference was statistically significant (t=15.47, P=0.000 1). The transwell assay shows RMVEC with the concentration of 4 μmol/L TTR [(140±7) cells] migrants faster than RMVEC with the concentration of 0 μmol/L TTR [(227±14) cells], the difference was statistically significant (t=5.44, P=0.000 6). The scarification test shows that the RMVEC with the concentration of 4 μmol/L TTR [(134.4±45.4) μm] heals faster than the RMVEC with the concentration of 0 μmol/L TTR [(330.0±23.1) μm], the difference was statistically significant (t=8.25, P<0.01). The cells in 48 hours and 4 μmol/L group were healed completely, but not healed in 0 μmol/L group. ConclusionTTR can promote the proliferation, migration and healing abilities of RMVEC.
Objective To observe the abnormal expression of transthyretin in serum and vitreous of high myopia patients. Methods 116 patients (50 males and 66 females) with high myopia, diagnosed by retinoscopic reflex, slit-lamp microscope, indirect ophthalmoscope and Iol-Master, were enrolled. The patients had an average age of (49.7plusmn;12.3) years and average diopter of (-10plusmn;4.2) D. The control group had 86 healthy individuals (42 males and 44 females) with an average age of (48.5plusmn;10.5) years. According to the findings of indirect ophthalmoscope and optical coherence tomography (OCT) examination, the patients were classified as foveal detachment, macular hole, choroidal neovascularization, epimacular membrane, retinal scar and no significant pathological changes. 2 ml peripheral blood samples were taken from all the subjects. The vitreous samples of high myopia patients with foveal detachment and macular hole (20 patients, 20 eyes) were collected before pars plana vitrectomy. LTQ-MASS was performed on 16 serum samples of patients and 16 control serum samples. Western blot and enzyme linked immunosorbent assay (ELISA) were used to detect serum transthyretin expression in high myopia groups（n=100）and control group (n=80). Vitreous samples (n=20) and corresponding serum samples were further analyzed by ELISA. Meanwhile, the relationship between visual acuity after surgery and transthyretin expression level in vitreous were analyzed. Results LTQ-MASS detected 4 proteins differentially expressed in the serum of patients and control group, transthyretin was the most significantly increased protein in high myopia patients. Western blot validated that serum transthyretin of high myopia group was much higher than that of the control group (t=3.68, P＜0.05). High myopia patients with macular hole and macular detachment had much higher serum and vitreous transthyretin than that of control group (F=9.28，P＜0.01). Patients with higher vitreous transthyretin had better postoperative visual acuity. Conclusions The transthyretin level increases significantly in serum and vitreous of high myopia patients, and it correlated with ocular pathologies and visual outcomes.
Objective To investigate the effects of docosahexenoic acid (DHA) on large conductance Ca2+-activated K+ (BK) channels in normal retinal artery smooth muscle cells (RASMCs). Methods Cultured human RASMCs (6 th-8 th generations) were used to patch clamp experiment. The open probabihties (NP0) in BK channels with different concentrations (0.0, 1.0, 3.0, 5.0, 7.5, 10.0 μmol/L) of DHA were recorded by patch clamp technique in single channel configuration. RASMCs were intervened by different concentrations (0.0, 1.0, 5.0 μmol/L) of DHA as control group, low and high doses of DHA groups, respectively. The protein expressions of β subunit of BK channels in RASMCs from three groups were measured by Western blot. Results The NP0 of BK channels were 0.044 4±0.001 2, 0.081 2±0.004 2, 0.209 0±0.006 1, 0.310 5±0.005 3, 0.465 0±0.007 8 and 0.497 7±0.014 5 with perfusate of 0.0, 1.0, 3.0, 5.0, 7.5, 10.0 μmol/L DHA. DHA activated BK channels in a dose-dependent manner (F=2.621,P＜0.05). There was no significant difference in the protein expression of control group, low and high doses of DHA groups (F=11.657,P＞0.05). Conclusion DHA can directly activate BK channels, no increasing in subunit expression of BK channels.
ObjectiveTo investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.MethodsThirty patients with DR (DR group), 30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study. Three groups of subjects were taken 5 ml of venous blood, and total plasma RNA was extracted and purified. The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip, and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR). Bioinformatics was used to predict potential target genes for miRNA regulation, and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened. Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L). hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model, which was divided into blank control group, high expression group and negative control group. The expression of miR-1470 was detected by RT-PCR. The expression of EGFR protein was detected by Western blot. The measurement data of the two groups were compared using the independent sample t test. The comparison of the measurement data between the two groups was analyzed by ANOVA. The comparison between the measurement data of the groups was compared by multiple comparisons.ResultsThe results of RT-PCR were consistent with those of the gene chip. The expression of miR-1470 in the plasma of the DR group, the DM group and the normal group was statistically significant (F=63.486, P=0.049). Compared with the DM group and the normal group, the expression of miR-1470 in the DR group was significantly decreased, and the difference was statistically significant (q=111.2, 73.9; P＜0.05). The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082, P=0.015). The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=−39.939, P=0.016). The expression of miR-1470 (F=637.069, P=0.000) and EGFR (F=122.908, P=0.000) protein expression in hREC of blank control group, negative control group and high expression group were statistically significant . Compared with the blank control group and the negative control group, the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7, 328.8; P＜0.05), and the expression of EGFR protein was significantly decreased (q=242.5, 234.6; P＜0.05). There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5, 7.9; P＞0.05).ConclusionThe expression of miR-1470 in the plasma of patients with DR is significantly down-regulated, and the increase of EGFR expression may be related to it.
ObjectiveTo observe the changes in open probability and protein expression of large conductance Ca2+-activated K+ (BK) channel in retinal vascular smooth muscle cells (RVSMCs) of diabetic rats. MethodsStreptozotocin (STZ)-induced rat diabetic animal model was established by STZ injection intraperitoneally.RVSMCs were isolated by enzyme digestion. The BK currents in control and diabetic groups were recorded by patch clamp technique in single channel configuration. BK channel protein expression in control and diabetic group were measured by Western blot. ResultsCompared with control group, the NP0 of BK channels in diabetic group were significantly increased (t=4.260, P < 0.05). Compared with control group, there was no significant difference inα-subunit protein expression in diabetic group in RVSMCs (t=10.126, P > 0.05); however, β1-subunit protein expression was remarkably increased in diabetic group (t=5.146, P < 0.05). ConclusionThe NP0 of BK channels andβ1-subunit protein expression are increased in RVSMCs of diabetic rats.
Objective To measure the concentration of serum transthyretin (TTR) of patients with different stages of diabetic retinopathy (DR). Methods A total of 176 patients with diabetes mellitus were included in this study. There were 104 males and 72 females. The patients aged from 21 to 74 years, with the mean age of (56±11) years. The diabetes duration raged from 1 to 30 years, with the mean diabetes duration of (10±7) years. The HbA1C was 5.2%−14.1%, with the mean HbA1C of (8.6±2.0)%. According to the fundus examination, 58 patients had DR (33.0%), but the other 118 patients not (67.0%). For these DR patients, 10 patients were in stage Ⅰ (5.7%), 26 patients in stage Ⅱ (14.8%), 8 patients in stage Ⅲ (4.5%), and 14 patients in stage Ⅳ (8.0%). The concentration of serum TTR was measured by enzyme-linked immunosorbentassay kit. The differences in the concentration of serum TTR between different DR stages were compared.Bivariate analysis was used to analyze the influencing factors of TTR. Results The concentrations of serum TTR of the patients without DR or with DR of stage Ⅰ to Ⅳ were (224.96±65.47), (383.68±102.99), (247.44±63.21), (228.2±45.89), (189.34±70.12) mg/L, respectively. The difference between different DR stages was statistically significant (F=14.690,P＜0.001).Bivariate analysis showed that the concentration of TTR was correlation to DR (r=0.179,P=0.017). There was no correlation between the concentration of TTR and diabetes duration (r=−0.027,P=0.727), hypertension (r=0.018,P=0.810), hyperlipoidemia (r=0.101,P=0.182), and the use of insulin (r=−0.032,P=0.675). Conclusion The concentration of serum TTR was increased in early DR patients, and gradually decreased with the progression of DR. The concentration of TTR is correlated to DR.
Objective To investigate the prevalence rate and risk factors of diabetic retinopathy (DR) in residents aged 50 and above in Binhu Area, Wuxi City. Methods Stratified cluster random sampling method was used to investigate the prevalence of DR of residents aged 50 and above from January to December 2010. Detailed medical history, general examination, visual acuity, slit lamp microscope, direct ophthalmoscopy and blood sample testing were conducted for all selected subjects. DR diagnostic criteria refer to the 2002 International DR classification criteria. Unilateral or bilateral DR was both considered as DR patients.χ2 test was used for statistical analysis of risk factors, and independent risk factors were screened by SPSS 17.0 software. Results A total of 6150 residents underwent the examination with a participating rate of 91.5%. Seven hundred and three residents were diagnosed as with diabetes, in which 40 were unable to record the fundus condition due to opaque refractive media. Among the rest 663 diabetic patients, 36 (5.4%) were DR patients. There were 34 non-proliferative DR (5.1%) and two proliferative DR (0.3%). The duration of diabetes and fasting blood-glucose were the independent risk factor of DR (r=0.008, 0.009; P＜0.05). Conclusions The prevalence rate of DR is low in the residents aged 50 and above in Binhu Area, Wuxi City. The duration of diabetes and fasting blood-glucose level were confirmed as the independent risk factor of DR.