Objective To explore the effect of irbesartan on cardiac endothelial-mesenchymal transition (EndMT) in diabetic rats.Methods The model of diabetic rat was induced by intraperitoneal injection with streptozotocin (STZ,35 mg/kg) in spontaneous hypertensive rats (SHR).Diabetic rats were divided into diabetic group and the Irbesartan treated group.The pathological changes were investigated by fluorescence microscope and electron microscope.The EndMT was studied in human aortic endothelial cells (HAEC) exposure to high glucose.The concentration of angiotensin Ⅱ in the supernatant was detected by radioimmunoassay.Immunofluorescence staining was performed to detect the co-localization of CD31 and FSP1.Results The significant myocardial fibrosis was presented in the diabetic group.Endothelial protrusions were prominent feature in myocardial microvascular of diabetic rat compared with the control group rats.Double staining of HAEC showed co-localization of CD31 and FSP1,which was decreased by the treatment of Irbesartan (P ＜ 0.05).When HAEC was exposed to high glucose,it showed some cells acquired spindle-shaped morphology and lost CD31 staining,and FSP1 and α-SMA protein expression levels were markedly upregulated,which attenuated by the treatment of Irbesartan.Conclusion Irbesartan might prevent diabetes from myocardial fibrosis via inhibition of EndMT in diabetic rats.

Objective To explore whether high glucose (HG)-induced endothelial-to-mesenchymal transition (EndMT) could be transitioned into mesenchymal stem cells (MSCs) and further differentiated into chondrocytes.Methods Human aortic endothelial cells (HAECs) were divided into three groups:normal glucose (NG,5.5 mmol/L glucose) group,HG (30 mmol/L glucose) group,and mannitol (5.5 mmol/L glucose + 24.5 mmol/L mannitol) group,and were cultured for 48 h.Immunofluorescence staining was performed to detect the co-expression of CD31 (endothelial markers),and fibroblast-specific protein 1 (FSP1,fibroblast markers).The expression of CD31 and FSP1 mRNA and protein was detected by real-time PCR and Western blotting.When endothelial-derived MSCs were grown in MSC medium for one week,the expression of the MSCs markers CD44,CD10 and the chondrocyte marker SOX9 was detected by Western blotting and RT-PCR.Chondrocyte expression was detected by alcian blue staining.Calcium deposit was analyzed by alizarin red staining.Pathological changes were investigated using electron microscopy.Results The expression of FSP1 mRNA and protein was significantly increased,but the expression of CD31 mRNA and protein was decreased (P ＜0.01),and the cells undergoing EndMT also significantly expressed CD10,CD44 and SOX9 in the HG group compared with those in normal glucose group (P ＜ 0.01).The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype,wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm.Double staining of the HAECs indicated a co-localization of CD31 and FSP1.After one week culture for chondrocyte medium,the expression of MSCs marker STRO-1 was significantly increased by immunofluorescence staining.Additionally,aleian blue staining in the HG group was positive compared to the NG group.Consistent with the elevation of SOX9 expression,calcium deposit also enhanced in the HG group.Conclusion HG can induce endothelial cells transdifferentiation into chondrocyte-like cells via the EndMT.

Objective To develop a model of type 2 diabetes with early renal injury on spontaneously hypertensive rats (SHR). Methods The 6-week old SHR were fed with the diets enriched with sucrose (20%, W/W), lard (10%, W/W), cholesterol (2.5%, W/W) and chleolate (1%, W/W) to induce insulin resistance. Hyperglycemia was developed by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg). Wistar-Kyoto rats (WKY) were used as normal controls. Rats with plasma glucose (PGL) ≥ 16.7 mmol/L were diagnosed as diabetes. Eight weeks after the induction of diabetes, plasma triglyceride (TG), cholesterol (CHO), glucose, systolic pressure(SP), 24-h urine protein excretion (Upro) were examined in all the rats, and the homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed. Renal pathological changes were studied by immunohistochemical staining and electron microscope. Results After 2 weeks on the high sucrose and fat diets, the model rats exhibited significant increase in basal PGL, TG and CHO levels as compared to control rats (P<0.05, respectively). The insulin resistance was developed in model rats demonstrated by the higher HOMA-IR (5.03±0.38 vs 2.61±0.34, P<0.05). At the end of the experiment, model rats were associated with hypertension. Upro level was significantly increased in model rats compared with that in controls [(57.58±16.54) mg/24 h vs (5.35±1.90) mg/24 h, P<0.01]. The kidney hypertrophy index (KWI) was significantly increased in the model rats compared to controls (P <0.05). Moreover, the diabetic model rats showed glomerular hypertrophy, foot process effacement, micro villous transformation, glomerular basement membrane (GBM) thickening. Conclusion A rat model is successfully established, which presents typical features of human type 2 diabetes and can be served as an ideal model to study the diabetic nephropathy.

Objective:To explore the effect of adipose derived mesenchymal stem cells to regulate the differentiation of macrophage RAW264.7.Methods:First,we used RAW264.7 cells to simulate macrophage and induced them to M 1 macrophage with lipopolysaccharide ( LPS,1 μg/ml) .Then we cultured these RAW264.7 cells in culture mediums which were previously used to culture adipose derived mesenchymal stem cells to imitate the transplantation of ADMSC .Last,the mRNA relative expression of IL-10, IGF-1,Arg-1,TNF-α,FIZZ1,SPHK-1 was detected by real-time PCR.The protein expression of IL-12 p40,IL-27 Rα,IL-10 was detected by Western blot.Results:After been cultured in ADMSCCM and induced by LPS ,M1 markers (TNF-αmRNA,IL-12 p40;P<0.05) of the RAW264.7 cells declined while M2 markers (IGF-1 mRNA,IL-10 mRNA,IL-10;P<0.05) rose.Conclusion: ADMSC can secrete soluble cytokines to induce the RAW264.7 cell,which have been induced to the M1 macrophages,to differentiate towards M2 macrophages.

[ABSTRACT]AIM:ToinvestigatetheeffectofimmunosuppressantFK506onserumglucoseinratsandtoex-plore its mechanism .METHODS: Sprague-Dawley rats ( n =12 ) were randomly divided into drug group and normal group.The rats in drug group were intraperitoneally injected with FK 506 at dose of 1 mg· kg-1 · d-1 and the rats in nor-mal group received saline (1 mL· kg-1 · d-1 , ip) for 14 d.The fasting weight and fasting glucose were regularly meas-ured every 2 d.Visceral fat was isolated from the rats at the end of experiment .The mRNA expression of adiponectin , lep-tin, visfatin, resistin, retinol-binding protein 4 ( RBP4) and peroxisome proliferator-activated receptors γ( PPAR-γ) was determined by real-time fluorescence quantitative PCR .The protein expression of PPAR-γand adiponectin was measured by Western blotting .RESULTS:Compared with normal group , the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05).At day 14, the fasting blood glucose of the model group increased from (5.10 ±0.62) mmol/L to (7.73 ±0.73) mmol/L.No significant change of blood glucose in normal group between the 10th day and the 14th day [from (4.66 ±0.32) mmol/L to (5.80 ±0.10) mmol/L] was observed.Compared with normal group , the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was signifi-cantly decreased ( P <0.01 ) , whereas the expression of visfatin , resistin and RBP4 was significantly increased ( P <0.05).Compared with normal group, the expression of PPAR-γand adiponectin in model group was decreased (P <0.01).CONCLUSION:FK506 may decrease the expression of PPAR-γto change the expression of adipocytokines and induce hyperglycemia in rats .

Objective To evaluate a new prenatal diagnosis model of chromosomal abnormalities and nine microdeletion syndromes by using both traditional karyotyping and a newly-developed rapid prenatal diagnosis technology, BACs-on-Beads (BoBs) technique. Methods From June 2012 to December 2014, 807 pregnant women with high risk after screening or with other indicators, were performed amniocentesis. Traditional karyotyping and BoBs were employed simultaneously for prenatal diagnosis. Results Thirty-two cases with chromosome aneupoidies were successfully detected both by BoBs and karyotyping, including 18 cases of trisomy 21, 6 cases of trisomy 18, 1 case of trisomy 13, and 7 cases with sex chromosome abnormality. All 8 fetuses with chromosome structural abnormalities detected by karyotyping were missed by BoBs;while BoBs contributed more in detection of five microdeletion syndrome cases, including 3 cases of DiGeorge syndromes (two with microduplication and one with microdeletion), one case of Miller-Dieker syndrome, and one case of Wolf-Hirschhorn syndrome. Conclusion Combined use of traditional karyotyping and BoBs, is a rapid and effective prenatal diagnosis model that may enlarge our horizon on chromosomal diseases and should be widely used in future clinical service.

OBJECTIVETo detect the expression of CXCL14 in human osteosarcoma cell lines and tissues and investigate its association with the prognosis of the patients.

METHODSRT-PCR, enzyme-linked immunosorbent assay (ELISA) and real-time PCR were used to detect the expression of CXCL14 in 4 osteosarcoma cell lines and in 40 pairs of osteosarcoma tissues and adjacent muscular tissues. CCK8 assay and colony formation assay was used to assess the effect of CXCL14 suppression mediated by two specific siRNAs on the proliferation of U2OS osteosarcoma cells. Immunohistochemistry was performed to detect the expression of CXCL14 in 58 osteosarcoma tissues, and Kaplan-Meier method and log-rank test were performed for survival analysis of the patients.

RESULTSSignificant up-regulation of CXCL14 expression was found in the osteosarcoma cell lines and in osteosarcoma tissues compared with the adjacent muscles (P<0.01). In U2OS cell, suppression of CXCL14 expression by siRNA significantly inhibited the cell proliferation (P<0.01) and colony formation rate (P<0.05). Kaplan-Meier survival analysis indicated that patients with high CXCL14 expression had worse prognosis than those with low CXCL14 expression (P=0.02).

CONCLUSIONCXCL14 is up-regulated in both osteosarcoma cell lines and primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high CXCL14 expression in osteosarcoma tissues is associated with a poor prognosis, suggesting the that CXCL14 serve as a potential therapeutic target for osteosarcoma.

Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Chemokines, CXC ; metabolism ; Humans ; Osteosarcoma ; metabolism ; pathology ; Prognosis

OBJECTIVE To delineate a deletional mutation of the Dystrophin gene on the short arm of chromosome X in a family affected with Duchenne/Becker muscular dystrophy. METHODS G-banded karyotyping, multiple ligation probe amplification (MLPA), array-based comparative genomic hybridization(array-CGH) and whole genome exon high-throughput sequencing were employed to delineate the mutation in the family. RESULTS GTG banding has demonstrated deletion of the terminal part of the short arm of chromosome X in the fetus. The same deletion was also found in its mother and maternal grandmother. MLPA analysis has revealed removal of exons 52 to 79 of the Dystrophin gene. A 30 Mb deletion in Xp22.33-p21.1 and a 10 Mb duplication in Xq27.2-q28 were identified by array-CGH and whole genome exon high-throughput sequencing. CONCLUSION The Xp deletion has led to deletion of exons 52 to 79 of the Dystrophin gene in the family. The female carriers also had certain features of Turner syndrome due to the same deletion.
Chromosome Deletion ; Chromosomes, Human, X ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Muscular Dystrophy, Duchenne ; diagnosis ; genetics ; Nucleic Acid Amplification Techniques ; Pregnancy ; Prenatal Diagnosis

OBJECTIVETo establish a strategy for screening and diagnosing common microdeletion and microduplication syndromes among children with idiopathic mental retardation and development abnormalities.

METHODSPotential chromosomal variations among patients with unexplained mental retardation, cardiac anomalies, particular facial features, learning disabilities and other clinical characteristics were detected with bacterial artificial chromosome BACs-on-Beads (BoBs) technique and karyotyping. Positive results were verified with array-based comparative genomic hybridization (Array-CGH).

RESULTSFifty eight of the 60 patients had a normal chromosome karyotype. Ten patients with microdeletion and microduplication syndromes were detected by BoBs, which included two positive cases identified through chromosome karyotyping. Two patients were respectively diagnosed as Smith-Magenis syndrome and Prader-Willi/Angelman syndrome by BoBs and the results were confirmed by Array-CGH.

CONCLUSIONBoBs is capable of detecting chromosome microdeletion and microduplication with high specificity and throughput, which can compensate the shortcomings of conventional cytogenetic technology and will be widely applied for clinical diagnosis.

Adolescent ; Child ; Child, Preschool ; Chromosome Deletion ; Chromosome Duplication ; Chromosomes, Artificial, Bacterial ; genetics ; Comparative Genomic Hybridization ; Cytogenetic Analysis ; methods ; Female ; Humans ; Infant ; Infant, Newborn ; Karyotyping ; Male ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo provide genetic analysis for a pregnant woman with chromosomal translocations and intellectual disability, and to provide prenatal diagnosis for her fetus.

METHODSRoutine G-banding was performed to analyze the karyotypes of the woman and her fetus. Copy number variants were determined with array comparative genomic hybridization (array-CGH).

RESULTSThe pregnant woman has carried an apparently balanced translocation involving chromosomes 1, 2, 6 and 7, with a karyotype of 46, XX, t(1;2) (p22;p23), t(6;7) (q21;p15). The karyotype of her fetus was ascertained as 46, XY, t(6;7) (q21;p15) mat. Array-CGH has detected a 4 Mb microdeletion at 6q22.1-q22.31 (115 311 507-119 332 956) in both individuals. As the 6q22.1-q22.31 microdeletion may be associated with the main clinical manifestations of the woman, the family decided to terminate the pregnancy. The fetus was male and appeared to have no obvious abnormality.

CONCLUSIONPrenatal diagnosis for pregnant women with translocations and mental retardation is a challenging task. Combined application of cytogenetic analysis and array-CGH may facilitate the diagnosis and genetic counseling.

Adult ; Female ; Fetus ; abnormalities ; Genetic Testing ; methods ; Humans ; Intellectual Disability ; genetics ; Male ; Pregnancy ; Prenatal Diagnosis ; methods ; Translocation, Genetic ; genetics ; Young Adult