Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene.Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Nontransfected NPE cells were set as control group.The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively.Results The opticin mRNA expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were decreased compared with the control group (F=10.239，P＝0.000)；the inhibitory rate were 85.7%，62.87%，54.87% and 48.77% respectively.The opticin protein expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were also decreased compared with the control group (F=17.870，P=0.000)；the inhibitory rate were 78.7%，34.6%，31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
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.
Objective To explore and evaluate the protective effects of soluble fms-like tyrosine kinase recptor-1(sFlt-1) gene 2-3 and 2-4 transcellular region on retinal vascular leakage and phosphatidyl inositol-3 kinase (PI3K)/Akt pathway under hypoxia and/or hyperglycemia. Methods The plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) were constructed.. Two of these plasmids with carboxy methylation glucan (CMD) magnetic particles were transfected into human umbilical vein endothelial cells (HUVEC), which were cultured under hypoxia and/or hyperglycemia. The blood retinal barrier was evaluated by Evans blue permeation (EBP). Then the expression of p-Akt mRNA and protein were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot separately. Results In the diabetic rabbits, The blood retinal barrier breakdown was detected by the retinal vascular leakage of EBP. The expression of p-Akt mRNA and protein in hypoxia and hyperglycemia groups were also obviously increased. These changes were largely prevented by transfection the plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) (P＜0.01 in both groups). Conclusion Both sFlt-1(2-3) and sFlt-1(2-4) can make the retinal blood vessels less leaky and may be beneficial in preventing vision loss from diabetic retinopathy.
Objective To investigate the relationship between expression of retinal intercellular adhesion molecule-1 (ICAM-1) and blood-retinal barrier (BRB) rupture and therapeutic effect of intravitreous injection with triamcinolone acetonide (TA) on blood-retinal barrier rupture in rats with diabetes mellitus (DM). Methods Diabetic model of Wistar rats was induced and were divided into normal control group, DM-4-month group and DM-6-month group. Each group was subdivided into immunohistochemcial staining and BRB measurement groups. BRB measurement group was further divided into non-TA treatment group, 1-week-TA treatment group, and 2-week-TA treatment group. The rats were intravitreously injected with 5 mu;l TA. The digested retinal preparation was stained by immunohistochemcial method to observe the expression of retinal ICAM-1 and morphological changes. The mean optic density (A) value of endothelial cells was measured by image-analyzing software to quantify the expression of ICAM-1. BRB changes were measured by content test of retinal Evans blue (EB). Results In the immunohistochemcial staining groups, there was no significant positive expression of ICAM-1 in retinal capillary in control group. Compared with the control, there was significant positive expression of ICAM-1 in DM-4-month group (P＜0.001) with some morphological changes such as irregular width of capillary caliber, and there was enhanced positive expression of ICAM-1 in DM-6-month group (P＜0.001) with aggravated morphological changes and even acellular capillary. In the BRB measurement groups, there was no significant difference of EB content(P＞0.05) among control groups. The EB content in two DM groups significantly increased compared with that in the controls (Plt;0.001), and higher in DM-6-month group than that in DM-4-month group (Plt;0.01). In TA treatment groups, the EB content in all the DM groups significantly decreased (Plt;0.001) but with no significant difference among the groups(P＞0.05). EB content in DM-4-month group after 2-week treatment almost reached to normal value (P＞0.05) while was higher in the rest of TA treatment groups than that in the control group (Plt;0.05). Rectilinear correlation between A value of endothelial cells and the retinal EB content(r=－0.959)was found. Conclusion There is a positive relation between the expression of ICAM-1 and BRB rupture in retina of DM rats, and intravitreous injection with TA can effectively alleviate BRB rupture. (Chin J Ocul Fundus Dis, 2006, 22: 24-27)
Objective To establish a rapid in vitro culture method of human choroidal endothelial cells (HCEC) and the cellular Characteristics to provide an in vitro model for researches of choroiretinal diseases which involved the HCEC. Methods The human choroidal tissues were digested in two steps by trypsin and collagenase, and the HCEC were obtained and cultured after the digested cell suspension was sorted and purified with magnetic beads of CD31 Dynabeads. The characteristics of HCMEC were observed by the morphologic observation method, transmission electron microscopy, and immunohistochemical staining with FⅧ factor, CD31, and CD34. Results The cultured HCEC were polygonal and oval, and after amalgamation, the cells had slabstone-like appearance. After the subculture, the configuration of HCEC remained the same, and represented cobblestone appearance with less magnetic beads attached on the cellular surface after HCEC converged into a single layer. The Weibel-Palade body which is the characteristic marker of endothelial cells was found. The staining of FⅧ fatcor, CD31, CD34 were positive. Conclusion HCEC can be cultured in vitro successfully with our method, which is easy to get sufficient number of highly purified HCEC. (Chin J Ocul Fundus Dis, 2007, 23: 126-129)
Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group,oxygen-induced retinopathy (OIR) group, empty vector group and treated group,14 mices in each group. Except for the control group,the mice in the other groups were exposed to (75plusmn;2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid.The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane（ILM） was observed and counted by optical microscope.Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group.The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups（F=39.006，Plt;0.001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinalneovascularization in OIR.
Objective To evaluate the diagnostic performance of thyroid imaging reporting and data system (TI-RADS)classification and elastography in differentiating benign and malignant thyroid nodules according to size. Methods A total of 222 thyroid nodules (209 cases) with solid or predominantly solid internal contentdiagnosed at pathological findings were enrolled in this study, all the 209 cases underwent surgery in our hospital from Jan.2014 to Jun.2014. The diagnostic performance of TI-RADS classification and elastography in differentiating benign and malignant thyroid nodules according to size nodules were evaluated (for≤1 cm and > 1 cm respectively). Results By using TI-RADS category and elastography, 178 thyroid nodules were diagnosed as malignant nodules, and 44 thyroid nodules were diagnosed as benign nodules. The high predictive factors for malignant thyroid nodules of > 1 cm were irregular shape(OR=6.376), microcalcification(OR=21.525), and capsule invasion(OR=3.852), P < 0.05. The factors for thyroid nodules of≤1 cm were anteroposterior to transverse diameter ratio≥1(OR=3.406), capsule invasion (OR=3.922), and high elastography score(OR=1.606), that suggested the possibility of malignant (P < 0.05). For nodules of > 1 cm, the sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Youden index of TI-RADS combining with elastography were 98.3%(59/60), 68.6%(24/35), 87.4%(83/95), 84.3%(59/70), 96.0% (24/25), and 66.9% respectively; for nodules of≤1 cm, the sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Youden index were 98.5%(67/68), 30.5%(18/59), 66.9%(85/127), 62.0%(67/108), 94.7%(18/19), and 29.0% respectively. Conclusion TI-RADS category combined with elastography for diagnosis of thyroid nodules in different size has just a bit differently diagnostic indicators, but that play a high diagnostic performance on the thyroid nodules with maximum diameter > 1 cm.
Objective To observe the effect of hypoxia inducible factor-1alpha;（HIF-1alpha;）to the expression of cell surface adhesion molecules CD18 and the adhesion ability of leukocytes and vascular endothelial cells under early stage of diabetic retinopathy condition.Methods The human promyelocytic leukemia cell line HL60 and the rhesus choroid-retina vascular endothelial cell line RF/6A were cultured in RPMI 1640 medium-10% human serum, which was collected from the subjects of early stage of diabetic retinopathy and age-matched healthy control. The cells were cultured in 4 groups as control group (group A), diabetic group (group B), HIF-1 anti-sense oligonucleotides (ASODN) group (group C) and HIF-1 sense oligonucleotides (SODN) group (group D). The percentages of CD18 positive cell in the HL60 cell were measured by flow cytometry and mRNA in the HL60 cell by realtime reverse transcriptionpolymerase chain reaction（RT-PCR）. Results The percentage of CD18 positive cell in the group A, B, C and D was 17.06plusmn;6.01, 42.23plusmn;2.60, 25.33plusmn;3.05 and 32.40plusmn;10.57, respectively, the differences among them were significant (F=36.47,P＜0.001). Compared to the group A,the expression of CD18 mRNA in the group B,C and D was increased about 21.05plusmn;2.07、2.23plusmn;0.96 and 25.07plusmn;2.27 times,respectively, the differences among them were significant (F=180.34, Plt;0.001). The adherent rates of HL60 to RF/6A in group A, B, C and D was 0.06plusmn;0.00,0.09plusmn;0.10,0.05plusmn;0.00 and 0.07plusmn;0.01, respectively,the differences among them were significant（F=13.06，P=0.002）.Conclusion In vitro, HIF-1 could regulate the expression of CD18 by HL60, and the adhesion of HL60 to RF/6A when the cells were exposed to diabetic serum. The effects of human serum weaken with the inhibition of HIF-1 expression.HIF-1 play regulatory role in the expression of CD18 and adhesion of leukocytes and vascular endothelial cells under early stage of diabetic retinopathy condition.
Purpose To investigate the association between the macular volume and thickness, as assessed by optical coherence tomography (OCT), with refraction state and axial length (AL) in children. Methods A total of 100 normal children whose right eyes were randomly selected were divided into five groups due to their refraction, comprising hyperopia, emmetropia, low myopia, moderate myopia, high myopia. The AL of the eyes was measured by IOL mas ter measuring machine. Then the macular volume and thickness were measured by OCT. Results Children with high myopia have smaller macular volume and thinner parafoveal retinal thickness (especially out-ring macular). The minimum thickness and average thickness of the fovea were not significantly different among the five groups. The macular volume ,total average macular thickness and quadrant-specific macular thickness(except the nasal inner quadrants) were positively associated with refraction and negatively associated with AL, while t here were no correlation between minimum foveal thickness, the average foveal th ickness and refraction or AL. Conclusions In Chinese children, increasing axial length was associated with reduced macular volume and thickness (except the foveal region and the nasal inner quadrant). (Chin J Ocul Fundus Dis,2008,24:114-117)
Objective To elucidate the new development, structural features and appl ication of the lumbar interspinous process non-fusion techniques. Methods With the review of the development course and important research works in the field of the lumbar inter-spinous process non-fusion techniques, the regularity summary, science induction, and prospect were carried out. Results The lumbar inter-spinous process non-fusion technique was a part of non-fusion insertof spinal division posterior surface. According to the design, it could be divided into two major categories: dynamic and static systems. The dynamic system included Coflex and device for intervertebral assisted motion; the static system included X-STOP, ExtenSure and Wall is. The lumbar inter-spinous process non-fusion technique was a new technique of spinal division, it could reserve the integrated function of intervertebral disc and zygapophysial joint, maintain or recover the segmental movement to a normal level, and have no adverse effect on the neighboring segments. A lot of basic and cl inical researches indicated that lumbar inter-spinous process insert had extensive appl ication to curatio retrogression lumbar spinal stenosis, discogenic low back pain, articular process syndrome, lumbar intervertebral disc protrusion and lumbar instabil ity and so on. Conclusion With the matures of lumbar inter-spinous process non-fusion techniques and the increased study of various types of internal fixation devices, it will greatly facil itate the development of treatment of lumbar degenerative disease. But long-term follow-up is needed to investigating the long-term efficacy and perfect operation indication.