Objective To study the role of endoplasmic reticulum stress in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.Methods Cultured human glomeruar mesangial cells were divided into three groups: control group,high glucose group and high glucose+ 4-phenylbutyric acid (4-PBA) group.Cell number of proliferation was assessed by MTT assay.Cell cycle was measured by flow cytometric analysis.Expression of α-SMA was assessed by immunohistochemistry and was observed by laser scanning confocal microscope.Involved mRNA and protein expression were measured by real-time PCR and Western blotting.Results (1)Cell number of proliferation and S transition proportion in high glucose group significantly increased than that in control group (P ＜ 0.05).High glucose could induce α-SMA expression significantly (P＜0.05).4-PBA could significantly inhibit human glomerular mesangial cells proliferation (P＜0.05),S transition arrest (P＜0.05) and expression of α-SMA (P＜0.05) induced by high glucose.(2) Compared with control group,high glucose could significantly increase the expression of glucose-regulated protein78(Grp78 ) mRNA and protein (P＜ 0.05),which could be inhibited by 4-PBA treatment (P＜0.05).(3)High glucose could induce the mRNA and protein expression of TGF-β1 and FN significantly,which could be inhibited by 4-PBA treatment (P＜0.05).Conclusion Endoplasmic reticulum stress plays an important role in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.

OBJECTIVETo access the feasibility of employing metabonomics method in clinical studies. This pilot study intends to introduce nuclear magnetic resonance (NMR)-based metabonomics method to elucidate the metabolism of non-syndromic cleft lip and/or palate (NSCLP) patients.

METHODSHigh-resolution 1H NMR spectroscopy was performed on blood plasma obtained from NSCLP and non-malformed children. All signal of 1H NMR spectra were recognized within MESTRE-v4.7, and the 1H NMR spectra integration into bins (or buckets) across the spectral regions of bin 0.04 was performed automatically in MESTRE-v4.7. The resulting data matrix was further analyzed, which was performed by SIMCA-P 11.0. The principal component analysis (PCA) was applied to the centered data to explore any clustering behavior of the samples.

RESULTSThe results demonstrated the metabonomic difference in plasma between NSCLP and non-malformed children at least lies in 3-Hydroxybutyrate gamma-CH3, arginine and valine. Arginine excretion appeared to be higher in the non-malformed children population, while NSCLP population excreted higher concentrations of 3-Hydroxybutyrate gamma-CH3 and valine.

CONCLUSIONThe present study clearly demonstrated the great potential of the NMR-based metabonomics approach in elucidating the NSCLP plasma metabolism and the possibility of application in clinic diagnosis and screening.

Child ; Cleft Lip ; Cleft Palate ; Humans ; Magnetic Resonance Imaging ; Magnetic Resonance Spectroscopy ; Male ; Metabolomics ; Pilot Projects

OBJECTIVESTo investigate the effects of newborn bull serum(NBS), vitamin C and vitamin E on cryopreservation of mouse seminiferous epithelial cells.

METHODSThe seminiferous epithelial cells from 7-day-old mice were cryopreserved in different freezing solutions. The cell recoveries were examined by Trypan blue exclusive staining after thawing. The freezing solutions composed of DMEM, 10% dimethylsulphoxide(DMSO), and 0, 5%, 10%, or 20% NBS, respectively, or composed of DMEM, 10% DMSO, 10% NBS, and 150 micrograms/ml vitamin C or 50 micrograms/ml vitamin E, respectively.

RESULTSThe cell recoveries in freezing solution containing 0, 5%, 10%, or 20% NBS were 83.4%, 84.7%, 85.7% and 83.6%, respectively. There were no significant differences between them. The cell recoveries in freezing solution containing vitamin C or vitamin E were 88.0% and 82.9%, respectively. There was no significant differences compared with that in freezing solution containing 10% DMSO and 10% NBS.

CONCLUSIONSNBS, vitamin C and vitamin E have no significant protecting effects on mouse seminiferous epithelial cells, and can not significantly improve the cell recoveries.

Animals ; Ascorbic Acid ; pharmacology ; Cattle ; Cryopreservation ; Epithelial Cells ; physiology ; Fetal Blood ; physiology ; Male ; Mice ; Seminiferous Epithelium ; cytology ; Vitamin K ; pharmacology

OBJECTIVETo study the value of 3D visualization technique in breast-preserving surgery for breast cancer with immediate breast reconstruction using laparoscopically harvested pedicled latissimus dorsi muscle flap.

METHODSFrom January, 2015 to May, 2016, 30 patients with breast cancer underwent breast-preserving surgery with immediate breast reconstruction using pedicled latissimus dorsi muscle flap. The CT data of the arterial phase and venous phase were collected preoperatively and imported into the self-developed medical image 3D visualization system for image segmentation and 3D reconstruction. The 3D models were imported into the simulation surgery platform for virtual surgery to prepare for subsequent surgeries. The cosmetic outcomes of the patients were evaluated 6 months after the surgery. Another 18 patients with breast cancer who underwent laparoscopic latissimus dorsi muscle breast reconstruction without using 3D visualization technique from January to December, 2014 served as the control group. The data of the operative time, intraoperative blood loss and postoperative appearance of the breasts were analyzed.

RESULTSThe reconstructed 3D model clearly displayed the anatomical structures of the breast, armpit, latissimus dorsi muscle and vessels and their anatomical relationship in all the 30 cases. Immediate breast reconstruction was performed successfully in all the cases with median operation time of 226 min (range, 210 to 420 min), a median blood loss of 95 mL (range, 73 to 132 mL). Evaluation of the appearance of the breast showed excellent results in 22 cases, good appearance in 6 cases and acceptable appearance in 2 cases. In the control group, the median operation time was 283 min (range, 256 to 313 min) and the median blood loss was 107 mL (range, 79 to 147 mL) with excellent appearance of the breasts in 10 cases, good appearance in 4 cases and acceptable appearance in 4 cases.

CONCLUSION3D reconstruction technique can clearly display the morphology of the latissimus dorsi and the thoracic dorsal artery, allows calculation of the volume of the breast and the latissimus dorsi, and helps in defining the scope of resection of the latissimus dorsi to avoid injuries of the pedicled vessels. This technique also helps to shorten the operation time, reduce intraoperative bleeding, and improve the appearance of the reconstructed breast using pedicled latissimus dorsi muscle flap.

OBJECTIVETo explore the impact of Foxp3 expression and CD(4)(+)CD(25)(+) regulatory T cells on pathogenesis of childhood asthma.

METHODSTotally 15 patients with acute asthma exacerbation, 15 children with asthma remission and 10 children who were hospitalized for skeleton deformity without atopic disorders or history of allergic diseases or respiratory infections within a month as controls were recruited in this study from Sep. 2004 to Mar. 2005. The percentage of CD(4)(+)CD(25)(+) T cells were detected by 2-color flow cytometry. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-gamma, transforming growth factor (TGF)-beta in plasma and supernatant were assayed by ELISA. Both the asthmatic children and the control children were selected to induce sputum by hypertonic saline. Sputum was processed for detecting the expression of Foxp3-mRNA. The expression of Foxp3-mRNA in both sputum and PBMC was detected by semi-quantitative RT-PCR with beta-actin as internal control.

RESULTSThe percentage of CD(4)(+)CD(25)(+) regulatory T cells in exacerbation and remission asthmatic children was significantly lower than that of the control children both prestimulation [(10.1 +/- 2.1)% vs. (15.5 +/- 2.7)%, (11.7 +/- 2.5)% vs. (15.5 +/- 2.7)%, P < 0.05] and poststimulation with PHA [(12.4 +/- 2.3)% vs. (26.9 +/- 3.8)%, (17.3 +/- 3.2)% vs. (26.9 +/- 3.8)%, P < 0.05]. The percentage of CD(4)(+)CD(25)(+) regulatory T cells was significantly higher after PHA stimulation in normal children [(15.5 +/- 2.7)% vs. (26.9 +/- 3.8)%, P < 0.01]. The expression of Foxp3-mRNA (Foxp3/beta-actin) in asthmatic children was significantly lower than that in the control children in both PBMC and induced sputum. The expression of Foxp3-mRNA in PBMC was significantly higher after PHA stimulation in the control children (0.77 +/- 0.22 vs. 1.07 +/- 0.21, P < 0.05). However, there was no significant difference in Foxp3-mRNA expression in asthmatic children pre and post PHA stimulation. A significant positive correlation between the Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells was detected. The levels of IFN-gamma and TGF-beta were significantly lower in asthmatic children than those in the control children, and the levels of IFN-gamma and TGF-beta correlated positively with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells. The level of IL-4 both in plasma and supernatant was higher in asthmatic children. The levels of IL-10 was higher only in exacerbation than in control children, the levels of IL-4 and IL-10 had no correlation with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells.

CONCLUSIONInsufficient secretion of TGF-beta, decreased Foxp3 expression, insufficient number of CD(4)(+)CD(25)(+) regulatory T cells and the defective ability of converting CD(4)(+)CD(25)(-) T cells to CD(4)(+)CD(25)(+) regulatory T cells might play an important role in pathogenesis of asthma.

Asthma ; physiopathology ; Case-Control Studies ; Child ; Cytokines ; analysis ; blood ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sputum ; chemistry ; T-Lymphocytes, Regulatory ; physiology

OBJECTIVETo identify zebrafish mutants with myelopoiesis defects by ENU mutagenesis and large-scale forward genetic screening.

METHODSMale zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in the spermatogonial cells to generate the founders, which were outcrossed with AB to raise F1 fish. The F1 fish from different founders were mated to generate the F2 families. The F3 embryos from F2 sibling crosses were screened by Sudan black B staining and neutral red staining.

RESULTSA total of 350 F2 families from F1 sibling crosses were screened, and 1424 F2 crosses were analyzed. Six mutations were identified resulting in abnormal Sudan black B staining and neutral red staining, indicating the involvement of neutrophil deficiency or macrophage abnormalities.

CONCLUSIONIt is simple and cheap to induce and screen myelopoiesis deficiency in zebrafish by ENU chemical mutagenesis and Sudan black B staining and neutral red staining. These mutants shed light on the identification of the genes important to myelopoiesis in zebrafish.

Animals ; Gene Expression Regulation, Developmental ; genetics ; Genetic Testing ; Male ; Mutagenesis ; Mutation ; Myeloid Progenitor Cells ; physiology ; Myelopoiesis ; genetics ; Zebrafish ; genetics

Objective:To explore the clinical application of a new high dielectric constant (HDC) to improve image quality in 3.0 T fetal head MR scans.Methods:Forty pregnant women who underwent 3.0 T fetal head MR examinations at the Drum Tower Hospital of Nanjing University School of Medicine from May to July 2021 were prospectively included and divided into a test group and a control group according to the placement and non-placement of HDC pads. After the scans were completed, qualitative and quantitative analysis were performed on the image quality of the two groups acquired in each case. Qualitative analysis: A 5-point scale was used to score the images of both groups by two diagnosticians and their scores were recorded. Quantitative analysis: Firstly, the overall radiofrequency specific absorption ratio (SAR) values of the two sets of fetal cranial cross-sectional scans of each pregnant woman were recorded separately, and the average rate of change of the overall SAR values was calculated; secondly, four regions of interest (ROIs) were placed on the standard level of the cross-sectional section of each fetal cranium (including the level of the basal ganglia region of the dorsal thalamus), and the minimum and maximum of the four ROIs of each of the two data sets were calculated separately. The ratio of minimum to maximum signal intensity (RSI), signal to noise ratio (SNR) and contrast to noise ratio (CNR) were calculated for each of the four ROIs in the two sets of data. Wilcoxon test was used to analyze the differences between the two groups of image quality score results; paired sample t-test or paired rank sum test was used to analyze the differences in SAR, RSI, SNR and CNR values between the two groups. Results:The fetal head image quality score was 4 (3, 4) in the test group and 3 (1, 4) in the control group, and the test group was significantly higher than the control group, with statistically significant difference ( Z=-3.62, P<0.01), and the images in the test group had a uniform signal compared with the control group, and none of them had significant artifacts. The results of quantitative analysis showed that the overall SAR value of the test group was significantly reduced, with a mean reduction rate of 32.1%, and the difference between the SAR values of the two groups was statistically significant ( Z=-2.78, P<0.01). The RSI, SNR and CNR in the frontal, temporal, thalamic and occipital lobes of the test group were all higher than those of the control group, and the differences were all statistically significant ( P<0.01). Conclusion:The HDC pads can significantly improve the image quality of 3.0 T fetal head imaging by reducing or eliminating the inhomogeneous artifacts in the RF field, which makes a good technical foundation for fetal head MR imaging.

Aim To study the effect of three kinds of active ingredients of traditional Chinese medicine (sinomenine, rhynchophylline and isorhynchophylline) on dre-miR-723-5p expression in morphine-induced zebrafish brain. Methods Morphine was injected intraperitoneally to zebrafish, conditional position preference (CPP) was trained and then the behavioral of animals were observed; the miRNA expression profiles of morphine-additive zebrafish were determined by small RNA sequencing; qRT-PCR was used to verify the expression of dre-miR-723-5p, three target gene databases (miRanda, miRDB, andRNAhybrid) were used to predict the target genes of dre-miR-723-5p; Kobas 3.0 was used to perform Gene Ontology (GO) and KEGG pathway analysis of these target genes. Results Morphine-induced CPP model was established successfully. Compared with control group, the resident time and movement map in drug-pair box of zebrafish in model group significantly increased. After drug administration, the resident time and movement map in drug-pair box of zebrafish decreased. The verification results of qRT-PCR were consistent with the results of small RNA sequencing. Ninety-nine putative target genes of dremiR-723-5p that were common to all three target gene databases, which were mainly enriched in biological process, cell composition and molecular function, involved in the positive regulation of MAPK signaling pathway, lysosome, cytokine-cytokine receptor interaction, and apoptosis. Conclusion Morphine can increase the expression of dre-miR-723-5p in the zebrafish brain, which can be reversed by sinomenine, isorhynchophylline, and rhynchophylline treatment, and dre-miR-723-5p may participate in the mechanism underlying morphine-induced damage of brain.

【Objective】 To investigate the effects of miR-126-3p targeting chemokine receptor 1 (CCR1) in exosomes derived from bone marrow mesenchymal stem cells (BMSC) on the proliferation, migration, and invasion of lung cancer cells. 【Methods】 BMSC cells were cultured; exosomes were extracted and identified by the exosomal marker proteins CD63 and TSG101. After exosome culture of A549 cells for different durations (0, 24, 48, and 72 h), cell survival rate was detected by CCK-8, mRNA levels of miR-126-3p and CCR1 were detected by qRT-PCR, and cell migration and invasion abilities were detected by Transwell assay. The relative expressions of CCR1, epithelial cadherin (E-cad), neural cadherin (N-cadherin), and Vimentin were detected by Western blotting. 【Results】 Exosomes had round or oval cup-shaped structures with bright edges and dark middle, with a particle size distribution of about 152 nm, expressing CD63 and TSG101 proteins. The expression of miR-126-3p in exosomes was higher than that in A549 cells. The expression of miR-126-3p was low in A549 cells and that of CCR1 mRNA was high. However, after co-culture with exosomes, the expression of miR-126-3p in A549 cells was increased, while the expression of CCR1 was decreased. A549 cells were cocultured with exosomes for 0, 24, 48, and 72 h. The survival rate, migration and invasion abilities, CCR1 gene and protein expression levels, and N-cad and Vimentin protein expression levels of A549 cells decreased gradually with the extension of culture time. The level of miR-126-3p and the expression of E-cad protein increased gradually with the extension of culture time. 【Conclusion】 The co-culture of exosomes derived from bone marrow mesenchymal stem cells with A549 cells can increase the expression level of miR-126-3p, and miR-126-3p can reduce the proliferation, migration, and invasion of A549 cells by targeting the inhibition of CCR1 expression.

Objective To explore the value of trans-lymphatic contrast-enhanced ultrasound(CEUS)in the diagnosis of cervical lymph node metastasis of thyroid cancer. Methods The patients with suspected thyroid cancer underwent conventional ultrasound and trans-lymphatic CEUS examinations before the biopsy.The differences in ultrasound and CEUS characteristics of cervical lymph nodes between the metastatic group and the non-metastatic group were compared,and pathological results were regarded as the golden standard. Results Twenty patients had thyroid cancer,including 12 cases with lymph node metastasis and 8 cases without metastasis.The diagnostic sensitivity(91.7%
Contrast Media ; Humans ; Lymph Nodes/diagnostic imaging* ; Lymphatic Metastasis/diagnostic imaging* ; Thyroid Neoplasms/diagnostic imaging* ; Ultrasonography