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Objective:To observe the expression and significance of Jagged1 in fibrovascular membranes of proliferative diabetic retinopathy (PDR).Methods:Sixty preretinal fibrovascular membrane specimens collected from fifty-seven patients (60 eyes) with PDR during vitrectomy in The Affiliated Hospital of Qingdao University from July 2014 to July 2015 were set as the PDR group.The patients were divided into the injection group (30 cases, 32 eyes) and non-injection group (27 cases, 28 eyes) according to whether they received anti-vascular endothelial factor drug intravitreally before surgery.Ranibizumab injections were administered to the patients in the injection group intravitreally 2-7 days before surgery.Eighteen macular epiretinal membrane specimens obtained from 18 non-diabetic patients were served as the control group.Hematoxylin-eosin staining was applied to observe the structural festures of specimers. The immunohistochemical staining was used to detect the expression levels of Jagged1, Delta-like 4(Dll4) and Notch1 in the injection and non-injection groups, and the real-time fluorescent quantitative polymerase chain reaction was performed to detect the relative expression levels of Jagged1, Dll4 and Notch1 mRNA in the three groups.Pearson linear correlation analysis was used to evaluate the relationships between the expression of Jagged1 mRNA and both Dll4 mRNA or Notch1 mRNA in the PDR fibrovascular membranes.This study protocol adhered to the Declaration of Helsinki and was approved by an Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL25645). Written informed consent was obtained from each patient.Results:The neovascularization was found in fibrovascular membranes of PDR with a light microscope, and the lumen of the new blood vessels in the injection group was narrow, but relatively dilated in the non-injection group.There was no neovascularization found in the macular epiretinal membranes.The immunohistochemical staining revealed that there was the positive expression of Jagged1, Dll4 and Notch1 proteins in all PDR membranes, mainly located in the vascular endothelium during neovascularization.The absorbance values of Jagged1, Dll4 and Notch1 proteins were 6.25±1.82, 6.87±1.89 and 5.12±2.14 respectively in the non-injection group, which were all higher than 1.46±0.37, 1.55±0.24 and 1.32±0.53 respectively in the injection group, showing statistically significant differences ( t=5.168, P=0.014; t=6.012, P=0.008; t=3.453, P=0.030). There were statistically significant differences in Jagged1, Dll4 and Notch1 mRNA relative expression levels among the three groups ( F=77.337, 62.305, 51.869; all at P<0.01). The relative expression levels of Jagged1, Dll4 and Notch1 mRNA in the fibrovascular membranes with PDR were significantly higher than those of control macular epiretinal membranes, and the relative expression levels of Jagged1, Dll4 and Notch1 mRNA of the injection group were significantly lower than those of the non-injection group (all at P<0.05). The expression level of Jagged1 mRNA was positively correlated with expression levels of both Dll4 and Notch1 mRNA ( r=0.925, 0.950; both at P<0.05). Conclusions:There is a high expression of Jagged1 in the vascular endothelium of fibrovascular membranes with PDR and the Jagged1 expression is positively correlated with the expression of Dll4 and Notch1.The effect of Jagged1 on the neovascularization in PDR may be related to Dll4 and Notch1.
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Objective:To observe and investigate the effect of HIF-2α in the process of neovascularization of proliferative diabetic retinopathy (PDR).Methods:Retrospective clinical study. From July 2014 to July 2015, 60 eyes of 57 PDR patients diagnosed in Ophthalmology Department of Affiliated Hospital of Qingdao University were included in the study. Twenty-eight eyes of 27 patients received intravitreal injections of 0.5 mg ranibizumab (0.05 ml) at 2-7 days before surgery (ranibizumab group) and other 32 eyes of 30 patients did not (group without ranibizumab). Eighteen eyes of 18 patients with epiretinal membranes were included as controls. Pathological specimens of PDR fibrovascular membrane and premacular membrane were obtained during vitrectomy. The immunohistochemical staining and real-time PCR (RT-PCR) were used to detecting the expression of HIF-2α, Dll4 and VEGF. Kruskal-wallis test was used to compare the expression differences of correlation factors between groups. Spearman correlation analysis was used to analyze the relationship between the two variables.Results:The immunohistochemical staining revealed that there were positive expression of HIF-2α, Dll4 and VEGF in all PDR membranes, regardless of the injection of the ranibizumab. The levels of HIF-2α, Dll4 and VEGF protein in the group without ranibizumab were higher than those of the ranibizumab group ( t=4.36, 6.01, 4.82; P=0.000, 0.008, 0.016). RT-PCR showed that the differences of the mRNA expression of HIF-2α, Dll4 and VEGF were all statistically significant among the PDR patients and controls ( H=18.81,19.60, 20.50; P=0.000, 0.000, 0.000). The expression of HIF-2α, Dll4 and VEGF in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR patients,the expression of HIF-2α, Dll4 and VEGF of the group without ranibizumab was higher than that of the ranibizumab group. The spearman correlation analysis showed that the expression of mRNA between HIF-2α and Dll4, HIF-2α and VEGF were both significantly correlated ( r=0.95, 0.87; P<0.05). Conclusions:The expression of HIF-2α in the PDR membranes was higher than that of the controls. It is positively correlated with the expression of the DLL4 and VEGF.
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Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP).Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A),diabetes mellitus (DM) only group (group B),DM + balanced salt solution (BSS) group (group C),DM + hUCMSC group (group D),with 10 rats in each group.DM rats were induced by intraperitoneal injection of streptozotocin.Apoptosis of retinal cells was assayed by dUTP nick end labeling.Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats.Results Compared with group A,large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C,however the apoptotic cells in group D were significantly reduced than group B and C.The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A,throughout the inner plexiform layer (IPL) in group B and C,only distributed in RNFL and GCL in group D.It was obvious that the expression of GFAP in group B and C was higher than group A.Compared with group B and C,the expression of GFAP in group D was significantly reduced.The difference of GFAP expression among the 4 groups was significant (F=79.635,P＜0.05).Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
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Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.
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Objective@#To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function.@*Methods@#Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes.@*Results@#The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 μg and 1.00 μg HSP70-antigen peptide and 1.00 μg HSP90-antigen peptide activated lymphocytes significantly. Their A490 values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 μg HSP70-antigen peptide and 1.00 μg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 μg HSP90-antigen peptide and 1.00 μg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048).@*Conclusions@#The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.
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<p><b>OBJECTIVE</b>To investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism.</p><p><b>METHODS</b>The recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA.</p><p><b>RESULTS</b>Recombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (<0.05). The RTKN proteins in MCF-7 cells were significantly decreased after transfection with siRNA-RTKN, and the proliferation and migration ability of MCF-7 cells were significantly reduced (all<0.05).</p><p><b>CONCLUSIONS</b>MiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.</p>
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[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .
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BACKGROUND:Studies have found that erythropoietin has a protective effect on embryonic development of the retina, but there are rare studies concerning the embryonic development of the lens. OBJECTIVE: To observe the difference in erythropoietin expression in the lens from Wistar rats at different embryonic periods and to investigate the effect and significance of erythropoietin in the embryonic development of the lens. METHODS:Clean Wistar rats with pregnancy for 10 days (n=5), 12 days (n=5), 14 days (n=5), 16 days (n=5), 18 days (n=5), 20 days (n=5) were randomly colected and divided into six groups. Every two embryonic ratsof the different 30 pregnant rats were obtained randomly by the caesarean operation under ketamine-induced anesthesia. The eye tissues of al the embryonic rats were isolated and cut into sections. The expression of erythropoietin protein and mRNA in rat lens was detected by immunohistochemistry and reverse t ranscription-PCR, respectively. RESULTS AND CONCLUSION:Erythropoietin was distinctly expressed at the six different embryo stages, and the expression of erythropoietin protein and mRNA gradualy increased from embryonic day 10 to embryonic day 16, and decreased from embryonic day 10 to embryonic day 20. There were significant differences between the six groups. These findings indicate that the expression of embryonic appears in a low to high to low fashion during the embryonic development of Wistar rats, which may be closely associated with the developing procedure of lens.
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AIM: To study the expression and the role of neurokinin-1 receptor(NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum during acute necrotizing pancreatitis(ANP).METHODS: 130 adult Sprague-Dawley rats were divided into 2 groups: the rats in ANP group( n=80 ) were induced by the retrograde intraductal infusion of 5% sodium taurocholate. The rats in sham operation control group ( n=50 ) received laparotomy only. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, Western blot was used to investigate expression level of NK-1R.RESULTS: NK-1R and NK-2R mRNA levels were enhanced in the distal ileum of ANP rats compared with controls. Western blot revealed stronger NK-1R immunoreactivity exited in intestinal mucosa in ANP rats. The overexpression of NK-1R was associated with mucosal pathological score( r=0.77,P
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AIM To observe the change of Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway in opioids dependent NG108-15 cells and the relationships between Ca 2+ /calmodulin dependent protein kinaseⅡ (CaMKⅡ) signal pathway and cAMP accumulation. METHODS NG108-15 cells were used as an in vitro model system. Competitive protein binding assay and radioimmunoassay were used to examine the intracellular cAMP accumulation. Calmodulin activity was assayed by PDE method. CaMKⅡ activity was assayed by ?- 32 P incorporation of syntide-2. mRNA expression of mDOR was measured by RT-PCR. RESULTS DPDPE long-term treatment also increased cAMP accumulation, and resulted in opioids dependence in NG108-15 cells; DPDPE long-term treatment increased calmodulin activity and CaMKⅡ activity in NG108-15 cells. Specific calmodulin antagonist W-7 was found to inhibit significantly the elevation of calmodulin and CaMKⅡ activity which resulted from DPDPE long-term treatment, and CaMKⅡ inhibitor KN-62 also inhibited elevation of CaMKⅡ activity by DPDPE long-term treatment. DPDPE long-term treatment increased cAMP accumulation, when W-7 or KN-62 was added at the same time, cAMP accumulation decreased. When naloxone was added to NG108-15 cells which were long-term treated by DPDPE, calmodulin and CaMKⅡactivity increased more. mRNA level of mDOR did not change significantly in opioids dependent NG108-15 cells. CONCLUSION Ca 2+ /CaMKⅡ signal pathway was involved in the mechanisms of opioids dependence through regulating cAMP level when DPDPE is long-term administered to NG108-15 cells.
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As for the ? opioid receptor, there are some disputes about the domains involved in the selective recognition of ligands, the relative spatial orientation of the amino acids within the TM affect the affinities of selective ligands. Regulation of opioid receptor activities does not appear to involve in their ability to promote the association of GTP onto the G proteins and the subsequent dissociation of heterotrimers. It is not very clear if phosphorylation correlates with agonist induced receptor desensitization. The celluar processing of ? opioid receptors requires the formation of multiple protein complexes, it is clear that interactions of ubiquitous transcriptional factors determine gene transcription.