Objective To explore the mechanism of malignant behavior of cervical cancer(CC)cells based on AMP-activated protein kinase(AMPK)/rapamycin target protein(mTOR)signaling pathway mediated by nucleo-tide-binding oligomerization domain receptor 2(NOD2).Methods Bioinformatics analysis was performed to deter-mine the expression of NOD2 in CC tissue.Plasmids targeting NOD2(shNOD2)and shRNAs negative control(shNC),NOD2 overexpression(NOD2)and vectors(Vec)were transfected into CC cells.The effect of NOD2 on the growth of CC cells was determined by cell counting kit-8 assay,colony formation and Transwell cell invasion as-say.Transcriptome analysis was performed by high throughput RNA sequencing(RNA-Seq).Western blot was used to detect the expression of NOD2,AMPK/mTOR signaling pathway and autophagy protein in the cell line.24 female BALB/c nude mice were randomly divided into four groups,with 6 mice in each group:vector group(Vec group),NOD2 overexpression group(NOD2 group),shNC group and shNOD2 group.The distant metastasis mod-el was established in mice,and the fluorescence intensity of lung metastasis was monitored and the number of lung metastasis nodules was counted.Results On-line database analysis showed that the expression of NOD2 in CC tis-sues was significantly higher than that in normal tissues,and there were significant differences in the mRNA expres-sion of NOD2 in different stages of CC(P＜0.05).In addition,the high expression of NOD2 was associated with poor overall survival and disease-free survival(P＜0.05).NOD2 overexpression promoted the proliferation,colony formation,migration and invasion of CC cells,while NOD2 knock-down was the opposite.Consistent with the re-sults in vitro,the lung colonization and lung metastasis of CC cells in NOD2 group were significantly higher than those in Vec group(P＜0.05),while those in shNOD2 group were significantly lower than those in shNC group(P＜0.05).RNA-Seq results showed that the expression of NOD2 was significantly related to AMPK signal activa-tion,mTOR signal inhibition,autophagy regulation pathway activation and autophagy formation.Compared with shNC group,the expression of phosphorylated AMPK and LC3 protein decreased significantly in shNOD2 group(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein increased significantly(P＜0.05).Compared with Vec group,the expression levels of LC3 and AMPK protein in NOD2 group increased significantly(P＜0.05),and the expression levels of phosphorylated mTOR and p62 protein decreased significantly(P＜0.05).Compared with shNC group,the point accumulation of GFP-mRFP-LC3 in shNOD2 group decreased signifi-cantly(P＜0.05).Compared with Vec group,the point accumulation of GFP-mRFP-LC3 increased significantly(P＜0.05).Conclusion NOD2 may promote the proliferation,migration and invasion of CC through AMPK/mTOR signal,and its mechanism partly involves autophagy activation.

Objective:To observe the influence of shared medical appointments on health-related quality of life and quality of sleep in patients after liver transplantation.Methods:By randomized controlled study, a total of 124 patients after liver transplantation were included from our hospital from January 2018 to January 2019, and according to the lottery method, all subjects were divided into the routine management group ( n=64) who received routine outpatient intervention and the shared medical management group ( n=60) who received shared medical appointments management. The health-related quality of life and quality of sleep were investigated and compared by post-liver transplant quality of life questionnaire (pLTQ) and Pittsburgh sleep quality index (PSQI) before intervention (the day of discharge) and after intervention (the end of the last shared outpatient service). Results:After intervention, the dimension scores of worry, economics, body function, emotional function, health service, complication and total score of pLTQ were improved in tow groups than before intervention [the routine management group: (41.90±7.61), (18.13±4.22), (22.22±5.10), (14.92±3.28), (20.39±4.87), (14.63±3.99), and (132.19±37.09) vs (32.25±5.55), (12.77±3.47), (17.58±4.72), (9.23±1.38), (15.17±4.81), (10.89±1.51) and (98.00±29.03) score, t=8.20, 7.85, 3.58, 12.79, 6.10, 7.01, 5.81, all P<0.001; shared medical management group: (46.12±7.92), (24.16±5.34), (25.55±5.42), (17.90±3.60), (24.81±5.12), (17.93±3.60) and (155.47±41.00) vs (32.57±5.69), (12.81±3.82), (17.00±4.70), (9.60±1.39), (15.39±4.84), (11.00±3.52) and (98.37±28.96) score, t=10.76, 13.39, 9.23, 16.66, 10.36, 10.66, 8.81, all P<0.001], and those in the shared medical management group were higher than those in routine management group ( t=3.03, 6.95, 3.53, 4.82, 4.93, 4.83, 3.32, all P<0.05). After intervention, the total score of PSQI scale were lower than before intervention in the routine management group [(10.48±2.14) vs (11.89±2.45) score, t=3.47, P=0.001], and the dimensions score of sleep quality, full-sleep time, sleep time, sleep efficiency, sleep disorders, daytime function, hypnotic and total score of PSQI were lower than before intervention in the shared medical management group [(1.41±0.32), (0.54±0.13), (1.17±0.26), (1.11±0.35), (1.21±0.27), (1.30±0.33), (1.08±0.21) and (8.05±1.75) vs (1.88±0.53), (0.86±0.37), (1.84±0.41), (2.05±0.56), (1.39±0.33), (1.47±0.43), (1.22±0.32) and (11.71±2.43) score, t=-5.88, -6.32, -10.69, -11.03, -3.27, -2.43, -3.65, -9.47, all P<0.05], and those in the shared medical management group were lower than those in routine management group ( t=-6.68, -6.39, -10.43, -10.97, -2.62, -2.12, -3.54, -6.90, all P<0.05). Conclusion:Shared medical appointments model can improve the health-related quality of life and quality of sleep in patients after liver transplantation, and improve the effectiveness of outpatient intervention.

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.

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.

Objective:To construct a Nomogram model in predicting recurrence-free survival (RFS) and overall survival (OS) at six months, one year and two years after hepatocellular carcinoma (HCC) resection by using inflammatory markers combined with other routine clinical indicators.Methods:The data of 314 patients with HCC who underwent first time hepatectomy at Beijing Chaoyang Emergency Rescue Center and Air Force Characteristic Medical Center from January 2013 to January 2018 were analyzed. HCC patients who underwent hepatectomy at the First Medical Center of PLA General Hospital from January 2011 to January 2016 ( n=106) were used as the external validation group. Univariate and multivariate Cox proportional risk model was used to analyze independent risk factors of recurrence and death in HCC patients. A Nomogram model was constructed based on independent risk factors. Validation of the efficacy of the Nomogram model was done based on external data. Results:In the experimental group, 174 patients relapsed. The median RFS was 26 months. The 6 months, 1 year and 2 years RFS were 26.8%, 43.9%, and 68.8%, respectively. A total of 142 patients had died. The median survival time was 30 months. The 6 months, 1 year and 2 years OS were 5.9%, 23.6% and 63.1%, respectively. In the external validation group, 63 patients had developed recurrence, with a median RFS time of 28 months. The 6 months, 1 year and 2 years RFS were 26.4%, 45.3%, 54.7%, respectively. The median survival time was 31 months. The 6 months, 1 year and 2 years OS were 7.5%, 25.5%, 46.6%, respectively. Tumor size (>6.0 cm, HR: 1.447), vascular invasion ( HR: 1.408), TBil (>0.94 mg/dl, HR: 1.949), NLR (>2.54, HR: 2.843), AGR (≤0.88, HR: 2.447) were independent risk factors of HCC recurrence ( P<0.05). Tumor size (>6.0 cm, HR: 2.207), vascular invasion ( HR: 1.529), and NLR (>2.54, HR: 2.708) were independent risk factors of death for HCC patients ( P<0.05). The C-indexes of half-year, one-year and two-year RFS were 0.764 (95% CI: 0.677-0.854), 0.710 (95% CI: 0.615-0.824) and 0.673 (95% CI: 0.601-0.786), respectively. The C-indexes of half-year OS, one-year OS and two-year OS were 0.729 (95% CI: 0.648-0.841), 0.708 (95% CI: 0.608-0.813) and 0.664 (95% CI: 0.618-0.771), respectively. Conclusion:In this study, the construction of a Nomogram model in predicting prognosis of HCC patients was helpful to guide clinicians in improving preoperative treatment plans and in providing ideas for individualized treatment of patients.

OBJECTIVE: To establish a digital subtraction angiography (DSA) information management and image analysis system to realize scientific management of DSA image information and efficient processing of image data. METHODS: Based on Java Web under Windows 7 environment, a dynamic Browser/Server mode system was constructed by JSP and Servlet on the network. Eclipse and MySQL were used as development tool and database development platform. Tomcat network information service was used as application server. Matlab codes were embedded to analyze DSA image. RESULTS: The system consists of five modules:image information management, image processing, image analysis, advanced retrieval and clinical data management. It may complete such process as storing, deleting, saving, analyzing of DSA image and basic information of patients. CONCLUSIONS The main interface of the system is user-friendly and easy to operate. The system will be helpful to the clinical, teaching and scientific research work related to DSA.
Angiography, Digital Subtraction ; statistics & numerical data ; Databases, Factual ; Humans ; Indonesia ; Information Management ; Internet ; Software ; User-Computer Interface

Objective: To study the correlation between Erα gene polymorphism and the distribution pattern of TCM meridian syndrome in women with knee osteoarthritis. Methods: A total of 120 Knee osteoarthritis (KOA) female patients admitted to our hospital between January 2018 and December 2018 were selected as the observation group, and 120 healthy female subjects accepted physical examination at our hospital over the same period were selected as the control group. The Erα gene distribution of estrogen receptor was compared between the two groups to identify the differences between KOA women and healthy women. The TCM syndromes and meridians types of the patients in the observation group were defined to evaluate the degree of KOA. The correlation among KOA status, Erαgenotypes, TCM syndromes and meridians was analyzed. Results: The distribution of Erα gene XbaI was significantly different between the observation group and the control group (χ²=35.339, P<0.001). There was a statistical difference in the distribution of XbaI genotypes between mild to moderate and severe patients in the observation group (χ²=29.088, P<0.001). There was a moderate correlation between the Lequesne score and the fascia type in KOA women, and a high correlation between the Lequesne score and the TCM syndrome differentiation type, and a moderate correlation between the Lequesne score and the Erα gene and the XbaI genotype (P<0.05). The Erα gene polymorphisms, meridians and syndrome differentiation were moderately correlated (P<0.05). Conclusions In female patients with knee osteoarthritis, the conditions are more serious in patients with complex type of tendons, deficiency of liver and kidney, phlegm and blood stasis, Erα gene xx genotype, and there is a clear correlation between the TCM type of tendons and ER gene polymorphism.

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.

OBJECTIVETo investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism.

METHODSThe 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.

RESULTSRecombinant 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).

CONCLUSIONSMiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.

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.