Abstract:Objective To investigate the regulatory effect of HIV-1 Tat on mitotic centromere-associated kinesin (MCAK） expression and the related mechanism. Methods Tat-expression TE671 cell model and purified prokaryotically expressed Tat protein were used to verify the down-regulated expression of MCAK using Northern blotting and Western blotting analysis. Various DNA fragments in the promoter region of the MCAK gene were amplified with PCR, linked to the luciferase reporter plasmid, and then transferred into TE671 cells. Luciferase activity analysis was performed to measure the promoter activity of various DNA fragments, so as to determine the active promoter region of MCAK gene. Moreover, HIV-1 Tat protein was co-incubated with TE671 cells, and the promoter activity was detected to analyze the modulating effect of Tat on promoter activity in vitro. Results The results of Northern blotting and Western blotting analysis indicated that the mRNA and protein levels of MCAK were strongly decreased by Tat protein. The core region of MCAK promoter was located in -399~+1 bp region, and the promoter activity was strikingly inhibited by Tat protein. Conclusion HIV-1 Tat has a marked inhibitory effect on MCAK expression, which might be related to the decreased promoter activity caused by Tat protein.

Abstract:Objective To investigate the effect of siRNA targeting TGF-β inducible early gene (TIEG) on expression of Smad2 in advanced glycation end products (AGEs)-mediated renal tubular epithelial cells. Methods The pshRNA-copGFP-lentivector containing target gene was used as a vector to construct siRNA-TIEG, which was then transfected into normal rat proximal tubular epithelial cells(NRK52E),which were then cultured in RPMI 1640 medium supplemented with AGE-BSA for 24 h and 48 h. The expression of Smad2 mRNA and protein was examined by PCR, Western blotting analysis, respectively. Blank vector group served as control.Results SiRNA-TIEG significantly reduced the expression of TIEG mRNA, Smad2 mRNA and Smad2 protein in normal rat proximal tubular epithelial cells in presence of AGE-BSA compared with those in the control group (P<0.05). Conclusion Silence of TIEG can effectively inhibit the expression of Smad2 mediated by AGEs in NRK52E cells.

Abstract:Objective To construct the eukaryotic expression vector of human survivin gene and stably transfect it into mouse melanoma B16 cell line. Methods The full length human survivin cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo; the restriction enzyme position and 6 his tag were added to obtain recombinant plasmid pIRES-neo-SUR-(his)6 , which was then transfected into B16 cells by Lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, and the transcription and expression of the human survivin gene were identified by RT-PCR, Western blotting analysis and immunofluorescence assay. Results The result of restriction enzyme digestion and the sequence analysis showed that the recombinant of pIRES-neo-SUR-(his)6 was successfully constructed. RT-PCR results showed survivin gene (about 530 bp) was amplified from the total RNA in the group stably transfected with pIRES-neo-SUR-(his)6. Western blotting analysis showed the expression of survivin protein in pIRES-neo-SUR-(his)6 transfection group, but not in the control group. FACS and immunofluorescence assay showed high fluorescence signal in pIRES-neo-SUR-(his)6 transfection group, with the fluorescence positive rate being 91.38% when No. 5 monoclonal antibody was used; no fluorescence signal was found in the control group(transfected with pIRES-neo). Conclusion We have successfully constructed the eukaryotic expression vector of human survivin pIRES-neo-SUR-(his)6, and established a B16 cell line stably and highly expressing human survivin gene.

Abstract:Objective To analyze the epitopes of cDNA sequences of Deinagkistrodon acutus snake venom metalloproteinases using bioinformatical method, and to observe the immune protective effect of the new immunogen designed according to the identified epitopes. Methods The cDNA sequences of Deinagkistrodon acutus snake venom metalloproteinases were amplified by PCR. The epitopes of the sequences were analyzed by Jameson-Wolf method and Clustal X software, then the sequences of the screened epitopes were artificially synthesized and linked to the vector pIRESneo. BALB/c mice were immunized by the resultant plasmid at 0, 2, and 4 weeks for three times, then the titers of the anti-serum were measured by ELISA. The immune protective effects of the anti-serum were tested by the neutralization of venom hemorrhagic activity and venom attacking test. Results Bioinformatical analysis yielded 6 epitopes (MPA-1-MPA-6). The ELISA results of anti-serum showed that these epitopes could induce immune reaction in mice, and the anti-serum was detectable even when it was diluted to 1100. The neutralization test and venom attacking test demonstrated that the anti-serum induced by the epitodes could neutralize the venom and protect the mice from haemorrhage. Conclusion Six epitopes of Deinagkistrodon acutus snake venom metalloproteinases have been obtained successfully using bioinformatical method, and the new immunogen designed based on these epitopes shows a primary immune protective effect.

Abstract:Objective To evaluate the role of decellularized vascular scaffolds coated with anti-CD34 antibodies in capturing endothelial progenitor cells from the circulation to participate in recellularization.Methods Fresh caprine forelimb arteries were treated with repeated frozen/thawing, ultrahigh pressure and SDS to prepare decellularized vascular scaffolds. After reaction with photochemical crosslinker SANPAH, anti-rabbit CD34 was coated onto the decellularized vascular scaffolds using ultraviolet ray. Twenty New Zealand white rabbits were performed for right lower-abdominal pedicled skin flap, which were supplied by branches of femoral artery. Whole defects (1 cm) were made in femoral arteries and end-to-end anastomosis repaired by 10 cross-linked and 10 non-cross-linked scaffolds in experimental group and control group, respectively. The patency of the pedicle was observed through color and appearance of the flap postoperatively. After transplantation, patency rate and cell seeding were detected by Doppler, DSA, and pathological test. Results Flaps of the experimental group had good blood supply after transplantation, while swelling and necrosis could be found in the control group. Doppler and DSA showed that the 7 of 10 cross-linked scaffolds remained patent for 4 weeks and there was no stenonsis,but all the scaffolds were obstructed in the control group after 1 week. Hematoxylin and eosin staining revealed that the inner layers of cross-linked scaffolds were partly covered with endothelial cells four weeks later, and there was no noticeable stenosis. In contrast, thrombosis formation was noticed in the control group and there was no cell coverage one week after operation. Conclusion Cross-linked scaffolds with anti-CD34 antibodies are superior to bare scaffolds in early postoperative anticoagulation and patency.

Abstract:Objective To investigate the effect of deguelin on mitochondrial permeability transition pore of human breast cancer cell line MDA-MB-231. Methods The inhibitory effect of deguelin on cell proliferation was determined by MTT assay; cell apoptosis rate was analyzed by flow cytometry with AnnexinⅤ FITC／PI double staining; MDA-MB-231 cells were stained by Rhodamine123 to detect the changes of mitochondrial transmembrane potential by FCM; alteration of protein of Cyt c outside of mitochondria was detected by Western blotting analysis; caspase-3 activity was assessed by colorimetric assay; and MDA-MB-231 cells were stained by Fluo-3/AM to detect changes of intracellular Ca2+ concentration by FCM. The expression of Bcl-2 and Bax was examined by RT-PCR and Western blotting analysis. Results Deguelin significantly inhibited the growth of MDA-MB-231 cells in a time- and dose-dependent manner(P<0.05). After treatment with deguelin, mitochondrial transmembrane potential was decreased, the expression of Cyt c outside of mitochondria was increased, and caspase-3 activity was significantly increased compared with negative control group(P<0.01). FCM analysis showed that the apoptotic rate of MDA-MB-231 cells and intracellular Ca2+ concentration increased gradually with the increase of deguelin concentration. RT-PCR and Western blotting analysis showed that the expression of Bcl-2 mRNA and protein was down-regulated and that of Bax was up-regulated after deguelin treatment. Conclusion Deguelin can inhibit proliferation and induce apoptosis of MDA-MB-231 cells, and the induction of apoptosis might be related to increased intracellular Ca2+ concentration and changes of mitochondrial permeability transition pore induced by altered Bcl-2, Bax expression.

Abstract:Objective To investigate the influence of tanshinone Ⅱ A on learning and memory ability, expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinase Ⅱ (MMP2) and release of free radicals in the hippocampus of rat Alzheimer’s disease (AD) model, and to explore the related mechanism. Methods Rat AD models were established by direct βamyloid protein (Aβ) injection method. For tanshinone Ⅱ A (Tan Ⅱ A) intervention test, the learning and memory ability of the AD rats were observed, the expressions of iNOS, MMP2 at gene and protein levels were determined and their correlation were analyzed, and the release of free radical in the hippocampus region of AD rats was quantified. Results The expressions of iNOS and MMP2 in the hippocampus region of AD rats were significantly higher than that in the control group (P<0.05), and the expressions of iNOS and MMP2 were positively correlated at both mRNA and protein levels (P<0.05). Meanwhile, nitric oxide (NO), ONOO and reactive oxygen species (ROS) release in the hippocampus of AD rats were significantly higher than those in the control rats (P<0.05). The learning and memory ability was obviously decreased in AD model rats. Tan Ⅱ A intervention (50 mg/kg) significantly reduced the ADinduced release of NO, ONOO, and ROS, and expression of iNOS (P<0.05) and MMP2 protein (P<0.01) in the hippocampus region; it also greatly improved the learning and memory ability of AD model rats. Conclusion Tan Ⅱ A can effectively alleviated the AD symptoms, possibly by inhibiting ADinduced iNOS and MMP2 expressions, reducing toxic free radical, and suppressing oxidative injury in AD rats.

Abstract:Objective To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E.coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes（P<0.05）. Conclusion Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening.

Abstract:Objective To observe the bone graft fusion of goat cervical model implanted with anterior cervical-adjustable fusion fixator(AC-AFF), laying a foundation for future studies.Methods Eighteen experiment goats were implanted with AC-AFF, titanium mesh and autogenous iliac bone combined cervical plate after corpectomy. Six months later, the fused cervical spine vertebrae were collected, prepared, and observed grossly, under microscope, by X-ray photographs and CT scan; the fusion of the graft was evaluated. Meanwhile, the fusion of AC-AFF with the adjacent vertebral surface was observed. Results All the experiment animals survived after operation; all the implants were stable, without displace or loosening. The interface where the titanium or AC-AFF contacts the vertebral body surface was bulged and ossified. All iliac bone graft fused well and a great deal of bony callus was found covering the vertebral body surface. Lucency area was not seen surrounding the bone graft, titanium and AC-AFF on the X-ray films, and there was new bone formation in the interface of internal fixation and bone. The central region of titanium and AC-AFF became vague and new bone formation was found in the intracavitary area of implants by CT scan. And part of the new bone transited the parietal reticulation and connected with normal ossea at titanium and AC-AFF. A large number of phoroblasts and chondrocytes were found microscopically in the bone fusion sites in all cases; however, the region without bony callus still existed in the titanium mesh lateral wall.Conclusion There are no differences in bone fusion between AC-AFF with titanium mesh and autogenous iliac bone combined cervical plate. New phoroblasts and chondrocytes can be generated in the bone graft interface. AC-AFF has been proven as an important method for cervical stability reconstruction after decompression operation.

Abstract:Objective To explore the changes of nuclear factor-κB (NF-κB) activity, TNF-α, IL-1β and IL-10 contents in the lung tissue of rabbit with seawater drowning-induced acute lung injury (SWD-ALI) and the effects of dexamethasone on the changes. Methods Forty-two New Zealand white rabbits were randomly allocated to control group (C group, n=18), seawater drowning group (S group, n=12) and dexamethasone treatment group (D group, n=12). The drowning model was established by instilling seawater (2 ml/kg body weight) into the endotracheal catheter of animals, then the rabbits received arterial injection of dexamethasone (D group, 1 mg/kg body weight) or 2 ml normal saline (S group). Blood gas analysis was done at predefined time points. The lung wet to dry weight ratio (W/D) and lung permeability index (LPI) were calculated. The activity of NF-κB was analyzed by non-radioactive electrophoretic mobility shift assay (EMSA). The contents of TNF-α, IL-1β and IL-10 were detected by ELISA. Meanwhile, the pathology changes of lung tissues were detected by H-E staining, and the semi-quantitative lung pathologic scores (LPS) was also calculated. Results The lung of rabbits in S group was obviously enlarged and had more severe edema and congestion compared with C group; furthermore, histopathologic findings indicated inflammatory cell infiltration and other pathologic signs of ALI; the oxygenation index (PaO2/FiO2) reached the bottom at 0.5 h and did not elevate to more than 300 mmHg(1 mmHg=0.133 kPa) until 6 h. The largest W/D ratio appeared at 3 h after seawater drowning, and the highest LPI and LPS appeared at 6 h; and NF-κB activity and the contents of TNF-α, IL-1β and IL-10 in lung tissues were significantly higher in S group than in C group（P<0.05,P<0.01）. The pathological changes of lung in the rabbits of D group were improved compared with S group but worse than those of group C. The W/D ratio, lung permeability index, and lung pathological score in D group were lower than those of the S group; and the oxygenation index was greatly improved 6 h after seawater drowning. NF-κB activity and the contents of TNF-α, IL-1β and IL-10 in lung tissues were significantly lower than those of the S group（P<0.05,P<0.01）. Conclusion Dexamethasone can inhibit the activity of NF-κB and the expression of TNF-α, IL-1β and IL-10 in the lung tissue of rabbits with SWD-ALI and relieve the inflammatory responses and pathological changes.

Abstract:Objective To observe the inhibitory effects of matrine, oxymatrine and sophordine on expression of CD91 and CD13 and secretion of TNFα in macrophage RAW264.7. Methods Macrophages were cultured in 6hole plate and coverslip was prepared. Then macropages were cultured one day with different concentrations(0,12.5,25,50,100,200 mg/L) of matrine, oxymatrine and sophordine; the expression of CD91 and CD13 by immunohistochemical dyeing was analyzed by ImagePro plus system (Media Cybemetic, Inc., 6.0 edition). Meanwhile, the levels of TNFα in the culture medium of macrophages were examined by ELISA. Results Matrine, oxymatrine and sophordine could all inhibit CD91 and CD13 expression in macrophage RAW264.7, and the inhibition varied with the concentrations of matrine, oxymatrine and sophordine; the inhibitory effect of matrine was the most prominent one. Moreover, matrine, oxymatrine and sophordine also inhibited the secretion of TNFα. Conclusion Matrine, oxymatrine and sophordine can greatly inhibit expression of CD91 and CD13 and secretion of TNFα in macrophage RAW264.7.

Abstract:Objective To investigate the expression of CDX2,PTEN,Ecadherin and NM23 in gastric cancer and its clinical significance. Methods Immunohistochemical SP method was used to detect expression of CDX2,PTEN,Ecadherin and NM23 in 77 gastric cancer specimens and 30 normal stomach mucosal tissues,and then the relationship of the expression with clinical pathology parameters was analyzed. Results The positive rates of CDX2,PTEN,Ecadherin and NM23 were significantly higher in gastric cancer tissues than those in the normal gastric tissues (P<0.05). The expression of CDX2,PTEN,Ecadherin and NM23 was correlated with the differentiation,histological type,infiltration,lymph node metastasis and TNM stage of the tumors (P<0.05),but not with patients’ gender, age or tumor size(P>0.05). The 1,3 and 5year survival rates of CDX2,PTEN,Ecadherin and NM23 positive patients were higher than those of negative patients（P<0.05, P<0.01）. Conclusion The expression of CDX2,PTEN,Ecadherin,NM23 in gastric cancer is related to the differentiation,infiltration,lymph node metastasis and TNM stage of gastric cancer, and detecting their expression in gastric cancer may help to judge the prognosis of gastric cancer.

Abstract:Objective To explore the feasibility of repairing opposite side ventriplanta soft tissue defects with the medial plantar artery combined flaps with saphenous nerve.Methods From Jan. 2007 to Apr. 2009, 12 patients with opposite side ventriplanta soft tissue defects were repaired using the medial plantar artery combined flaps with saphenous nerve. Five cases used cross leg flaps, and 7 cases used free skin flaps. All the cases were men, with an age range of 640 years (only one child case). The sizes of flaps were 12 cm × 7 cm13 cm×8 cm for adults and 8.5 cm×6 cm for child. The pedicles of cross leg flaps were cut off after 34 weeks.Results All the 12 flaps survived. The patients were followed up for 6 months to 1 year. The color，texture and appearance of the flaps were satisfactory．The flaps of the two point discrimination were 69 mm on ventriplanta of reconstruction, and the affected limb could exercise freely, without ulcer on the flap.Conclusion The medial plantar artery combined flaps with saphenous nerve can be used to repair opposite side ventriplanta vast soft tissue defects, with large flap and good texture, appearance. It can effectively recover the two point discrimination, but it has a complicated protocal.

Abstract:Objective To assess the efficacy and safety of thiazolidinediones combined with metformin in treatment of polycystic ovary syndrome.Methods Electronic database searching was performed on Medline, Cochrane Library, EMbase，EBSCO，ScienceDirect，OVID，Springer LINK，Wiley，Chinese biology and medicine(CBM), China National Knowledge Infrastructure (CNKI), and Chongqing VIP Database; meanwhile, unpubished literature, conference papers and dissertations were also searched manually. The data included those from the establishment to November 2009. Randomized or clinical controlled trials concerning the thiazolidinediones and metformin combination therapy for PCOS were selected and assessed for the methodological quality, and Meta-analysis was performed using statistical software Revman 5.0.Results Five randomized controlled trials met the inclusion criteria and were included. Compared to metformin group, thiazolidinediones combined with metformin significantly improved insulin resistance(P<0.000 01,WMD=-0.21,95%CI［-0.27，-0.15］) and ovulation rates(P=0.003, OR=2.76,95%CI［1.14,5.42］) . There was no difference in WHR(P=0.51,WMD=0.02,95%CI［-0.04,0.08］) and serum testosterone(P=0.20,WMD=-31.14,95%CI［-79.21，16.92］) between the combination and metformin groups. However, single metformin therapy was shown to have more benefit in ameliorating BMI(P=0.02,WMD=0.54,95%CI［0.08，1.00］) than the combination group. There were no serious adverse events during the treatment. Conclusion Thiazolidinediones combined with metformin can greatly improve of insulin resistance and ovulation rates in PCOS women; besides, it is comparatively safe.

Abstract:Objective To try to perform single-port laparoscopic nephrectomy in pigs, and to summarize and improve the basic skills, so as to decrease risks in clinical practice. Methods From Apr. to May 2009, we performed 10 times (5 pigs) of nephrectomy on porcine model using Triport system. The experience was summarized with respect to the following issues: the choice of endoscopy and instruments, insertion of Triport single port system, and optimization of the basic operation procedure. Results All the procedures were successful, without bleeding, organ injury or other complications. Operative time decreased from 75 min at the first time to 23 min for the last one. Clashing of instruments was the biggest frustration in the process; “X” shape crossing can partially resolve the problem, based on which four kinds of basic operating modes were designed. Conclusion Single-port laparoscopy nephrectomy in pigs can help the new learners to quickly improve their skills and decreases clinical risks.

Abstract:Objective To study the chemical constituents of Anthogorgia sp. collected from Beihai area of Guangxi Zhuang Autonomous Region, China. Methods The Et2O extract of Anthogorgia sp. was subjected to repeated silica gel (eluted with Pe∶Ac=99∶1, 49∶1, 39∶1, 34∶1, 29∶1, 24∶1, 19∶1, 14∶1, 11∶1, 9∶1, 8∶1, 7∶1, 6∶1, 5∶1, 4∶1, 3∶1, 2∶1, 1∶1, 100%Ac; CHCl3∶MeOH=24∶1, 19∶1, 14∶1, 9∶1, 7∶1, 5∶1, 3∶1, 1∶1, 1∶4, 1∶9, 1∶39, 100% MeOH) and Sephadex LH-20 column chromatography(n-hexane∶CHCl3∶MeOH=2∶1∶1) for isolation and purification. The structures of the resultant compounds were elucidated using spectroscopic analysis(1H NMR, 13C NMR and MS).Results Six steroids and one ceramide were isolated and identified as: Ergost-5,24(28)-dien-3β-ol(1),(22E,24R)-ergost-7,22-dien-3β,5α,6β-triol(2),(22E,24S)-ergost-5,22-dien-3β-ol(3), cholesta-5-en-3β,7β,19α-triol(4),(22E)-cholesta-5,22-dien-3β-ol(5), cholesterol(6), and N-palmitoyloctadecasphinga-4(E)-ene(7). Conclusion All the six compounds are isolated from Anthogorgia sp. for the first time.

Abstract:Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death worldwide; meanwhile, it is also one of the fastest growing malignancies. The causes of HCC are multiple; HBV is the most important cause in China, with about 25%-30% of HBV infection patients finally develop hepatic cirrhosis and liver cancer. At present, large scale epidemiological studies revealed that the metabolic syndrome (MS) was closely related to liver cancer, and it even served as an independent risk factor for the occurrence and progression of liver cancer.Non-alcoholic fatty liver disease is a metabolic syndrome clinically manifested in the liver; recently it has been indicated to be closely related with HCC development and progression. This paper reviews the recent researches on metabolic syndrome and liver cancer, so as to provide literature for preventive and therapeutic studies on non-virus-related liver cancer.

Abstract:The vast majority of traumatic cranial nerve injuries are associated with compression of fragment fracture, and microsurgery outside the epidural can be used for most of the cases. Therefore, early and accurate diagnosis of cranial nerve injury is especially important. As the cranial nerves go out of the cranial cavity through the holes and cracks of the skull base, and there are a number of special structures through which cranial nerve goes into the human skull, and they include the optic canal, superior orbital fissure, facial nerve canal, jugular foramen and so on. Most traumatic cranial nerve injuries are associated with these structures; however, the common imaging examination is very difficult for these structures due to their deep location. To further study the imaging diagnosis of cranial nerve injury associated with traumatic brain injury, this paper reviews the radiological technology for examination of the special positions in the skull.

Abstract:Establishment of a hairy root culture system from the medicine plants transformed by the Agrobacterium rhizogenes has unique advantages in large scale production of secondary metabolites and provides an effective solution to the shortage of resources. Meanwhile, root-induced(Ri) plasmid is also an ideal vector for plant genetic engineering. Insect-resistant, disease-resistant genes or genes encoding key enzymes involved in biosynthesis of plant secondary metabolites can be harbored by Ri plasmid and integrated into the host plant genome for expression, thus improving the plant traits and enhancing the secondary metabolite content. Establishment of plant transgenic hairy root culture system has laid a solid foundation for regulating plant secondary metabolites content and for industrial production of pharmaceutical active ingredients by using of genetic metabolic engineering strategies. This paper reviews the latest research advances in this field and the related applications.

Abstract:Objective To develop an effective preparation method to improve the sensitivity and accuracy of real-time PCR in detection of BK virus’s (BKV’s) load in urine samples. Methods A total of 24 samples documented as positive probes in primary detection were enrolled in this study. The candidate samples were prepared by 4 different approaches: unprocessed urine, BKV’s DNA extracted from urine, 110 diluted urine, and 1100 diluted urine; and then they were subjected to real-time PCR examination to obtain the viral load. The data obtained were analyzed by SPSS 11.0. Results The four different preparation processes for urinary specimens had significant impact on detection results of real-time PCR. Three samples were negative in the unprocessed urine group and 66.7% of its samples had the lowest viral loads compared with the other three groups. Two samples in the 1100 diluted urine group were negative and 79.2% of its samples had the highest viral loads, but its median load was similar to that of the 110 group. Viral gene was detected in all samples in the DNA extraction group and 110 diluted urine group, but the loss of the target gene was more severe in the DNA extraction group. Conclusion The 110 diluted urine is better for real-time PCR detection of BKV’s load, as it lose less viral gene and is more efficient, easy to perform and economical.

Abstract:Obesity is the most important risk factor of primary hypertension.Rapid increase of obesityrelated hypertension has become a worldwide problem. But the specific mechanism of the condition remains to be fully understood. This paper reviews the latest literatures and tries to explain the mechanism of obesityrelated hypertension from the following four aspects:hypoadiponectinemia, leptin and resistance of leptin, insulin and resistance of insulin and the activation of reninangiotensin system(RAS).

Abstract:目的 构建人亲环素A（CypA）基因的原核表达载体，诱导表达、纯化蛋白，制备抗体，为进一步研究其生物学作用奠定基础。方法 以人肺癌组织总RNA为模板，RT-PCR扩增CypA基因片段，克隆到pMD18-T载体中。扩增产物与原核表达载体pET30a(+)连接，构建表达质粒pET30a-CypA，转化大肠杆菌BL21(DE3)，IPTG诱导表达，用镍树脂亲和层析柱High-Affinity Ni-IDA Resin纯化表达产物，SDS-PAGE 检测纯化蛋白。用获得的蛋白免疫大白兔，制备抗CypA蛋白多抗，采用蛋白印迹法检测抗体特异性。结果 成功构建了CypA的原核表达载体，经大肠杆菌诱导表达、镍亲和层析柱纯化，得到纯度达80.2%的融合蛋白，免疫大白兔后得到多抗血清。蛋白印迹结果显示此多克隆抗体能与CypA蛋白特异性结合。结论 成功获得CypA纯化蛋白及CypA多克隆抗体，为进一步研究CypA在肺癌中的作用机制奠定了基础。

Abstract:目的 探讨潘妥洛克减轻胃缺血-再灌注（I/R）损伤和凋亡途径的关系。方法 C57BL/6小鼠随机分为假手术组、模型组（溶剂+手术组）、给药组（潘妥洛克+手术组）,每组10只。夹闭小鼠腹腔动脉30 min后松开动脉夹再灌注1 h制作胃I/R模型，根据分组情况于手术前1 h腹腔注射溶剂10 ml/kg或2 mg/ml的潘妥洛克溶液10 ml/kg。再灌注1 h后取胃标本铺平，拍照软件分析计算出血百分比。应用蛋白免疫印迹法检测比较各组Caspase-8、分裂型Caspase-3、Bax、Bcl-2、丝氨酸/苏氨酸蛋白激酶（AKT）以及磷酸化AKT的表达水平。结果 给药组胃黏膜糜烂出血面积明显减少，与模型组比较差异有统计学意义（P<0.01）；给药组凋亡相关蛋白Caspase-8、分裂型Caspase-3、Bax及Bcl-2与模型组相比无明显差异，而上游促修复抑制凋亡的关键信号蛋白AKT的磷酸化水平比模型组明显降低（P<0.01）。结论 潘妥洛克减轻胃缺血-再灌注损伤不依赖于抑制Caspase-3促凋亡途径，而可能与潘妥洛克抑制AKT的磷酸化有关。

Abstract:目的 研究子宫内膜癌中SFRP1基因启动子甲基化及其对SFRP1 mRNA表达的影响。方法 应用甲基化特异性PCR技术检测36例子宫内膜癌组织和对应癌旁组织中SFRP1基因启动子甲基化状态，RT-PCR方法检测上述样本的mRNA表达水平，并对结果进行统计学分析。结果 子宫内膜癌组织中SFRP1基因启动子甲基化的阳性率为47.2%，癌旁组织中为11.1%，两者比较差异有统计学意义 （P=0.001）。14例存在启动子甲基化和5例无启动子甲基化的子宫内膜癌组织中缺乏SFRP1 mRNA表达或表达较癌旁组织明显下降。结论 SFRP1基因启动子甲基化在子宫内膜癌中常见，部分成为其表达下降的原因。

Abstract:目的 研究夏枯草对淋巴瘤细胞（Raji细胞）生长的影响及可能的机制。方法 参考临床常用剂量,采用50 g/L夏枯草（夏枯草组）、50 g/L夏枯草+20 μmol/L JNK特异抑制剂SP600125（SP600125组）、50 g/L夏枯草+1 μmol/L Akt特异抑制剂Wortmannin（Wortmannin组）处理Raji细胞,以同体积生理盐水作为对照组,应用MTT法检测各组的细胞增殖率,蛋白免疫印迹法检测各组的JNK和Akt磷酸化水平。结果 夏枯草组细胞增殖率低于对照组（P<0.05）,SP600125组细胞增殖率比夏枯草组增高（P<0.05）,Wortmannin组细胞增殖率比夏枯草组降低（P<0.05）；JNK磷酸化水平在夏枯草组中明显升高（P<0.05）,SP600125可以抑制其磷酸化（P<0.05）,Wortmannin不能抑制其磷酸化（P>0.05）；Akt磷酸化水平在夏枯草组中降低（P<0.05）,SP60015及Wortmannin均可抑制其磷酸化（P<0.05）。结论 夏枯草可以明显抑制Raji细胞的生长,这种抑制作用可能是通过激活JNK信号转导通路,抑制Akt通路激活实现的。

Abstract:目的 采用高效液相色谱法测定复方大黄颗粒中盐酸小檗碱、淫羊藿苷及5种大黄蒽醌的含量。方法 采用Diamonsil C18色谱柱（200 mm×4.6 mm，5 μm），流速1.0 ml/min，柱温35℃，测定盐酸小檗碱与淫羊藿苷的流动相为乙腈-0.05 mol/L KH2PO4缓冲液（24∶76），检测波长为270 nm；测定5种大黄蒽醌的流动相为甲醇-0.1%H3PO4水溶液（75∶25），检测波长为254 nm。结果 盐酸小檗碱、淫羊藿苷、芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚基线分离，线性良好（r≥0.999 8）；方法学考察表明，日内及日间精密度符合相关标准，加样回收率分别为97.4%、97.8%、98.7%、97.1%、97.8%、99.8%、100.2%。测定了3批样品中盐酸小檗碱、淫羊藿苷及5种大黄蒽醌的含量（平均值）分别为18.77、1.54、0.490、0.678、0.786、1.56、1.41 mg/g。结论 该方法简便、快速，稳定可靠，为复方大黄颗粒的质量控制提供了依据。

Abstract:多奈哌齐为新一代中枢乙酰胆碱酯酶抑制剂,能够增加大脑乙酰胆碱含量,因其半衰期长、不良反应较小,是目前治疗早、中期阿尔茨海默病（AD）的有效药物；苯磺酸氨氯地平是第二代长效的双氢吡啶类钙拮抗药,用于治疗高血压和心绞痛,目前已成为临床治疗高血压的一线药物。由于临床治疗的需要经常要进行联合用药，目前，有关两药在药代动力学上的相互作用未见报道。为更好地指导临床医生合理用药，本试验采用HPLC-MS法\[1\]对两药在健康人体内的药代动力学相互作用进行研究，为临床治疗提供理论依据。

Abstract:炎性细胞因子在神经病理性疼痛的发生机制中具有重要作用\[1\]。我们的前期研究证实成人膝关节滑膜组织中存在μ阿片受体,且在慢性炎症期间表达上调,局部外源性阿片类药物的应用能够缓解慢性炎症的疼痛\[2\]；后续研究发现犬急性膝关节炎炎症期滑膜组织μ阿片受体同样表达上调\[3\]。因此,本研究在前期实验的基础上进一步观察膝关节腔内注射金黄色葡萄球菌诱发的急性犬膝关节炎模型中血清和膝关节滑膜组织中炎症因子（IL-1β、IL-6、IL-10、TNF-α）的表达,观察炎性因子可能的致痛作用,为后续镇痛研究奠定基础。

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