Table of Content

    05 June 2012, Volume 32 Issue 6
    A clinical report about distributions of hepatic veins in ex vivo adult to adult split liver transplantation
    2012, 32(6):  603-607. 
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    0bjective:To investigate clinical respective effects about different distribution of hepatic veins in adult to adult split liver transplantation(SLT) ex vivo.Methods: A retrospective analysis was conducted on 12 adult to adult split liver transplantations performed at Tianjin First Centra1 Hospital between Jan 2007 and Oct 2011.The analysis was focused on the distribution and reconstruction of hepatic veins. Monitor the vascular complications and patient’s prognosis. Results: There were four types about distribution of hepatic vein in 6 right hemi-liver. Grafts. Only 1 graft got RHV( right hepatic vein)+MHV(middle hepatic vein)+Vena cava; 2 case was with RHV+5,8 vein reconstruction +Vena cava; another 2 grafts obtained RHV+5,8 segmental vein reconstruction; the last one was consist of RHV+ hemi-MHV +Vena cava. For split liver 6 corresponding left grafts had 4 variations as: LHV(left hepatic vein)+vein of 4 segment reconstructed; LHV+MHV; LHV+MHV+ Vena cava; LHV+hemi-MHV. One recipient with LHV+vein of 4 segment reconstructed left liver graft occurred small for size syndrome for occlusion of reconstructed 4 segmental vein and eventually died. Other recipients had no complications related with hepatic vein. Conclusions: Multiple methods existes when we face with distribution and reconstruction of hepatic vein in adult to adult split liver transplantation. Before we make a decision on how to split hepatic vein in clinical procedure, we should prove the functional graft volume enough first, then reference patient’s conditions and feasibility of surgical procedures.
    Identification of a novel DNA aptamer against HER2
    Zhe Liu Xian-da YANG
    2012, 32(6):  608-612. 
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    Abstract: Objective To develop HER2 DNA aptamers that may potentially serve as tumor-binding ligand in novel HER2-targeted therapeutic strategies. Methods A single-stranded 86nt DNA library containing 40 random oligonucleotides was synthesized in vitro. A new aptamer HA5 were developed by SELEX technique with HER2 epitope peptide as target. Flow cytometry were performed to monitor the enrichment of aptamer pool and to evaluate the binding characterization of HA5. The structure of HA5 was performed by Mfold. Results The selected HA5 bound to the HER2 structure and had minimal cross reactivity to trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative cells. Conclusion The results suggest that HA5 may have application potentials in targeted therapy against HER2-positive malignancies.
    Comparation of infection efficiency of H1N1 virus in human airway epithelial cells
    2012, 32(6):  613-617. 
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    Abstract: Objective Define the infection efficiency of 2009 novel swine-origin influenza A virus and seasonal influenza virus in human airway epithelial cells. Methods Infection efficiency of H1N1 virus in A549, CNE-2Z, H1650, Hep-2 and MRC-5 was examined by indirect immunofluorescence assay. Effect of inhibitors in human airway epithelial cells infected by H1N1 virus was examined by indirect immunofluorescence assay. Results (1)Infection efficiency of swine-origin H1N1 virus: H1650>A549>CNE-2Z>MRC-5>Hep-2;Infection efficiency of seasonal H1N1 virus: A549>CNE2Z>H1650>MRC-5>Hep-2;(2)Swine-origin H1N1 and seasonal H1N1 both entered into airway epithelial cells by the way of endocytosis. Conclusion Infection efficiency of seasonal H1N1 virus was clearly higher than swine-origin H1N1 virus in A549 cells; Infection efficiency of swine-origin H1N1 virus was clearly higher than seasonal H1N1 virus in H1650 cells as infection time increased.
    The effects of HCV core protein on the expression of microRNAs in HepG2 cells
    2012, 32(6):  618-622. 
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    Abstract:Objective To investigate the effects of HCV core protein(HCV-Core)on the expression of microRNAs in HepG2 cells. Methods pCDNA3.1(+)/HCV-Core eukaryotic expression plasmid was transfected into HepG2 cells. The levels of mRNA and protein expression of HCV core protein in HCV-Core transfected HepG2 cells were validated by RT-PCR,immunohistochemistry and Western blot. The differences of microRNAs expression between pCDNA3.1(+)/HCV-Core and pCDNA3.1(+)transfected HepG2 cells were analyzed by miRNA Array GeneChip(Affymetrix,USA), followed by the verification by real-time Tagman-Fluoresence quantitative RT-PCR. Results miRNA array analysis demonstrated that there were eight microRNAs altered in HepG2 cells transfected with HCV core protein, and quantitative RT-PCR confirmed that miR-29,miR-146a, miR-149, miR-192, miR-221, miR-222, miR-193b were up-regulated, while miR-196a was down-regulated. Conclusion HCV core protein could induce the up-regulation or down-regulation of some microRNAs in HepG2 cells, which may play important roles in the pathogenesis of hepatocellular carcinoma.
    Mesenchymal stem cells stimulate epithelial to mesenchymal transition in breast cancer cell line MCF7
    Qi-Lin XU Chun-hua ZHAO Liang WANG
    2012, 32(6):  623-627. 
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    Objective To study the effect of human adipose tissue derived MSCs (haMSC) in breast tumor micro-environment on breast cancer cells. Methods Breast cancer cell lines (MCF7) were cultured alone or indirectly co-cultured with MSCs using transwell system. The co-cultured MCF7 cells were collected for analysis of the changes in morphology, EMT related marker and tumor characteristics. Results Following indirect co-culture with MSCs, breast cancer cells morphology and EMT specific markers altered in response to MSCs. The invasive capacity and migration ability of MCF7 cells post co-cultured with MSCs were significantly enhanced, while no changes were observed in cell cycle and proliferation. Conclusion Human adipose tissue derived MSCs can stimulate epithelial to mesenchymal transition of breast cancer cells.
    Identification of down-regulated miRNAs in extrahepatic and intrahepatic cholangiocarcinoma by using microRNA-microarray and Real-Time PCR analysis.
    2012, 32(6):  628-633. 
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    Objective We intend to investigate the expression profile of miRNAs down-regualted in human extrahepatic and intrahepatic cholangiocarcinoma tissues and probe the effect on cell growth of four of these miRNAs in QBC939 cell line. Methods Down-regulated miRNAs in extrahepatic or intrahepatic cholangiocarcinoma tissues were analyzed by using miRNA-microarray, which was confirmed by using miRNA Real-Time PCR analysis. Based on these findings, four of these down-regulated miNRAs were chosen to perform function investigation. The miRNAs mimics were transfected into QBC939 cells and cell proliferation assay was performed by using MTT, which would demonstrate the relationship between down-regulated miRNAs and cholangiocarcinoma cell growth. Results 25 miRNAs and 15 miRNAs were down-regulated in extrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma, respectively. There were 6 miRNAs down-regulated both in two types of cholangiocarcinoma. miR-181a, miR-596, miR-492, and miR-602 were overexpressed in QBC939 cells, which indicated a significant inhibitory effect on cell growth. Conclusion The miRNAs expression patterns in human extrahepatic and intrahepatic cholangiocarcinoma tissues are different and Down-regulated miRNAs act as suppressors on cholangiocarcinoma cell growth.
    DNA shuffling and bioinformatics analysis of the dust mites group 2 allergen genes, Der f2 and Der p2
    2012, 32(6):  634-638. 
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    Objective A chimeric gene library of the dust mites group 2 allergen, Der f2 and Der p2, was constructed by using DNA shuffling method and analyzed by bioinformatics. Method Der f2 and Der p2 genes were amplified by polymerase chain reaction (PCR), respectively; the PCR products were mixed equally (v/v) and digested with DNase I. DNA fragments of 50bp-100bp were purified. DNA shuffling was performed through three rounds of primerless PCR and additional PCR with Der f2 specific primers. A single product of correct size was inserted into pUCm-T vector and transformed into E. coli DH-5α. Sixty clones were picked out randomly, sequenced and analyzed by multiple sequence alignment using DNAMAN software. T helper cell epitopes were predicted by NetMHCII 2.2 Server. Results Ten recombinants were obtained. The amino acid sequences deduced from these recombinants were predicted to not only share the common Th cell epitopes with Der f2 and Der p2 but also possess new epitopes. Conclusion A chimeric gene library of Der f2 and Der p2 was successfully established, which could provide the basis for selection of vaccine with high immunogenicity and low allergenicity for asthma treatment in future.
    Differentiated hepatocytes derived from human bone mesenchymal stem cell in 3-dimentional culture and the polarity characteristic
    2012, 32(6):  639-643. 
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    Abstract: Objective To explore hepatic differentiation of human bone mesenchymal stem cell (BMSC) in 3-dimentional (3D) culture system of rat type-I collage and the polarity of differentiated hepatocyte. Methods BMSCs were isolated from adult bone marrow and differentiated to hepatocyte in 3D culture system of rat type-I collage. The morphological and structural characteristics of differentiated hepatocyte derived from BMSC in 3D culture were observed using HE staining. The expression of hepatocyte-specific markers of ALB and AFP were analyzed using immunohistochemical staining. The expression of hepatocyte polarity protein BSEP and SRB1 were assessed using the Immunofluorescence. Results Differentiated hepatocyte derived from BMSC formed tight junction and tubular or spherical structures in 3D culture system. The expression of ALB and AFP in hepatocytes differentiated from BMSC was positive. The expression of BSEP and SRBI in the 3D culture was positive and characteristic. Conclusion BMSC can differentiate to hepatocyte in 3D culture. The differentiated hepatocyte can form hepatocyte-specific polarity in 3D culture.
    Stable maintain and propagation of mouse embryonic stem cells-derived neural stem cells in vitro
    Rui-Zhu LIN Chun-hua ZHAO Qi-Lin XU
    2012, 32(6):  644-649. 
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    Objective To establish an efficient way to induce the mouse embryonic stem cells into homogeneous neural stem cells and to develop culture conditions that support the viability and propagation of stable NSCs in vitro. Methods In the serum-free medium, firstly, we induced ESCs to neuroepithelial progenitor cells (NPCs). Then, NPCs were cultured in a suspension medium supplemented with EGF and FGF2 and aggregates were collected and replate to differentiate to neural stem cells (NSCs) in monolayer. We used 46C for monitoring and quantitating the transition from ESCs to NPCs. Expression of NSCs marker was determined by quantitative PCR and immunoflurescence. Results After 5 days of induction, mESCs differentiated into NPCs with high expression of Sox1+. NPCs can further differentiated in to NSCs after culture in suspension. As determined by real-time quantitative PCR (qPCR), Pax6、Nestin、Mash1、BLBP(FabP7) genes expressed highly in NSCs. The immunoflurescence demonstrated that 90% of NSCs express Nestin, RC2 and Pax6. Conclusion ESCs can successfully be induced to homogeneous NSCs, and NSCs can maintain self-renewal status and propagate in vitro after several passage.
    Effect of MicroRNA-26a expression on proliferation of acute myeloid leukemia Cells
    2012, 32(6):  650-655. 
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    【Abstract】0bjective Construct the recombinant lentivirus vector expressing has-miR-26a, and to observe the effect of overexpression of miR-26a on acute myeloid leukemia cell proliferation. Method The lentiviral vector of MicroRNA-26a (LV-miR-26a-GFP) was constructed and transfected into human acute myeloid leukemia cells.The cells were divided into non-transfected group, transfected group (transfected with LV-miR-26a-GFP) and negative control group( transfected with LV-GFP).The expression level of miR-26a was measured by real-time PCR. The expression of target gene PTEN was tested by Western blot. The cell growth curves and the effects of the miR-26a on the ability of cell proliferation were respectively analyzed by XTT and soft agar colony formation assay.Result The expression vectors of miR-26a restructuring lentivirus have been successfully achieved. After transfected acute myeloid leukemia cell NB4, successfully achieved the over expression o f miR-26a. Compared with non-transfected group and negative control group, the ability of cell proliferation for transfected with LV-miR-26a-GFP group was significantly increased (P < 0.01). The miR-26a overexpressed can inhibit the expression of target gene PTEN in NB4 cells. Conclusion MicroRNA-26a can inhibit the expression o f target gene PTEN, enhance the ability of acute leukemia cell proliferation, and promote cell proliferation.
    A Chinese girl with Primary hypertrophic osteoarthropathy (PHO) caused by homozygous novel deletion mutation in HPGD and literature review
    2012, 32(6):  656-659. 
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    Abstract:Objective Primary hypertrophic osteoarthropathy, associated with mutations in HPGD gene, is a rare autosomal recessive disorder. The authors report the clinical characterization and the novel mutation in HPGD of a Chinese girl with PHO in order to improve the clinical recognition of this disease. Methods Total genomic DNA was extracted from peripheral blood leukocytes of the patient and her father. The HPGD exons 1-7 were amplified by polymerase chain reaction (PCR) and were analyzed by direct sequencing. Review previously published literature about HPGD mutations. Results A novel homozygous mutation c.308_309delCT(p.Thr103Thrfs4X)was identified in the patient, who is a 5 years girl with PHO manifest characteristic digital clubbing, furrowing of the skin of the face and hyperhidrosis etc. Conclusion Testing for mutation of HPGD can identified the diagnosis of PHO patients. Clinical manifestation, biochemical testing and X ray can help to make a diagnosis of PHO ,Testing the HPGD mutation can identify it.
    A novel Compound Heterozygous Mutations causing 21-Hydroxylase Deficiency
    2012, 32(6):  660-663. 
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    Abstract: Objective To investigate the clinical and molecular genetic characteristics of a Chinese patient with 21-hydrosylase deficiency (21-OHD). Methods Clinical features and laboratory data were collected from that patient. Ten exons and the boundaries of exon-intron of CYP21A2 gene were amplified by PCR and sequenced. The correlation between clinical characteristics and genotype was analyzed. Results The female patient was diagnosed as 21-OHD with simple-virilizing form according to the clinical manifestation. DNA sequencing results showed a heterozygous mutation. One allele of the patient contained a base transversion (T>A) at c.515 and led to a missense mutation of Ile to Asn at the 172th amino acid. The other allele had one base transversion (T>G) at c.593 and led to a nonsense mutation at the 198th codon, which is a new mutation. The novel compound heterozygous mutations cause the simple-virilizing form of congenital adrenal hyperplasia. Conclusion Through CYP21A2 gene analysis, a new mutation causing 21-OHD was found, which broadened the mutation database, and the clinical diagnosis of 21-OHD was confirmed.
    Promotion of apoptosis on ovarian cancer SKOV3 Cells by silencing NEK2
    2012, 32(6):  664-668. 
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    Abstract: Objective To study the effect of silencing NEK2 via RNAi on proliferation and apoptosis of ovarian cancer SKVO3 cells and the possible underlying molecular mechanism. Methods Three pairs of siRNA were designed according to the NEK2 gene sequence and synthesized chemically. The siRNAs were transfected into the ovarian cancer SKOV3 cells. After forty-eight hours of transfection, Real-time PCR and Western blotting were performed to detect the expression levels of NEK2 mRNA and protein so as to screen the most effective siRNA. MTT and FCM were performed to detect the proliferation and apoptosis of SKOV3 cells respectively. Western blotting assay was used to determine the expression level of proteins BAD,BCL-2 and CASPASE-3. Results Real-time PCR and Western blotting revealed that Nek2-siRNA notably down-regulated NEK2 expression on both mRNA and protein levels. The proliferation of Nek2-siRNA transfected SKOV3 cells was inhibited while the apoptotic cells were increased. Western blotting assay revealed that silencing NEK2 in SKVO3 cells up-regulated expression level of BAD and CASPASE-3, while down-regulated BCL-2 significantly. Conclusion Nek2-siRNA can effectively promote apoptosis of ovarian cancer SKOV3 cells by down-regulating NEK2.These data suggested that NEK2 plays an important role in apoptosis of ovarian cancer cells, which might be a potential target.
    The effectiveness of preoperative intravenous iron alone for patients of gastrointestinal tumor with anemia
    2012, 32(6):  669-672. 
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    Objectives: To evaluate the effectiveness and security of preoperative intravenous iron alone in patients of gastrointestinal tumor with anemia and find out the validity to reduce the perioperative blood transfusion. Methods: to collect the information of patients with preoperative anemia admitted to Beijing Union Medical College Hospital From June, 2010 to February, 2012 for gastrointestinal tumor. Patients with iron deficiency anemia were administrated of intravenous iron by the way of intravenous drip. In the morning of operation or 14th day after treatment, we review the blood routine, serum iron, ferritin, transferrin protein, liver and kidney function tests. Perioperative blood transfusion dose of those subjects was compared with that of the same period of hospitalization in patients with anemia. Results: A total of 121 patients entered and completed the study, in which 13 cases of gastrointestinal stromal tumor, 59 cases of gastric cancer and 49 cases of colorectal cancer. Application of preoperative intravenous iron improved hemoglobin level rapidly, and also increased red blood cell count. MCV, MCHC and MCH which are indicators of red blood cell were improved after treatment. Shortly after intravenous iron treatment, serum iron and serum ferritin levels were significantly increased compared with that before treatment(P<0.05). And the patients with a hemoglobin levels before treatment <100g/L had a better response to intravenous iron compared to which with pre-treatment hemoglobin level ≥ 100g/L. Iron treatment can reduce the perioperative transfusion of red blood cell dose compared with the non treatment anemia patients with cancer(P<0.05). Conclusion: In the gastrointestinal cancer patients with pre-treatment anemia, intravenous iron alone can increase the hemoglobin, decrease the iron deficiency of preoperative anemia patients safely and rapidly, and can reduce perioperative blood transfusion. The efficacy of intravenous iron may be affected by the degree of anemia.
    Different cell growth inhibition effects of cardiac glycosides on lung cancer cell lines A549 and H1975
    2012, 32(6):  673-677. 
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    Objective: To explore the inhibitory effects of cardiac glycosides(Digoxin,Gitoxin, Digitoxin) on human lung cancer cell lines A549 and H1975.Methods: After treated by Gitoxin,Digitoxin and Digoxin ,the drug sensitivity of human lung cancer cells (A549 and H1975) to cardiac glycosides were detected by MTS assay. The change of cell apoptosis and proliferation were analyzed by using flow cytometry. Results: A549 and H1975 were exposed to cardiac glycosides for 48h at different concentrations. H1975 with a 50% inhibiting concentration of about 100nM was more sensitive to A549 with a 50% inhibiting concentration above 10μM. Flow cytometry demonstrated that cardiac glycosides could induce apoptosis to A549 and H1975.And the apoptosis rate of H1975 showed dose-depended manner. Also, the cardiac glycosides could inhibit the proliferation of both cell lines at different concentrations. Conclusions:Cardiac glycosides could induce apoptosis and inhibit proliferation of A549 and H1975.But it showed great differences for the different genetics background of them.
    The role of ACE2 in ventricular remodeling and effect of valsartan treatment on the spontaneously hypertensive rats
    2012, 32(6):  678-681. 
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    Abstract:Objective To investigate the expression of angiotensin converting enzyme 2 (ACE2) in the SHR,and the effect of valsartan on the expression of ACE2 mRNA.Methods Twenty-four Twelve-week-old male SHR were randomly divided to SHR group and valsartan group;Twelve -week-old male Wistar rats were served as control group.After 10 weeks,determine the heart weight index and the 1eft ventricular weight index; determine the concentration of AngⅡ in plasma by ELISA; determine the hydroxyproline content in myocardium by buck solution; The expression of ACE2mRNA in myocardium was measured by RT-PCR. Results Compared with the control group,the LVWI、the hydroxyproline content and the concentration of AngⅡ were significantly increased in the SHR group,but the ACE2 expression was markedly decreased; Compared with SHR group, the above indexes were decreased except for ACE2 was increased in valsartan group.Conclusions Valsartan can reverse left ventricular remoding in hypertension. The possible mechanism is that valsartan can incerease the expression of ACE2.
    Cell fusion reduced melanoma cell proliferation through PI3K-AKT pathway
    2012, 32(6):  682-686. 
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    Abstract: Objective To investigate the effect of cancer cell fusion in melanoma proliferation and the underlying mechanism. Methods Stable melanoma fusion cells were obtained by the polyethylene glycol (PEG) fusion method and isolated by the fluorescence activated cell sorting (FACS). Cell proliferation rate was detected by cell culture in vitro and counting the cell number everyday. Differentially expressed genes between the stable melanoma fusion hybrids and the parental cells were detected by the Affymatrix gene chip following the gene function analysis by Ingenuity Pathways Analysis (IPA). Protein expression was analyzed by western blot. Results Stable melanoma fusion cells were successfully obtained by the PEG fusion method. The proliferation rate of fusion hybrids was significantly decreased compared to the parental melanoma cells. Gene chip and the following gene function analyses showed that PI3K-AKT pathway related to cell proliferation was significantly down-regulated in the melanoma fusion hybrids. Moreover, the expression of phospho-S6 in the fusion hybrids significantly decreased. Conclusions Cell fusion reduced melanoma cell proliferation through the PI3K-AKT pathway.
    Rosiglitazone inhibits neointimal hyperplasia and the expression of MMP-2 of carotid arteries after balloon injure in rats
    2012, 32(6):  687-692. 
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    Objective To investigate the effects of PPARγ ligand rosiglitazone on neointimal hyperplasia and the expression of MMP-2 and TIMP-2 in rat carotid arteries after balloon injure. Methods Treatment group (rosiglitazone 3 mg/kg?d,n=5 ) and control group (saline,n=5) are included in the study. Vascular restenosis in rat carotid arteries was established by balloon denudation. The thickness and area ratios of intima to media (I/M and IA/MA) were measured by using hematoxylin and eosin staining. The mRNA levels and protein expression of MMP-2 and TIMP-2 in vascular tissues were measured by RT-PCR and Western-blotting, respectively. Results Both I/M and IA/MA were significantly reduced in rosiglitazone group compared to that in control group at different time points after balloon injury (P<0.0001). Rosiglitazone markedly suppressed the mRNA and protein expression of MMP-2 in carotid arteries induced by balloon injury(P<0.0001). But there were no difference in the mRNA and protein expression of TIMP-2 between rosiglitazone group and control group. Conclusion This study demonstrates that rosiglitazone inhibits neointimal hyperplasia in carotid arteries after balloon injury. Our results suggest that rosiglitazone suppresses neointimal hyperplasia by modulating some taget gene transcription, down regulating MMP-2 expression and breaking the balance of MMP-2 and TIMP-2 in sequence.
    The analysis of factors on the number of sentinel lymph nodes in breast cancer patients
    2012, 32(6):  693-696. 
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    Abstract:Objective To analysis the factors on the number of sentinel lymph nodes (SLN) in breast cancer patients and explore the appropriate cut-off value for SLNs. Methods Between January 2007 and December 2011, 578 cases of sentinel lymph node biopsy (SLNB) profiles were gathered retrospectively. The logistic regression model was used to evaluate the relationship between number of SLNs and clinical-pathological characteristics. Results The study group was consisted of 578 female patients with an average of 49.9 years in age (range 21 to 90). 2222 SLNs were resected in total by surgeon. The mean number of sentinel lymph nodes removed was 3.8 (range 1 to 15). Sentinel lymph nodes involved in metastasis were found in 17.8% of patients. There was no difference in the number of SLNs between the SLN-positive and the SLN-negative. In univariate analysis, variables associated with sentinel lymph nodes number were operation type, tracer technique and BMI (P<0.05 for all variables). In multivariate analysis, variables associated with number of sentinel lymph nodes were simple mastectomy, dual tracer technique and BMI≤30 (P<0.05). All the SLN-positive were screened out when the cut-off value was set to no more than 5 SLNs. Meanwhile, 18.7% of patients need no further lymph node dissection and 298 lymph nodes were spared. Conclusion The patients with the dual tracer technique, BMI≤30 or simple mastectomy are prone to harvest more sentinel lymph nodes. It is appropriate for surgeons to set cut-off value to 5 sentinel lymph nodes in breast cancer.
    An Expression and Purification of Tagless Cystatin C
    2012, 32(6):  697-701. 
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    Objective To construct a prokaryotic expression vector of human Cystatin C(CysC)gene, express TF-CysC fusion protein; prepare tagfree CysC used as protein standard for immunodiagnosis test. Methods A cDNA fragment coding for the full-length human CysC was inserted into pCold TF expression vector, followed by sequencing analysis. The constructed recombinant plasmid was transformed to E.coli BL21(DE3) for expression under induction of IPTG. After purified by Ni-Sepharose affinity chromatography, The TF-CysC was treated with GST-HRV 3C protease, and the TF tags and GST-HRV 3C proteases were removed by size-exclusion chromatography(SEC)in one step. The prepared CysC was identified by SDS-PAGE and Western blot. New Zealand rabbits were immunized with the fusion protein and the antiserum was obtained. The titers and reactogenicity of the prepared polyclonal antibody were determined by indirect ELISA and Western blot respectively. Results Restriction analysis and sequencing proved that recombinant plasmid pCold TF-CysC was constructed correctly. Tagfree CysC was high expressed in a soluble form and its molecular weight is approximately 13.3 kD, reached a purity of 95%. The titers of the prepared antiserum against CysC reached more than 1:4×106 and the good reactogenicity of antiserum was confirmed. CysC was stored in 4 ℃, and the concentration of the CysC was found no obvious decrease in one month. Conclusion The purity tagfree CysC was obtained as protein standard for immunodiagnosis test, and the method of the protein expression and purification was available and efficient.
    Exploration of vibration response imaging in evaluating diffuse parenchymal lung disease
    2012, 32(6):  702-706. 
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    Abstract:Objective To explore the value of vibration response imaging (VRI) system in evaluating the patients with diffuse parenchymal lung disease (DPLD).Methods 35 DPLD patients that their CT show reticulation and honeycombing in the base of the lung and 33 healthy volunteers were enrolled,Clinical examination and VRI test were performed.The parameters of VRI included vibration energy graph grades, dynamic image grades,areas of Maximal energy frame(MEF), quantitative lung data(QLD) and crack .The statistical analysis was performed using 2 Independent Samples Tests and Independent-Samples T test.Results The VRI parameters of the DPLD group were as follows,ie.graph grades of 2(1~3),dynamic image grades of 3(2~4),MEF areas of (71.13±4.94)kilo-pixels,QLD of (54.31±11.34)%(left).The VRI parameters of the control group were as follows,ie. graph grades of 0(0~1),dynamic image grades of 0(0~1),MEF areas of (71.61±3.42)kilo-pixels,QLD of(56.70±6.02)% (left) .There were significant differences in graph grades and dynamic image grades(P<0.05) ,otherwise MEF areas and QLD were not.Energy move down during inspiration in DPLD patients.Crack detectation was consistent with auscultation in 89% subjects.Conclusion VRI results of DPLD patients that their CT show reticulation and honeycombing in the base of the lung have its characters.there was a statistical difference between the VRI results of DPLD patients and healthy subjects.VRI will be helpful in diagnosing DPLD during its early stage.
    Clinical study of 246 cases of ectopic pregnancy with the analysis
    2012, 32(6):  707-708. 
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    Abstract Objective Discussion on the main causes of ectopic pregnancy and the various methods of treatment of ectopic pregnancy treatment clinical value, improve understanding on ectopic pregnancy. Method Retrospective analysis of 2010 January to December year the hospital treated 246 cases of ectopic pregnancy cases, the conservative treatment of 136 cases including 33 cases of operation treatment, turn. Operation treatment of 141 cases, 55 cases of laparoscopic resection of lesions or tubal ostomy. Result The study of 246 cases of patients with ectopic pregnancy, abortion or medical abortion history has 190 cases, accounting for 77.24%, laparoscopic resection of lesions or salpingostomy in 55 cases, accounting for the proportion of operation 41.78% conclusion abortion or medical abortion if concurrent infection can cause chronic tubal inflammation of pelvic inflammatory disease and increase the incidence of ectopic pregnancies, thus reduce the artificial abortion on the prevention of ectopic pregnancy occurs extremely important. Laparoscopic technique of little injury, quick recovery, shorter hospitalization time, use less, appearance in treatment of ectopic pregnancy has played an increasingly important role in.
    Clinical characteristics and outcome of infective endocarditis in patients with congenital heart disease
    2012, 32(6):  709-712. 
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    Objective: To summarize the clinical characteristics and management of infective endocarditis(IE) in adult patients with congenital heart disease (CHD) and to identify the predictors of outcome. Methods: From January 2001 to December 2010, fifty-one adult patients with CHD and definite IE based on the Duke criteria were enrolled; the clinical data were retrospectively reviewed.Results: 20.6% IE patients had shunt CHD. Ventricular septal defect and patent ductus arteriosus were the most common underlying defects. Streptococci were the most common causative organisms (47.1%). 27 patients(52.9%)had complications. The frequent complications were valve destruction and embolic events. 38 patients (58.8%) underwent cardiac surgery, the operation were performed during the active phase of infective endocarditis in 21 cases. The overall mortality rate was 19.6%. Logistic regression analysis identified presence of severe heart failure [OR 0.293(0.045-0.347),p =0.012] and central nervous system complications [OR 0.308(0.074-0.63),p =0.014] as predictors of overall mortality. Surgical intervention was independently associated with lower overall mortality [OR 0.238(0.009-0.183),p =0.031]. Conclusion: Ventricular septal defect and patent ductus arteriosus were the most common underlying defects in IE patients with CHD. The mortality of IE in patients with CHD was high. Presence of severe heart failure and central nervous system complication were independently predictors of mortality. Surgical intervention reduces the risk of mortality of IE in patients with CHD .
    Isolation and Purification and Primary Culture of Lung Fibroblasts from Adult Mouse
    2012, 32(6):  713-714. 
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    Abstract: Objective To explore a reasonable method for isolation, purification and primary culture of lung fibroblasts from adult mouse lungs and research the growth curve of lung fibroblast. Methods Adult mice lung fibroblasts were isolated and cultured using three methods of lung tissue directly adherent, trypsin digestion, mixed culture method of lung tissue adherent and trypsin digestion. Lung fibroblasts were purified in different centrifugal force and different adherence. Lung fibroblasts were identified according to cell shape and the expression of vimentin and α-SMA by immunohistochemistry and optical microscope. Their growth curve was measured by MTT. Results Single purified lung fibroblasts were elongated spindle-shaped, cord-like. Monolayer covered lung fibroblasts were swirling, palisading, radially arranged. The expression of vimentin in lung fibroblasts was positive and that of α-SMA was weakly positive. Cells were appeared exponential growth after seeded 48 h and reached their peak at 120 h. Conclusions Lung fibroblasts from adult mouse were successful isolated, purified and cultured by above three methods. This study provided reliable and feasible technical approaches to obtain adult mouse lung fibroblasts.
    Progress of the related susceptibility genes on altitude illnesses
    2012, 32(6):  719-723. 
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    The gene polymorphism is one of the key factors that induce high altitude illnesses,some people are more of susceptibility to it. More understanding about the related susceptibility genes could offer reference for its prevention and treatment.
    Application of PBL mode with research subjects in Histological Techniques
    2012, 32(6):  724726-724726. 
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    Histological Techniques is a practical course, which can help medical students to master some useful research methods. Application of PBL mode with research subjects can enhance the research ability of students,and promote finishing research subjects,which have the win-win effect.The teaching mode play an important role in training students of high quality.