Objective To evaluate the heterozygous ambiguity resolution primers (HARPs) method in resolving ambiguous genotyping results of human leukocyte antigen (HLA) genes in Chinese Hart population, and choose some appropriate HARPs primers. Methods HLA-A, HLA-B and HLA-DRB1 genes of 416 southern Chinese Han individuals were genotyped by sequence-based-typing(SBT) method and then the ambiguous genotyping samples were sequenced again by HARPs primers provided by American Atria company. Results The percentage of ambiguous genotyping samples resolved by HARPs for HLA-A, HLA-B and HLA-DRBI locus was 86.3% (132/153), 73.9% (130/176) and 38.1% (85/223) respectively. Among them, 48.5% (64/132)HLA-A, 80.0% (104/130)HLA-B and all HLA-DRB1(85/85)samples only need one primer, 47.7 % (63/132)HLA-A and 20.0% (26/130)HLA-B samples need two primers. Three to six different HARPs primers can resolve more than 90% ambiguities. Conclusion HARPs is a convenient method and could be a routine method to resolve ambiguities for HLA-A, HLA-B and HLA-DRB1 genes genotyped by SBT in Chinese Han population.

BACKGROUND: The judgment of the engraftment of hematopoietic stem cells after transplantation mainly depends on various genetic labeling in vivo, which are different in sensitivity and effectiveness, thus a method with powerful differential ability, high sensitivity and not restricted by sex is to be established.OBJECTIVE: To observe the DNA genetic loci of short tandem repeat in the blood samples of both donors and recipients before allo-hematopoietic stem cell transplantation and those of recipients at different time points after transplantation.DESIGN: An observation measurement.SETTING: Laboratory of Immunogenetics, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center.PARTICIPANTS: Blood samples of 18 pairs of donors and recipients, who were successfully matched and accepted hematopoietic stem cell transplantation, were selected from the Laboratory of Immunogenetics, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center from February 2004 to December 2005. Among the 18 patients, there were 10 males and 8 females, with a mean age of 35 years old, including 6 cases of them were donated by relatives with blood relationship, and 12 cases by volunteers without blood relationship. Informed consents were obtained from all the participants.METHODS: The blood samples of both donors and recipients before transplantation and the blood samples of recipients after transplantation were collected, and the fluorescence labeling short tandem repeat technique was used to detect the 15 loci for short tandem repeat and Amelogenin sex locus, so that the differential loci between the donor and recipient could be screened. The engraftment and dynamic changes of the short tandem repeat genes of the donors in the recipients after transplantation were observed, the times for the earliest occurrences of short tandem repeat genes of the donors and the complete chimerism were recorded.MAIN OUTCOME MEASURES: ① Differential genes between the donors and recipients before transplantation;②Times for the earliest occurrences of short tandem repeat genes of the donors and the complete chimerism.RESULTS: All the 18 pairs of donors and recipients were involved in the final analysis of results. Satisfactory results of the typing at the 15 loci for short tandem repeat and 1 sex locus in the 18 pairs of samples of both donors and recipients before transplantation and the sample of the recipients after transplantation respectively. Averagely 12.4 (8-15) differential loci for short tandem repeat could be distinguished between the donors and recipients. ②After transplantation, short tandem repeat genes could be detected the earliest at 8 (5-14) days averagely, It took 14 (9-23) days averagely for short tandem repeat loci to convert from recipient type completely into donor type, and the engraftment converted from the recipient chimerism types completely into the donor types.CONCLUSION: The fluorescence labeling compound amplification of short tandem repeat technique can precisely measure the number of PCR products, describe the engraftment of hematopoietic stem cells and the whole process of development. It can also provide accurate and timely information for the early judgement of engraftment, predicting failure of transplantation and controlling recurrence.

Objective To evaluate wall histological abnormalities 2 to 3 cm to the end in high or intermediate anal atresia in order to identify features that explain postoperative bowel dysfunctions.Methods Sixty anal atresia patients treated in the Capital Pediatric Institution between January 2008 and December 2012 were recruited in our study.36 patients were resected the terminal anal segment (3 cm).Compared with those 24 cases who were not.Resected samples were fixed for HE and immuno-histochemical stainings.Clinical data including sacral ratio (SR),age at operation,gender,bowel function were evaluated.Results There was no significant difference in patients' SR value,gender and age at operation between resected group and control group.The median follow-up period was 4.5 years.The rates of voluntary bowel movement,soiling (grade 1,2,3) were similar in both groups,however,the rates of severe constipation in resection group was significantly lower that in control group (3 ％ vs.21％,P ＜ 0.05) In the bowel wall of distal 2 cm anrectal canal,the connective tissue was found to be irregular and abnormally represented.Muscle coat was abnormal in all cases,showing the dysplasia circular and longitudinal layers.The number of enteric nervous system was significant fewer in distal 2cm anrectal canal than that in distal 3 cm(1.6 ±0.9 vs.5.6 ±1.8,t=11.715,P＜0.01).Conclusions Resection of terminal 3 cm at least of the atresia anal canal benefits postoperative bowel defecation function.

OBJECTIVE: To explore the effects of novaferon on dextran sulfate sodium (DSS)-induced colitis and expression of TNF-α in mice and to evaluate the efficacy of novaferon on ulcerative colitis and the possible mechanisms. METHODS: A total of 70 BALB/C mice [weight (20.0±2.0) g, 8-week years old, female, pathogen free] were randomly divided into 7 groups: a normal group, a model group, a mesalazine treatment group, a prednisone treatment group, a low-dose novaferon group, a middle-dose novaferon group and a high-dose novaferon group (10 mice per group). The normal group-mice were given distilled water. The ulcerative colitis model was established by treated the mice with 4% DSS for 7 continuous days. At the 8th day, the mice in the all of drug treatment groups were injected corresponding drugs (i.p.). During the experiment, the general situation, daily weight, stool trait and occult blood were recorded, and the mice were killed on the 14th day. The disease activity index (DAI), colon length, histological scores were assessed. Immunohistochemistry was used to measure the expression of TNF-α in colonic mucosa. RESULTS: 1) The mice treated with DSS solution showed diarrhea, mucous stool and bloody stool, and the DAI score increased gradually. The mesalazine, predinison and nofaferon could ameliorate the general situation of the mice, reduce the DAI and histological scores, and reverse the decrease in the colon length. 2) Compared with the model group, the DAI scores were significantly decreased in the novaferon groups (at low, middle or high dose), the mesalazine group or the prednisone group (all P<0.01), but there was no difference among the mesalazine group, the prednisone group and the low-dose novaferon group (all P>0.05). The efficacy of novaferon in the middle-dose group and the high-dose group are better than that in the mesalazine group, the prednisone group and the low-dose novaferon group (all P<0.01). The efficacy of novaferon showed a dose-dependent manner. 3) The injury of colonic mucosa was relatively mild in the novaferon groups (at low-dose, middle-dose or high-dose), the mesalazine group and the prednisone group, and there were partial glands and less inflammatory cells. Compared with the model group, there was statistics difference (all P<0.05). The tissue injury was significantly alleviated, and the DAI score was decreased in the high-dose novaferon group compared the middle-dose novaferon group (P<0.05), but there was no significant difference between the low-dose novaferon group and the middle-dose novaferon group or between the mesalazine group and the prednisone group (both P>0.05). 4) The TNF-α expression was significantly down-regulated in the novaferon groups (at low-dose, middle-dose or high-dose), the mesalazine group and the prednisone group compared with model group (all P<0.01); but there was no significant difference between the mesalazine group and the prednisone group (P>0.05); the decrease of TNF-α expression by novaferon displayed a dose-dependent manner. Compared with the mesalazine group or the prednisone group, the TNF-α expression in novaferon groups at all dosages was dramatically reduced (all P<0.01). CONCLUSION Novaferon can improve the DAI scores and colonic tissue injury in ulcerative colitis induced by DSS in mice, and down-regulate the TNF-α expression in dose-dependent manner.
Animals ; Colitis, Ulcerative ; chemically induced ; drug therapy ; Dextran Sulfate ; adverse effects ; Female ; Interferons ; therapeutic use ; Intestinal Mucosa ; drug effects ; Mesalamine ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Prednisone ; therapeutic use ; Recombinant Proteins ; therapeutic use ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo detect the expression of human telomerase reverse transcriptase (hTERT) protein and mRNA in bile duct carcinomas and the adjacent tissues and to elucidate its role in bile duct carcinogenesis.

METHODSThe expression of hTERT protein and hTERT mRNA in the formalin-fixed paraffin-embedded specimens of 71 cases of bile duct cancers and 39 cases of adjacent tissues was detected by streptavidin-peroxidase immunostaining and in situ hybridization. The correlation was analysed statistically between the expression of hTERT protein and mRNA and clinicopathological parameters bile duct carcinomas.

RESULTSThe positive rate of hTERT protein expression and mRNA expression in malignant specimens was 78.9% (56/71) and 67.6% (48/71), while that in the adjacent tissues was 35.9% (14/39) and 23.1% (9/39), respectively. All the positive signals were found in the hyperplastic biliary epithelia. No significant correlation was established between hTERT expression and clinicopathological parameters.

CONCLUSIONhTERT gene transcription and protein expression is most likely involved in the proliferation and malignant transformation of bile epithelia and the malignant progression of bile duct carcinomas. The detection of hTERT expression may serve elucidating the carcinogenesis of bile duct.

Adult ; Aged ; Aged, 80 and over ; Bile Duct Neoplasms ; enzymology ; pathology ; DNA-Binding Proteins ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Telomerase ; analysis ; genetics

OBJECTIVETo investigate the dynamic expressions of TGF-beta 1 and TGF-beta 1 mRNA at different stages of hepatocellular carcinoma (HCC) development and their use in clinical diagnosis.

METHODSHepatoma models were developed with 2-FAA using male Sprague-Dawley (SD) rats. Morphological changes of the rat liver histological preparations (H and E stained) were studied. The fragment of TGF-beta 1 gene obtained was amplified by nested RT-PCR. Dynamic change of TGF-beta 1 level was quantitatively analyzed by ELISA. The distribution of TGF-beta 1 in the cells and its gene expression were detected in human HCC tissues.

RESULTSThe progressive increases of hepatic TGF-beta 1 and TGF-beta 1 mRNA were observed in rat hepatocytes which progressed from granular degeneration, atypical hyperplasia and finally to HCC development induced by 2-FAA. The expression levels in HCC tissues were significantly higher than those in the normal and degenerative ones. TGF-beta 1 was shown in rat hepatocytes by immunohistochemistry. Plasma TGF-beta 1 was detected in 89.5% of all the patients with HCC, but it was detected in 93.3% of them who had an AFP less than 400 microg/L. TGF-beta 1 mRNA showed a stronger expression in HCC tissues. TGF-beta 1 mRNA was found in peripheral blood mononuclear cells from all HCC patients with extrahepatic metastasis.

CONCLUSIONTGF-beta 1 may participate in hepatocyte canceration. The overexpression of TGF-beta 1 and TGF-beta 1 mRNA could be useful markers for early diagnosis and predicting prognosis of HCC.

Adult ; Aged ; Animals ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; diagnosis ; pathology ; Female ; Humans ; Liver Neoplasms, Experimental ; blood ; diagnosis ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; prevention & control ; Prognosis ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; blood

OBJECTIVETo investigate the expression of Slingshot-1L (SSH-1L) in peripheral blood eosinophils of asthmatic patients.

METHODSPeripheral vein blood sample of 30 ml was collected from 15 healthy volunteers, 15 asthmatic patients with glucocorticoid treatment and 15 asthmatic patients without the treatment. The eosinophils were isolated, purified and counted for each sample, and SSH-1L/beta-actin gene fragments were amplified simultaneously by RT-PCR for the total RNA. SSH-1L protein was detected by Western blotting from the total protein of the peripheral eosinophils. The expressions of SSH-1L at both mRNA and protein levels are compared between different groups.

RESULTSSSH-1L/beta-actin ratio significantly increased in untreated patients with asthma attacks in comparison with healthy volunteers (P<0.05), which did not occur in patients treated with glucocorticoids (P>0.05). The optical density of SSH-1L protein significantly increased in untreated asthmatic patients (P<0.05), but not in patients treated with glucocorticoids (P>0.05), as compared with the healthy volunteers.

CONCLUSIONSignificantly increased SSH-1L expression in peripheral eosinophils may play an important role in the activation and migration of eosinophils.

Actin Depolymerizing Factors ; metabolism ; Actins ; metabolism ; Adult ; Asthma ; blood ; Blotting, Western ; Eosinophils ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Phosphoprotein Phosphatases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo detect the expression of HBV X gene (HBx mRNA) in extrahepatic biliary tract carcinomas and the adjacent non-cancerous tissues, and to analyzed the relationship between HBV infection and incidence of biliary tract carcinomas, thereby to elucidate the possible role of HBx in the carcinogenesis of biliary tract.

METHODSThe plasmid pSPX46 was digested by appropriate restriction enzyme. HBx fragment was obtained through gel extraction kit. The digoxigenin-labeled DNA probes for HBx mRNA were prepared by a random prime technique. The expression of HBx mRNA was detected in formalin-fixed- paraffin-embedded specimens from 71 cases of biliary tract carcinomas and 39 specimens of non-cancerous tissues adjacent to cancer by in situ hybridization. The correlations between HBx mRNA expression and clinicopathological parameters were statistically analysed in 71 cases of biliary duct carcinomas.

RESULTSForty-three of 71 malignant specimens had detectable HBx mRNA expression with a positive rate being 61%. Only 7 of 39 specimens of non-cancerous tissues adjacent to cancer had weak HBx mRNA expression, with a positive rate being 18%, and all these positive signals were found in the hyperplastic biliary epithelium. No significant correlation was found between HBx mRNA expression and clinicopathological parameters, but a strong positive correlation was found between HBx mRNA and protein expression.

CONCLUSIONThere is a high frequency of HBx mRNA expression in extrahepatic biliary tract carcinomas. HBV infection and its gene integration might play a role to certain extent in the development of biliary tract carcinomas.

Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Bile Ducts, Extrahepatic ; pathology ; Biliary Tract Neoplasms ; complications ; virology ; Female ; Hepatitis B ; complications ; virology ; Hepatitis B virus ; genetics ; Humans ; In Situ Hybridization ; Male ; Middle Aged ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Trans-Activators ; genetics

Objective To study mental characteristics of children with light lead poisoning. Methods Eighty - six children with light lead poisioning and 33 control children were evaluated with WISC - R and social adaptive capacity(SAB). Results 1. Intelligence quotient( IQ)and SAB of children with light lead poisoning were lower than normal children. 2. There was also deficiency of perception factor and memory/ concentration factor among children with lead poisioning and the blood lead level had negative association with perception factor. Conclusion Light lead poisioning may affect children's IQ, especially in the perception factor and memory /concentration factor.