Objectives To investigate the effects of combined radiation and thermal burn injury on the survival of skin allografts and to analyze the relationship between the prolongation of allograft survival and the changes of immune functions of the thymocytes and splenocytes in rats.Methods Wistar rats were irradiated with 3, 4, 5, 6 and 8 Gy of gamma rays. Thirty minutes after radiation, 15% TBSA Ⅲ-degree burn was inflicted to the rats. Twenty-four hours after the burn injury, allografts were used to cover the burn wounds. In the 8 Gy group, 1 hour before skin grafting, the bone marrow cells (4×108) from the same donor were also transplanted. All rats were carefully observed after injury. The rats with single radiation injury of 5 Gy gamma rays, with single burn injury and with combined radiation-burn injury were killed 3, 7, 10, 15 and 30 days after skin grafting for immunological assay and pathological study.Results All the allografts in the single burn group were rejected in 10 days. In the combined injury groups, the survival rates of the allografts in rats undergoing 3 and 4 Gy radiation were 20% and 30%, respectively. In the combined injury groups undergoing 5, 6 and 8 Gy radiation, the 10-day survival rates of the allografts were 69%, 88% and 100% respectively, and the 30-day survival rates in the three groups were 36%, 42% and 100% separately. The grafted allogenic skin, with normal epithelial cells and good vascularity, healed well with the recipient's skin. Hairs grew well from the allografts 30 days after grafting. Three, 7 and 15 days after allografting, in the single burn group, the proliferative activities of the thymocytes were 90%, 185% and 130% of the preinjury level, and the antibody forming capacities of the splenocytes were 200%, 171% and 300% of the preinjury level, respectively; in the combined injury groups, the proliferative activities were 6%, 99% and 91% of the preinjury level, and the forming capacities were 2%, 36% and 90% of the preinjury level.Conclusions The survival rate of allograft in rats undergoing combined radiation and thermal burn injury rises with the increase in radiation dosage. The allograft covering single bun injury is severely rejected by immune reaction. The prolongation of the survival of allograft in combined injury group mainly results from radiation that suppresses immune functions.

The S1 gene hypervariable region I (HVR I) of 22 infectious bronchitis virus (IBV) strains isolated in Guangxi during the period of 1985-2007 were sequenced and compared to that of the other IBV reference strains and the pigeon coronavirus isolates. A phylogenetic tree based on nucleotide sequences of HVR I of all the IBV showed that they were classified into 5 distinct Clusters. 16 out of 22 IBV isolates were grouped into Cluster I, and had higher homology with pigeon coronavirus isolates but lower homology with the Massachusetts (Mass) type vaccine strains. There were 4 and 3 amino-acid residues inserted at the sites of 33-34 and 34-35 respectively within HVR I in 15 isolates, except in isolate GX-NN6 there had 4 amino-acid residues inserted at the both sites; isolates GX-YL1 and GX-NN2 had close relationship with Mass type vaccine strains, and they shared Cluster II; isolates GX-G and GX-XD of Cluster III had close relationship with the Japanese strain JP Miyazaki 89 which was isolated at the same period; isolates GX-YL6 and GX-NN7 of Cluster V had close relationship with the European strain 4/91. The results showed that there were high phylogenetic diversity among the IBVs prevailed in the field in Guangxi resulting from the commonly occurred mutation or insertion within the S1 gene HVR I of the viruses, and majority of the isolates had lower homology with the commonly used Mass type vaccine strains. There was much higher homology among viruses isolated in the same period of time, but without distinct difference in geographical origins.
Amino Acid Sequence ; Animals ; Chickens ; virology ; Genetic Variation ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Membrane Glycoproteins ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; chemistry ; genetics

OBJECTIVETo explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell.

METHODSAfter whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing.

RESULTSThe rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%.

CONCLUSIONWGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

Base Sequence ; DNA Mutational Analysis ; Female ; Genotype ; Humans ; Lymphocytes ; metabolism ; pathology ; Middle Aged ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Preimplantation Diagnosis ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; blood ; genetics

OBJECTIVETo detect the expressions of miR-122 and miR-224 in hepatocellular carcinoma (HCC) by real-time fluorescence quantitative RT-PCR and investigate the significance of miRNAs in early diagnosis of HCC.

METHOD2(-Delta Delta CT) method was used for quantitative analysis of the expression pattern of miR-122 and miR-224 in 35 HCC and adjacent normal tissues. All the quantitative results were confirmed by Northern blotting.

RESULTSCompared with adjacent normal tissues, the HCC tissues showed significant miR-122 down-regulation (P<0.01) and miR-224 over-expression (P<0.001).

CONCLUSIONSHCC has obvious alteration in the expression patterns of miR-122 and miR-224, and real-time fluorescence quantitative RT-PCR provide a new means for early, efficient, and accurate diagnosis of HCC.

Adult ; Aged ; Base Sequence ; Blotting, Northern ; Carcinoma, Hepatocellular ; diagnosis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; diagnosis ; genetics ; Male ; MicroRNAs ; genetics ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Spectrometry, Fluorescence ; Time Factors

OBJECTIVETo generate a gene delivery plasmid carrying the dominant negative form of the protein phosphatase 2A catalytic subunit a (DN-PP2Aca) driven by a hepatocellular carcinoma (HCC) tissue-specific promoter and investigate its ability to inhibit growth of cultured hepatoma cells.

METHODSThe gene delivery plasmid was constructed by PCR-amplifying DN-PP2Aca from wild-type PP2Aca using site-directed mutagenesis and then ligating the sequence-verified amplicon downstream of an alpha-fetoprotein enhancer and phosphoglycerate kinase promoter (AFpg) in the luciferase reporter vector pGL3-Basic. Following transfection into two AFP+ hepatoma cell lines (HepG2 and HepG3) and two AFP- hepatoma cell lines (SK-HEP-1 and L02), the transcriptional activity of the AFpg-driven DN-PP2Aca plasmid was tested using luciferase reporter gene assay and western blotting. The effect on cell growth was tested using MTT assay. Between group differences were assessed by t-test.

RESULTSThe AFpg-driven DN-PP2Aca plasmid showed high transcriptional activity and protein expression in both HepG2 and Hep3B cells. At 72 h after transfection, the proliferation capacities were repressed by 42.65%+/-3.99% (P = 0.0002) and 39.87%+/-3.91% (P = 0.0002) in AFP+ HepG2 and Hep3B cells, respectively (vs. untransfected). In contrast, the plasmid was transcriptionally inactive in and had no effect on proliferation of AFP- cells.

CONCLUSIONThe AFpg-driven DN-PP2Aca plasmid exhibits selective cytotoxicity against AFP+ hepatoma cells, and may represent a useful gene therapy strategy to treat HCC.

Carcinoma, Hepatocellular ; genetics ; metabolism ; Enhancer Elements, Genetic ; Genetic Therapy ; Genetic Vectors ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Mutation ; Promoter Regions, Genetic ; Protein Phosphatase 2 ; genetics ; alpha-Fetoproteins ; genetics

OBJECTIVE We have recently reported that cysteinyl leukotriene (CysLT) signaling plays an important role in microglial interleukin (IL)-1β secretion and subsequent neurotoxicity. The present study aimed to examine microglial morphological changes and the upstream molecular underlying IL-1βproduction in CysLT receptor agonist leukotriene D4 (LTD4)-treated BV2 microglia in vitro. METHODS Twenty-four hours after murine microglial BV2 cells were stimulated with LTD4 (1-100 nmol·L- 1), the cell proliferation and morphology were observed. The expression level of cysteinyl aspartate-specific protease 1 (CASP1) protein was measured by Western blotin BV2 cells. In addition, BV2 cells were pretreated with or without CysLT1 receptor antagonist montelukast for 1 h and the effects of monte-lukaston LTD4-stimulated microglial activation and CASP1 expression were evaluated. RESULTS The number of BV2 cells had an increasing tendency after 24 h treatment with LTD4, but no significant differences were observed between the control and LTD4-treated cells (P>0.05). Under basal and resting conditions, BV2 microglial cells displayed a ramified morphology. However, LTD4 at 100 nmool · L- 1 drove microglial morphological changes from a ramified towards an amoeboid shape. The expression of CASP1 protein was significantly upregulated in 100 nmool·L-1 LTD4-treated BV2 microglia (P<0.01). Furthermore, pretreatment with CysLT1 receptor antagonist montelukast prevented cell morphological changes and suppressed the increased CASP1 expression in LTD4-treated BV2 cells (P<0.05). CONCLUSION CysLT receptor agonist LTD4 induces morphological changes and CASP1 expressionin BV2 microglia, which can be inhibited by CysLT1 antagonist. These results suggest the involvement of CysLT signaling in microglial morphological changes and CASP1 expression.

BackgroundAn ideal animal model is very important for the investigation of the immune mechanism of high-risk rejection corneal transplantation.ObjectiveThis study was to compare three methods of creating a high-risk corneal transplantation model in rabbits to study high-risk rejection corneal transplantation.MethodsForty-five New Zealand white rabbits were utilized and assigned randomly to three groups of different modeling methods,with 15 rabbits for each group.The high-risk corneal transplantation models were created by suturing with 5-0 silk thread in 4 quadrants,inducing alkali burn with 1 mol/L NaOH or corneal xenotransplantation.In the suturing group and alkali burning group,the rabbits received a unilateral 7.25 mm diameter corneal allograft after corneal neovascularization was induced,and in the xenotransplantation group,corneas from cats were used as donors.Rabbits were followed-up for 4 weeks in all groups.Corneal neovascular area was calculated and compared among the three groups.The amount of rejection,inflammatory index ( IF),neovascularization and histology of grafts were clinically scored to calculate the reject index (RI).ResultsThere were 14,15 and 15 rabbits that survived the high-risk penetrating corneal transplantation,respectively,in the suturing group,alkali burning group and xenotransplantation group.Two weeks after operation,the IF scores were 0.543 ± 0.103,0.811 ± 0.054 and 0.191 ±0.087,and the RI were 2.111±0.928,7.0±0.816 and 3.182±0.751 in the suturing group,alkali burning group and xenotransplantationgroup,respectively,showingstatisticallysignificantdifferencesamongthethreegroups (x2 =25.736,22.432,P =0.000).The IF value was lower in the xenotransplantation group compared with the suturing group and alkali burning group (Z =3.841,3.993,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.568,P =0.000).The RI value of the xenotransplantation group was significantly raised in comparison with the suturing group and declined in comparison with the alkali burning group (Z =2.373,P =0.018;Z =3.936,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.729,P =0.000 ).The survival times of the grafts were ( 17.9±2.0 ) days,( 13.4 ±2.4) days and ( 15.5 ±2.0 ) days in these three groups with a significant difference among them ( F =9.474,P =0.001 ).The neovascularization area in the xenotransplantation group was smaller than the suturing group and alkali burning group (P＜ 0.05 ).Histological examination revealed a large number of inflammatory cells infiltration in the grafts 2 and 4 weeks after transplantation in the suturing group and alkali burning group,but less inflammatory cells were seen in the xenotransplantaion group.Immunofluorescence staining showed abundant CD4+ T positive cells in the grafts in the three groups.Conclusions The cat-rabbit corneal xenotransplantation can induce stable and moderate immune rejection.This animal model has milder inflammatory response and less corneal neovascularization than the suture and alkali burn models.This method therefore is an ideal model for high-risk corneal transplantation.

Spine cord injury is a kind of severe central nervous system trauma causing motion and sensation dysfunction. Treatment fo-cuses on controlling secondary injury cascade and improving regeneration which are heavily regulated by microRNAs (miRNAs). This re-view discussed the effect of miRNAs with different subtypes on spine cord injury, and investigated their potential roles as therapeutic agents in the personalized treatment of patients with spine cord injury.

AIMTo report the experience with single stage dorsal inlay buccal mucosal grafts using the Snodgrass technique for complex redo cases.

METHODSFrom May 2004 to December 2005, a total of 53 patients aged from 3 to 34 years old (average 11.62 +/- 7.18 years) with failed previous hypospadias surgery were included in the present study. Indications included urethral strictures and repair breakdown. The unhealthy urethra was unroofed from the meatus in the ventral midline, a buccal mucosal graft was inlayed between the incised urethral plate and fixed to the corpora cavernosa. The neourethra was tubularized, and covered with subcutaneous (dartos) tissue and penile skin. Glanuloplasty was also performed in all cases. Outcome analysis included clinical follow-up, and endoscopy in 2 selected cases.

RESULTSThe buccal mucosal graft was 3.0-7.5 cm in length and 0.7-2.0 cm in width. All patients required glanuloplasty, with buccal mucosal grafts extended to the tip of the glans. After a follow-up of 14-30 months (mean 22.6 months), the total complication rate was 15.1%, with five cases of fistula and three cases of stricture.

CONCLUSIONInlaying dorsal buccal mucosal grafts applying the Snodgrass technique is a reliable method for creating a substitute urethral plate for tubularization. The recurrent rate of urethral stricture and fistula is at an acceptable level for redo cases. This approach represents an effective, simple and safe option for reoperations.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Male ; Mouth Mucosa ; transplantation ; Secondary Prevention ; Transplants ; Treatment Outcome ; Urethra ; surgery ; Urethral Stricture ; prevention & control ; Urinary Fistula ; prevention & control ; Urologic Surgical Procedures, Male ; adverse effects ; methods

OBJECTIVETo explore the role of survivin and PI3K/AKT pathway in the pathogenesis of psoriasis vulgaris (PV).

METHODSPlaque-like lesions collected from 22 patients with PV in progressive stage and 18 normal control skin specimens were examined using immunohistochemical staining, Western blotting and real-time quantitative PCR for expressions of survivin, PI3K and AKT in the keratinocytes, and their correlation was analyzed. A small interfering RNA (siRNA) was used to knock down AKT in cultured HaCaT cells, and Western blotting was used to detect the changes in the expression of survivin. RESULTS Compared with normal skin, PV lesions showed obviously up-regulated expressions of survivin, PI3K and AKT in the keratinocytes. Survivin expression was positively correlated with PI3K (r=0.4510, P=0.0351) and AKT (r=0.4423, P=0.0393) in the keratinocytes in PV lesions. In cultured HaCaT cells, siRNA-mediated knockdown of AKT caused down-regulation of survivin expression.

CONCLUSIONSurvivin and PI3K/AKT signaling pathway may participate in the occurrence and progression of PV.