Objective:To study the conditions of in vitro proliferation and differentiation of rat bone marrow stromal cells(MSC) for its application in transplantation.Methods:MSC were obtained by flushing the shaft with DMEM using a sterile syringe,dissociated by density gradient centrifuge,and cultured in the medium containing DMEM and 10% fatal bovine serum(FBS).Then we added neural growth factors into the medium and observed their influence on MSC proliferation,differentiation and survival by phase contrast microscopy.Immunocytochemistry was used to identify cell types.Results:MSC cultured in medium containing DMEM/ 10% FBS/ neural growth factors proliferated quickly.The neuron-like cells appeared at 7-9 d with the expression of neurone specific enolase(NSE) at(52?9.2)% and glial fibrillary acidic protein(GFAP) at(15?7)%,and could survive more than 7 days;In contrast,cells induced by ?-mercaptoethanol(BME) died gradually after 24 hours.Conclusion:MSC can not only proliferate and differentiate into neuron-like cells effectively in the medium containing neural growth factors but also survive longer.

Objective: To construct a recombinant expression vector containing the VP1 whole gene of human parvovirus(HPV) B19-XA strain by DNA recombinant technology and to induce the expression of the VP1 fusion protein in E.coli.Methods: The gene of interest was amplified by PCR from the viral DNA genome extracted from the patient infected with B19 virus,and inserted into the pET28(a) expression vector.The positive recombinants pET28(a)-VP1 were transformed into E.coli BL21(DE3),and then the VP1 proteins were expressed after induced by IPTG and analyzed by SDS-PAGE and immunoblotting.In addition,rabbits were immunized with the recombinant VP1 fusion proteins as antibodies,and their valence was detected by ELISA.Results: The positive bacteria strains containing the recombinant expressive vector pET28(a)-VP1 were constructed successfully,and IPTG induced a high expression of the VP1 fusion proteins.The valence of multiclone antibodies of anti-VP1 proteins was 1∶12 800.Conclusion: The VP1 fusion proteins of the HPV B19-XA strain can be expressed efficiently in E.coli,and these proteins possess satisfactory immunogenicity and play an important role in the preparation of diagnostic reagents and the development of vaccines.

Objective To compare the analgesia effect of transversus abdominis plane(TAP) block and caudal block after radical surgery of Hirschsprung’s disease.Methods Sixty pediatric pa-tients,52 males,8 females,aged 1-3 yr,of ASA physical status Ⅰ or Ⅱ,scheduled for elective rad-ical surgery on Hirschsprung’s disease,were equally randomized into TAP block group(group TAP) and caudal block group (group CA).0.2% ropivacaine 1 ml/kg was used in both blocks.FLAAC scores were measured at 6,12,18 and 24 h after surgery.The requirement of sufentanil every 6 h, the first passage of flatus,the time of removing urine tube and the incidence of nausea and vomiting within 24 hours were recorded.Results Compared with group TAP,FLAAC scores and the require-ment of sufentanil were significantly decreased at 6 h after operation in group CA(P <0.05).FLAAC scores and the requirement of sufentanil were similar at 12,18 and 24 h in the two groups.The first passage of flatus,the time of removing urine tube in group TAP were significantly shorter than group CA(P < 0.05 ).The incidence of nausea and vomiting were not significantly different in the two groups.Conclusion Although both TAP block and caudal block can provide effective analgesia for the pediatric patients after radical surgery on Hirschsprung’s disease,caudal block provided superior anal-gesia at early stage after operation,however,TAP block is more beneficial for earlier recovery of bowl function.

Objective To observe the clinical efficacy of Yaoyi-Shenhuojiu combined with synthetic rehabilitation therapy on shoulder-hand syndrome after stroke (SHSAS). Methods 100 SHSAS patients were randomly divided into an observation group and a control group, with 50 patients in each group. Patients in the observation group were treated with Yaoyi-Shenhuojiu on the basis of comprehensive rehabilitation training of the control group. After treated for 2 consecutive weeks, the pain, motor function, activities of daily living in patients of both groups were compared and improvement of the constitution according to VAS, FMA, MBI was also compared. Results After treatment, the decrease of VAS score in the observation group (3.43 ± 1.76 vs. 5.21 ± 2.15, t=4.530) and the increase of FMA score (55.66 ± 4.33 vs. 40.45 ± 3.75, t=18.776) was significantly better than the control group (P<0.01); Physiological function in SF-36 health survey questionnaire (95.78 ± 7.68 vs. 87.88 ± 7.21, Z=5.042), physiological functions of (50.78 ± 21.44 vs. 36.89 ± 18.42, Z=7.241), body pain (83.22 ± 13.45 vs. 75.52 ± 13.23, Z=4.055), overall health (50.25 ± 18.45 vs. 39.01 ± 18.63, Z=8.043), vigor (79.28 ± 11.92 vs. 70.22 ± 10.69, Z=3.216), social function (85.78 ± 20.12 vs. 74.71 ± 19.22, Z=4.128), emotional function (36.66 ± 23.79 vs. 28.11 ± 18.21, Z=6.052) and mental health (81.17 ± 9.72 vs. 74.61 ± 11.25, Z=5.367) eight dimensions of higher score in the observation group were also significantly better than the control group (P<0.05). The total effective rate (94.0% vs. 76.0%) in the observation group was significantly better than the control group (χ2=5.020, P=5.020). Conclusion Yaoyi-Shenhuojiu combined with comprehensive rehabilitation therapy can effectively improve limb movement function in patients with SHSAS, besides its improving the life self-care ability and life quality.

Objective To investigate the psychological status of leprosy people with disability and their expectation towards surgical outcomes before surgical operation.Methods 937 cases from nine provinces，which were covered by the China National Commonweal funded surgical camps, were surveyed by a structure questionnaire designed by the Department of Leprosy Control and Rehabilitation, Institute of Dermatology, the Chinese Academy of Medical Sciences (CAMS).Results Reasons for the patients to refuse the surgical operation included concerns in medical cost (79.19%), unawareness of the free medical surgery program (64.78%), scaring of surgery (48.88%), unwilling to expose their disabilities (46.43%), unwilling to expose their history of leprosy (45.57%), despairing with disability (43.86%), having no ideas on surgical Results (37.46%), believing that disability is an inevitable outcome of the disease (32.98%), and rejecting the operation becasue of age(30.63%). More than 90% of PALs who are willing to accept surgery expected that surgery can improve their physical ability and they may contribute more to their family or become less independent after surgery; however, they thought there was little opportunity to return to work after surgery.Conclusion It is helpful and necessary to understand the psychological status of the leprosy people with disability before surgery.

Objective To observe the effect of stump ulcer repairing in people affected by leprosy (PALs). Methods Bone shortening and osteophyte removal were performed in 73 PALs amputees with stump ulcers. Results All the incisions were healed without any delay. During a follow-up period of 3～12 years, no relapse or new ulcers were found in 70 PALs. 3 PALs developed new ulcers, which were healed after debridement, resting, and prosthesis repairing. Conclusion Surgical revision of the bone in amputation stump can change the local distribution of pressure. Postoperative self-care teaching to the PALs and their sustaining daily self-care will finally prevent them from stump ulcer recurring.

ObjectiveTo study the effect of tetramethylpyrazine cooperated with autogenous periosteum graft on fibrous scar formation in the epidural space after laminectomy. Methods48 SD rats were divided randomly into 4 groups. Laminectomy was performed in lumbar 2 segment, and the exposed duras were covered with saline solution (group A), tetramethylpyrazine (group B), autogenous periosteum (group C), and tetramethylpyrazine and autogenous periosteum (group D) respectively. 12 weeks postoperatively, they were evaluated with the gross and histopathological observation, biochemistry examination. ResultsThe assessment of Rydell-Balazs and Nussbaum's Criterion of group B, C, D were obviously better than that of group A (P<0.01), and that of group D was superior to group B and C (P<0.05), no significant difference between group B and C (P>0.05). ConclusionTetramethylpyrazine cooperated with autogenous periosteum graft has a better effect on prevention peridural adhesion after laminectomy.

By engineering the subsite +1 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans, we improved its maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) synthesis. Specifically, we conducted site-saturation mutagenesis on Leu194, Ala230, and His233 in subsite +1 separately and gained 3 mutants L194N (leucine --> asparagine), A230D (alanine --> aspartic acid), and H233E (histidine --> glutamic acid) produced higher AA-2G yield than the wild-type and the other mutant CGTases. Therefore, the 3 mutants L194N, A230D, and H233E were further used to construct the double and triple mutations. Among the 7 obtained combinational mutants, the triple mutant L194N/A230D/H233E produced the highest AA-2G titer of 1.95 g/L, which was increased by 62.5% compared with that produced by the wild-type CGTase. Then, we modeled the reaction kinetics of all the mutants and found a substrate inhibition by high titer of L-AA for the mutants. The optimal temperature, pH, and reaction time of all the mutants were also determined. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the three positions (194, 230 and 233) and the substrate sugars.
Ascorbic Acid ; analogs & derivatives ; chemistry ; Glucosyltransferases ; genetics ; metabolism ; Hydrogen Bonding ; Kinetics ; Mutagenesis, Site-Directed ; Paenibacillus ; enzymology ; Polysaccharides ; chemistry ; Protein Engineering ; Substrate Specificity ; Temperature

Obgective To investigate the impact of different ways and doses of recombinant human interferon o1b(rhIFN-α1b) on antiviral effect on respiratory syncytial virus(RSV)-effected mice and signal pathway of JAK/ STAT.Methods Forty-eight mice were divided randomly into 6 groups,negative control group,RSV-infection model group,12.5,25.0,50.0 μg rhIFN-α1 b atomization inhalation of intervention group,and 12.5 μg rhIFN-α 1 b injection of intervention group.After continuous drug therapy for 5 days,the left lung tissues of mice were aseptically dissected in the sixth day.Then it were observed lung tissue pathology changes by optical microscope,and expression of STAT1 and STAT2 protein with laser scanning confocal microscopy.Results It was observed that RSV-infection model mice's lungs had significant inflammatory injury under light microscope.After treatments of rhIFN-α 1b,it showed that the mice lung tissue had recovery of inflammation on different degrees.The group of inhaled rhIFN-o1b 50.0 μg was damaged on lightest degree.There were statistically significant differences between each group (all P ＜ 0.05).The expression of STAT1 and STAT2 protein in RSV-infection model mice's lungs decreased remarkably under fluorescene microscopy.Treatments of rhIFN-α 1b increased the expression of STAT1 and STAT2 inhibited by RSV-infection.The expression of STAT1 and STAT2 protein of the inhaled rhIFN-α1b 50.0 μg group increased significantly.The differences between groups were statistically significant(all P ＜ 0.05).Conclusions rhIFN-α 1 b played an important role in anti-RSV effects,simultaneously,it could improve the activity of JAK/STAT signal pathway inhibited by RSV-infection.The treatment effect of rhIFN-α 1b deliveried by atomization inhalation was better than that of intraperitoneal injection.And curative effect is proportional to the atomization inhalation dose within a certain range.

Human beta defensin 4 is a small cationic peptide with a broad range of antimicrobial activity. It plays an important role in innate immunity of human body, especially in mucosal and epithelial defense. In this study, the full-length encoding gene of HBD4 was synthesized by overlap extension polymerase chain reaction and inserted into cloning vector pMD18-T. The gene encoding mature peptide of HBD4 was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-2. Then pGEX-4T-2/mHBD4 was transformed into E. coli DH5 alpha, which was induced by isopropy-beta-D-thiogalactoside (IPTG). The identification was made by means of endonuclease digestion, DNA sequencing, sodium dodecyl sulphate-polyacrylamine gel electrophoresis (SDS-PAGE). The results showed that the synthesized gene and cloned gene were identical to the HBD4 gene sequence registered in GenBank and were successfully cloned into cloning vector pMD18-T and prokaryotic expression vector pGEX-4T-2. After IPTG induction, the GST-HBD4 fusion protein was successfully expressed in E. coli.
Anti-Infective Agents ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics