Objective To evaluate the effect of alIoHCT on recurrence and metastasis of HCC after hepatic radical resection. Methods Umbilical cord blood were collected after labor. The efficacy of separa-tion by 6% hydroxyethyl starch (6% HES) and NH4CL lysing solution were examined. Twenty-two SCID mice were randomized into the scheduled transplantation group (n=8), the single transplantation group (n =8) and the normal saline group (n=6). Human nucleated cells (NC)at a amount of 5×107 were transfused through the tail vein into the 2 groups of transplantation. All the mice in the 3 groups received cyclophospha-mide (CTX) as conditioning regimen before tansplantation and Methylprednisolone (MP) for 1 week continu-ously after trasplantation. Hematopoietic and immune recovery, graft versus host disease (GVHD), engraft-ment and survival rate were observed after transplantation. Six weeks after alloHST, the orthotopic tumor model in SCID mice was established by implanting histologically intact tissue under the embrane of liver. Ten days later, the mice received radical resection of lobe bearing tumor. The condition of recurrence and metas-tasis was observed 4 weeks after operation. Results A murine model of umbilical cord blood transplantation using CTX and MP as conditioning regimen could be successfully established in SCID mice. The percentage CD34+ cells of peripheral blood NC in scheduled transplantation group and single transplantation group was 1.66%±0.47% and 0.68% + 0.56%, respectively. There was significant statistical significance (P<0.01). The intrahepatic recurrence rate after operation was 100% in all the 3 groups. However, the recur-rent tumor volume was (367.18±31.86) mm3 , (648.26±155.22) mm3, (811.38±127.36) mm3, re-spectively in the 3 groups. There was marked difference among the 3 groups (P<0.01). The inhibitory rate of group A and B was 54.7% and 20.1%. The incidence of lung metastasis was 14.3 (1/7), 6.7% (4/6) and 100% (5/5), respectively in the 3 groups and there was remarkable difference (P<0.01). The inci-dence of celiac lymph node metastasis was 14.3 (1/7), 33.4% (2/6) and 40% (2/5), respectively in the 3groups and there was no significant difference (P=0.58). Conclusion AlloHSCT is a useful method for de-creasing metastasis and recurrence of liver cancer after radical resection in early stage.

Objective To evaluate the efficacy and safety of simulect (basiliximab) after orthotopic liver transplantation (OLT). Methods Forty adult recipients with benign end-stage liver disease between November 2003 to November 2004 were assigned randomly in a 1∶1 ratio to receive either two doses of simulect or matching placebo. The patients in the two groups received baseline triple immunosuppression with the calcineurin inhibitor (CsA or FK506), MMF and steroids. A total of 40 mg simulect was given in two doses of 20 mg each on the day 0 before inferior vena was opened and the day 4 after transplantation respectively. Acute rejection, infection and serum ALT, AST, TBIL, DBIL and ALP in both groups were observed in the first 30 days after OLT. Results In Simulect group had less frequent incidence of acute refection during the first 30 days after OLT. In Simulect group and matching placebo, incidence of acute refection was 10 % (2/20) abd 45 % (9/20) respectively (P= 0.034), and that of infection was 40 % (8/20) and 45 % (9/20) respectively (P= 0.749). Bilirub and aminopherase in both groups were declined gradually and ALP increased. There were significant difference in ALT and TBIL between two groups. Conclusion The application of simulect in combination with CsA/FK506, MMF and prednisone is safe and well tolerated, and can effectively reduce the incidence of acute refection, and does not lead to increased opportunistic infections.

HIV-1 envelope glycoprotein gp41,which is a hopeful target for HIV-1 fusion inhibitors,plays a critical role in the fusion of viral and cellular membranes.In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41,HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells.Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR,enzyme digestion and ligation taking the clade B HIV-1 genome as a template.The plasmid was transferred into E.coli BL21(DE3)and then induced by IPTG.The expressed protein was purified by affinity chromatography after denaturation and renaturation.The SDS-PAGE analysis was used during expression and purification.Native-PAGE was used to identify the interaction between gp41 5-helix and T-20.The result will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

The pol gene of HIV-1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′-processing step and the 5′-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was puriied sy the Co+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV-1 integrase.

OBJECTIVETo investigate the clinical effect of the nervus cutaneus femoris posterior pedicle flap on repairing large soft tissue defects at the heel or inferior segment of the shank.

METHODSTotally 14 cases were followed up for 8-22 months (mean 15.5 months) to observe the clinical effects of nervus cutaneus femoris posterior pedicle flap on repairing large soft tissue defects of the heel or inferior segment of the shank. Among them, there were 3 patients afflicted with infection and cutaneous defects in the middle and inferior segment of the shank after internal fixation of open fracture, 4 patients with soft tissue defects of the ankle and uncovered tendo calcaneus, and 7 patients with soft tissue defects of the heel and exposed calcaneus.

RESULTSThe flaps survived well in 13 cases and partial necrosis occurred in 1 case that was thereafter cured with changing dressing. Various extents of pain and stiffness of the knee joints were present in all cases and disappeared through 1-8 weeks' (mean 3.2 weeks) functional exercises. The last follow-up showed that all the flaps kept good texture and satisfactory appearance.

CONCLUSIONSThe nervus cutaneus femoris posterior pedicle flap, having the advantages of simple surgical procedures, anastomosing the nerves and restoring the sensation of recipient site, can be used for recovering large soft tissue defects of the shank and ankle.

Adolescent ; Adult ; Aged ; Female ; Heel ; surgery ; Humans ; Leg ; surgery ; Male ; Middle Aged ; Soft Tissue Injuries ; surgery ; Surgical Flaps

OBJECTIVETo study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo.

METHODSTo construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods.

RESULTSAfter transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited.

CONCLUSIONGrowth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Blotting, Western ; Brain Neoplasms ; pathology ; therapy ; Cell Line, Tumor ; Cell Proliferation ; DNA, Antisense ; genetics ; metabolism ; Flow Cytometry ; Genetic Therapy ; methods ; Glioma ; pathology ; therapy ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; PTEN Phosphohydrolase ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection ; Tumor Burden ; Xenograft Model Antitumor Assays

OBJECTIVETo study the strategy of the treatment for dislocation of cervical vertebra.

METHODSThe clinical data of 39 cases with dislocation of cervical vertebra were analyzed. Among them,29 were male and 10 were female. The average age was 40 years old (range from 6 to 74 years old). Segment of dislocation: 15 cases in C(1,2), 1 case in C(3,4), 9 cases in C(4,5), 9 cases in C(5,6), 5 cases in C(6,7). Spinal injury according to Frankel grade, 9 cases were A grade,8 were B, 5 were C, 8 were D, 8 were E, 1 case had radicular symptom. Thirty-two cases were early and rapidly treated with traction (progressive weight). Seventeen cases were treated with operation.

RESULTSTraction-reduction was successful in 90% of patients. According to Frankel grade, 32 cases averagely improved 0.63 grades. Six cases of severe spinal injury accompany with interlocking of zygopophysis died.

CONCLUSIONInspecting weight of traction is important in rapid traction-reduction for dislocation of cervical vertebra. The choice of surgical treatment depends on the degree of reduction, the result of MRI,the grade of spinal trauma and the status of patients.

Adolescent ; Adult ; Aged ; Cervical Vertebrae ; injuries ; surgery ; Child ; Female ; Humans ; Joint Dislocations ; surgery ; therapy ; Male ; Middle Aged ; Traction ; Treatment Outcome ; Young Adult

Objective To evaluate the protective effect of Yersinia pestis capsular antigen F1 and recombinant rV270 on mice after immunization with them.Methods According to body weight,40 female Balb/c mice aged 6 to 8 weeks were randomly divided into four experimental groups(Fl-10 μg + aluminum adjuvant,F1-20 μg + aluminum adjuvant,rV-10 μg + aluminum adjuvant,and rV-20 μg + aluminum adjuvant) and a control group,8 in each group.Mice in experimental groups were immunized with the natural antigen F1 and recombinant antigen rV270 adsorbed to 25％ aluminum adjuvant and the control group was immunized with the same amount of aluminum adjuvant.Each mouse was immunized at the hind leg muscle with 100 ml immunizing agent,then a booster immunization was done once on the 21st day after the first immunization.The blood of all mice was collected on the 8th week after the first immunization,serum antibody titers were detected by ELISA and the data of antibody titers were analyzed by t test for comparison between groups.At the same time the mice were injected subcutaneously with 2000-fold LD50 of Yersinia pestis virulent strain 141,after 14 days,the protective effect of immunization was analyzed.Results The control group did not produce antibody.Antibody geometric mean titers (GMT) of the F1-10 mg + aluminum adjuvant and F1-20 mg + aluminum adjuvant groups were 1 ∶ 30443.9,and 1 ∶21527.8,respectively,and compared between the two groups,the difference was not statistically significant (t =1.1282,P ＞ 0.05).The GMTs of the rV-10 μg + aluminum adjuvant and rV-20 μg + aluminum adjuvant groups were 1 ∶ 13957.3 and 1 ∶18100.9,respectively,and compared between the two groups,the difference was not statistically significant(t =0.9408,P ＞ 0.05 ).After subcutaneous injection with Yersinia pestis virulent strain 141,all mice died in the control group but all survived in the experimental group.Conclusion The immune activity of natural antigen F1 and recombinant rV270 is high,which can be used as the main component of subunit vaccine in the plague subunit vaccine study.

Objective To explore the effect of homemade restraint strap on restless patients of neurosurgery.Methods The newly admitted 76 restless patients were randomly divided into observation group and control group,each with 38 cases.The control group was taken ordinary restraint strap,and the observation group was taken homemade restraint strap.Then the broken rate of restraint strap,the rate of accidental extubation,the rate of skin lesions and the iatrogenic injury frequency were compared within these two groups.Results The broken rate of restraint strap and the rate of accidental extubation were respectively 7.89％ (3/38),2.63％ (1/38) in the observation group,which were lower than 26.32％ (10/38),18.42％ (7/38) in the control group,and the differences were statistically significant (x2 =4.547,5.029,respectively; P ＜0.05).The rate of skin lesions and the rate of swelling in the constraint limb were respectively 7.89％ (3/38),5.26％ (2/38) in the observation group,which were lower than 28.95％ (11/38),31.58％ (12/38) in the control group,and the differences were statistically significant (x2 =5.604,8.756,respectively ; P ＜ 0.05).Conclusions Homemade restraint strap supplies patients with effective and safe constraints in neurosurgery,and it is worthy to be widely used in our clinical work.