OBJECTIVETo investigate the main factors that influence the results of sperm alkaline single-cell gel electrophoresis (SCGE), optimize the conditions, and standardize its procedures.

METHODSUsing alkaline SCGE, we detected the DNA fragments of sperm treated with different concentrations of H2O2 and determined the influences of the number of agarose gel layers, pH during DNA unwinding and electrophoresis, the time of DNA unwinding and electrophoresis, and cumulative sperm number on the results of sperm alkaline SCGE. Then we optimized the procedures, analyzed the repeatability of the optimized method, and examined 40 semen samples using the method.

RESULTSThree agarose gel layers could reduce the background. The optimal pH during DNA unwinding and electrophoresis was 10, and the best times for DNA unwinding and electrophoresis were 40 min and 30 min, respectively. Fifty sperm were adequate to ensure the reliability of the results. Based on the percentage of tail DNA, the intra- and inter-assay repeatabilities of the optimized sperm alkaline SCGE were 3.12% and 7.13%, and by the DNA damage score, they were 2.38% and 6.09%, respectively. Sperm DNA fragments were significantly increased in the infertile patients with oligoasthenoteratozoospermia as compared with healthy fertile males (P <0.05).

CONCLUSIONThe optimized sperm alkaline SCGE, highly repeatable and easy to be standardized, can be applied to the clinical detection of sperm DNA fragmentation in infertile men.

Asthenozoospermia ; genetics ; Comet Assay ; standards ; DNA Damage ; DNA Fragmentation ; Humans ; Hydrogen Peroxide ; toxicity ; Male ; Oligospermia ; genetics ; Oxidants ; toxicity ; Reproducibility of Results ; Sperm Count ; Spermatozoa ; drug effects ; enzymology ; Time Factors

OBJECTIVETo develop a tight tetracycline-controlled HCV-C double transgenic mouse model.

METHODSBy crossbreeding of ApoE-rtTA-tTS transgenic mice with TRE-HCV-C transgenic mice, the double transgenic mice were produced in the F1 generation. The presence of HCV-C and tTS gene in the F1 generation was confirmed by PCR, followed by further identification and quantification of the transgene using Southern blot hybridization. The expression of HCV-C in the liver of the mouse model was detected immunohistochemically.

RESULTS AND CONCLUSIONTwo transgenic mice were obtained, which contained ApoE-rtTA-tTS and TRE-HCV-C genes in the genome. Five founders contained HCV-C gene as confirmed by PCR and Southern blot hybridization. The tight tetracycline-controlled system may facilitate further study of HCV-C gene expression and gene therapy of hepatic cellular carcinoma.

Animals ; Apolipoproteins E ; genetics ; Blotting, Southern ; Breeding ; Crosses, Genetic ; Female ; Gene Expression Regulation, Viral ; drug effects ; Hepacivirus ; genetics ; immunology ; Hepatitis C Antigens ; genetics ; immunology ; Male ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction ; Tetracycline ; pharmacology ; Trans-Activators ; genetics ; Viral Core Proteins ; genetics

OBJECTIVETo investigate the effect of new bone formation in sinus augmentation with guided bone regeneration (GBR) using collagen membranes.

METHODSThe first maxillary molars of 18 adult female Beagle dogs were extracted and the sinus floors of both sides were lifted with simultaneous implantation. A combination of autografts and Bio-Oss in a 2:1 ratio was placed in the space under the membrane. On the experimental side in each dog, the collagen membrane was folded at the lateral osteotomy window, the apex of the implants and a certain part of palatal bone. On the contralateral control side, the collagen membrane only covered the osteotomy window. Six animals were sacrificed at 4, 12, and 24 weeks respectively after surgery. Gross observation, biomechanical testing and histological examinations were performed.

RESULTSThe translocation of grafted materials and bone absorption were found on the top of implants in the control side, and the grafted materials kept original shape at the experimental side at 4th week. The granule of Bio-oss absorbed obviously at 12th and 24th week. The pull-out force increased with time. At 24th week, the force of pull out was 558.1 ± 37.4 N at the study side, and 471.4 ± 31.5 N at the control side. There was a significant difference in the pull-out force was noted between the two groups (P < 0.01). Histological examination showed new bone formation on the sinus floor, and the grafted materials gradually reduced with time

CONCLUSIONSGBR with the enfolded-coverage of the membrane can effectively decrease absorption of the grafted materialon the apical surface of implants and stimulate new bone formation in the sinus augmentation.

Animals ; Collagen ; Dental Implantation ; Dental Implants ; Dogs ; Female ; Guided Tissue Regeneration ; Male ; Maxilla ; Maxillary Sinus ; surgery ; Minerals ; Molar ; Osteogenesis ; physiology ; Sinus Floor Augmentation ; methods ; Tooth Extraction

OBJECTIVETo explore a method for the treatment of the skin defects at the distal phalanges of 2-5th fingers.

METHODSThe island flap at the dorsum of the middle phalange was designed with the pedicle of dorsal branches from the digital proper artery. When the flap was used to repair defect at finger pulp, the dorsal branch of the digital proper nerve in the flap was kept to be anastomosed to the digital proper nerve at the recipient finger. From Feb. 2005 to May. 2010, 54 cases with skin defects at the distal phalanges of 61 fingers were treated with the flap, including 35 defects at finger pulp and 26 defects at finger tip.

RESULTSThe maximum size of defects and flaps was 2.2 cm x 2.5 cm and 2.4 cm x 2.7 cm, respectively. 61 flaps survived completely. Blister was happened in 3 flaps 2 days after operation, which healed spontaneously without necrosis. 54 cases were followed up for 5 to 22 months (average, 11 months). The flaps had good texture and color match with normal sensation (grade S4). The 2-point discrimination distance was 6-9 mm. The interphalangeal joint had normal movement.

CONCLUSIONSThe island flap at the dorsum of the middle phalange is an ideal method for the skin defect at the distal phalange of finger.

Adolescent ; Adult ; Aged ; Arteries ; Female ; Fingers ; blood supply ; surgery ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Skin Transplantation ; methods ; Surgical Flaps ; blood supply ; Young Adult

OBJECTIVETo study the therapeutic effect of collapsed and comminuted distal radius fracture.

METHODSTwenty-six patients with collapsed and comminuted distal radius fracture were hospitalized from July 1998 to June 2003. All fractures were treated by the methods of open reduction, sustained bone grafting and passing joint external fixator to restore the anatomic shape of distal radius.

RESULTSAll 26 cases were followed up, and the results showed that the fractures have been united radiographically. The joint surfaces were intact and there was no length discrepancy occurred in patient's radius. The average volar tilt was 6 to 15 degrees and the average ulnar tilt was 18 to 25 degrees. According to the Dieust criterion, 19 cases were rated as excellent and 7 as good.

CONCLUSIONSThe method that applying passing joint external fixator and bone grafting for the treatment of collapsed and comminuted distal radius fracture could maintain the stability of fracture and restore the length of radius and the intact of joint surface.

Adult ; Aged ; Bone Transplantation ; methods ; Cohort Studies ; Combined Modality Therapy ; External Fixators ; Female ; Follow-Up Studies ; Fracture Fixation ; instrumentation ; methods ; Fracture Healing ; physiology ; Fractures, Comminuted ; diagnostic imaging ; surgery ; Humans ; Injury Severity Score ; Male ; Middle Aged ; Radiography ; Radius Fractures ; diagnostic imaging ; surgery ; Recovery of Function ; Retrospective Studies ; Risk Assessment

OBJECTIVETo investigate the pathological changes of liver in children with hepatitis B virus associated glomerulonephritis (HBV-GN).

METHODSThirteen children with HBV-GN (aged from 2-14 years) underwent renal and liver biopsy. The biopsy findings were analyzed.

RESULTSDifferent degrees of hepatic lesions were seen in all of the 13 patients, mild lesions accounting for 69.2% (9/13). HBSAg positive was the most common in the liver tissue [76.9% (10/13)]. Among the renal lesions, membranous glomerulopathy accounted for 69.2%( 9/13), followed by membranoproliferative glomerulonephritis 30.8% (4/13). HBsAg and HBcAg positive were presented in all patients' kidney tissues. HBV antigens were detected in stroma between nephric tubule in all samples. Four patients presented with HBcAg positive in both live and kidneys.

CONCLUSIONSThe children with HBV-GN couple with liver lesions. The severity of the renal lesions is not always accord with that of the liver lesions. The appearance of HBcAg in both kidneys and liver indicates severe lesions of the two organs. It is suggested that a liver-kidney holistic treatment is necessary for children with HBV-GN.

Adolescent ; Child ; Child, Preschool ; Female ; Glomerulonephritis ; pathology ; Hepatitis B ; complications ; pathology ; Hepatitis B Core Antigens ; analysis ; Humans ; Kidney ; pathology ; Liver ; pathology ; Male

OBJECTIVETo investigate fibronectin synthesis in SD rat mesangial cells after transforming growth factor-beta1 (TGF-beta1) is silenced by the short interfering RNA (siRNA) expressed by reconstructed pGEFP-C1 vectors.

METHODSDepending upon the 538th - 556th (A) and 895th - 913th (B) nucleotides of rat TGF-beta1 gene, a nucleotide (A or B) was constructed into a small hairpin nucleotide which was separately (A or B) or together (A plus B) inserted into a pGEFP-C1 vector with three reconstructed pGEFP-C1 vectors separately expressing the siRNAs for A or/and B. TGF-beta1 and fibronectin were dynamically investigated for their interrelationship by ELISA in the supernatant and RT-PCR in their extracted total RNA.

RESULTSThe siRNA hairpin-like molecules were constructed according to the 538th - 556th nucleotides of rat TGF-beta1 gene were able to markedly silence the expression of TGF-beta1 mRNA (P < 0.01) and protein (P < 0.01) at 48 h. Lipfectamin 2000 transfection stimulated the peak secretion of fibronectin at 24 h in the control and the experimental group whose TGF-beta1 was not silenced, but the silence of TGF-beta1 in both experimental groups delayed the top values of fibronectin to 48 h (P < 0.01).

CONCLUSIONThe silence of TGF-beta1 by siRNA decreased the fibronectin expression, but the latter was possibly not completely TGF-dependent.

Animals ; Cells ; Cells, Cultured ; Fibronectins ; metabolism ; Mesangial Cells ; drug effects ; metabolism ; RNA Interference ; drug effects ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; chemistry ; genetics

OBJECTIVETo investigate the role of mast cells in the development of renal interstitial fibrosis in children with Henoch-Schonlein purpura nephritis (HSPN) and possible mechanisms.

METHODSParaffin-embedded renal biopsy tissue sections from 20 children with HSPN were examined for the levels of tryptase-beta and transforming growth factor-beta1 (TGF-beta1) by immunohistochemical staining. Mast cells were counted by toluidine blue staining. Masson staining was used to assess the level of renal interstitial fibrosis and renal histopathological scores. Normal renal tissue sections from 5 nephrectomized children for nephroma were used as control group.

RESULTSThe percentages of positive tryptase-beta cellsand mast cells and the TGF-beta1 expression in the HSPN group were significantly higher than those in the control group (P < 0.05). The percentages of positive tryptase-beta cells and mast cells and the TGF-beta1 expression in renal tissue were positively correlated with the glomeruli histopathological score (r =0.940, 0.920, 0.937, respectively; P < 0.05) and were also positively correlated with the histopathological score of renal interstitium (r=0.903, 0.859, 0.948, respectively; P < 0.05). The level of renal interstitial fibrosis was positively correlated with the percentages of positive tryptase-beta cells and mast cells and the expression of TGF-beta1 (r =0.790, 0.766, 0.858, respectively; P < 0.05). There was a positive correlation between the percentages of positive tryptase-beta cells and mast cells (r =0.941, P < 0.05), between the percentage of positive tryptase-beta cells and the TGF-beta1 expression (r =0.897, P < 0.05) and between the percentage of positive mast cells and the TGF-beta1 expression (r=0.942, P < 0.05).

CONCLUSIONSTubulointerstitial mast cell infiltration is associated with the development of renal interstitial fibrosis in children with HSPN. Mast cells together with TGF-beta1 and mast cell-derived tryptase-beta may be involved in the development of the renal interstitial fibrosis in HSPN.

Adolescent ; Child ; Female ; Fibrosis ; Humans ; Kidney ; chemistry ; pathology ; Male ; Mast Cells ; physiology ; Nephritis ; pathology ; Purpura, Schoenlein-Henoch ; metabolism ; pathology ; Transforming Growth Factor beta1 ; analysis ; Tryptases ; analysis

OBJECTIVE: To investigate the difference of Pax2 and P53 expressions in children with primary nephritic syndrome (PNS) and the effect of Pax2 on glucocorsteroid (GC)-resistance. METHODS: Renal Pax2 and P53 expressions in children with PNS (40 patients) were detected by immunohistochemistry. A semiquantitative score was used to evaluate the injury degree of the glomeruli and the tubulointerstitium, and correlation analysis was done among Pax2, P53 and pathologic score. RESULTS: Pax2 and P53 expressions were not found in the control group. Pax2 expression of renal tubule epithelia exsisted in children with PNS and there was weak or no expression of Pax2 in the podocytes. Pax2 expressions in the proximal tubule and the distal tubule in the GC-resistant group were more intense than those in the GC-intensive group (P <0.01). The more the Pax2 expression in the tubule, the more abnormal structure such as dilation and atrophy. Pax2 expression in the tubule epithelia was positively correlated with pathologic score of tubulointerstitium (P < 0.01). There was no P53 expression in the GC-intensive group, but there exsisted P53 expression in parts of the patients from the GC-resistant group, mainly distributing in the renal tubular epithelia. P53 expression was positively correlated with P53 expression and the pathologic score of tubulointerstitium (P < 0.01). CONCLUSION Over-expression of Pax2 in the renal tubule epithelia may improve P53 expression to a certain degree, which may aggravate the lesion of the renal tubule. It may be one of the mechanisms resulting in GC-resistant in children with PNS.
Adolescent ; Child ; Child, Preschool ; Drug Resistance ; Female ; Glucocorticoids ; therapeutic use ; Humans ; Male ; Nephrotic Syndrome ; drug therapy ; metabolism ; PAX2 Transcription Factor ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

Objective To summarize the clinical features,natural history and causes of death of mitochondrial encephalomyopathy,lactic acidosis and stroke-like episodes (MELAS).Methods We retrospectively evaluated the clinical findings of 64 patients diagnosed as MELAS more than 3 years (death cases excluded) in Huashan Hospital from January 2005 to March 2017 and analyzed the natural course and the causes of death of the disease.Results Among 64 patients,the male-to-female ratio was 1.3 ∶ 1.Median onset age was 20.5 (16.8) years.The peak of incidence age was from 14 to 22 years.The most common features of MELAS in acute phase were seizures (48/64,75.0％),headache (41/64,64.1％),blurred vision (37/64,57.8％),nausea and vomiting (27/64,42.1％),fever (25/64,39.1％),mental and behavioral disorder (24/64,37.5％).Lactate dehydrogenase (31/60,51.6％),resting blood lactic acid (43/58,74.1％) and cerebral spinal fluid lactic acid (9/9) were elevated.Abnormal findings in electroencephalogram (36/40,90.0％),electrocardiogram (37/47,78.7％),electromyography (25/41,61.0％) were detected.In this cohort,20 patients (20/64,31.3％) with MELAS were dead.A Kaplan-Meier survival curve showed the estimated overall median survival time was 12 years.The median survival time of the group onset before sex maturity (≤ 14 years) was 8 years and that in the group onset after sex maturity (＞ 14 years) was 21 years.The causes of death were cardiogenic incidence (4/20,20.0％),pulmonary infection (4/20,20.0％),lactic acidosis (2/20,10.0％) and status epilepticus (2/20,10.0％).Conclusions MELAS is usually presented in young people associated with high mortality rate.The leading causes of death are cardiogenic,pulmonary infection and lactic acidosis.