Objective:Making single chain antibody(scFv)-alkaline phosphatase(Ap)fusion protein.Methods:An expression vector pSTE2-C66-Ap was constructed by sequentially inserting the Ap coding region into plasmid pSTE2-8E5 and replacing the VH-VL fragment of 8E5 by VH-VL fragment of C66.The fusion protein scFvC66-Ap was expressed in E.coli.and analysed by SDS-PAGE and immunoblotting.Results:Have obtained the scFvC66-Ap fusion protein with a molecular weight of 75 kD.It bound a 60 kD molecule from KG1a cell proteins on immunoblotting membrane detected directly by Ap enzymatic activity.Conclusion:A method permits the production of scFv-Ap conjugates in E.coli.which can replace conventionally prepared Ap-labeled antibodies in immunoassays.

Aim The present study was designed to investigate the growth inhibition and anti-angiogenesis of arsenic trioxide (As 2O 3) on B16 cells xenografts in C57BL/6J mice and observe the effects of As 2O 3 on cell proliferation, cell morphology, cell cycle and apoptosis in vitro. Methods Mice melanoma cell line B16 was transplanted into C57BL/6J mice. The weight of tumor and percent of tumor development were examined after As 2O 3 intraperitoneal administration. HE staining and immunohistochemistry for Ⅷ-R Ag were performed to detect the microvessel density in tumor tissues. CellTiter 96 Aqueous One was used to determine the cell proliferation. Giemsa and Feulgen staining were used to observe the morphological changes of the cells. Cell cycle and apoptosis were analyzed by flow cytometry (FCM). Results As 2O 3 significantly inhibited the tumor growth in vivo, with an inhibitory rate of 81.61%. The microvessel density in tumor tissues was obviously reduced after treated with As 2O 3. There was obviously a concentration-dependent relationship between As 2O 3 and the inhibition of B16 cells proliferation (P

To investigate the expression of osteopontin (OPN) and its receptor integrin alphanubeta3 in the placental tissue from pregnant women complicated with preeclampsia, the expression of OPN and alphanubeta3 in the placenta of the pregnant women with preeclampsia and healthy pregnant women was detected by immunohistochemistry, Western blotting and RT-PCR. Our results showed that OPN and alphanubeta3 protein were expressed in the placenta from normal pregnant woman and those with preeclampsia. OPN was located in the placental syncytiotrophoblasts and the cytoplasm of capillary endothelial cells and integrin alphanubeta3 was mainly expressed on the surface of trophoblast cells. Expression of OPN and integrin alphanubeta3 in the placental tissue from preeclampsia subjects was significantly lower than that from the control group (P<0.05). Compared with the control group, expression of OPN in the placental tissue from preeclampsia group was significantly lower (P<0.05) but there was no significant difference in the expression of alphanu and beta3 between the preeclampsia group and the controls. It is concluded that OPN and its receptor integrin alphanubeta3 may be involved in the pathogenesis of preeclampsia.

OBJECTIVE:To establish the quality standard for Gentiana scabra dispensing granule. METHODS:TLC was ad-opted to identify G. scabra in the preparation;HPLC was adopted to determine the content of gentiopicroside in the preparation:the column was Dionex C18 with mobile phase of methanol- water(25∶75,V/V)at a flow rate of 1.0 ml/min,the detection wave-length was 270 nm,column temperature was 30 ℃,the injection volume was 10 μl. RESULTS:TLC showed clear spots and good separation. The linear range of gentiopicroside injection volume was 0.302 6-3.026 μg (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2%;recovery was 98.27%-99.56%(RSD=0.68%,n=6). CONCLUSIONS:The estab-lished standard can be used for the quality control of Gentiana scabra dispensing granule.

Objective To explore the genetic mechanism of Yang-deficiency constitution by detecting genomic copy number variations (CNVs). Methods Thirty cases of Yang-deficiency constitution and 30 cases of balanced constitution were included according to the standards of Classification and Determination of Constitution in Traditional Chinese Medicine. DNA was extracted from white blood cells in peripheral blood. A genome-wide association study was conducted by using Affymetrix SNP 6.0 platform. CNVs of each sample were analyzed using PennyCNV software. The Yang-deficiency constitution-specific copy number variation regions (CNVRs) of each autosome were identified. CNVR-related genes and their annotations were searched at online Human Genome Browser. Results The mean number of CNVs in balanced constitution group was 12.63±3.39, ranging from 8 to 20. After stepwise elimination of two Yang-deficiency constitution subjects, the mean number of CNVs in Yang-deficiency constitution group was 15.04±8.95, ranging from 2 to 38. A total of 26 CNVRs were identified from 28 Yang-deficiency constitution subjects, including 19 duplicated CNVRs, 6 deleted CNVRs, and 1 mixed type CNVR. Most CNVRs were shared by a few Yang-deficiency constitution subjects, and only 7 CNVRs were shared by more than 5 Yang-deficiency constitution subjects. The functions of representative genes in Yang-deficiency constitution-specific CNVRs were related with extracellular and intracellular signal transduction, metabolic regulation, and immune response, etc. Conclusion Yang-deficiency constitution subjects have some specific genomic CNVs, which might result in Yang-deficiency constitution phenotypes by influencing the expression of genes associated with extracellular and intracellular signal transduction, material metabolism (energy metabolism), and immune response, etc.

Objective To investigate whether paclitaxel can efficiently induce the apoptosis of ovarian cell HO-8910,and to study the relationship between the apoptosis of cells and the cell cycle.Methods With the treatment of paclitaxel with different concentrations and different time,the morphologic change of HO-8910 ovarian cells was observed using fluorescence microscopy and transmission electron microscopy(TEM),and the apoptosis of cells and the changes of cell cycle were determined by flow cytometry(FCM). Results The typical changes of HO-8910 cell apoptosis were observed by TEM and Fluorescence microscopy.With the treatment of paclitaxel,the HO-8910 ovarian cells were firstly arrested in G_2/M phase,and the typical ultrastructural changes of apoptosis were appeared only after the cells were apparently arrested in G_2/M phase.Conclusion Paclitaxel can induce the apoptosis of HO-8910 cells and the apoptosis is associated with the blockage of G_2/M phase in cell cycle.

AIM: To clone the extracellular domain of N-cadherin cDNA, and to observe the antigenicity of the expressed protein. METHODS: Total RNA was extracted from CD34+ cells separated from fetal liver and bone marrow cells. The extracellular domain of N-cad cDNA was amplified with RT-PCR and inserted into a vector pOPE101-215. The recombinant pOPE-N-cad was expressed with IPTG induction. Then, mice were immunized with the protein. RESULTS: The extracellular domain of N-cad cDNA from CD34+ cells was identified by DNA sequencing. The recombinant pOPE-N-cad in host XL1-blue expressed a 70 kD protein after induced with IPTG, and anti-N-cad antibody was detected in serum of the immunized mice after 5 times injection of the recombinant N-cad protein. CONCLUSION: CD34+ cells bore N-cad gene and the recombinant protein of the extracellular domain of N-cad cDNA shows good immunogenicity.

Objective To analyze the setup errors of bone metastases patients by tomotherapy with megavoltage CT (MVCT) and calculate the CTV-PTV margins.Methods 30 patients with bone metastases were enrolled.All patients received tomotherapy,fixed with body net and received MVCT scanning before radiation.The MVCT images were registered with the kilovoltage CT (kVCT) images,the setup errors of X (lateral),Y (vertical),Z (longitudina) and Roll (transverse profile rotation) were obtained according to the formula M =2.5Σ+0.7σ calculated CTV-PTV margin.Results 30 patients were received 494 MVCT images.The errors of systemic±random were (2.85±0.77) mm,(3.11±0.95) mm,(2.21±0.55) mm,and (0,55±0.24)° on X,Y,Z and Roll directions,respectively.The CTV-PTV margins were 3.64 mm,4.17 mm,and 2.86 mm on X,Y,Z directions,respectively.Conclusion The application of image-guided technology for bone metastases can correct positioning in time,which greatly reduces setup errors of the fractionated treatment,further improves the treat accuracy and has a positive value in guiding clinical radiotherapy.

Objective To compare the efficacy of conventional radiotherapy and Tomo radiotherapy com -bined with zoledronic acid in spinal metastase .Methods Seventy -nine patients with spinal metastases in our department of Air Force General Hospital were recruited from January 2011 to December 2012 .All patients were divided into two groups:group A:39 cases were treated with conventional radiotherapy combined with zoledronic ( PTV:30~40 Gy/10~20 f);40 patients with target in target Tomo radiotherapy combined with zoledronic acid were separated into group B(PTV/GTV/40~50 Gy/50~60 Gy/15~20 f).Results The overall following -up rate was 96.2%.The total response rate was 88.61%,the analgesic effect of radiotherapy in group A and group B were 84.62%、92.5%,respectively.The average pain scores were decreased by 2.55 and 3.54 in two gourps u-sing a numerical rating scale;the average pain relieve time were 7.25d and 8.32d,respectively;the median last-ing time of pain relieves were 6.68 and 8.41months;and the pain recurrence rate were 25.81% and 5.41% in corresponding group A and group B .The main acute side effects were hematologic toxicity , in which occurring rates were 12.82%and 12.5%in RTOG gradeⅠ,23.08% and 20%in RTOG grade Ⅱ,2.56% and 2.5% in RTOG gradeⅢ,2.46%and 0%in RTOG grade Ⅳin group A and group B,respectively.There were no signifi-cant differences in pain relief rate ,start-effect time,lasting time and acute side effects between the two different radiotherapy methods .However ,the decreased degree of pain relief and recurrence rate were obviously decreased in group B when compared with group A ,and the differences were statistically significance (P<0.05).Conclu-sion Target in target Tomo radiotherapy combined with zoledronic acid in the treatment of spinal metastasies is safe and effective,mild toxicity,and the degree of pain relief was significantly improved .Moreover,pain recurrence rate is lower than conventional radiotherapy .Hence ,it is worth of recommendation in clinical practice .

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
AC133 Antigen ; Animals ; Antibodies ; genetics ; Antigens, CD ; immunology ; Antigens, CD34 ; immunology ; Base Sequence ; Cadherins ; immunology ; Female ; Glycoproteins ; immunology ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Library ; Peptides ; immunology