Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.

BACKGROUND:Anterior cervical discectomy and intervertebral disc replacement have been extensively used in treatment of cervical degenerative disease. Its short-term therapeutic effects are encouraging, but it lacks of long-term and comprehensive evaluation.
OBJECTIVE:To observe the folow-up results of a group of cases of anterior cervical discectomy and Mobi-C intervertebral disc replacement for more than 5 years.
METHODS:A total of 25 cases of cervical spondylosis were treated in the 309 Hospital of Chinese PLA from January to September 2009. 19 of them were folowed up. Al patients received anterior cervical discectomy and Mobi-C intervertebral disc replacement. Twenty-three Mobi-C intervertebral disc prostheses were implanted. The range of motion was measured using lateral X-ray films during extension and flexion. Ectopic ossification was assessed by McAfee method. According to the cervical vertebra MRI images, the degeneration of adjacent segments was determined using Pearce classification. NDI score was used to evaluate the function of cervical spine. Pain improvement was evaluated using visual analogue scale score.
RESULTS AND CONCLUSION: A total of 19 patients were folowed up for 59-65 months, averagely 62 months. No significant difference in range of motion was detected before surgery and during final folow-up (P > 0.05). Ectopic ossification in two cases and the degeneration of adjacent segments in one case were found during final folow-up. No prosthesis loosening or displacement appeared. Visual analogue scale score and NDI score were significantly lower during final folow-up compared with that before surgery (P < 0.05). These results indicate that under the premise of reasonable choice of indications, the therapeutic effect of Mobi-C intervertebral disc replacement for degenerative cervical spondylosis was satisfactory in five-year folow-up. The range of motion was good, and the incidences of ectopic ossification and the degeneration of adjacent segments were low.

From January 2005 to January 2008, 23 cases of fractures and dislocations of cervical spine treated with anterior decompression, reconstruction of bone graft with titanium mesh, and titanium locking plates fixation in Department of Orthopaedics, the 309 Hospital of Chinese PLA were retrospectively analyzed, including 16 males and 7 females, aged 41.9 years (ranging 25 ?65 years). The clinical effect and application value of this treatment was evaluated; the conditions at preoperative, immediate postoperative and follow-up were evaluated by JOA grade, and these radiographs were taken. Lordosis (kyphosis) of the fusion segment with Cobb's angle, and lordosis (kyphosis) of cervical spine with D value were measured to evaluate effect of reconstruction and cervical spine physiological curve. All cases were followed-up for 20.5 months (ranging 1-3 years). Clinical stability was reached in all cases during the follow-up. There were significant differences between preoperative and immediate postoperative, between preoperative and follow-up groups in terms of JOA grade, Cobb's angle and D value (P < 0.05). The JOA grade of immediate postoperative was improved compared with that during follow-up (P < 0.05). The mean improvement rate was 63% at 1 year of follow up. A good curative effect was obtained by using anterior locking plate with titanium mesh systems in treatment of cervical fractures and dislocations and a good effect of restoring and maintaining the lordosis of cervical spine can be also obtained.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Marine actinomyces LYG-1 was isolated from marine mud flats in Lianyungang,China.Strain LYG-1 was identified using the methods of morphology,physiological and ehemotaxonomic characterization and 16S rRNA gene sequence analysis.The results showed that strain LYG-1 was a marine variable species of Streptomyces roseosporus.The fermentation broth of strain LYG-1 exhibited conspicuous antitumor activity against HepG2,MCF-7,HCT116 and MDA-MB-231 cell lines,and the IC50 values were defined by MTT method respectively.

To investigate the effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer. The peripheral blood lymphocytes of patients with lung cancer and healthy persons were separated by the density gradient centrifugation method for subsequent experiments, with those from healthy persons as the positive control. The effect of Tanreqing injection on stimulating the proliferation of lymphocytes with phytohemagglutinin (PHA) was determined by MTT method. The effect of Tanreqing injection on the lymphocyte secretions of IFN-γ and TNF-α and the subset ratio of lymphocytes cultured separately or with Tanreqing injection of different concentrations were examined by ELISA and flow cytometry (FCM) respectively. In addition, the LDH release assay was used to detect the cytotoxicity of cytotoxic T cells (CTL) and natural killer cells (NK). According to the findings, all of immunological indexes of lymphocytes from patients with lung cancer were weaker than that of healthy persons, but with the obvious increases in proliferation activity and IFN-γ and TNF-α secretions of lymphocytes co-cultured with Tanreqing Injection (P < 0.05). Among lymphocyte subsets co-cultured with Tanreqing Injection, CD3+, CD3+ CD4+ and CD3- CD16 + 56+ cell ratios notably increased, whereas CD4+ CD25+ Treg cell ratio obviously decreased (P < 0.05). In the meantime, Tanreqing injection can markedly promote the cytotoxicities of CTL and NK (P < 0.05). In conclusion, Tanreqing injection shows a significant effect in promoting the immune activity of lymphocytes from patients with lung cancer and their anti-tumor immunity.
Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interferon-gamma ; genetics ; immunology ; Killer Cells, Natural ; drug effects ; immunology ; Lung Neoplasms ; drug therapy ; genetics ; immunology ; physiopathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

Objective To evaluate the feasibility of intra-operative ultrasound guide in breast conservative-surgery and its effect on positive margin rate and breast tissue volume.Methods Fifty-five cases of invasive breast cancer staged T1-2N0-1 were randomly assigned to palpation-guided group(control group) 26 cases and ultrasound-guided group(experiment group) 29 cases.Before and during operation,high-frequency ultrasound was used to guide the resection of breast cancer with 1 cm margin in experiment group.The resection margin positive rate deteced by tissue section frozen biopsy and resected breast tissue volume were compared between experiment and control group.Results The clinicopathologic features were accordant between two groups.The margin positive rate and near-margin positive rate in experiment group were 0 and 3.45％ respectively,slightly lower than that of control group 3.85％ and 15.38％,but with no statistical significance (P ＞ 0.05).The same situation occurred at the re-exision rate.The resected breast tissue volume in experiment group (32.40 ± 10.93) cm3 was lower than that of control group (55.11 ± 12.88) cm3,and with statistical significance (P ＜ 0.01).Conclusions Intra-operative ultrasound guide could reduce the margin positive rate and unnecessary resected brcast tissue volume in breast conservative-surgery,and improve cosmetic effect.

Objective To study the therapeutic effect and mechanism of invigorating kidney and activa-ting blood(IKAB)recipe,a Chinese herb compound,on diabetic erectile dysfunction (DED)in rats. Methods 40 male SD rats were randomly divided into four groups (each n =10):normal group,model group,low-dose IKAB group (low-dose group)and high-dose IKAB group(high-dose group).The rat model with 2 type of diabet mellitus was induced by intraperitoneal injection of streptozotocin (STZ). Normal group and model group were intragastrically administered (ig.)with normal saline,low-dose group was ig.3 g/kg of IKAB and high-dose group was ig.6 g/kg IKAB for consecutive 8 weeks.The penis viscera index was measured;the intracavernous pressure (ICP)were detected by Biopack electro-physiological instrument;the expressions of pAKT and AKT were detectd by Western blotting;cell apop-tosis of corpus cavernosum smooth muscle were measured by using TUNEL method and the pathological changes in penile tissue were observed by using Masson trichrome staining.Results Compared with model group,the penis viscera index of both IKAB groups had no statistical differences (P >0.05);the expressions of pAKT and AKT increased significantly(P <0.05);although the ratio of cell apoptosis of corpus cavernosum smooth muscle decreased,the statistical differences were not shown (P >0.05).Fur-thermore,ICP and ICP/MAP increased markedly in high-dose group and the collagen fiber proportion de-creased in low -dose group compared with model group (P <0.05).Conclusion The mechanism of in-vigorating kidney and activating blood recipe increasing ICP in rat model with DMis related to increasing the expression of pAKT,inhibiting cell apoptosis of corpus cavernosum smooth muscle and reducing colla-gen fibers formation in penis.

Objective To ascertain the usage of Toxoplasma Gondii(T.gondii) test kit,and analyze the trends of T.gondii infection in Shanghai pregnant women so as to provide scientific evidence for effective intervention measures to prevent congenital toxoplasmosis.Methods The data were collected on methods and results of screening T.gondii infection in pregnant women in Shanghai from 2003 to 2010,and analysis of T.gondii infection was made on pregnant women in this city.Results During 2003 and 2010,in 30 hospitals of the City was carried out the screening test for pregnant women T.Gontii infection.The overall serological positive rate to T.gondii was 1.46% from the 30 hospitals.The positive rates of IgG antibody was 2.46%(1.48%-4.96%) in 277 523 persons,that of IgM antibody was 0.83%(0.38%-1.48%) in 439 868 person.The IgM or both IgG and IgM positive pregnancy cases accounted for 41.74% of the total positive cases which appeared in pregnant women from 2008 to 2010.There were also significant differences in the positive rates among different hospitals so as seasons.ELISA and MEIA were more detected for T.gondii antibody detection,and their detection rates of IgG and IgM were significantly different.Imported kit has higher positive rate of detection T.gondii IgM antibody,but lower positive rate of screening IgG antibody than domestic kit.Conclusion The seroprevalence of T.gondii in pregnant women from 2003 to 2010 were close to the average level investigated in the past in Shanghai,but more attention should be paid to the higher proportion of IgM antibody positive,and stable and reliable test kits should be selected for detecting T.gondii IgG and IgM antibodies to avoid leak and mistaken diagnosis.

Objective To study the inhibitory effect of small interference RNA(siRNA) ofcyclin E gene on the growth ofK562cells. Methods siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification.The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via LipofectamineTM2000.The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. Results Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G1-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels.Conclusion RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation ofK562 cells.