Circulating endothelial cells(CEC)are mature endothelial cells,which have been shed from the vascular cell lining and enter into blood circulation.Rare in healthy individuals,increased CEC in peripheral blood reflects significant vascular damage and dysfunction.Increased CEC have been documented in many human diseases characterized as vascular damage,including different types of tumors.Clinical data suggest that CEC count is a promising tool for monitoring disease activity with potential to assess tumor prognosis and response to treatment.

Objective:To clone soluble human anti-digoxin antibodies from large phage antibody library and construct a vector that expresses diabody.Methods:Soluble ScFvs were prepared through infecting E coli.HB2151 with the selected phage antibodies and induced with IPTG.The diabody vector was modified by enzyme digestion and checked by SDS-electrophoretogram.Results:It was showed by ELISA that soluble ScFv had specific binding ability to digoxin.The vector to express a soluble diabody was obtained by genetic modification,which was shown by Western blot.Conclusion:Soluble human anti-digoxin antibodies were successfully obtained from phage antibodies.The vector is efficient in creating diabody.

Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.

Objective To compare the effects of propofol and ketamine on long-term memory and the expression of brain N-methylgroup-D-aspartate receptor 2B(NMDAR2B) and Gamma-aminobutyric acid receptor 1(GABAR1) in aged rats, and preliminary investigate the relation between the long-term memory and expression of neurotransmitter receptors in different cerebral areas. Methods The aged male rats were randomly divided into control group,propofol group and ketamine group. Morris water maze training was performed in all the rats of three groups for 5 days. On the 6th day, intraperitoneal injection of 50 mg?mL-1 propofol was administrated in propofol group,80 mg?mL-1 ketamine was intraperitoneally injected in ketamine group,and blank control group was given the same dose of saline.Seven days after the administration,space exploration experiment and navigation experiment test were performed to test the impact on the learning and memory ability of rats. After that, the expression levels of GABAR1 and NMDAR2B in temporal lobe and hippocampal CA1 region of the rat brain were detected by immunofluorescence and FISH technique. Results The results of Morris water maze showed there was no significant difference between propofol group (9.49±1.24) s and blank control group (8.82±2.22) s.There was statistically significant difference between ketamine group (12.04±2.67) s and blank control group (P<0.05),with longer latency time and less number of times of passing through target as compared with blank control group.By using immunohistochemistry and FISH technique,the expression of GABAR1 in temporal lobe and hippocampal CA1 region of the rat brain was not significantly different between propofol group and blank control group,but it was significantly up-regulated in ketamine group as compared with blank control group ( P<0.05) . The expression of NMDAR2B in temporal lobe and hippocampal CA1 region of the rat brain was not significantly different between propofol group and blank control group,but it was significantly down-regulated in ketamine group as compared with blank control group (P<0.05). Conclusion Propofol anesthesia alone had no effect on long-term learning and memory,but ketamine anesthesia can result in long-term learning and memory impairment. The mechanism may be related with down-regulation of the expression of NMDAR2B receptor and up-regulation of GABAR1 not only in CA1 region hippocampus,but also in temporal lobe.

Objective:The treatment of late pregnancy complicated with utedne leiomyoma was investigated.Methods:193 Cases of Iate pregnancy complicated with uterine leiomyoma from January 2003 to August 2008 were recruited in our hospital.According to the delivery route,size and subtype of fibroid,blood loss,operation hours and postoperative inpatient period were compared.Results:104 cases of pregnancy complicated with uterine leiomyoma were diagnosed before cesarean section(CS).No significant differences on blood losses and operation hours were found between CS group and CS+myomectomy group(P>0.05).The operation heurs of leiomyoma in corpus uteri was significantly shorter than leiomyoma in lower uterine segment and cervix(P=0.007).Leiomyoma bigger than 8 cm needed significantly Ionger operative hours and lose more blood than the smaller leiomyoma.Operation hours,blood loss and postoperative inpatient period were significantly different between submucous leiomyoma and subserosal leiomyoma(P<0.05).Conclusions:Pregnancy complicated with uterine leiomyoma should be diagnosed as early as possible.During cesarean section on when leiomyoma is bigger than 8 cm,locating at lower uterine segment or cervix or submucous,the treatment should be cautious.

Objective To construct the competitiveness evaluation index system of listed TCM pharmaceutical companies and provide efficient technology and methods for the evaluation in related field.Methods Index base was founded by the means of the literature research method at first. Then 20 experts were asked to score all these indexes according to the importance of each index. Dimensionality and index base of competitiveness evaluation index system of listed TCM pharmaceutical companies were screened. With two rounds of questionnaires, the evaluation index system was constructed finally.Results The positive coefficients of two rounds of expert consultation were 95% and 100%;the cooperative coefficients were 0.659 and 0.639;the authoritative coefficient was 0.713 2. Evaluation system consisted of 5 first grade indexes and 26 second grade indexes.Conclusion The positive coefficients and the authoritative coefficients are both high enough through Delphi method. Opinions of all the experts in the two round of expert consultation tend to be uniform, which reveals that the evaluation index system of listed TCM pharmaceutical companies is relatively scientific.

E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.

Objective:To construct bispecific minibody by using anti HBsAg and anti RBC single chain Fv(ScFv).Methods:A “knob” variant T366W was first obtained by replacement of a small amino acid with a larger one in the human IgG1 CH 3 domain.The knob was designed to insert into a “hole” in another CH 3 domain which was created by replacement of three large residues with three smaller residues:T366S:L368A:Y407V.The “knob into hole” mutation:S354C:T366W/Y349C:T366S:L368A:Y407V.Then a disulfide bond was engineered in combination with previously designed “knob” or “hole” CH 3 was connected to anti HBsAg or anti RBC ScFv genes respectively.Then the two genes were combined together to form a bispecific minibody expression vector.The bispecific minibodies were expressed in E.coli.Results:Three different form of anti HBsAg and anti RBC minibody expression vectors were constructed.They contained wild type CH 3,“knob into hole”CH 3 or “knob into hole” plus disulfide bond CH 3 respectively.The results indicated that these three different types of bacterially expressed minibodies had similar HBsAg and RBC binding activities.The second and third type of minibody could cause agglutination of human RBC when HBsAg was present,which demonstrated bispecific function.Conclusion:Engineered interface of CH 3 can promote formation of heterodimers of different antibodies and faciliates the formation of bispecific antibodies(bispecific minibody) in E.coli expression system.

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that: (1) Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear membrane of cytotrophoblast; (2) Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly (P<0.01); (3) After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls (P<0.05). It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotrophoblast invasion and migration activity.

This study examined the association of expression of vascular endothelial growth factor (VEGF), a promoter of angiogenesis, with endothelium dysfunction in preeclampsia. The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. VEGF expression in the human umbilical vein endothelial cells (HUVECs) was blocked by small interfering RNAs (siRNAs). The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers. The cell proliferation and cell-secreted nitric oxide (NO) level were detected by MTT method and nitrate reductase assay, respectively. The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue. The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects, although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high. Moreover, VEGF deficit could lead to endothelium cell dysfunction, and the administration of VEGF could protect endothelium cells from injury. It was concluded that lack of VEGF contributes to endothelium dysfunction, which may lead to the occurrence and development of preeclampsia.