Objective To investigate the influencing factors of the controlled virtual touch tissue quantification (VTQ) in liver of patients with type 2 diabetes mellitus (T2DM) complicated withnon-alcoholic fatty liver disease (NAFLD). Methods Two hundred and twenty-seven patients with T2DM complicated with NAFLD were enrolled in this study,and the shear wave speed of the liver was measured by VTQ. Levels of the fasting plasma glucose, glycated hemoglobin,total cholesterol three acids glyceride, low density lipoprotein, high density lipoprotein , aspertate aminotransferase , alanine aminotransferase , gamma glutamyl transpeptidase and uric acid were measured. The relationships among VTQ and the severity of NAFLD , and those quantitative indexes were analyzed. Results Univariate analysis showed that the value of shear wave speed was negatively correlated with age,duration of diabetes,INS and HDL-C,while was positively correlated with HbA1c and AST. Conclusion Age, sex and levels of serum HbA1c,INS,AST and HLD-C might affect the values of shear wave speed in patients with T2DM complicated with NAFLD.

Objective To investigate a Chinese pedigree suffering from Leydig cell hypoplasia ( LCH) based on clinical data and genetic diagnosis. Methods The patient was diagnosed by means of clinical data, hormone profiles, and human chorionic gonadotropin ( hCC) test. The luteinizing hormone/chorionic gonadotropin receptor(LHCGR) gene of the patient and family members was amplified and sequenced. Results The patient presented with male pseudohermaphroditism, low level of testosterone, which did not respond to hCG. Genetic analysis of the LHCGR revealed two novel mutations: a missense mutation located in exon 5, resulting in Ile replaced by Thr in the extracellular domain; and a splice site mutation in the 3' terminal of intron 6( IVS6-3 C→A). Proband's sister (46, XX) who lacked clinical manifestations showed the identical genotype with the patient. Conclusions A mutation in the consensus sequence of 3' splice site, in addition to a missense mutation (Ile 152Thr)in the extracellular ligand-binding domain is the cause of inactivation of the LHCGR gene in patient with Leydig cell hypoplasia.

OBJECTIVEThe aim of this experiment is to find proper cell carrier for skin tissue engineering.

METHODSVarious concentration of fibroblasts were seeded onto HA-ECM and cultured in vitro. The performance of cells' growth, array, adhesion and collagen secretion on HA-ECM was observed with light microscope and transmission electron microscope.

RESULTSThe fusiform fibroblasts oriented radiantly or longitudinally and closely packed onto the HA-ECM, they attached firmly and proliferated to confluence on the stromal surface of HA-ECM.

CONCLUSIONThe optimal cell concentration is 3.5 x 10(6)/ml, HA-ECM is ideal carrier for fibroblasts because of its excellent scaffold and diffusion characteristics. Futhermore, it has the bioactive molecules such as fibronectin and laminin which play an important role in fibroblasts attachment and proliferation on HA-ECM.

Amnion ; cytology ; Animals ; Cell Culture Techniques ; methods ; Cells, Cultured ; Extracellular Matrix ; Female ; Fibroblasts ; cytology ; Hematoxylin ; Humans ; Microscopy, Electron, Scanning ; methods ; Rabbits

To investigate the clinical features, genetic diagnosis, and treatment of a patient with Prader-Willi syndrome(PWS). For a case with clinically suspected PWS, methylation specific PCR(MSPCR)amplification was applied to CpG islands of SNRPN(exon α)gene locus in the 15q11-q13. Furthermore, the diagnosis was comfirmed by the method of bisulfite sequencing PCR(BSPCR). Metabolic status before and after the operation of sleeve gastrectomy were compared. Absence of amplification of paternal allele on chromosome 15q11-q13 was detected in the case by MSPCR, different from the normal control. Results of BSPCR further proved a full methylation of CpG islands in the SNRPN gene locus. Four months after sleeve gastrectomy, systemic metabolic status and ventricular function were improved. MSPCR and BSPCR were both consistent with genetic diagnosis of PWS. Weight loss surgery is expected to be a major therapy of this disease.

Objective To investigate the adrenal steroidogenic function in genotype-proven heterozygotes carrying mutations in CYP17A1 gene in vivo. Methods Eight patients and 14 family members from 5 families with 17-hydroxylase/17,20-lyase deficiency (17OHD) were recruited. The mutations of the CYP17A1 gene in these individuals were screened by direct sequencing of PCR products. The hormonal response to ACTH was evaluated in the 14 genotype-proven carriers and 45 age- and sex-matched normal subjects. Results Three mutations were found in 5 unrelated families. 14 carriers with CYP17A1 mutation were identified, including 7 heterozygotes with D487_F489del, 6 with Y329fs, and 1 for H373L. Compared to the normal subjects, the carriers exhibited lower basal and ACTH-stimulated cortisol levels, but higher ACTH-stimulated corticosterone level. The ratios of corticosterone to cortisol in the genotype-proven heterozygotes were higher than those of normal individuals at baseline and following ACTH-stimulation. Similarly, progesterone level and ratios of progesterone to 17-hydroxyprogesterone in the male heterozygotes were also higher than that of normal individuals before and after stimulation. No significant differences were observed in the hormone levels between two genotypes (D487_F489del vs Y329fs). Conclusions Genotype-proven carriers of 17OHD without apparent clinical symptoms exhibit decreased enzyme activity,analogous to mildly impaired adrenal 21-hydroxylase activity in the carriers of CYP21 A2 gene mutation.

OBJECTIVETo repair rabbit tendon defects with tissue engineering method.

METHODSThe third passage of fetal skin fibroblast cells was labeled with 5-bromo-2' deoxyuridine (Brdu) and then seeded on human amnion extracellular matrix (HA-ECM). Using 1 cm-long-Achilles tendon defects as repairing models in the experimental group, tendon defects were core bridged with polydioxanone (PDS) and then capsulated with the complex of fibroblasts-HA-ECM. In the control group I, defective tendons were sutured with PDS following the former procedure and capsulated with HA-ECM (without fibroblasts). In the control group II, only PDS was applied to connect the defective tendons. Gross examination, light microscopy, scanning electronmicroscopy and biomechanical measurement of the repaired tendons were respectively performed at postoperative 1, 2, 3 month as well as immunohistochemical examination.

RESULTSThe optimal cell concentration for seeding fibroblasts was 3.5 x 10(6) cells/ml. Cells grew well and radiated or paralleled on HA-ECM. Immunohistochemistry showed that the labeled seed fibroblasts played an important role in tendonization. The results of light microscopy, electron microscopy, and biomechanical assessment suggested that the rate and quality of tendonization in the experimental group was superior to those of the control group I and II. The tensile strength in the experimental group was the greatest, the next was in the control group I, and the worst in the control group II (P<0.05).

CONCLUSIONSHA-ECM is the excellent carrier for fibroblasts. Fibroblasts-HA-ECM complex has the capability to repair tendon defect and to tendonize with rapid rate and good performance three months after operation. Its tensile strength is 81.8% of that of normal tendon.

Amnion ; transplantation ; Animals ; Cells, Cultured ; Extracellular Matrix ; Fibroblasts ; cytology ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Polydioxanone ; Rabbits ; Suture Techniques ; Tendons ; surgery ; ultrastructure ; Tensile Strength ; Wound Healing ; physiology

Objective To analyze CYP17A1 gene mutation in a patient with 46,XY disordered sex development and to explore the possible influence on the phenotype of the patient.Methods Eight exons of CYP17AI gene in the patient and her parents were amplified and directly sequenced.In order to construct Mini-gene system,PCR fragments containing wildtype and mutant splicing sites were inserted in expression vector,and then transfected into cells.RT-PCR was used to observe the influence of splicing site mutation.Wildtype and aberrant splicing CYP17A1 cDNA expression plasmids were constructed and transfected into cells respectively,and CYP17A1 enzyme activity was tested in vitro.Results Mutation analysis revealed compound heterozygous CYP17A1 mutations,with Y329fs in one allele and a synonymous substitution( c.1263G＞A:GCG＞GCA) in another allele.In vitro analysis showed that the synonymous substitution induced a novel splicing site,which resulted in aberrant splicing of CYP17A1 mRNA and lacked six or seven amino acids after 415 in splicing product.In vitro transfection and enzyme activity experiment showed that the aberrant splicing product abolished the enzyme activity completely.However,this mutation did not completely influence splicing.The patient also had a part of normal splicing product,which was a coincidence to the phenotype of the patient.Conclusion This is the first description of an exonic splicing mutation in CYP17A1 relevant to the 17ot-hydroxylase deficiency phenotype.The functional study of the aberrant splicing variant has been initiated.

Objective The aim of this study is to explore the hypoglycemic effect of active components of Anoectochilus roxburghii on zebrafish models. Methods Anoectochilus roxburghii components were extracted and separated into three groups: the alcohol extraction group, macromolecular polysaccharide group (≥ 5 ×103) and small molecular polysaccharide group (＜5×103). Zebrafish embryos were exposed to 2% glucose solution (2% Glu) at 24 h to imitate acute hyperglycemia phenotype, and then treated with the three Anoectochilus roxburghii components. Based on this high-glucose model, the zebrafish embryos at 72 h were collected to detect the whole tissue glucose value. Furthermore, semi-quantitative PCR and whole mount in situ hybridization were performed to detect the expression of mRNA levels of glycometabolism-related genes. Results An acute diabetic zebrafish model was induced by high glucose stress. In this model, some key factors during glycometabolism such as insulin, pck-1 and pdx-1 were significantly affected, while the alcohol extracts of Anoectochilus roxburghii obviously reversed these abnormalities induced by high glucose stress, even to normal levels. Conclusions The alcohol extracts of Anoectochilus roxburghii has obvious hypoglycemic effect on diabetic zebrafish model. Our result suggest that Anoectochilus roxburghii has a potential application in the hypoglycemic drug screening.

Objective To analyze the clinical features and genotypes of adult patients with simple virilizing form of 21-hydroxylase deficiency (SV 21-OHD).Methods This is a retrospective study including 33 patients with SV 21-OHD from January 2015 to March 2018 in the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine.Results The diagnostic age of the patients was (26.3± 6.5) years old.All patients presented with signs of masculinization,such as short stature (100％),clitoromegaly/microphallus (89.65％,26/29),undeveloped breasts (82.76％,24/29),deep voice (55.17％,16/29) and primary amenorrhea (89.65％,26/29).The serum levels of 17-hydroxyprogesterone (17-OHP),androstenedione (AD) and testosterone were significantly elevated in 90.9％,93.9％ and 91.2％ of the patients,respectively.Thirteen types of mutations were identified in CYP21A2 from these patients.Among them,I173N accounted for 40％ and I2 G accounted for 18.33％.Four patients were found with multiple mutations in CYP21A2.Conclusions Short stature,clitoromegaly/microphallus and primary amenorrhea are the most common clinical features in adult patients with SV 21-OHD.Serum levels of 17-OHP and AD are important indices for the diagnosis and monitoring of the patients.I173N and I2 G are the two most prevalent mutations in patients of the present study.Limitation of clinical recognition and delay in treatment contribute to the short stature of the SV 21-OHD patients.

Objective To investigate the effect of flash glucose monitoring (FGM) on ambulatory glucose profile of only oral antidiabetic drugs treated patients with type 2 diabetes mellitus. Methods Twenty-eight type 2 diabetic mellitus patients with only oral antidiabetic drugs treatment from August 2017 to January 2018 were enrolled. All the patients were exposed to FGM for 14 d without changing the original treatment and encouraged to manage self-behavior by adjusting diet and activity based on the blood glucose data obtained from the real-time scanning. The changes in glucose profile during the FGM period were observed, including estimated glycated hemoglobin (HbA1c), standard deviation of blood glucose, variable coefficient of blood glucose, mean amplitude of glycemic excursions, time in range (blood glucose 3.9 to 10.0 mmol/L), area under the curve hyperglycemia (blood glucose> 10.0 mmol/L) and area under the curve hypoglycemia (blood glucose<3.9 mmol/L). The blood glucose levels on second day and thirteenth day were used as baseline and end point respectively. Results All of the 28 patients did not change their anti-diabetic drug therapy and there were no adverse events occurred. The estimated HbA1c was significantly lower than the baseline HbA1c: (6.90 ± 1.48)% vs. (7.57 ± 1.35)%, and there was statistical difference (P = 0.004). The standard deviation of blood glucose, variable coefficient of blood glucose, mean amplitude of glycemic excursions, area under the curve hyperglycemia and area under the curve hypoglycemia at end were significantly lower than those at baseline: (2.07 ± 0.86) mmol/L vs. (2.44 ± 0.86) mmol/L, 0.26 ± 0.11 vs. 0.30 ± 0.11, (5.32 ± 2.75) mmol/L vs. (6.76 ± 3.06) mmol/L, 265 (0, 1 310) vs. 351 (107, 2 177) and 0 (0, 0) vs. 0 (0, 19), the time in range at end was significantly higher than that at baseline: (1 069 ± 386) min vs. (921 ± 449) min, and there were statistical differences (P＜0.05 or＜0.01). The rate of scanning was (12.92 ± 4.87) times/d. Conclusions FGM could be applied by type 2 diabetic mellitus patients to make self-glycemic management without changing therapy, reduce the estimated HbA1c,and hypoglycemia, and improve the glucose fluctuations, which may result from real-time scanning to find abnormal glycemia and adjust daily behavior.