Establishment and validation of bioactivity measurement method for recombinant human midkine / 药学学报

To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).

Modified Erchentang Alleviates Inflammation in Chronic Obstructive Pulmonary Disease via Midkine/Notch2/Hey1 Signaling Pathway in Rats / 中国实验方剂学杂志

ObjectiveTo investigate the molecular mechanism of the anti-inflammatory effect of Erchentang in the lung tissue of the rat model of chronic obstructive pulmonary disease （COPD） via the heparin-binding factor （Midkine）/transmembrane receptor protein （Notch2）/Hey1 signaling pathway. MethodSixty SD rats were randomized into normal group， model group， modified Erchentang （5， 10， 20 g·kg-1·d-1） groups， and Notch1 pathway inhibitor （γ-secretase inhibitor， DAPT， 0.02 g·kg-1） group， with 10 rats in each group. The rat model of COPD was established by cigarette smoke combined with lipopolysaccharide （LPS）. After the modeling， the rats were administrated with corresponding drugs by gavage， and those in the normal and model groups were administrated with normal saline by gavage for 21 days. The levels of Midkine， cytokine-induced neutrophil chemoattractant-1 （CINC-1）， macrophage-derived chemokine （MDC）， chemokine ligand 5 （CXCL5）， neutrophil elastase （NE）， and nuclear factor-kappa B （NF-κB） p65 in bronchoalveolar lavage fluid （BALF） were determined by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and immunohistochemistry were respectively employed to determine the mRNA and protein levels of Midkine， Notch2， and Hey1 in the lung tissue. ResultCompared with the normal group， the modeling increased the levels of Midkine， CINC-1， MDC， CXCL5， NE， and NF-κB p65 in BALF （P<0.01） and up-regulated the mRNA and protein levels of Midkine， Notch2， and Hey1 in the lung tissue （P<0.01）. Compared with the model group， medium- and high-dose modified Erchentang and DAPT lowered the levels of Midkine， CINC-1， MDC， CXCL5， and NF-κB p65 in BALF （P<0.01） and down-regulated the mRNA levels of Midkine， Notch2， and Hey1 （P<0.01）. ConclusionModified Erchentang may inhibit the inflammation in COPD rats by down-regulating the expression of Midkine， Notch2， and Hey1 and reducing the content of Midkine， CINC-1， MDC， and CXCL5.

The auxiliary diagnostic value of combined detection of serum midkine and thyroid stimulating hormone for differentiated thyroid cancer / 中国医师进修杂志

Objective:To investigate the auxiliary diagnostic value of serum midkine (MK) and thyroid stimulating hormone (TSH) for differentiated thyroid cancer (DTC).Methods:Seventy-one postoperative DTC patients (DTC group) treated with 131I were selected, and 143 patients with benign thyroid lesions (benign thyroid disease group) treated with surgery in Center Hospital of Xiaogan from March 2019 to December 2020 at the same period were also selected. Clinical data such as liver and kidney function indexes, positive rate of anti thyroglobulin antibodies (TGAb) and positive rate of thyroid peroxidase antibody (TPOAb) were collected before treatment, and their fasting blood samples were collected before treatment. Fully automated electrochemiluminescence immunoassay was used to measure free thyroxine (FT 4), free triiodothyronine (FT 3), TSH levels in patients′ serum. The serum MK levels were measured by enzyme-linked immunosorbent assay (ELISA). Binary Logistic regression model was used to screen for independent risk factors for the development of DTC. A receiver operating characteristic curve (ROC) was drawn to evaluate the efficacy of MK, TSH and MK combined with TSH, in aiding the diagnosis of DTC and its staging. Results:Serum TSH and MK levels in DTC group were higher than those in benign thyroid disease group: (3.55 ± 0.61) mU/L vs. (2.97 ± 0.46) mU/L, (394.25 ± 63.36) ng/L vs. (311.45 ± 42.66) ng/L, and the differences were statistically significant ( P<0.05). Elevated serum TSH and MK levels were independent risk factors for DTC. When MK combined with TSH was used to diagnose DTC, the area under the curve (AUC), sensitivity and specificity were higher than those of MK and TSH alone (0.925 vs. 0.859 and 0.783, 83.10% vs. 78.87% and 73.24%, 89.51% vs. 85.31% and 79.02%), and the differences were statistically significant ( P<0.05). Serum TSH and MK levels in stage Ⅲ and Ⅳ patients in DTC group were higher than those in stage Ⅰ and Ⅱ patients: (3.79 ± 0.65) mU/L vs. (3.42 ± 0.56) mU/L, (427.88 ± 52.73) ng/L vs. (311.45 ± 42.66) ng/L, and the differences were statistically significant ( P<0.05). The AUC, sensitivity and specificity of MK combined with TSH in the diagnosis of different stages of DTC were higher than those of MK and TSH alone (0.822 vs. 0.657 and 0.666, 73.90% vs. 56.52% and 56.52%, 83.33% vs. 77.08% and 79.17%), and the differences were statistically significant ( P<0.05). Conclusions:Serum TSH and MK levels are independent risk factors for the occurrence of DTC in patients, and the combination of them has certain auxiliary diagnostic value for the identification and staging of DTC.

Relationship between serum KLK11 and MK levels and the efficacy of the first 131I ablation after surgery for differentiated thyroid cancer / 中华内分泌外科杂志

Objective:To explore and analyze the relationship between serum KLK11 and MK levels and the effect of first iodine 131 ( 131I) ablation after operation for differentiated thyroid cancer. Method:108 patients with differentiated thyroid cancer who underwent total thyroidectomy in our hospital from Jun. 2020 to Jun. 2021 were consecutively selected, and received radioactive ablation after surgery. There were 37 males and 71 females. The age was (48.32±4.25) years, ranging from 28 to 79 years. The patients were divided into successful ablation according to whether the ablation was successful after treatment. There were 64 cases in the group and 44 cases in the unsuccessful group, and 60 healthy people with no abnormality in physical examination during the same period were selected as the control group. The patients were divided into a metastasis-positive group of 20 cases and a metastasis-negative group of 88 cases according to whether lymph nodes occurred. After surgery, serum samples of all subjects were taken, and enzyme-linked immunosorbent assay was used to detect the levels of serum kallikrein-related peptidases 11 (KLK11) and midkine (MK) , and the levels of serum KLK11 and MK were analyzed. Gender, age, BMI, TNM stage, TSH, maximum diameter of lesion, and duration of nail removal were collected. Univariate analysis and logistic regression analysis were used to analyze the independent risk factors of postoperative efficacy.Result:The levels of serum KLK11 and MK in the successful and unsuccessful groups were higher than those in the control group, while the levels of KLK11 and MK in the unsuccessful group were higher than those in the successful group (KLK11: t= 2.642, P<0.05; MK: t=11.906, P<0.05) . The serum levels of KLK11 and MK in the metastasis-positive group were higher than those in the metastasis-negative group (KLK11: t= 2.908, P<0.05; MK: t=14.907, P<0.05) . Univariate analysis showed that BMI ( χ2=6.780, P=0.009) , maximum diameter of lesions ( χ2=14.819, P=0.001) , TSH ( χ2=13.627, P=0.001) , serum KLK11 ( t=2.642, P=0.01) , and serum MK ( t=11.906, P<0.001) were associated with the effect of first 131I ablation after surgery for differentiated thyroid cancer ( P<0.05) . Taking the success of ablation as the dependent variable, a multivariate logistic regression analysis was performed. The results showed that the maximum diameter of the lesions greater than 2 cm ( OR=10.740, 95%CI: 7.033-16.401) , increased level of TSH ( OR=8.559, 95%CI: 2.812-26.057) , increased serum KLK11 level ( OR=16.710, 95%CI: .548-32.666) and increased serum MK level ( OR=10.580, 95%CI: 6.294-17.786) were the factors affecting the first 131I ablation effect after DTC surgery ( P<0.05) . Conclusion:The elevated levels of serum KLK11 and MK are independent risk factors affecting the efficacy of the first 131I ablation after surgery for differentiated thyroid cancer.

H. sinensis mycelium inhibits epithelial-mesenchymal transition by inactivating the midkine pathway in pulmonary fibrosis / 医学前沿

The medical fungus Hirsutella sinensis has been used as a Chinese folk health supplement because of its immunomodulatory properties. Our previous studies established the antifibrotic action of Hirsutella sinensis mycelium (HSM) in the lung. The epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of idiopathic pulmonary fibrosis. The present study investigates the role of HSM in mediating EMT during the development of pulmonary fibrosis. HSM significantly inhibits bleomycin (BLM)-induced pulmonary fibrosis by blocking the EMT. In addition, the expression levels of midkine are increased in the lungs of the BLM-induced group. Further analysis of the results indicates that the mRNA level of midkine correlated positively with EMT. HSM markedly abrogates the transforming growth factor β-induced EMT-like phenotype and behavior in vitro. The activation of midkine related signaling pathway is ameliorated following HSM treatment, whereas this extract also caused an effective attenuation of the induction of EMT (caused by midkine overexpression) in vitro. Results further confirm that oral medication of HSM disrupted the midkine pathway in vivo. Overall, findings suggest that the midkine pathway and the regulation of the EMT may be considered novel candidate therapeutic targets for the antifibrotic effects caused by HSM.

Urinary Biomarkers may Complement the Cleveland Score for Prediction of Adverse Kidney Events After Cardiac Surgery: A Pilot Study

BACKGROUND: The ability of urinary biomarkers to complement established clinical risk prediction models for postoperative adverse kidney events is unclear. We assessed the effect of urinary biomarkers linked to suspected pathogenesis of cardiac surgery-induced acute kidney injury (AKI) on the performance of the Cleveland Score, a risk assessment model for postoperative adverse kidney events. METHODS: This pilot study included 100 patients who underwent open-heart surgery. We determined improvements to the Cleveland Score when adding urinary biomarkers measured using clinical laboratory platforms (neutrophil gelatinase-associated lipocalin [NGAL], interleukin-6) and those in the preclinical stage (hepcidin-25, midkine, alpha-1 microglobulin), all sampled immediately post-surgery. The primary endpoint was major adverse kidney events (MAKE), and the secondary endpoint was AKI. We performed ROC curve analysis, assessed baseline model performance (odds ratios [OR], 95% CI), and carried out statistical reclassification analyses to assess model improvement. RESULTS: NGAL (OR [95% CI] per 20 concentration-units wherever applicable): (1.07 [1.01–1.14]), Interleukin-6 (1.51 [1.01–2.26]), midkine (1.01 [1.00–1.02]), 1-hepcidin-25 (1.08 [1.00–1.17]), and NGAL/hepcidin-ratio (2.91 [1.30–6.49]) were independent predictors of MAKE and AKI (1.38 [1.03–1.85], 1.08 [1.01–1.15], 1.01 [1.00–1.02], 1.09 [1.01–1.18], and 3.45 [1.54–7.72]). Category-free net reclassification improvement identified interleukin-6 as a model-improving biomarker for MAKE and NGAL for AKI. However, only NGAL/hepcidin-25 improved model performance for event- and event-free patients for MAKE and AKI. CONCLUSIONS: NGAL and interleukin-6 measured immediately post cardiac surgery may complement the Cleveland Score. The combination of biomarkers with hepcidin-25 may further improve diagnostic discrimination.

Changes and significance of serum midkine in children with allergic purpura and purpura nephritis / 中国小儿急救医学

Objective@#To investigate the role of midkine(MK)in the pathogenesis of Henoch-Schonlein purpura(HSP) and Henoch-Schonlein purpura nephritis(HSPN).@*Methods@#In the case group, 35 cases were hospitalized in the pediatric kidney rheumatism immunology ward of Shengjing hospital affiliated to China Medical University from December 2016 to January 2018.Among them, 10 cases were HSP, 25 were HSPN.According to quantitative level of 24-hour urine protein, HSPN group was divided into HSPN(nephrotic level of proteinuria)group of 15 cases and HSPN(non-nephrotic level of proteinuria)group of 10 cases.The control group consisted of 12 healthy cases who underwent physical examination at outpatient department in the same period in the developmental pediatric clinic of our hospital.Blood samples were collected to detect MK.The other clinical datas including renal function, 24-hour urine protein quantitative, immunoglobulin, etc were collected.The serum MK and renal function indexes were compared among groups.The correlation between MK and various clinical indicators was analyzed, and the receiver operating characteristic(ROC) curve was used to analyze the diagnostic significance of MK for HSP and HSPN.@*Results@#MK level of case group was higher than that of healthy control group[(289.34±160.70)pg/ml vs.(100.03±56.75)pg/ml, P<0.05]. Moreover, the difference of MK concentration among the HSPN(nephrotic proteinuria)group, the HSPN(non-nephrotic proteinuria)group and the HSP group was still statistically significant[(449.91±141.91)pg/ml vs.(244.04±89.15)pg/ml vs.(175.94±46.30)pg/ml, P<0.05]. MK was positively correlated with urine microalbumin(r=0.54), IgA(r=0.132), IgE(r=0.304), urine β2 microglobulin(r=0.483), 24-hour urine protein /body weight(r=0.503), and urine transferrin level(r=0.509)in the case group(P<0.05). According to the ROC curve, the area under ROC of MK for predicting the diagnosis of HSP was 0.908(95%CI 0.828-0.988). The optimal value in predicting the diagnosis of HSP was 182.762 pg/ml, with sensitivity and specificity of 81.4% and 91.7%.The area under ROC of MK in predicting HSPN was 0.947(95%CI 0.888-1.000), and the optimal value of predicting HSPN was 218.186 pg/ml, with sensitivity and specificity of 84.0% and 95.5%.@*Conclusion@#MK may be involved in the pathogenesis of HSP and HSPN.It can provide the basis for clinical diagnosis of HSP and HSPN, and has significance in evaluating the degree of renal damage of HSPN.

Effect of midkine on expression of non-coding RNA in ovarian cancer / 临床检验杂志

Objective@#To explore the correlation of non-coding RNA and the tumor-associated antigen midkine (MK) in SKOV3cells and the clinical significance for diagnosis of ovarian cancer. @*Methods@#The Agilent′s gene chips (miRNAs chip and lncRNAs chip) were used to analyze the differential expression of miRNAs and lncRNAs in both MK-overexpressing SKOV3-MK cells and the control SKOV3-Con cells to screen the potential biomarkers in ovarian cancer. The clinical significance of midkine in the serum and tissues samples was analyzed for the patients with ovarian cancer by quantitative PCR combined with clinical data. @*Results@#Compared with control SKOV3-con cells, MK overexpression significantly promoted the expressions of 11 miRNAs and 7 lncRNAs in SKOV3 cells (P＜0.01, ratio＞3 fold), reduced the expressions of 8 miRNAs and 13 lncRNAs (P＜0.01, ratio＜0.3). Results of qPCR showed that the expression level of miR489 was significantly lower in ovarian cancer tissues than that of the contralateral normal ovarian tissues, while HOTAIR was significantly elevated (P＜0.05). The expression level of HOTAIR in the serum of ovarian cancer patients was significantly higher than that in healthy controls group with same age (0.036±0.024 vs 0.019±0.020, P=0.002). ROC curve analysis of HOTAIR showed that the specificity was 66.7%, the sensitivity was 75.6% and the AUC value was 0.749 as a marker for serum detection of ovarian cancer when the cutoff value was 0.017 6. @*Conclusion@#Long-chain non-coding RNA HOTAIR may be served as a potential biomarker in serum of ovarian cancer patients.

Progress in development and application of FNA washout detection in thyroid and parathyroid surgery / 中华内分泌外科杂志

Recently,the detection of FNA washout has been widely spread due to its high diagnostic efficiency,which then derived several detection indicators so far.Based on the relative literature,this paper aims to review the progress in development and application of FNA washout detection in thyroid and parathyroid surgery.We will summary the diagnosis applications from the following five parts:1.FNA-Tg in lymph node metastasis of thyroid cancer,2.FNA-CT in medullary thyroid carcinoma and lymph node metastasis,3.FNA-PTH in hyperparathyroidism and intraoperative protection of parathyroid,4.FNA-FT4 in thyroid adenoma,5.FNA-MK in distinguishing between benign and malignant thyroid nodules.

Expression of copeptin and midkine in diabetic retinopathy and their significance / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the expression of copeptin and midkine(MK)in patients with diabetic retinopathy and their clinical significance. <p>METHODS: Totally 180 patients with type 2 diabetes mellitus(T2DM)admitted to our hospital from June 2016 to October 2017 were divided into three groups according to the criteria of diabetic retinopathy staging. Among them, there were no retinopathy group(68 cases), non-proliferative retinopathy group(72 cases), proliferative retinopathy group(40 cases)and 90 healthy subjects in the same period as the control group. The copeptin level was detected by double antibody immunosorbent assay and the MK level by ELISA. The glycosylated hemoglobin(HbA1c)was detected by TOSOH automatic glycosylated hemoglobin analyzer and the whole blood hs-CRP was detected by using blood routine instrument. Fasting plasma glucose(FPG), triglyceride(TG), cholesterol(TC), low density lipoprotein(LDL-C)and high density lipoprotein(HDL-C)were detected by automatic biochemical analyzer and the systolic blood pressure(SBP)and diastolic blood pressure(DBP)of each group were recorded. The relationship between copeptin and MK levels and blood lipid, blood pressure, biochemical parameters and the course of diabetes mellitus were also analyzed. <p>RESULTS: The levels of copeptin and MK in patients with diabetic retinopathy were significantly higher than those in controls, and the levels of copeptin and MK were gradually increased with the progress of disease(<i>P</i><0.05). The levels of copeptin and MK were correlated with the levels of HbA1c, hs-CRP and course of T2DM(<i>P</i><0.05), but no significant correlation with plasma lipids, blood pressure and FPG(all <i>P</i>>0.05). <p>CONCLUSION: The copeptin and MK levels are closely related to the course of diabetic retinopathy and the severity of the disease, both of which may be involved in the development of diabetic retinopathy.

Influence of Combined Detection of Serum Midkine and TBIL Levels in Diagnostic Efficacy of Diabetic Retinopathy / 医学研究杂志

Objective To explore the diagnostic efficacy of combined detection of serum midkine (MK) and total bilirubin (TBIL)levels of diabetic retinopathy(DR),and determine the value in diagnosis of DR.Methods Clinical data of 215 cases of patients with type 2 diabetes mellitus (T2DM) treated in our hospital from July 2014 to December 2016 were collected,and diabetes mellitus without retinal lesions (NDR) in 79 cases,non-proliferative diabetic retinopathy (NPDR) in 81 cases,proliferative diabetic retinopathy (PDR) in 55 cases.Another 70 cases of physical examination persons in the same period were as the control group.Serum MK and TBIL levels were detected in the four groups,and the relationship between indexes and DR was done by Person correlation analysis and Logistic regression analysis,and diagnostic efficacy of Midkine and TBIL was evaluated in DR.Results Serum MK and TBIL levels were compared between the four groups,with statistically significant differences (P ＜ 0.05).MK level increased in normal control group,NDR group,NPDR group and PDR group,while TBIL levels decreased in each group.MK was positively correlated with DR (r =0.779,P =0.005),and TBIL was negatively correlated with DR (r =-0.657,P =0.012),and both were independent factors influencing DR (OR =2.257,0.681,P ＜ 0.05).The sensitivity of combined detection of MK and TBIL in diagnosis of DR was higher than that of each index detected separately (P ＜ 0.05).Conclusion Serum MK and TBIL levels are closely related to the occurrence of DR and severity of the disease,and the combined detection has high diagnostic efficacy for DR.

The effect of MK gene overexpression in hepatic carcinoma cells on multidrug resistance / 中华普通外科杂志

Objective To study the effect of enhanced MK gene expression in hepatic carcinoma cells.Methods The recombinant plasmid pIRES2-EGFP-MK was transfected into SMMC 7721 cells.The mRNA and protein expression levels of MK gene in these cells were determined by real-time PCR,Western blotting and flow cytometry.The intracellular DNR accumulation of these cells was measured by flow cytometry.To investigate the effect of MK gene mediated multidrug resistance,MTT assay was employed to determine the cellular sensitivity of different chemotherapeutic drugs in MK-overexpressed SMMC 7721 cells.Results The mRNA and protein expression levels of MK gene significantly increased after the recombinant plasmid pIRES2-EGFP-MK transfected into SMMC 7721 cells,suggesting that the recombinant plasmid pIRES2-EGFP-MK can enhance the transcription of MK effectively.The DNR accumulation of MK transfected cells decreased significantly (4.06 ± 0.88,P ＜ 0.05),and IC50 of MK transfected cells to ADM/5-FU increased significantly (15 ± 3,27 ± 4,P ＜ 0.05).Conclusions After the recombinant plasmid pIRES2-EGFP-MK transfected into hepatic carcinoma cells,expression of midkine increased,enhancing the resistance of hepatic carcinoma cells to chemotherapeutic drugs.

Predictive value of serum midkine for metastatic lesions in thyroid cancer patients with positive thyroglobulin antibody / 天津医药

Objective To investigate the predicative value of midkine (MK) as a cancer biomarker for metastatic lesions in differentiated thyroid cancer (DTC) patients with positive thyroglobulin antibodies (TgAb) before the first 131Ⅰ therapy.Methods MK levels were measured by enzyme-linked immunosorbent assay in 151 recruited DTC patients included in this study according to strict inclusion and exclusion criteria.There were 28 TgAb positive DTC patients with metastases and 123 DTC patients without metastases.The value of pre-131Ⅰ-ablative MK to predict metastasis was assessed by receiver operating characteristic (ROC) curves in these two groups of patients.Results MK levels were significantly higher in TgAb positive DTC patients than those in DTC patients without metastases.MK levels showed good diagnostic value,with an area under the curve of 0.856 (P＜0.001),and a diagnostic accuracy of 83％ at the optimal cut-off value of 550 ng/L.Conclusion Results show that MK can potentially be used as a surrogate biomarker for predicting DTC metastases when thyroglobulin is not suitable due to TgAb positivity.

Midkine gene silencing enhances the sensitivity of liver cancer Bel/Fu cells to 5-fluoreouracil and its possible mechanism / 肿瘤

Objective: To investigate the effect of midkine (MK) gene silencing on chemosensitivity of human liver cancer 5-fluorouracil (5-Fu)-resistant cell line Bel/Fu, and to explore its possible mechanism. Methods: The specific sequence targeting MK gene (MK-siRNA) was transfected into Bel/Fu cells, then the expression levels of MK mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Bel/Fu cells were transfected with MK-siRNA and treated with 5-Fu, then the proliferation and apoptosis of Bel/Fu cells were detected by MTT and FCM assay, respectively. Furthermore, the expression levels of phosphorylated-phosphoinositide 3-kinase (p-PI3K), phosphorylated-protein kinase B (p-PKB, p-Akt), nuclear factor-kappa B (NF-?B), Bcl-2, survivin and caspase-3 proteins were detected by Western blotting. Results: The expression levels of MK mRNA and protein in Bel/Fu cells transfected with MK-siRNA were significantly down-regulated (both P < 0.05). The half inhibitory concentration (IC50) of 5-Fu in Bel/Fu cells transfected with MK-siRNA was (472± 21) μg/mL, significantly lower than that in untransfected blank control group (2 035 ± 34) μg/mL (P < 0.05). The apoptotic rate of Bel/Fu cells transfected with MK-siRNA was (32.10±3.61)%, significantly higher than that in untransfected blank control group (7.90±0.91)% (P < 0.01). The expression levels of p-PI3K, p-Akt, NF-?B, Bcl-2 and survivin proteins were significantly down-regulated (all P < 0.05), while the expression level of caspase-3 was significantly up-regulated (P < 0.05) in Bel/Fu cells transfected with MK-siRNA. Conclusion: MK gene silencing can enhance the chemosensitivity of hepatocellular cancer Bel/Fu cells to 5-Fu. The mechanism may be involved in activation of PI3K/Akt signal pathway.

Diagnostic value of serum midkine combined with total bilirubin for diabetic retinopathy / 中国糖尿病杂志

Objective To investigate the diagnostic value of serum MK (Midkine)and total bilirubin (TB)in diabetic retinopathy (DR). Methods A total of 148 patients with type 2 diabetes were divided into three groups:diabetes without retinopathy (NDR group,n = 50),diabetic with non proliferative retinopathy (NPDR group,n = 52 )and diabetic with proliferative retinopathy (PDR group,n = 46 ) according to whether retinal lesions were detected. The diagnostic value of serum MK and TB were investigated. Results Age,gender,body mass index (BMI),FPG,HbA1 c,TG,TC,LDL-C,HDL-C, SBP,DBP were not statistically different among the three groups (P >0.05).Duration,UAlb/Cr,SOD, MDA,AOPP,MK,TB and DB were statistically different among the three groups (P<0.05). Duration, ALB/CR,MDA,AOPP,and MK were higher,SOD,TB,DB were lower in PDR and NPDR group than in NDR group (P<0.05). Duration,UAlb/Cr,MDA,AOPP,and MK were higher,and SOD,TB,DB were lower in PDR group than in NPDR group (P <0.05).Logistic regression analysis showed that duration, ALB/CR,MDA,AOPP,and MK were risk factors (OR =1.36,1.71,1.27,1.65,2.35,P <0.05 )and SOD,TB,DB were protective factors for DR (OR =0.46,0.31,0.46,P <0.05). Diagnosis sensitivity, specificity,positive predictive value,negative predictive value and accuracy of TB combined with MK were higher than TB,MK alone(AUC=0.918,0.735,0.762,P <0.05). Conclusion DR may be associated with increased MK and decreased TB.Diagnostic efficacy of MK combined with TB is better than MK,TB alone.

The expression of helper T cells 17/regulatory T cells balance associated factors in rheumatoid arthritis patients and their correlation with serum midkine / 中华风湿病学杂志

Objective To investigate the expression of helper T cells (Th)17/regulatory T cells (Treg) balance associated factors in rheumatoid arthritis (RA) patients and their correlation with serum midkine (MK).Methods A total of 60 RA patients were divided into active RA patients (n=32) and inactive RA patients group (n=28).MK level in sera was detected by enzyme linked immunosorbent assay (ELISA) in 60 patients with RA and 30 healthy controls (HCs).The fraction of CD4+CD25+FOXP3+ Treg cells and IL-17+CD4+ Th17 cells in RA patients and healthy controls were determined by flow cytometry (FCM), and the expreasion of Foxp3, RORγt, Signal transducer and activator of transcription (STAT) 3 and STAT5 mRNA were detected with real-time polymerase chain reaction (PCR).Results were evaluated using ANOVA followed by q tests for comparisons of Th17 population between active RA patients, inactive RA patients and HCs, t test was used for comparing of Foxp3, RORγt, STAT3, STAT5 mRNA between RA group and HCs.The correlations between serum MK concentration and peripheral Treg cells, Th17 cells, Foxp3, RORγt, STAT3, STAT5 mRNA were analyzed by Pearson's correlation analysis.Results The percentages of Treg cells from active RA patients, inactive RA patients and HCs were significantly different (F=129.6, P＜0.01), the percentages of Treg cells of active RA patients [(1.41±1.05)％] were lower than that of the inactive RA patients [(3.6±1.6)％;q =7.92, P＜0.05] and healthy group [(7.7±1.7)％;q=22.45, P＜0.05], and there was significant difference between healthy group and inactive RA group (q=14.53, P＜0.05).The percentages of Th17 cells of the three groups were also significantly different (F=36.3, P＜0.01),the percentage of Th17 cells of active RA patients [(1.84±1.01)％] was significantly higher than that of inactive RA patients [(0.71±0.28)％;q=9.59, P＜0.05] and healthy group (0.53±0.16)％ [(P＜0.05;q=1 1.10, P＜0.05], there was no significant difference between the inactive RA group and healthy group (q=1.51, P＞0.05).The expression of RORγt and STAT3 mRNA in RA patients was higher than that of healthy controls (t=5.84, P＜0.01;t=4.52, P＜0.01).The expression of Foxp3 and STAT5 mRNA in RA patients were lower than healthy controls (t=6.01, P＜0.01;t=2.18, P＜0.05).Serum MK values were correlated with STAT5 (r=-0.55, P＜0.01), but not with Foxp3, RORγt, STAT3 mRNA or the percentage of Treg/Th17 cells.Conclusion Serum MK expression and the percentage of Th17 cells increase, while the percentage of Treg cells decrease in RA patients.Serum MK values are negatively correlated with STAT5 mRNA which is associated with Th17/Treg balance.This may be important in the pathogenesis of RA.

Midkine-a inhibits zebrafish embryonic heart growth by limiting the cardiomyocyte pool / 中国实验动物学报

Objective To investigate the functional role of a soluble secretion factor midkine-a in the process of zebrafish embryonic heart development .Methods Whole-mount in situ hybridization was performed to detect whether midkine-a is expressed in the embryonic heart .In the transgenic embryonic heart of Tg ( pmidkine-a:EGFP) , the expres-sion of EGFP in the heart was monitored .Multiple heat shock treatments were applied to Tg (phsp:midkine-a:EGFP) em-bryos in order to overexpress midkine-a, and the phenotype of the heart was observed .The heterozygous Tg ( phsp:mid-kine-a:EGFP) fish were crossed with homozygous Tg (pcmlc2:dsRed) fish, which specifically expresses RFP in the nucle-oli of embryonic cardiomyocytes to facilitate myocyte number counting .To get Tg ( phsp:midkine-a:EGFP/pcmlc2:dsRed)embryos, and the total number of cardiomyocytes in their heart was counted and compared with the controls when the heat shock induced overexpression of midkine-a.Morphonino knocked out of midkine-a was performed to observe the heart phe-notype.Results Midkine-a was expressed in the zebrafish embryonic heart during development .Overexpression of mid-kine-a led to a smaller heart and reduced total number of cardiomyoctes in a single heart , which might be associated with the smaller heart phenotype .Morphonilo knocked out of midkine-a had no effect upon the heart development .Conclusions Midkine-a impedes zebrafish embryonic heart growth by limiting its cardiomyocyte pool .

Study of MK promoting breast cancer cell MDA-MB-231 angiogenesis through EPCR/PAR1 pathway in vitro / 临床与实验病理学杂志

Purpose To observe the effects of midkine ( MK) on human breast cancer cell line MDA-MB-231 angiogenesis in vitro, and to explore its mechanism. Method shRNA interference was performed to silence the expression of MK in MDA-MB-231 cells, and Western blot was used to identify the expression of MK and EPCR. After MK and EPCR knockdown, or treated with anti protease-activated receptor 1 (PAR1) antibody, the culture medium of MDA-MB-231 cells were collected and the conditioned medium were pre-pared. Human umbilical vein endothelial cells ( HUVECs) were cultured with conditioned medium, and the endothelial cells prolifera-tion was detected by CCK-8 assay, cell migration was detected by transwell method, vasculogenic activity was assessed by Matrigel-based tube formation assay. Results After knockdown of MK, the protein level of EPCR was decreased in MDA-MB-231 cells. Com-pared with control, knockdown of MK and EPCR decreased the proliferation, migration and angiogenesis ability of HUVECs significant-ly (P<0. 05), and the effect of EPCR knockdown group was stronger than MK knockdown group (P<0. 05). After treated with anti-PAR1 antibody, the proliferation, migration and angiogenesis ability of HUVECs were decreased compared with control and EPCR knockdown group (P<0. 05). Conclusion MK promotes human breast cancer cell line MDA-MB-231 angiogenesis through EPCR /PAR1 signaling pathway in vitro.

Immunohistochemical evaluation of midkine and nuclear factor-kB as markers for diagnosis and prediction of synchronous metastasis in papillary thyroid cancer / 中华内分泌代谢杂志

Objective Midkine ( MK ) and nuclear factor-kappa B ( NF-kB ) play pivotal roles in tumorigenesis, which are considered as promising cancer biomarkers. The efficacy of MK and NF-kB as markers for diagnosis and prediction of synchronous metastasis in papillary thyroid cancer ( PTC ) was the aim of present investigation. Methods Seventy six cases of PTC and seventy cases of multi-nodular goiter ( MNG ) were retrieved. The PTC group was further divided into subgroup 1 (16 cases with synchronous metastasis) and subgroup 2 (60 cases without metastases). A retrospective review of clinical information, radiological examinations,131 I treatments and post-131 I-therapy scans were done. Immunohistochemistry of MK, NF-kB p65, and Ki-67 was performed on paraffin-embedded specimens and results were quantified. Diagnostic values of the parameters were conducted by receiver operating characteristic (ROC) curves. Diagnostic sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were determined. Protein levels of MK and NF-kB p65 were then confirmed by Western blot. Results Immunoreactivities of MK and NF-kB p65, and positive percentage of Ki-67 were significantly higher in PTC group than in MNG group (all P<0. 01). ROC showed good differential diagnostic capabilities of all three parameters with diagnostic accuracies of 82. 192% , 80. 137% , and 84. 091%respectively. Moreover, all three parameters were significantly higher in subgroup 1 than those in subgroup 2 (all P<0. 01). ROC showed good predicting efficacies in synchronous metastasis of all three parameters with diagnostic accuracies of 82. 895% , 80. 263% , and 76. 316% respectively. By one-way analysis of variance, Western blot showed that MK and NF-kB p65 protein levels in lesions from subgroup 1 were significantly higher than those from subgroup 2, both were significantly higher than those in MNG lesions ( P<0. 01). Conclusion MK and NF-kB immunohistochemistry can potentially be used for differential diagnosis between PTC and MNG, and for prediction of synchronous metastases.

The quantitative measurement of midkine in serum of patients with acute leukemia and its clinical significance / 白血病·淋巴瘤

Objective To explore the meaning of midkine (MK) levels in serum in different development stage of acute leukemia,and to explore the relationship between MK and WT1.Methods The levels of the MK in serum of 86 cases of acute leukemia and 30 cases of normal people were detected by ELISA.Real-time quantitative PCR (RQ-PCR) method was used to determine the expression of WT1 at mRNA level in 15 AML patients.Results The MK level in serum in the new diagnosed group was higher than that in the complete remission group and the normal control group [7.52 (5.44,10.55) ng/ml vs 3.52 (1.56,5.20) ng/ml vs 2.44 (1.89,3.12) ng/ml].There' s no statistical difference between midkine level in new diagnosed acute B cell leukemia (B-ALL) group and acute myeloid leukemia (AML) group [7.88 (5.78,15.78) ng/ml vs 6.25 (4.59,16.33) ng/ml].The clear correlation was found between the level of serum MK and quantities of marrow WT1 gene (r =0.529,P =0.043).Conclusions The level of MK in serum of acute leukemia patients is increased at the time of new diagnosis and decreased at complete remission.ELISA may be a way to measure the status of AL.The location of MK gene is adjacent to WT1 gene and MK' s clinical significance is similar to WT1' s,furthermore,there is a clear correlation between MK in serum and WT1 of marrow in quantities.