Objective To evaluate the performance of a DNA microarray method for detecting HBV antiviral drug-resistant mutations. Methods Two hundred and twenty four serum samples from patients with CHB were tested in parallel by DNA microarray and direct sequencing for the mutations within the HBV reverse transcriptase (rt) region, which included rtL180, rtA181, rtM204 and rtN236. Samples with discrepant results were retested by clonal sequencing. Results Complete concordance between DNA microarray and direct sequencing results was observed in 214 out of 224 samples (95. 5% ). The presence of mixed viral populations in the other 10 samples detected by DNA microarray but not by direct sequencing was confirmed by clonal analysis. The DNA microarray could detect minor viral populations which constituted 5.0%-15. 0% of the total viral load. Conclusion DNA microarray is highly consistent with direct sequencing in detecting HBV mutations conferring drug resistance and more sensitive in detecting mixed mutant and wild-type sequences than direct sequencing, which makes it a useful tool for early detection of drug resistance early.

Objective A method was established for genotyping of hepatitis B virus (HBV A-D genotype),based on the PCR-restriction fragment length polymorphism (RFLP) created by Hinf Ⅰ,Ear Ⅰ,Apo Ⅰ action on an amplified segment of the S region.Methods Clinical diagnosis research.One hundred and twenty-eight HBV S sequences obtained from GeneBank were analyzed for restriction enzyme sites that would be genotype-specific.Restriction patterns following digestion with restriction enzymes Hif Ⅰ,Ear Ⅰ,Apo Ⅰ were determined to identify A-D HBV genotypes.The method was used to detect the HBV genotype of fifty severe hepatitis patients due to chronic hepatitis B in China.Then the detection results were confirmed by direct sequencing.Results The new genotyping method was established,named simple PCR-RFLP,which could identify HBV genotypes A to D.Genotypes B,C,B/C and A or D could be determined by a single step digestion with Hif Ⅰ.Eight patients of genotype A/B/C classified by single step digestion with Hif Ⅰ were conformed as genotype B variant by further digestion and direct sequencing.Extracted randomly and diluted into different concentration,three specimens were tested for genotype of HBV repeatedly and respectively.The results were all in accord with the originals,and the lowest detection limit of HBV DNA was 7 ～ 9 IU/ml.This was particularly useful in China where genotypes B and C were predominant.Twenty-three of genotype B and ten genotype C patients were classified from these fifty severe hepatitis B patients by a single step digestion with Hif Ⅰ through the simple PCR-RFLP method.The same results were also obtained by direct sequencing of PCR products (Kappa =1.00,P =0.001).The simple PCR-RFLP method was superior to direct sequencing in detecting HBV B/C polyinfection (9 cases and 0 case; x2 =18.00,P =0.001).Conclusions Both the sensitivity and repetitiveness of Simple PCR-RFLP method are satisfactory.It is superior to direct sequencing in detecting HBV B/C polyinfection,and simple,convenient.

Objective:To study the antiviral effect of ISG20 on HCV replicon.Methods:Wild type ISG20/mutated ISG20 cDNAs were obtained by RT-PCR/two step-PCR directed mutagenesis, and wild type ISG20 and dominant negative mutated ISG20 mammal expression vectors were consuructed. The constructed pISG20wt and pISG20m expressing vectors were transfected into Huh7 cells or Huh7 cells containing HCV replicon to investigate its effects on HCV replicon replication.Results:The ISG20wt/ISG20m expression vectors were constructed and the expressions of these two vectors were confirmed at both mRNA and protein levels. The effects of ISG20wt on HCV replicon replication were evaluated by Northern blot and Western blot. The results showed that expression of ISG20wt had significant inhibitory effect on HCV RNA replication.Conclusion:ISG20 participates in the anti-HCV action of IFN-? on HCV replicon system.

Objective:To investigate the cellular immunological regulation mechanism of Zhongjiling tablet on the myasthenia gravis(MG) patients.Methods:The myasthenia gravis patients were randomly divided into 2 groups, the curing group and the control group, 30 cases per group. Patients of curing group administered Zhongjiling tablet and prednisone placebo, patients of control group administered prednisone tablet and Zhongjiling tablet placebo. Course of treatment was 12 weeks. The distribution of T lymphocyte subgroup of myasthenia gravis patients was detected by flow cytometry. The content of IFN-?, IL-4 and TGF-? of the patients’ peripheral blood mononuclear in vitro were detected by ELISA kits.Results:After treatment, CD4+T cell percentage and the ratio of CD4+/CD8+ decreased significantly(P0.05). The IFN-? and IL-4 of treating group depressed, compared with that of pre-treatment, significant difference exited(P

To investigate the patient-specific dose verification method using ArcCHECK for total marrow irradiation (TMI) with Volumetric Modulated Arc Therapy (VMAT) and Helical Tomotherapy (HT). The kVCT images collected from 8 patients were respectively designed for RapidArc and Tomotherapy plans in total marrow irradiation. ArcCHECK was used for dose verification for the head-neck, chest-abdomen and pelvic. The merging function of ArcCHECK was used in VMAT and the method of double plans (reference and delivery plans) were used in HT. The γ-analysis passing rates for the head-neck, chest-abdomen, pelvic were 98.9% ± 1.9%, 98.4% ± 1.8%, 97.4% ± 2.1% for VMAT plans and 94.3% ± 1.5%, 96.5 ± 1.2%, 94.1% ± 1.9% for HT plans. The results show that using the merging function of ArcCHECK can achieve the dose verification well for VMAT plans with TMI. The method of double plans was done for the dose verification of HT plans with TMI as well as the plans with the targets keeping away from the set-up center.
Bone Marrow ; radiation effects ; Humans ; Radiotherapy Dosage ; Radiotherapy Planning, Computer-Assisted ; Radiotherapy, Intensity-Modulated

Objective To study the mutations of Env sequence of SIVmac239 after infection of Chinese rhesus monkeys, and compare the differences and characteristics of Gp120 sequences of enterotropic and neurotropic SIV strains. Methods Six strains of simian immunodeficiency virus were analyzed in this study: four separated from peripheral blood mononuclear cells of SIVmac239-infected monkeys and two neurotropic SIVmac251 strains.Isolated and cultured monoclonal virus was obtained by limiting dilution assay.Gp120 sequences were amplified after the RNA extraction and phylogenetic analysis was processed thereafter.So did the Gp120 amino acid sequence and N-glycosylation sites analysis of the enterotropic and neurotropic strains.Results SIVmac239 had different mutations in four rhesus monkeys.The diversity in amino acid sequences of the enterotropic and neurotropic strains concentrated in the V1 and V4 regions of Gp120.The enterotropic strains had an addition of glycosylation site in V4 but the glycosylation site changes of neurotropic strains were located in the conservative regions of C1, C2 and C3.Conclusions The addition of one glycosylation site in V4 region of GP120 and loss of one glycosylation site in C1 region are associated with enhanced enterotropism and neurotropism.The differences between the enterotropic and neurotropic strains are not dipicted in Gp120 V3 region which is closely related with the tropism of strains.

BACKGROUND:Exosomes are membrane vesicles secreted by mesenchymal stem cells. Increasing studies have shown that mesenchymal stem cells can secrete exosomes via paracrine function to play a role in tissue injury. However, reports on how to isolate and identify exosomes derived from human umbilical cord blood mesenchymal stem cells are few. OBJECTIVE:To extract, purify and identify exosomes derived from human umbilical cord blood mesenchymal stem cells. METHODS:The cellculture supernatant of human umbilical cord blood mesenchymal stem cells was col ected. Exosome was extracted and purified with ultrafiltration and gradient centrifugation methods. The morphology of exosome was observed by transmission electronic microscope, and the expressions of CD63, CD81, CD90, CD73, CD105, CD29, and CD166 in exosome of mesenchymal stem cells were analyzed by fluorescent activated cellsorting. RESULTS AND CONCLUSION:Mesenchymal stem cells from human umbilical cord blood secreted exosome which exhibited el iptic or saucer-like shape and its diameter ranged from 40 to 100 nm with membrane structure. Exosome could express the common surface adhesion molecules CD63, CD81 and the surface adhesion molecules CD90, CD73, CD105, CD29, CD166 of mesenchymal stem cells. These findings indicate that exosome may be secreted by mesenchymal stem cells of human umbilical cord blood, which contains plasma membrane proteins of umbilical cord blood mesenchymal stem cells.

Objective To investigate the effect of HBV on the expression of fibrosis-related factors in hepatic stellate cells(HSC)and its relation with liver fibrosis.Methods HSCs were co-cultured with HepG2 or HepG2.2.15 in vitro and HSCs cultured alone served as the control.The mRNA expression of matrix metalloproteinase(MMP)-2 and tissue inhibitor of metalloproteinase(TIMP)-1 was detected by realtime PCR.The protein expression of MMP-2 and TIMp-1 was detected by Western-blot.Results Compared with the control and the HSCs co-cultured with HepG2,the expression of MMP-2 and TIMP-1 mRNA in HSCs co-cultured with HepG2.2.15 was increased remarkably and the most significant difference was found at 72 h(F=11.91,23.13;P=0.008,0.001);the expression of MMP-2 and TIMP-1 protein in HSCs co-cuhured with HepG2.2.15 was also increased remarkably and the most significant difference was found at 72 h(F=20.70,6.54;P=0.002,0.003)too.Conclusion The expression of fibrosis-related factors in HSCs increased significantly after co-cultured with HepG2.2.15,which suggests that HBV could promote liver fibrosis.

Objective To study the effect of endothelial progenitor cells (EPCs) transplantation on CCl4 induced hepatic cirrhosis in rats. Methods Eight male SD rats were used as normal control. Thirty rats were induced liver cirrhosis by feeding with 25% CCl4/olive oil for 12 weeks, and then were subdivided into cirrhosis group (n = 10), EPCs transplanted group (n = 10) and saline control group (n = 10). EPCs were transplanted into the portal vein for 4 weeks in EPCs transplanted group. Rats in EPCs nontransplanted group were sacrificed at the beginning of the 12th week. Rats in EPCs transplanted group and saline control group were killed at the beginning of 16th week. Serum biochemical parameters were examined. The degree of liver cirrhosis was evaluated by Masson staining and by detecting the expression of α-SMA, Collagen Ⅲ and Ki67. Results The volumes of liver in cirrhosis group were twice as much as that in normal rats. 12 weeks after CCl4 administration, compared with saline control group, in EPCs transplanted group, hepatic activity index (HAI) ( F = 75. 062, P < 0. 01 ), the levels of ALT( F = 29. 942, P<0.05), AST(F=16.618,P<0.05) and TBIL(F=9.911 ,P<0.05) in serum decreased, the level of Alb ( F = 4. 944, P < 0. 05 ) and Ki67 ( F = 45. 966, P < 0. 01 ) was increased, the expression of α-SM A ( F = 7.86,P<0.05) and collagen Ⅲ (F = 135.787,P <0.01) decreased (P <0.05). Compared with untransplanted group, in EPCs transplanted group, the levels of ALT, AST and TBIL in serum were lower; In saline control group, the levels of ALT, AST and TBIL in serum were higher, the level of Alb and Ki67was lower, the expression of α-SMA and collagen Ⅲ were higher( P < 0. 05 ). Compared with normal rats, in saline control group, the levels of INR were higher (P < 0. 05 ). Conclusion EPCs transplantation improves hepatocye regeneration and ameliorates established hepatic cirrhosis.

Objective To investigate the methods and results of perforator pedicled flaps by anastomosis of superficial veins for reconstructing the extremity defects.Methods From February, 2012 to September, 2012, a total of 14 patients with traumatic skin and soft tissue and artery defects in extremities were repaired by perforator pedicled flaps anastomosed superficial veins with the recipient vessels.Fourteen flaps were based on five kinds of perforator arteries, the posterior interrosseous artery in 5 cases, the ulnar artery in 3 cases, the metacarpal artery in 2 cases, the peroneal artery in 3 cases, the lateral supramalleolar artery in 2 cases, the ulnar artery in 2 cases,and the posterior interrosseous artery in 1 case.After the operation, the blood supplies of flaps were observed severely,postoperative flap swelling were evaluated by edema level classification.After 1 month the blood flow of vein anastomosis were examed by color Doppler ultrasound.Questionnaire of the flap aesthetic satisfactory were performed during fellow-up periods.Results Twelve flaps were all survived well, 2 flaps had partial marginal skin necrosis in the distal, which was managed with surgical debridement, and wound healed in 1 month.Flap swelling were light, edema level classification were one degree in 4 cases,two degree in 10 cases postoperation 7 days, one degree in 11 cases and two degree in 3 cases, after 6 months.Color Doppler ultrasound examinations showed 9 vein anastomosises were all bypassed well.Eleven cases had 6-12 months' fellow-up periods.The flaps had like-like appearance, good contour, high aesthetic satisfactory.Conclusion Perforator pedicledr flaps by anastomosis of superficial veins can reduce the flap venous pressure obviously, avoid transientvenous venous congestion, improve the survival quality of the flap.It's a safe and effective method for soft tissue coverage of traumatic extremity wounds.