·AIM: To detect the effect of CTGF on the apoptosis in the diabetic retina with small interfering RNAs (siRNA) targeting with CTGF. ·METHODS: A total of 60 rats were divided into six groups including control group, diabetic 4,8,12,16 weeks group, and interference group. Diabetic rats were induced by STZ intra-peritoneal. At 4, 8, 12, 16 weeks after diabetic setting up, retinas were obtained from control, diabetic rats and diabetic animals treated by intravitreal injection of CTGFsiRNA to suppress the expression of CTGF mRNA. Retinal cells apoptosis was detected by Tunnel staining and mRNA expression of CTGF was analyzed by RT-PCR.·RESULTS: The levels of CTGF and the apoptosis in the retinas of diabetic rats were significantly higher than those in the controls. Apoptosis occurred at 4 weeks after a diabetic model setting up, became serious with the diabetes developing, while CTGF elevated at 8 weeks. The cell apoptosis counts increased to 25.8cells/mm2 at 24 weeks of diabetes. SiRNA-mediated inhibition of CTGF mRNA resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and apoptosis in the retina.·CONCLUSION: These results suggest that CTGF might be involved in retinal cells apoptosis which is a characteristic of early diabetic retina. siRNA targeting CTGF seems to have the advantage of ameliorating retinal cells lost.

The microbial transformation of buflomedil by Cunninghamella blakesleana AS 3.153 was studied, as well as a microbial model which can be used to mimic metabolism of buflomedil in mammal was established. Experiments were conducted to screen the capabilities of four strains of Cunninghamella species to transform buflomedil, in which C. blakesleana AS 3.153 was selected for a preparative biotransformation. Furthermore, the microbial model was established based on the transformation condition optimization. The parent drug and its metabolites produced by C. blakesleana AS 3.153 were detected by liquid chromatography-mass spectrometry method and three metabolites were identified while two of them were new found metabolites. Two major metabolites, para-O-desmethyl buflomedil and 12-C-oxidated buflomedil, were isolated by semi-preparative HPLC. Based on the comparison between different species, the microbial transformation of buflomedil by C. blakesleana AS 3.153 is more similar to the metabolism of buflomedil in human and Beagle dog than that in rat.
Adult ; Animals ; Biotransformation ; Chromatography, High Pressure Liquid ; Cunninghamella ; metabolism ; Dogs ; Female ; Humans ; Male ; Molecular Structure ; Pyrrolidines ; chemistry ; pharmacokinetics ; Rats ; Rats, Wistar ; Spectrometry, Mass, Electrospray Ionization ; Young Adult

OBJECTIVETo investigate the advantages and disadvantages of dual-color silver-enhanced in-situ hybridization (DSISH) and fluorescence in-situ hybridization (FISH) for determination of HER2 gene status in gastric carcinoma and to evaluate the feasibility of DSISH.

METHODSEighty cases of primary gastric or gastroesophageal junction adenocarcinomas diagnosed and treated surgically from January to March, 2009 at the West China Hospital were enrolled in the study. Automated immunohistochemistry (IHC) staining, FISH and automated DSISH were carried out to detect the HER2 status, respectively, and the concordance of the three techniques was then evaluated.

RESULTSDSISH and FISH failed initially, but repeated detection was successful in 5 cases. Gene amplification was detected in 12/13 IHC 3+ cases in DSISH and in 11/13 IHC 3+ cases in FISH. In 6 IHC 2+ cases, the amplification rate was both 1/6; in 18 IHC 1+ cases, the amplification rate was both 2/18. No amplification was observed in 43 IHC 0 cases. Only one of the 80 cases showed discrepancy, and therefore the overall concordance between FISH and DSISH was 98.8% (κ = 0.958, P < 0.01).

CONCLUSIONSDSISH represents a novel approach for the determination of HER2 status in gastric carcinoma, and the overall concordance between DSISH and FISH is excellent. Despite their advantages and disadvantages, DSISH is more feasible and practical for routine application in surgical pathology.

Adenocarcinoma ; genetics ; Esophagogastric Junction ; Gene Amplification ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; methods ; In Situ Hybridization, Fluorescence ; Sensitivity and Specificity ; Silver ; Stomach Neoplasms ; genetics

OBJECTIVETo suppress the expression of CCR5 and CXCR4, the co-receptors for human immunodeficiency virus type 1 ( HIV-1), and thus inhibit HIV-1 from entering cells.

METHODSDNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells (PBMCs) by reverse transcript polymerase chain reaction (RT-PCR) and sequencing was performed. Correct fragments were inserted into Shuttle plasmid inversely, which was recombined with backbone plasmid containing homologous adenoviral genome in E. coli BJ5183. The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified. Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR, and the titres of them were determined by cytopathic effect (CPE) method. The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5 (multiplicity of infection, MOI = 100) and CXCR4 (MOI = 200), respectively. The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24, 48, 72 hours and 10 days after infection. In addition, the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively.

RESULTSWe constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively. The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5 x 10" PFU/ml and 7 x 10(10) PFU/ml, respectively. The expression rate of CCR5 on U937 cells decreased from 82. 10% (blank control) to 1.12% (Ad-antiR5 infected) , and that of CXCR4 on MT4 cells decreased from 42% (blank control) to 1.03% (Ad-antiX4 infected) 24 hours later. The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02% , 1.26% , 1.23% at 48 hours, 72 hours, and 10 days later, respectively. The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%, 1.17%, 1.22% at 48 hours, 72 hours, and 10 days later, respectively. Moreover, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells.

CONCLUSIONThe recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.

Adenoviridae ; genetics ; Cell Line, Transformed ; Cell Proliferation ; Chemotaxis ; Down-Regulation ; Genetic Vectors ; Humans ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, CXCR4 ; biosynthesis ; genetics ; Transfection ; U937 Cells

The aim of the present study was to gain an insight into the thiopurine methytransferase (TPMT) genotyping assay, which was based on polymerase chain reaction (PCR), allele-specific PCR, restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and in combination with direct DNA sequencing. Among the f our methods to test TPMT genetic SNPs based on PCR, allele specific PCR was not able to differentiate wild type from varied type. BsiYI, MwoI and AccI to digest PCR products were used so that SNP in TPMT exon 5, 7 and 10 tested. It showed that there were no differences between the results of digestion of PCR products and those of DNA sequence analysis. Therefore, this method was reliable. But some other methods were still needed to look for a compensation, because no restriction map changing resulted from the 2 SNPs in TPMT promotor was found. As to the results of DHPLC, those for the screening of TPMT exon-5 and -10 for SNPs were the same as restriction analysis of PCR products and direct DNA sequencing. But the variation of the heterozygotes in exon-7 was high, which was different from the results of direct DNA sequencing. After changing the Tm of DNA step by step, It was found that all the samples showed single peak when the temperature was 54 degrees C. But this result was unbelievable because a heterozygote in exon 7 as positive control could not be found. Therefore, it was necessary to test the sensitivity and accuracy of DHPLC, though DHPLC could be used as an effective method of SNPs screening. The results of the SNaPshot sequencing were also same as those of restriction analysis of PCR products and direct DNA sequencing. And the results showed that the bases of TPMT promoter -91 and -168 were G, instead of A and T. The results of the four methods to detect TPMT genetic SNPs based on PCR showed that SNPs analysis technique should be a combination of the techniques above-mentioned. One technique alone could not satisfy the need in clinics and research. The compensation of each other was very important.
Chromatography, High Pressure Liquid ; Exons ; Humans ; Methyltransferases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Aged ; Aged, 80 and over ; Cerebrovascular Disorders ; epidemiology ; Diabetes Mellitus ; epidemiology ; Female ; Humans ; Hyperlipidemias ; epidemiology ; Hypertension ; epidemiology ; etiology ; Male ; Military Personnel ; Prevalence ; Renal Insufficiency, Chronic ; epidemiology ; Retirement ; Systole

OBJECTIVEHearing impairment or loss is a common disorder at birth. Many perinatal conditions may also cause hearing impairment. The present study aimed at investigating the incidence and morbidity of hearing impairment in critically ill neonates with high risk factors in neonatal intensive care unit (NICU).

METHODSPatients were evaluated using the auditory brain-stem response (ABR) to detect hearing impairment or loss in 248 neonates stabilized or before discharge from the NICU during the period from Dec. 1999 to Aug. 2001 in SCMC. In this article, high risk factors of hearing loss included severe asphyxia, hyperbilirubinemia and very low birth weight, persistent pulmonary hypertension or on a ventilator, exposed to ototoxic medications such as aminoglycosides and diuretics, a family history of hearing loss, meningitis and craniofacial anomalies, etc. Screening test was performed with a 70 dB (SPL) hearing level click stimulus to each ear.

RESULTSSeventy-two newborns had detectable hearing impairment in all the patients studied, the incidence of hearing impairment was 29.03%. Among them 3 cases had serious hearing loss. Twenty cases were among the 50 asphyxiated newborns (40.0%); 24 were from the 91 neonates with hyperbilirubinemia (26.37%); 15 were from the 44 premature infant group (34.09%), three of 4 premature cases with birth weight less than 1 500 g had hearing impairment. Six cases were among the 15 newborns who used mechanical ventilation (40.0%); 19 cases were among the 46 newborns who received ototoxic agents (41.30%).

CONCLUSIONSCritically ill neonates with some specific high risk factors had a significantly high incidence of hearing impairment. Early hearing screening is necessary for neonates who are discharged from NICU.

Evoked Potentials, Auditory, Brain Stem ; Female ; Hearing Loss ; diagnosis ; physiopathology ; Hearing Tests ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Male ; Risk Factors

OBJECTIVETo analyze serum levels of non-esterified fatty acids (NEFA) and albumin (ALB) in children with nephrotic syndrome (NS) and investigate the clinical significance of altered serum NEFA to ALB ratio in children with NS in acute and remission phases.

METHODSSerum levels of NEFA and ALB were measured in 55 NS children in acute phase, in 33 NS children in remission and in 122 healthy control children, and the ratio of NEFA to ALB was calculated. The other lipid/lipoprotein and renal function parameters were also analyzed in these children.

RESULTSCompared with the healthy control children, children with NS had a significantly decreased serum ALB level (t=11.152, P<0.001) and a significantly increased NEFA to ALB ratio (t=4.991, P<0.001). Compared with NS children in remission, those in acute phase showed a significantly decreased ALB (Z=7.822, P<0.001) and an increased NEFA to ALB ratio (t=4.991, P<0.001). In all the NS children, NEFA to ALB ratio was positively correlated with the levels of TC (r=0.564, P<0.001), TG (r=0.444, P<0.001), LDL-C (r=0.625, P<0.001), urea (r=0.437, P<0.001), creatinine (r=0.278, P=0.013), and uric acid (r=0.397, P<0.001), while negatively correlated with the level of total protein (r=-0.461, P<0.001). Multiple linear regression analyses showed that NEFA to ALB ratio was independently associated with serum urea levels (β=0.703, P=0.001; adjusted R=0.494) after adjustment of other related factors.

CONCLUSIONSerum NEFA to ALB ratio is significantly increased in NS children in close association with impaired kidney function, and may function as a novel parameter for assessing the development of NS.

Case-Control Studies ; Child ; Fatty Acids, Nonesterified ; blood ; Humans ; Nephrotic Syndrome ; blood ; Regression Analysis ; Serum Albumin, Human ; analysis

OBJECTIVESTo evaluate the relationship between the operative methods and the therapeutic results of the patients with hypospadias.

METHODSNine operative types and different tissue materials taken in hypospadias operations in the past 12 years were retrospectively analyzed.

RESULTSThe operative types and tissue materials had significantly effects on the therapeutic results besides the clinical experience of the operators. The cure rates of Tunneltron Urethroplasty, Preputial island flap urethroplasty and Bladder mucosa graft urethroplasty were 86.4%, 83.3% and 83.0% respectively.

CONCLUSIONSAll the nine types and different tissue materials of Hypospadias operations have its own advantages and disadvantages. They are worth further study and improvement.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Infant ; Male ; Mucous Membrane ; transplantation ; Penis ; surgery ; Surgical Flaps ; Treatment Outcome ; Urethral Stricture ; surgery ; Urinary Bladder ; transplantation ; Urinary Fistula ; surgery ; Urologic Surgical Procedures, Male ; methods

OBJECTIVETo evaluate the practical method of vacuum sealing drainage (VSD) technique combined with sural neurovascular pedicle fasciocutaneous flap to repair deep wounds in the foot near the ankle joint with exposed bone and tendons.

METHODSFrom January 2006 to January 2009, 79 patients with deep wounds in the foot near the ankle joint with exposed bone and tendons were treated by VSD technique combined with sural neurovascular pedicle fasciocutaneous flap including 58 males and 21 females with an average age of 34 years old ranging from 7 to 59 years. There were 17 cases in low 1/3 part of leg and achilles tendon, 28 in lateral malleolus and lateral dorsum of foot, 21 in medial malleolus and medial dorsum of foot, 13 in heel and pelma. Firstly the wounds were debrided and cultivated by using VSD technique, then the soft tissue defections were repaired with sural neurovascular pedicle fasciocutaneous flap.

RESULTSThe area of flap was from 6 cm x 5 cm to 18 cm x 15 cm; All patients stayed in hospital for 14 to 30 days, 18 days in average. Living flaps of all patients were followed-up from 6 months to 3 years, the flaps of 2 patients were mostly necrotic, 3 were necrotic, 5 cases appeared obstacle of venous back streaming. The others survived with no infections.

CONCLUSIONThe wound would become fresh and clean as soon as possible with VSD. The sural neurovascular pedicle fasciocutaneous flap could provide a good covering for the exposed wound. Therefore the wound healed faster with friction resistance and fine appearance. The time of hospitalization were greatly shortened after combined application.

Adolescent ; Adult ; Ankle Joint ; surgery ; Child ; Drainage ; methods ; Female ; Foot Injuries ; pathology ; surgery ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Soft Tissue Injuries ; surgery ; Surgical Flaps ; Vacuum