Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro.Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 μmol/L hypericin with or without preincubation of phorbol 12-myristate 13-acetate (PMA). The activities of cytosolic PKC (c-PKC) and membranous PKC (m-PKC) were assayed by PKC kit. Results The original activities of c-PKC and m-PKC of RPE cells were (35.34±4.10) pmol·min-1·mg-1and (62.52±8.80) pmol·min-1·mg-1.The activity of c-PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c-PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m-PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c-PKC and m-PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down-regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis，2003，19：55-58)
Objective To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot test. Results The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH Ⅰ and Hind Ⅲ), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2×103 and 30.3×103 respectively. The results of Westernblot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.
Objective To investigate the alteration of protein kinase C (PKC) and endothelin system in early diabetic rats, and the effect of specific PKC inhibitor on the expression of retinal endothelin-1 (ET-1). Methods The rats model with streptozotocin(STZ)-induced diabetes were set up. The expression of retinal PKC was detected by enzyme-linked immunoabsorbent assay (ELISA). The expression of retinal ET-1, ET-3, ET-A and ET-B receptor mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The alteration of retinal ET-1 mRNA after intravitreal injection of PKC inhibitor GF109203X in diabetic rats was also observed. Results The activities of membranous PKC were significantly increased in 2-week diabetic rats compared with that in normal rats（t=3.296 , P=0.008）, while activities of cytosolic PKC were unchangeable（t=0.138, P=0.894）. The expression of retinal ET-1 mRNA was significantly increased（P=0.008）, while no change was found in expression of ET-3, ET-A and ET-B mRNA（P=0.918，P=0.889，P=0.500）. After intravitreal in jection of 10-5、10-6、10-7 mol/L PKC inhibitor GF109203X in diabetic rats, the expression of retinal ET-1 mRNA was decreased in a dose-dependent manner compared with the control rats. Conclusion Activation of PKC and increased expression of ET-1 could be found in the retina of early diabetic rats, and PKC inhibitor could inhibit the expression of retinal ET-1. (Chin J Ocul Fundus Dis，2004，20：168-171)
Objective To investigate the role of ephrin A genes in the development of oxygen induced retinalneovascularization (OIR) in mice.Methods The OIR model was established by oxygen induction in new born C57BL/6J mice.Reversed transcript polymerase chain reaction (RT-PCR) was used to measure the expression levels of ephrin A1-A5 in retinas of mice in experimental and normal control group.Results All of the ephrin A family genes expressed in normal retinas. Ephrin A1 mRNA was significantly higher in OIR group(t=3.19,P=0.019); ephrin A2 mRNA was higher in the 15-day-old OIR retinas(t=3.71,P=0.033）; ephrin A3-A5 mRNA decreased or disappeared in 12 and 13-day-old RNV mice, and increased in 15-day-old OIR mice. Conclusion Ephrin A genes are involved in the development of retina and OIR.
ObjectiveTo evaluate the screening performance of commonly used renal function indicators for chronic kidney disease (CKD) in subjects of different ages, so as to explore the appropriate screening regimen for Chinese population.MethodsA total of 2 131 adult subjects in Sichuan Orthopaedic Hospital or Mianyang Central Hospital from May 2016 to October 2017 were selected. They were divided into three groups according to age: group A (18–39 years, n=278), group B (40–64 years, n=1 167), and group C (≥65 years, n=686). Serum levels of creatinine (SCr), urea, and cystatin C [CysC; to calculate estimated glomerular filtration rate (eGFR) based on this index], as well as urine levels of albumin, total protein and creatinine were measured, and urine albumin to creatinine ratio (ACR) and urine protein to creatinine ratio (PCR) were calculated respectively. CKD was diagnosed according to the Kidney Disease: Improving Global Outcomes (KDIGO) Guideline (2012 Edition). The receiver-operating characteristic (ROC) curve analysis was finally performed to investigate the diagnostic performance of each indicator for CKD.ResultsThe prevalences of CKD in group A, B, and C were 10.8% (30/278), 16.4% (191/1 167), and 45.8% (314/686), respectively, and the difference among these groups was statistically significant (χ2=233.525, P<0.001). In addition, the levels of the six renal function indicators between CKD and non-CKD subjects also had statistically significant differences in different age groups (P<0 05="" otherwise="" roc="" curve="" analysis="" revealed="" that="" the="" diagnostic="" values="" of="" these="" indicators="" were:="" acr="" or="" pcr=""> eGFR or CysC > serum urea or SCr (AllP<0 05="" except="" that="" egfr="" cysc="" serum="" urea="" and="" scr="" in="" group="" a="" assessed="" the="" similar="" diagnostic="" performance="" among="" the="" three="" indicators="" recommended="" by="" kdigo="" guideline="" the="" diagnostic="" performances="" of="" acr="" and="" pcr="" in="" different="" age="" groups="" were:="" group="" b="" 0="" 915="" 0="" 914=""> group A (0.885, 0.890) > group C (0.841, 0.846), while the diagnostic performance of eGFR was group C (0.807) > group B (0.728) > group A (0.696). The best boundary values of ACR and PCR were lower while the value of eGFR was higher than the corresponding KDIGO recommended medical decision levels.ConclusionsACR is the first choice for screening CKD when the renal function index creatinine is normal. Moreover, eGFR can further improve the diagnostic value, especially in subjects over 65 years old.
ObjectiveTo study the effects of Ghrelin for glucose metabolism and insulin sensitivity of L6 rat myoblasts in palmitic acid induced, and to explore its possible mechanisms. MethodsThe L6 rat myoblasts were cultured until differentiation, then using palmitic acid(0.3 mmol/L) for 16 hours. The experimental group was treated with different doses of Ghrelin(1, 10, and 100 nmol/L) for 8 hours, then the glucose uptake was detected by using glucose oxidase peroxidase method(GOD-POD), the cell membrane glucose transporter 4(GLUT-4) protein staining was observated under confocal microscopy, and the expressions of total protein kinase B(Akt), phosphorylated protein kinase B(pAkt), total glycogen synthase kinase-3β(GSK-3β), and phosphorylated glycogen synthase kinase-3β(pGSK-3β) were detected by using immunoblotting(Western blot). ResultsGhrelin enhanced the glucose uptake of L6 rat myoblasts with insulin resistance, the cell membrane Glut-4 stain was deepen, the expressions of pAkt and pGSK-3βprotein increased, and this effect could be PI3K blocker(LY294002) eliminated. ConclusionGhrelin promotes the glucose uptake of L6 rat myoblasts through PI3K/Akt/GSK-3βsignaling pathway, so as to improve the sensitivity of insulin in L6 rats muscle cells.
Objective To observe the effect of high glucose on the expression of activating transcription factor 4 (ATF4) in cultured retinal Muuml;ller glia cells. Methods The retinal tissue of Sprague-Dawley (SD) rats was collected, and Muuml;ller cells were isolated and cultured. The glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) of Muuml;ller cells were identified by streptavidin-biotin-peroxidase complex. Cultured rat Muuml;ller cells were divided into control group (5.5 mmol/L glucose), group A (20 mmol/L glucose), group B (30 mmol/L glucose) and group C (40 mmol/L glucose). ATF4 protein expressions in Muuml;ller cells of four groups were measured by Western blot four days after cultured. Results GFAP and GS expressed in more than 95% of Muuml;ller cells. Over 95% of Muuml;ller cells of group A, B and C were positive for GFAP and GS. Western blots indicated that ATF4 protein in group A, B and C increased obviously compared with the control group (q=0.293, 0.754,0.484;P＜0.05). Conclusion High glucose can increase the expression of ATF4 protein and cause endoplasmic reticulum stress in retinal Muuml;ller glia cells in vitro.
Mutations in the BEST1 gene are associated with a range of retinal diseases collectively referred to as "Best diseases", including Best vitelline macular dystrophy. More than 300 mutations at different sites of the BEST1 gene have been found, which may cause a series of functional disorders such as the mistransport of the calcium-activated anion channel protein-1 protein encoded by it, protein oligomerization defects, and abnormal anion channel activity, leading to different clinical phenotypes. Although it has been established that the BEST1 gene mutation is associated with at least one different type of Best disease, the relationship between the specific gene mutation site and the specific clinical phenotype has not been fully defined. For the time being. Drugs and gene therapy for the Best diseases are still in the basic research stage, which provides a broad development space for future treatment exploration. In the future, when selecting gene therapy in clinical applications, it is necessary to combine the clinical phenotype and molecular diagnosis of patients, and clearly define their mutation types and pathogenic mechanisms in order to achieve better personalized treatment effects.
PURPOSE:To verify existance of a-,~-,and 3'-protein kinase C(PKC)subspecies and their localization in rabbit retina. METHODS: Using an immunohistoehemical technique with mono- elonal antibodies against PKC isozymes- I (a),-I[ (13),and -~[ (Y) to characterize the distribution of PKC in rabbit retina. RESULTS:There is a positive immunostaining for a-,13-,and ~-PKC in rabbit retina. The immunoreactivity of a-PKC was observed mainly in the bipolar cells of inner nuclear layer and the outer segments of photorecptors. The positive immunostaining of 13-PKC could be seen in the ganglion cells,inner plexiform layer,inner nuclear layer,and the outer segments of photoreceptors. A diffuse and weak staining of Y-PKC is recognized in the ganglion cell layer,inner plexifrom layer,inner nuclear layer, and the outer segments of photoreceptors. CONCLUSION:The protein kinase C sub- speeies-a,-~,and-'Y are present in retina which is a part of the central nervous system
ObjectiveTo detect the expression of Liver X receptors (LXRs), protein phosphatase 1A (PPM1A), transforming growth factor-β1 (TGF-β1) and Smad2 in peripheral blood of bronchial asthma patients, and to explore whether LXRs and PPM1A are related to airway remodeling.MethodsSubjects were divided into healthy control group, mild and moderate asthma group and severe asthma group, with 30 subjects each. Lung function and high-resolution computed tomography examination were performed on patients with bronchial asthma to define airway remodeling. Peripheral blood was extracted and the serum levels of LXRα, LXRβ, PPM1A, TGF-β1 and Smad2 were detected after centrifugation. Then the data were analyzed.ResultsThe airway remodeling level of the mild and moderate asthma group was significantly higher than that of the control group. The airway remodeling level of the severe asthma group was significantly higher than that of the mild and moderate asthma group. Serum LXRα, LXRβ in asthma group were higher than those in the control group. The levels of LXRα and LXRβ in severe asthma were higher than those in mild and moderate asthma group. There was no significant correlation between LXRs and airway remodeling. The PPM1A level in mild and moderate asthma group was lower than that in the control group. The levels of PPM1A in severe asthma were lower than that in mild and moderate asthma group. PPM1A level was negatively correlated with airway remodeling.ConclusionsThe level of PPM1A in asthma patients is lower than that in healthy subjects, and is negatively correlated with the degree of airway remodeling. Serum LXRs in asthmatic patients are higher than that in healthy subjects, but LXRs are not significantly correlated with airway remodeling.