Table of Content

05 May 2014, Volume 34 Issue 5

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Generation of induced neural stem/progenitor cells from human fibroblasts

2014, 34(5):  579-582. 

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Objective To investigate induced neural stem/progenitor cells (iNSCs) from human fibroblasts. Methods Human fibroblasts were infected with lenti-virus expressing green fluorescent protein (GFP) under Pax6 promoter, followed by a hygromycin-resistant gene. Hygromycin-selected fibroblasts were used as starter cells and infected with virus encoding 10 transcription factors Sox2, Klf4, c-Myc, Tcf3, Ascl1, Brn2, Neurog2, Foxg1, Hes1, Id1, which play critical roles in the development, differentiation, functional stability, and plasticity of neural cells. The reprogrammed cells that were GFP-positive were sorted by using flow cytometer and characterized by fluorescence staining and differentiation assay. Results iNSC expressed NSC markers (Nestin) and could differentiate into neurons(β Ⅲ- tubulin) and astrocytes(GFAP). Conclusion Human fibroblasts can be converted to NSC-likes cells by 10 transcription factors.

Generation of full-sized and ear-shaped cartilage with passaged remnant ear chondrocytes from microtia individual

2014, 34(5):  583-588. 

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Objective To test the feasibility of in vitro generation of a full-sized and ear-shaped cartilage with remnant ear chondrocytes derived from individual microtia patient. Methods The initial cell yield of remnant ear chondrocytes was analyzed from 40 cases of microtia. Proliferation was tested by MTS and expansion efficiency was calculated. The chondrocytic phenotype altering after continuous passages was characterized by immunofluorence staining and PCR. P3~P4 chondrocytes were seeded onto the PGA/PLA scaffold with the shape and full size of adult human ear. The complex was cultured with a redifferentiation system composed of chondrogenic factors, alginate gel, and a rotating culture device. Results The initial cell yield from remnant ear tissue was（3.90±1.27）×106/g. The proliferative ability of remnant ear chondrocytes from P1~P4 passages was enhanced by adding bFGF, and the amplification of the cells expanded to P4 could reach around（328.4±50.4）folds. However, the COLII and ACAN expressions gradually declined with passages and became negative in P4 chondrocytes whereas COLI expression showed stronger. The neo-cartilage in the experimental group maintained the ear shape well and formed cartilaginous structure with positive staining of Safranin O, Toludine Blue, and COLII, while the control group failed to form cartilage tissue and only showed fibrous structure. Conclusion A full-sized and ear-shaped cartilage could be engineered in vitro by the passaged remnant ear chondrocytes derived from individual microtia under a redifferentiation culture system.

Effects of focal adhesion kinase on mechanical stress-induced airway mucin hypersecretion in human bronchial epithelial cells

2014, 34(5):  589-594. 

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Objective To explore effects of focal adhesion kinase on mucin(MUC)5AC hypersecretion induced by mechanical stress. Methods Normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface and were exposed to chronic intermittent compressive mechanical stress via Transwell mechanical stress apparatus. Cells were treated with FAK siRNA. Western blot was performed to determine FAK and phosphorylation of ERK1/2 protein contents. Real-time PCR and ELISA were underwent to test MUC5AC mRNA and protein contents respectively. Results Relative expression level of phosphorylation of FAK at Tyr397 (p-FAK-Y397) in mechanical group was significantly higher than those in control group. Compared with the control group, mechanical stress could increase expressions of MUC5AC mRNA and protein contents significantly and also the protein expression of p-ERK1/2（P <0.01）. FAK siRNA could significantly attenuated the stress-induced increase in MUC5AC mRNA and protein expression and also the protein expression of p-ERK1/2（P <0.01）. But expressions of MUC5AC mRNA and protein and p-ERK1/2 protein in mechanical + FAK siRNA group were still higher than those in FAK siRNA control group（P <0.05）. PD98059 could significantly decrease the stress-induced increase in MUC5AC mRNA and protein expression（P <0.01）. Combination of FAK siRNA and AG1478 could significantly inhibit the stress-induced increase in MUC5AC mRNA and protein expression（P <0.01）. AG1478 alone could effectively decrease stress-induced increase in MUC5AC mRNA and protein expression（P <0.05），but when compared with AG1478 control group，differences were still significant. Conclusion The signal transduction pathway of stress-induced overexpression of MUC5AC is ERK-dependent，FAK is one of the upstream signal molecules of ERK.

Orai2 participated in the CaR Mediated Ca2+ Entry and NO Generation in Human Umbilical Vein Endothelial Cells

2014, 34(5):  595-601. 

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Objective To study the roles of calcium release-activated calcium modulator 2(Orai2) in extracellular Ca2+-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC). Methods 1) We silenced the expression of Orai2 genes in HUVEC by transfection constructed Orai2 RNA interference plasmids. The expression of Orai2 protein and mRNA levels were determined by Western blotting and real time RT-PCR, respectively. 2) The second to fifth passage of HUVEC were incubated with CaR agonist spermine(activating store-operates cation channels (SOC) and receptor-operated channels (ROC)), CaR negative allosteric modulator Calhex231(blocking SOC and activating ROC) and ROC analogue TPA (activating ROC and blocking SOC), protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967 (activate SOC and blocking ROC ), respectively .Those cell models were divided into three groups: Orai2-71 short hairpin RNA group (Orai2shRNA); control group and vehicle group. Intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, the production of NO was determined by DAF-FM (NO fluorescent probe) of every group in HUVEC. Results 1) Compared with the control group, the results of transfection constructed Orai2 RNA interference plasmids demonstrated that shRNA targeted to the Orai2 genes decreased Orai2 protein and mRNA levels by 70.58% and 75.75%, respectively (P<0.05). (2) Compared with the control group and vehicle group, the [Ca2+]i ratio and the net NO fluorescence intensity values of Orai2shRNA group in four different treatments were significantly reduced (P<0.05). Conclusions Orai2 participates in CaR-mediated Ca2+ influx and NO production by SOC and ROC activation in HUVEC.

Construction of the Targeted uPA-shRNA Lentiviral Vector and Its Promotion on proliferation of rabbit chondrocytes

2014, 34(5):  602-609. 

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Objective To construct the siRNA expression Lentiviral vector for uPA gene，infect to chondrocytes and identify its interference effect. Methods First, going through designing, constructing, restriction enzyme digestion, transformation, PCR identification, positive clone sequencing and lentivirus packaging, to obtain four different types of shRNA sequence (P1, P2, P3 and P4) from the targeted uPA gene of New Zealand rabbit based on siRNA theory. Secondly, to transfect the primary culturing cartilage cells of the New Zealand rabbit with P1, P2, P3 and P4, to observe the infection rate under fluorescence microscope, exam the expression level of the uPA-mRNA gene in cartilage cells by using the RT-PCR, and assay the expression level of the uPA protein in cartilage cells by conducting the Western-blot technology. The effect of proliferation of chondrocytes were detected by CCK-8. Results Constructed four different types of uPA-shRNA lentiviral vectors successfully, and these four types of vectors were all able to be transfected into the primary culturing cartilage cells. The infection rate can be as high as 85% in the experiment when MOI=100. It was showed that P1, P2, P3 and P4 were all capable of inhibiting expressing of the uPA gene and protein in chondrocytes. Moreover, among these four different sequences, the P2 had the highest silencing rate, which was 70%. It was further approved that this study has the statistical significance (P<0.05) when analyzing the result together with the control group. The significant Promotive effect of uPA-siRNA on the reproduction of chondrocytes was observed as determined by CCK-8 after 96 hours infection. Conclusions The most efficient targeted uPA-shRNA sequence was found after screening，It is also strongly verified that the siRNA lentiviral vector can be transfected into the cartilage cells, and can further inhibit expressing of the uPA gene efficiently and steadily, and can enhance proliferation of chondrocytes.

Exogenous BMMSCs ameliorate osteoporosis of ovariectomized rat via improving osteogenesis of endogenous BMMSCs

2014, 34(5):  610-614. 

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Objective To witness whether systemic administration of bone marrow mesenchymal stem cells (BMMSCs) can preventing osteoporosis of ovariectomized rat via ameliorating osteogenesis of endogenous BMMSCs. Methods Twelve female Sprague-Dawley rats were randomly divided into group Sham, OVX, High-dose and Low-dose. Established OVX and Sham model. 24 hours after operation, rats in group high-dose and low-dose were systemically injected BMMSCs via tail vein at the dose of 1.5×107 and 0.375×107 cells/kg, representatively. Proximal femurs were analysed by MicroCT. Osteogenesis of BMMSCs was examined by ALP, alizarin red staining and real-time PCR. Results Bone mineral density (BMD) and trabecular bone number (Tb.N) of group OVX were lower than those from group Sham. BMD and Tb.N of group High-dose were higer than group OVX. Compared to group OVX, osteogenesis of endogenous BMMSCs derived from group High-dose and Low-dose were much better, and that from group High-dose turned out to be better than group Low-dose. Conclusion It is effective to relieve symptoms of osteoporosis in ovariectomized rats through treating them with exogenous BMMSCs by the way of rescuing osteogenesis of endogenous BMMSCs.

Probable role of A1AR in renin secretion stimulated by low salt diet in mice

2014, 34(5):  615-621. 

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Objective To observe the pathophysiological characteristics of adenosine A1 receptor (A1AR) gene knockout mice and to investigate the role of A1AR among renin secretion stimulated by low salt diet. Methods After A1AR+/- mice of C57BL/6J mating, multiplication and gene identification, offspring mice were used to experiments. The differences of following items between A1AR+/+ (n=13)and A1AR-/- (n=14)mice were observed: body weight, blood pressure, heart rate, renal function and electrolyte of serum and urine; Pathology features were observed after regular staining on kidney tissue samples. Renin expression at the kidney was stained by Immunohistochemistry. Plasma aldosterone level was also examined(A1AR+/+group, n=5 A1AR-/-group, n=5). Results After low salt diet, much higher renin expression of the kidney cortex and plasma aldosterone level were observed among A1AR-/- mice, accompanied with increasing of 24-hour urine potassium excretion (P<0.05). Conclusion A1AR may mediate the renin secretion stimulated by low salt diet in mice.

The tumor pH microenvironment enhance stemness properties via β-catenin/TCF4 activation in human breast cancer cells

2014, 34(5):  622-627. 

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Objective To investigate the effects of acidic tumor microenvironment on maintaining stemness and underneath mechanism in breast cancer stem cells. Methods Cultured the human breast cancer cell MDA-MB-231 with either pH 6.8 or pH 7.4 medium, the percentage of ALDHhigh subpopulation, the expression of SOX2, OCT4, Lin28b and the stemness properties were detected by using FCM, Real-time PCR, Western blot, sphere test and scratch experiments. The activation of signaling pathway was also detected. Results All stemness properties of MDA-MB-231 cell cultured with pH 6.8 conditional medium were increased including the percentage of ALDHhigh subpopulation（P<0.05）, the expression of stemness gene（P<0.01）, the capability of sphere formation（P<0.01） and cell mobility（P<0.05）. These stemness properties of MDA-MB-231 cell were regulated via β-catenin/TCF4 signaling pathway that activated by pH 6.8 cultured medium. Conclusion The acidic tumor microenvironment increases the percentage of ALDHhigh subpopulation and enhances stemness phenotypes of breast cancer cells.

Selection and Identification of a Novel DNA aptamer against CD20 molecule

2014, 34(5):  628-632. 

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Objective To develop a CD20 aptamer that may potentially serve as a tumor-homing ligand for targeted therapy against Non-Hodgkin’s lymphoma (NHL). Methods A single-stranded 59nt DNA library containing 21nt random oligonucleotides was synthesized. A new CD20 aptamer termed CE4-1 was developed with SELEX technique, using a CD20 epitope as the target. Flow cytometry was performed to monitor the enrichment of the selected DNA pool, and the binding properties of CE4-1 towards CD20 structure and CD20-positive cells. The structure of CE4-1 was predicted by MFold software. Results The DNA aptamer CE4-1 could selectively bind with CD20 structure and CD20-positive cells, with minimal cross reactivity to BSA and CD20-negative cells. Additionally, trypsin treatment greatly reduced the binding of CE4-1 to CD20-positive cells. Conclusions A novel CD20 aptamer CE4-1 could recognize CD20 structure and CD20-positive cells selectively, which may have application potentials in targeted therapy against the CD20-positive tumors.

Identification of Epitopes for Neutralizing Monoclonal Antibodies against Highly Pathogenic Avian Influenza H5N1 Virus Hemagglutinin

2014, 34(5):  633-637. 

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Objective To generate and characterize neutralizing monoclonal antibodies against highly pathogenic avian influenza (HPAI) H5N1 strains in China and corresponding epitopes. Methods 9 candidate peptides of HAs were designed tandem epitopes to generate monoclonal antibodies using a patent technology of SEAL (Surface Epitope Antibody Library). Ascites were purified by affinity chromatography. Neutralizing monoclonal antibodies were characterized by ELISA, western blot and micro neutralization assays. Results 23 hybridomas clones anti-HA epitope monoclonal antibodies were obtained，in which 3 IgG3 subtype clones showed efficient neutralizing activity to H5N1 pseudotype virus and recognized corresponding epitopes " LIKKNNAYPT " and " R/KSSFFRNVVW " in a dose dependent manner. These 3 mAbs recognized rHA from 4 different branches of H5N1 demonstrated by immunoblot. Conclusions 3 high levels of neutralizing monoclonal antibodies were characterized. Two important neutralizing epitopes of HA were identified which might serve as a strong potential candidate for an effective vaccine to protect from highly pathogenic avian influenza H5N1 virus in China.

The combination of small-molecule inhibitors targeting the key proteins in the ER stress response and anticancer drugs show the inhibition effects to HeLa cells

2014, 34(5):  638-643. 

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Objective To investigate the therapeutic potential application of EeyarestatinI (EerI) and 4μ8C, the small-molecule inhibitors of UPR signaling pathway in the cervical cancer. Method The protein expression of GRP78/BiP, P97, Ubiquitin and IRE1α in normal cervical epithelia and cervical cancer were examined by immunohistochemistry. The effect of EerI and 4μ8C on HeLa cell proliferation and apoptosis were measured by MTS assay and flow cytometry, respectively. The combined antiproliferative effects of small-molecule inhibitors were carried out in combination of Bortezomib (BTZ) and Cisplatin (CDDP), respectively. Result GRP78/BiP, P97, Ubiquitin and IRE1α were significantly up-regulated in cervical cancer compared with normal cervical epithelia and displayed variability in the cervical cancer of different clinical stages (P<0.05). EerI and 4μ8C inhibited HeLa cell proliferation in a dose-dependent manner with inducing HeLa cell apoptosis in a similar fashion (P<0.05). MTS assays indicated that combination treatment significantly enhanced the antiproliferative effect of single chemotherapy drugs (P<0.05). Conclusion Small-molecule inhibitors targeting the key players of UPR show synergistic effects with other anticancer drugs, suggesting a new therapeutic strategy towards cervical cancer.

Enhancement of thermal damage to HER2-positive breast cancer cells by aptamer-guided magnetic nanoparticles

2014, 34(5):  644-647. 

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Objective To develop aptamer-modified nanoparticles (AptNPs) for targeted enhancement of thermal damage to HER2-positive breast cancer cells Methods HER2 aptamer was connected to NPs via biotin-streptavidin reaction. AptNPs were characterized by Dynamic Light Scattering (DLS). The binding feature of the aptamer was evaluated by flow cytometry, and the affinity of AptNPs to target cells by phase-contrast microscopy. Thermal damage under alternative magnetic field was measured by MTS assay. Results The average size of AptNPs was 333.7 nm. AptNPs exhibited strong binding to the HER2-positive but not the HER2-negative cells. Importantly, AptNPs enhanced the thermal damage to the HER2-positive tumor cells, but not that to the HER2-negative cells. Conclusions Aptamer-guided iron particles may have potential utility in development of novel HER2-targeted thermal therapies.

Effect of HIFU on hypoxia-inducible factor (HIF-1α) expression and cell apoptosis in liver cancer cell xenografts of nude mice

2014, 34(5):  648-653. 

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Objective To study the effects of hypoxia-inducible factor (HIF-1α) and apoptosis of the residual tumor cells in nude mice after treatment by high- intensity focused ultrasound (HIFU).Methods Human HCC implantation was established in 30 nude mice. CZF-Ⅱ HIFU therapeutic apparatus were used for treatment .Nude mice were randomly divided into control group and treatment group (included 1d group, 3d group, 5d group, 1w group,and 2w group). Pathological changes were observed with HE staining;Cell apoptosis were detected by DNA agarose gel electrophoresis and terminal deoxynucleotidyltransferase-mediated Dutp nick end-labeling (TUNEL) assay; SP immunohistochemistry was used to detect HIF-1α protein;Western blot and Real-time quantitative PCR were used to detect HIF-1α, P53 protein and mRNA expression levels. Results HE showed that there were residual tumor cells and large necrotic areas after treatment. Compared with other groups, the apoptosis index in 3d group was significantly higher (P <0.05),in the 5d group,1w group,2w group gradually decreased. Immunohistochemistry showed that the expression of HIF-1α, P53 protein was different in each group. Compared with other groups,Western blot and RT-PCR showed an increase of HIF-1α, P53 protein and mRNA levels in groups after treatment of 1 to 3 days, and peaked in 3d group(P <0.05). Conclusion HIFU treatment can induce consistent apoptosis in residual tumor, which may be related with disordered expression of HIF-1α and P53.

Pulsatilla saponin D –induced cell apoptosis in the U87MG cells

2014, 34(5):  654-660. 

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Objective To investigate the proliferation inbibition and apoptosis induction of the Pulsatilla saponin D on the human malignant glioma cell lines U87MG in vitro. Method The proliferation—inhibiting effect and apoptosis of the U87MG cells treated with the different concentrations of the Pulsatilla Saponin D for different times in vitro were detected by MTT assay and the flow cytometry respectively，and the cell apoptosis were examined by Hoshest 33342、the Fluorescence fiber mirror、TUNEL/PI、DNA-ladder，and the apoptotic pathways were analysed by the Western blot analysis. Result The proliferation inhibition of the U87MG by the Pulsatilla saponin D were 94.034%(n=6, p<0.05). The nuclear shape of the U87MG cells was changed after being treated with Pulsatilla saponin D, and stained by Hoechst 33342 in a time-and-dose dependent manner. The total protein was extracted from the U87MG cells and the expression of the caspase-3、8、Bax and Fas-L were showed to be increased, while the level of Bax expression reduced, and the level of Bcl-2 changed unobviously. Conclusion Pulsatilla saponin D can inhibit the proliferation of human malignant glioma cell lines U87MG at time-dose dependent manner and induce the apoptosis of U87MG cells through the Fas/Fas-L pathway.

Effect of Roux-en-Y gastric bypass surgery on renal gluconeogenesis in type 2 diabetes mellitus rats

2014, 34(5):  661-666. 

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Objective To investigate the effect of Roux-en-Y gastric bypass surgery on renal gluconeogenesis in type 2 diabetes mellitus rats, and explore its possible hypoglycemic mechanism. Method Build diabetes mellitus animal models . Diabetic rats were divided into four groups randomly: control group , diabetes model group (DM group),sham Roux-en-Y gastric bypass group (SRYGB group), Roux-en-Y gastric bypass group (RYGB group). Plasma TG、TC and FFA were measured respectively before and 4th week after the operation in each group rats; the fasting insulin was measured and OGTT was taken before and 1st、2nd and 4th week after the operation, calculate the AUC of blood glucose concentration - time and ISI. The key enzymes of renal gluconeogenesis: G-6-P and PEPCK were detected using RT-PCR and Western blot respectively 4th week after the operation in each group rats. Results 4th week after surgery, RYGB group rats compare with DM and SRYGB group rats, Roux-en-Y gastric bypass surgery can reduce the level of blood lipid (P< 0.05),decrease the blood glucose of fasting and 2h after OGTT significantly (P<0.05), reduce the AUC (P<0.05), increase insulin sensitivity(P<0.05) ,decrease expression of G-6-P and PEPCK mRNA and protein in some degree. Conclusion: Roux-en-Y gastric bypass surgery can reduce blood glucose in diabetic rats significantly,improve the glucose tolerance, its mechanism may be related to decrease of G-6-P and PEPCK mRNA and protein levels in renal tissue，weaken renal gluconeogenesis.

1,25-dihydroxyvitamin D3 inhibits of TRPC6 expression in kidney of rat induced by puromycin aminonucleoside

2014, 34(5):  667-673. 

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Objective To evaluate Effect 1,25-dihydroxyvitamin D3 ［1,25(OH)2D3］on TRPC6 expression in kidney of rat model in puromycin aminonucleoside nephropathy(PAN). Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: PAN model group (Mon), 1,25(OH)2D3 treated group(Vit D) and normal control group (Con). Con and Vit D rat medols were constructed by consecutive three time intravenous injection PAN of 100 mg.kg-1 body weight every ten days and Vit D rats were gavaged 1,25(OH)2D3(0.2μ g.kg-1.d-1 ). The rats were sacrificed at one and three months respectively after PAN injection. 24-hour urinary protein excretion was determined. Renal Function and blood lipid were determined by an autoanalyzer. The renal tissue morphology was observed with PAS staining by light and electron microscope. The expression of nephrin, TRPC6 mRNA were evaluated by RT-PCR. The protein expression and location of nephrin and TRPC6 were decided by confocal microscope. Results PAN administration caused heavy proteinuria, hydroperitoneum, hyperlipoidemia, hypoproteinemia and as well as loss of renal function--a classics nephritic syndrome symptoms. Inflammatory cell infiltration, a devil of a protein cast, renal interstitial edema, partly renal tubule atrophy and fibrosis and focal segmental gloumerular sclerosis was obvious in pathology. 24h urinary protein［One month, (338±120)mg vs (669±142)mg, three months (432±83)mg vs (601±95) mg, P <0.01］; Index of gloumeruslocis in three months［(2.3±0.6）vs (3.4±0.4), P<0.01］and renal function［Cr（40.2±3.4）ummol/L vs（53.4±6.3）ummol/L, BUN(9.4±3.0)mmol/L vs（17.3±2.9)mmol/L, P <0.01］were significant lower in Vit D group compared with Mod group, but was not significant difference in blood fat. TRPC6 mRNA expression was increased and Nephrin mRNA expression were decreased in PAN rats model. Compared with Mod group rat, TRPC6 mRNA expression［1 month, (0.42±0.10)vs(0.75±0.14), three months (0.35±0.07) vs (0.68±0.10), P <0.01］were significant lower, and Nephrin mRNA［one month, (0.81±0.19) vs (0.33±0.09), three months(0.77±0.10)vs(0.44±0.10), P <0.01］were significant higher. Conclusions TRPC6 mRNA expression was increased and Nephrin mRNA expression were decreased in PAN rats model. The renoprotective of 1,25(OH)2D3 may be partly attributable to TRPC6 suppress.

Construction and functional identification of miR-196b lentiviral vector

2014, 34(5):  674-678. 

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Objective To construct miR-196b lentiviral expression vector to lay the foundation for the study of miR-196b function in chronic myeloid leukemia and its role in the development of CML. Methods The precursor sequence of miR-196b was amplified by PCR with human bone marrow genomic DNA as a template, vector plVTHM-miR-196b was recombinant with lentiviral vector plVTHM, packaged and titered. The transfection efficiency and reporter gene expression efficiency of K562 cells infected with the virus solution were observed by fluorescence, and the GFP-positive cells with a green fluorescence were sorted by flow cytometry, and intracellular miR-196b expression level was detected using real-time quantitative PCR. Finally, proliferation of cells in each group was detected by CCK-8 assay. Results plVTHM-miR-196b lentiviral vector was successfully constructed; the carrier had a better expression of GFP activity; compared with control cells, miR-96b expression levels of the test group were significantly increased. The proliferation levels of K562 cells stably over-expressing miR-196b decreased significantly. Conclusion The miR-196b overexpression vector had been successfully constructed and highly and stably expressed in K562 cells, and miR-196b can inhibit the proliferation of K562 cells, these results conduct a foundation for the corresponding function of miR-196b in CML cells and in vivo.

Screen for chemical mutagens based on the recombinant Lac Z gene yeast cells regulated by RNR3 promoter and 96 - well micro plates

2014, 34(5):  679-683. 

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Objective To develop a rapid screening method for chemical mutagens in vitro. Methods RNR3 promoter was amplified using polymerase chain reaction (PCR) from yeast genome and inserted into the yeast report vector of pMP206 / ERE to construct yeast Lac Z gene report vector regulated by RNR3. This vector was transformed into the yeast cell of W303-1A. When the yeast cell was treated by the chemical mutagenesis, the expression of Lac Z gene was induced. So it can be used to screen the chemicals with DNA toxicity by measuring the beta - galactose enzyme activity. Results Various chemicals that cause DNA damaged can induce RNR3 expression. The mutagens alkylating DNA (methyl methanesulfonate and chlorambucil), the cleavages of DNA (cisplatin, 4- nitro -N- oxidation of quinoline, bleomycin and phleomycin) and the inhibitor of DNA polymerase or topoisomerase (5- fluorouracil, hydroxyurea and camptothecin) can induce RNR3 expression. Conclusions The recombinant Lac Z report gene yeast cells regulating by RNR3 can be used for the screening of many chemical mutagen and this screen can be carried out in 96 well micro plate, so it has a high throughput.

Determination of moxifloxacin hydrochloride in beagle dog plasma by LC-MS/MS and its application to pharmacokinetics

2014, 34(5):  684-689. 

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Objective To establish a sensitive and specific LC-MS/MS method for determination of Moxifloxacin Hydrochloride in Beagle dog plasma and to evaluate the pharmacokinetic characteristic in dog. Method The concentration of Moxifloxacin Hydrochloride was determined by LC-MS/MS with XTerra@MS C18 column (5μm, 2.1mm×150mm). The mobile phase consisted of methanol-water (50:50, v/v) with the flow rate of 0.2 ml/min. The injection volume was 10 μl. Ions of moxifloxacin and Ciprofloxacin were m/z 402.1→m/z 384.3 (F=120V, CE= 25), m/z 332.2→m/z 314.2(135V, CE=20). Results Standard calibration graph for Moxifloxacin Hydrochloride was linear over a curve range of 10μg/L -2000μg/L (R2>0.99) and the lower limit of quantification was 10 μg/L using a plasma sample of 100μL. The intra-day and inter-day precision (RSD) were in line with analysis of the biological samples. RSD were less than 11.6%, the extraction recoveries were greater than 80%. The concentration-time curves of Moxifloxacin Hydrochloride were best fitted to a two compartment model. The main pharmacokinetic parameters of t1/2, tmax, AUC0–t, AUC0–∞ and CL were 10.7h, 2.1h, 3943μg/L?h, 4177μg/L?h and 10.5L/h?kg respectively. Conclusion The analysis method was specific and suitable for the determination of Moxifloxacin Hydrochloride in Beagle dog plasma and to study the pharmacokinetic character.

Inhibitory Effects of Nodakenin on the Airway Inflammation and NF- κB Signaling Pathway in a Murine Asthmatic Model

2014, 34(5):  690-694. 

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Objective To observe the effects of nodakenin on airway inflammation in a mouse model of allergic asthma. Methods BALB/c mice were assigned randomly to one of the following four experimental groups: control, model, nodakenin and dexamethasone. All mice, except for those in control group, were sensitized and challenged by OVA to induce airway inflammation. One hour before every OVA challenge, nodakenin group was administered intragastrically with nodakenin at a dose of 10 mg/kg, and dexamethasone group was injected intraperitoneally with dexamethasone a dose of 1 mg/kg. Airway responsiveness was measured by a lung function analysis systems. The number of total leukocytes in BALF was counted using a hemocytometer, and differential cells were counted using Diff-Quick-stained smears. Histopathology of lung tissue was analyzed by Hematoxylin-eosin staining. Levels of inflammatory mediators in serum or BALFs were measured by ELISA. The activity and expression of proteins in NF- κB signaling pathway was respectively evaluated by EMSA and western blot analysis. Results Compared with control group, the model group exhibited obvious airway inflammation, airway reactivity were significantly increased, the levels of total cells and differential cells were significantly increased, levels of IL-4, IL-5, IL-13, IgE were significantly increased, levels of nuclear p65 and p-p65 protein was significantly enhanced, levels of cytoplasmic p65 and IκBα protein were significantly decreased, and the NF-κB DNA binding activity was significantly increased. Compared with model group, nodakenin significantly suppressed airway inflammation, airway hyperreactivity, reduced levels of IL-4, IL-5 and IL-13 in BALF, and IgE in serum, decreased levels of nuclear p65 and p-p65 protein, increased cytoplasmic p65 and IκBα protein, and increased the NF-κB DNA binding activity. Conclusion Nodakenin efficiently inhibited antigen-induced airway inflammation in asthmatic mouse.

Ten years experience of perioperative management of pheochromocytoma: 307 cases

2014, 34(5):  695-698. 

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Objective To summarize the experience of perioperative management of pheochromocytoma in Peking union medical college hospital in the past decade. Methods 461 cases scheduled for surgical removal of pheochromocytoma were involved in this study. At last, Data from 307 cases were finally analyzed. Cases from September 2002 to august 2012 were treated as an experimental group, while cases from September 1992 to august 2002 were treated as a control group. We just focus on preoperative preparation, intraoperative anesthetic management, intraoperative organ injury, postoperative ICU stay, postoperative hospital stay in the past 10 years. Result In the past 10 years, laparoscopic adrenalectomy for pheochomocytoma accounted for 84.1%. Preoperative preparation time was shortened by 8.9 days, the drainage tube removal time was shortened by 3.4 days, postoperative ICU stay time was shortened by 3 days, postoperative hospital stay time was shortened by 8.4 days， p<0.01. Conclusion Multidisciplinary collaboration to optimize treatment strategies can significantly improve perioperative prognosis of pheochromocytoma.

Two different buffer systems of paraformaldehyde effect on mouse brain tissue histochemistry experiment results

2014, 34(5):  699-700. 

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Substance P attenuates hyperoxia induced lung injury in neonatal rats

2014, 34(5):  701-703. 

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Lignans promots human gastric cancer MGC - 803 cell apoptosis

2014, 34(5):  704-706. 

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Human recombinant protein thymosin β10 promote apoptosis of ovarian cancer cell line SKOV3

2014, 34(5):  707-708. 

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Effect of intercostal nerve cryoanalgesia on post-thoracotomy pain

2014, 34(5):  709-710. 

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The role of microRNAs in dibetic nephropathy

2014, 34(5):  711-714. 

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Diabetic nephropathy is one of the important complications of diabetes mellitus. Although the mechanisms of diabetic nephropathy are not fully understood, recently a large number of studies demonstrated a role of epigenetic factors in the development of this disease. Among them, microRNAs (miRNAs) have been shown to be functionally important in the progression of diabetic nephropathy, such as maintaining the normal function of podocytes and the homeostasis of extracellular matrix.

The molecular mechanisms of exercise exerting prophylaxis and treatment of obesity

2014, 34(5):  715-718. 

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The mechanisms underlying the therapeutic effects of exercise on obesity has remained unknown until irisin, a novel protein-hormone, was identified. Exercise—PGC-1α—FNDC5/irisin—UCP1 pathway is the molecular mechanism of the exercise that can exert prophylaxis and treatment effect on obesity.

Clinical value of monitoring the extravascular lung water and pulmonary vascular permeability in patients with ARDS

2014, 34(5):  719-722. 

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The change of Extravascular Lung Water can quantitatively reflect the existence and severity of early pulmonary edema for the patients with ARDS,it is significant correlation with the prognosis and good for early diagnosis and making treatment guidline for ASDS. Pulmonary Vascular Permeability Index can indicate pulmonary vascular permeability directly which can be used to identify the reasons of pulmonary edema and reflect the injury severity of lung and to judge the prognosis of ARDS patients.