Objective Two autosomal recessive forms of muscular dystrophy:LGMD2B and Miyoshi myopathy may be indused by dysferlin gene mutation.The purpose of this study was to define molecular defects in dysferlin gene in a family with Miyoshi myopathy.Methods mRNA from peripheral blood in a Chinese Miyoshi myopathy pedigree was amplified by RT-PCR and the mutation was determined by sequencing the amplified products.Results The results of sequencing revealed a novel homozygous mutation,a 6429delG,on exon 53 of the dysferlin gene for the patients.Conclusion The 6429delG mutation in the dysferlin gene of patients creates a frameshift mutation which induces a stop codon at 2035 on exon 54 and the premature dysferlin contributes to the Miyoshi myopathy in the Chinese pedigree.

BACKGROUND: The transplantation of allogeneic cartilage has local immunological rejection, and it is necessary to further reduce the rejection to promote its application in clinic, thus it is significant to perform a series of experiments to induce local immune privilege.OBJECTIVE: To observe the in vivo growth of tissue engineered allogeneic cartilage reconstructed by chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL.DESIGN: A randomized controlled observation.SETTING: Shanghai Jiao Tong University.MATERIALS: Thirty-six allogeneic New Zealand rabbits as recipients and 45 1-week-old chinchillas as donors, either sex,were purchased by the experimental animal center of Chinese Academy of Sciences. Amphotropic recombinant retrovirus coated cell line PT67 was purchased from Clontech Company; Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), G418 and Polybrene were bought from GIBCO BRL.METHODS: The experiment was carried out in original Shanghai Second Medical University from January 2000 to July 2005. The New Zealand rabbits were randomly divided into three groups: FasL-transfected group (n =12), untransfected group (n =12) and blank control group (n =12). The rabbit allogeneic cartilages were constructed by the compound of pLNCX2-FasL transfected chondrocytes and tissue engineered material of pluronic F-127. ① Gross observation and mass changes of the grafts: Corresponding materials were infused subcutaneously, the grafts were removed at 1, 2 and 3 months after transplantation for gross observation and the mass changes. ② Staining observation: The grafts were removed at 1, 2 and 3 months after transplantation, then prepared into sections, and observed by hematoxylin and eosin (HE), Safranin'O and Masson's trichrome stainings. ③ Antibody detection: Blood samples (1 mL) were collected at 1 and 2 months after transplantation, the chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, and separated by electrophoresis in agarose medium, then acted with serum of recipient to observe whether corresponding antibody generated. ④ Complement dependent cytotoxicity (CDC) test: The chondrocytes of chinchillas were prepared into cell suspension (2×109/L), and then seeded into 96-well plate, attached grew for 24 hours, then recipient serum was added for the CDC test, and the percentage of apoptotic cells was counted under microscope.MAIN OUTCOME MEASURES: ① Gross observation and mass changes of the grafts;② Histological changes; ③ Results of the antibody detection; ④ Percentage of apoptotic cells.RESULTS: All the 81 rabbits were involved in the analysis of results. ① Gross observation and mass changes of the grafts: Two weeks after inoculation, there were obvious nod formations at the inoculated sites, but no nod formed in the blank control group. The new cartilage tissues became smaller gradually and completely disappeared at 4 months in the untransfected group, whereas those in the FasL-transfected group became smaller, but still existed after 7 months. The masses of grafts in the FasL-transfected group were higher than those in the untransfected group (P ＜ 0.05). ②Histological observation: Plenty of lymphocytic infiltrations around cartilage tissue could be observed in the untransfected group, and obviously decreased in the FasL-transfected group. No lymphocyte was observed inside the chondrocytes.Masson's trichrome staining was performed, and it was observed under light microscope that the small white parts in the middle were immature chondrocytes, and there were green collagen around most of the mature chondrocytes. Safranin O staining showed strong positive reaction, suggested that there were rich glycosaminoglycan in matrix. ③ Antibody detection: The chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, then acted with serum of recipient, and the results showed that no corresponding antibody generated. ④ Percentage of apoptotic cells: The percentages of serum CDC apoptotic cells in the FasL can ransfected group, untransfected group and blank control group were 5%, 6% and 1%, which were all negative.CONCLUSION: Rabbit allogeneic chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL can reconstruct tissue engineered cartilage, and can postpone the degeneration by 3 months.

BACKGROUND: At present,enhanced green fluorescent protein(EGFP)is proved to be the best labeled molecule,with unique advantages,such as high fluorescence specificity,easy to be detected,and so on.Recombined retroviral vector EGFP-pLNCX2,which can stably express EGFP,can be construct using gene-engineering technique.Transfecting allogenic chondrocytes is indeed very useful to investigate the target gene expression and process of constructing tissue engineered cartilage in vivo.OBJECTIVE:To construct recombined retroviral vector EGFP-pLNCX2 which can stably express EGFP,and investigate optimal conditions for retrovirus transfection of chondrocytes.DESIGN:Randomized controlled observation.SETTING:Shanghai Jiao Tong University.MATERIALS:Thirty New Zealand rabbits of either gender,1 week of age,were purchased from Experimental Animal Center of Chinese Academy of Sciences.Amphotropic retrovirus package cell line PT67 pLNCX2 and pEGEP-C1 were purchased from Clontech Company;NIH 3T3 cell line was purchased from ATCC Company;DH5a Bacterium coli was preserved by the laboratory of Shanghai Jiao Tong University;Retroviral vector pLNCX2 was purchased from Clontech Company;pEGFP-C1 plasmid with EGFP were donated by professor Cong Xiao-qian from Chinese Academy of Sciences.METHODS:This experiment was carried out in the Shanghai Jiao Tong University in August 2005.Chondrocytes of New Zealand rabbits were isolated and cultured.The recombinant retroviral vector EGFP-pLNCX2,which can stably express EGFP,was constructed to transfect cultured chondrocytes from New Zealand rabbits by using gene engineering technique.Transfection results were observed under fluorescence microscope.Altogether 6×105 chondrocytes incubated in 10 cm-diameter flat plate were used to transfect retrovirus-EGFP immediately and at 12,24 and 48 hours after inoculation.One week later,EGFP expression efficiency was measured with flow cytometer,and best occasion for retrovirus transfecting primary chondrocytes.Enzyme-digested chondrocytes were inoculated for 24 hours to transfect retrovirus.After 250 mg/L G418 was added,chondrocytes were screened on the 2nd,3rd,4th,5th and 6th days separately.After phosphate buffer solution(PBS)washings,the transfection efficiency of chondrocytes was detected by flow cytometer,and the best occasion for G418 screening was observed.MAIN OUTCOME MEASURES:①EGFP-pLNCX2 transfection efficiency.②The best occasions for retrovirus transfecting primary chondrocytes and G418 screening.RESULTS: ①Retroviral vector EGFP-pLNCX2 transfected primary chondrocytes of rabbits,and high expression of transfected chondrocytes could be obtained through preliminary screening of G418.After being screened and expressing EGFP,chondrocytes kept normal morphology,with pseudopod adhering to the wall and matrix secreting vigorously.②The best occasion for retrovirus transfecting primary chondrocytes was at 24 hours after cell inoculation.The transfection efficiency determined with flow cytometer was 19.14% on the 5th day.The best occasion for G418 screening was on the 5th day after culture.Transfection efficiency of G418 screening was 55.75% on the 7th day.CONCLUSION:Recombinant retroviral vector EGFP-pLNCX2 can effectively transfect chondrocytes.The best occasion for retrovirus transfecting primary chondrocytes is at 24 hours after inoculation, and the best occasion for G418 screening is on the 5th day after culture.

Objective To assess the application value of REP-PCR in genotyping of candida glabrata strains in clinical pratice. MethodsFrom 2009 to 2010, thirty-eight candida glabrata strains were isolated from Shanghai Ruijin Hospitals, Shanghai Renji Hospital, Shanghai Huashan Hospital, Anhui Medical University Hospital, Shenzhen People's Hospital. Six loci in housekeeping genes (FKS, LEU2,NMT1, TRP1, UGP1 and URA3 ) were amplified and sequenced. The sequences were compared with the MIST database and allele profile and sequence type (ST) were obtained. With primers Ca21, Ca22 and Com21 used to amplify the adjacent variable gene regions,the amplicons were analyzed through electrophoresis to generate different REP-PCR types. Finally, the results of these two genotyping methods were compared. ResultsFor REP-PCR, Ca22-Com21 has the best genotyping effect. REP-PCR and MLST have the same genotyping results. Five REP-PCR types were found in 38 candida glabrsta isolates. Type A,B, C, D and E strains from REP-PCR were genotyped as ST 7, 3, 19, 45 and new type respectively byMIST. REP-PCR saves time compared with MIST. Conclusions REP-PCR offers a simple and rapid method for molecular typing, with similar discriminatory power with MIST. Therefore, REP-PCR can be the preferred choice in laboratory, especially for a large number of isolates.

Objective To obtain conversion formulae of cyclosporine test results between TDX and AXSYM system and evaluate the influence of different sample diluents in over-range samples on AXSYM system.Methods One hundred samples with different concentration were analyzed by TDX and AXSYM system, respectively. The results were compared. Fifty over-range samples were diluted with prepared diluents such as whole blood, phosphate buffer, self-prepared diluents and zero point calibrator and then analyzed on AXSYM system.Results There was a significant difference in the results between TDX and AXSYM system. Reasonable formulae were concluded based on different concentration ranges. Results obtained by using whole blood and phosphate buffer as diluents differed from that got by using zero point calibrator. In contrast, there was no significant difference between self-prepared diluents and zero point calibrator.Conclusion The conversion formulae are helpful to evaluate the variation of the results while the analysis system was changed. Self-prepared diluents could be used to replace zero point calibrator as sample diluents.

Objective To evaluate the contribution of gyrA and parE detection in Ureaplasma genotyping.Methods Sixty Ureaplasma isolates were selected with the Mycoplasma IST kit.The gyrA and parE were amplified by PCR.The DNA was sequenced and compared with the corresponding sequences in GenBank.Results The nucleotide sequence of gyrA had 100% identity in serovar 1,3,6,14 and 100%identity in serovar 2,4,5,7～13,too.But the sequence had 91%identity between the two groups.The nucleotide sequence of parE had 98%～99% identity in serovar 1,3,6,14.And it had 100% identity in erovar 2,5,7,8,11 and 100% identity in serovar4,12,13.But it had only 90% identity between the two groups.Ureaplasma parvum(Up),Ureaplasma urealyticum(Uu)and Up+Uu infection were found 68.3%(41/60),21.7%(13/60)and 10%(6/60) of clinical specimens,respectively.In Up isolates,serovar 3 was 48.8%(20/41).Conclusion Ureaplasma can be divided into two genotypes(Up and Uu)by gyrA analysis.And Up can be divided into four subtypes which correspond to serovar 1,3,6,14,respectively.Serovar 3 is the main isolate in our research.

Objective To explore the matrix effect on cyclosporine A (CsA) test by fluorescence polarization immunoassay (FPIA) and enzyme-multiplied immunoassay technique (EMIT), explain the discrepancy of external quality control results between these two methods and find the corrective action.Methods One hundred whole blood samples with various concentrations were adopted and CsA levels were detected by FPIA and EMIT.The results were compared with each other.Moreover, the influence of residual metal ions upon immunoreactions was assessed by adding Cu2+ and Zn2+.The effect of non-whole blood matrix on extraction efficiency for quality control materials and CsA calibrator was evaluated by adding identical volume of Hb-rich reagents followed with re-extraction.Results There is good correlation between results measured with FPIA(X) and EMIT(Y) methods ( Y=0.926 8X -8.115,R2 =0.996 9).Neither FPIA nor EMIT was affected by residual metal ions ( P ＞ 0.05 ). Non-whole blood matrix decreased the extraction efficiency of two methods, but it could be corrected by supplementation of the Hb-rich reagents (≥30 g/L).Conclusions Non-whole blood matrix may be the main reason for the inconsistent results measured by FPIA and EMIT methods.It could be corrected by using Hb-rich reagents.In addition,we should consider the influence of low lib on CsA test,espocially for organ transplant patients with lower Hb ( ＜30 g/L).

Objective To assess the sequence variations in preS/S regions of occult hepatitis B virus (OHB) and their relationship to severe chronic hepatic injury. MethodsWe collected samples from HBsAg negative patients, and evaluated their HBV-DNA by nest-PCR. HBV-DNA positive samples were used for analysis of preS/S region by PCR sequencing. Results Sixty-nine cases with HBV-DNA were identified in 468 cases without HBsAg. The positive percents were 16％, 8.7％, 36.4％, 18.3％ and 0％in group of only HBcAb positive, only HBeAb positive, only HBeAg positive, both HBcAb and HBsAb positive and all indexes negative, respectively. The level of HBV-DNA of OHB was significant lower than that in HBsAg positive patients. Compared with HBsAg positive controls, preS/S deletion, M1I and Q2K in preS2 region, Q129N/R/P,G185R and S210R in S region were more common in OHB. Moreover, M1I and Q2K in preS2 region, G185R and S210R in S region in OHB with severe chronic hepatic injury were more common that those in OHB without severe chronic hepatic injury. Compared with HBsAg positive patients with severe chronic hepatic injury, the level of HBV-DNA was lower, while the frequency of M1I and Q2K mutation in preS2 region, G185R and S210R in S region were more common in OHB patients with severe chronic hepatic injury. ConclusionThe virological factors were different between OHB and HBsAg positive patients. The M1I and Q2K in preS2 region, G185R and S210R in S region might be useful for prognosis evaluation of OHB patients.

OBJECTIVETo establish a simple, rapid genetic diagnostic system for haemophilia B.

METHODSThe polymorphisms of eight STR loci in 87 normal persons and 8 haemophilia B families were assayed by PCR and genescan, and the linkage relations were analysed.

RESULTSSix of the eight STR loci can provide genetic information for haemophilia B, and the heterozygosity is 0.50 approximately 0.83, PIC 0.39 approximately 0.80, and DP 0.66 approximately 0.94.

CONCLUSIONCombination of multiple STR loci analysis could be effective method for genetic diagnosis of haemophilia B.

Female ; Genetic Carrier Screening ; methods ; Genetic Linkage ; Genetic Predisposition to Disease ; Hemophilia B ; diagnosis ; genetics ; Humans ; Male ; Microsatellite Repeats ; genetics ; Pedigree ; Polymorphism, Genetic

ObjectiveToinvestigate the prevalence, awareness and risk factors of chronic kidney disease (CKD) among community adult population in Shanghai, China, in order to provide early diagnosis and treatment of CKD, and informations for national health policy makers.MethodsTwo thousand five hundred and ninety six residents (≥ 18 years old) were randomly selected from community population in Changning district of Shanghai, China. They were interviewed and tested for albuminuria -morning spot urine albumin to creatinine ratio [ACR, abnormal: ≥ 17 mg/g (male), ≥25 mg/g (female)], reduced renal function-estimated GFR by abbreviated MDRD equation [abnormal: <60 ml ·rain-1 (1.73 m2)-1] and hematuria-morning spot urine dipstick confirmed by urine microscopy. The associations among demographic characteristics, healthy characteristics (e.g. diabetes and hypertension) and indicators of kidney damage were examined. The investigators and neighborhood committee were well trained. Those who had semiquantitative positive were detected again by albuminuria-morniag spot urine albumin to creatinine ratio after three months. ResultsTwo thousand five hundred and fifty four residents with complete data were enrolled in the study. Albuminuria was detected in 6.3% of subjects, reduced renal function in 5.8%, hematuria in 1.2%. Approximately 11.8% of these subjects had at least one indicator of kidney damage. The awareness rate of CKD was 8.2%. The Logistic regression model showed that hyperuricemia, nephrolithiasis, anemia, diabetes, central obesity, hypertension and age contributed to the development of CKD. ConclusionsThe prevalence of CKD in community adult population in Shanghai is 11.8%, And the awareness rate of CKD is 8.2%. Hyperuricemia, nephrolithiasis, anemia, diabetes, central obesity, hypertension and age are risk factors of CKD.