Objective To express and preliminary identify scavenger receptor B2 (SCARB2)protein.Methods SCARB2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR)from mRNA extracted from RD cells,and cloned into pMD19-T vector.Expression vector pET28a(+)/SCARB2 was constructed,and SCARB2 protein was expressed in prokaryotic ex-pression system.Obtained recombinant proteins were identified by detection of Western blot using His labeled monoclonal antibody.Results Recombinant SCARB2 proteins,expressed by induction of isopropy-β-D-thiogalactoside (IPTG),were success-fully purified and specifically recognized by His labeled monoclonal antibody.Conclusion SCARB2 proteins could be expressed and identified successfully,which could provide reference for researching mechanism of enterovirus type 71 (EV71)and scavenger re-ceptor protein,and for preparation of SCARB2 monoclonal antibodies.

Objective To study the genomic molecular organization and genogroup of human metapneumovirus(hMPV) infected infants in Guangzhou of China. Methods Primers were designed on the basis of the genomic sequence of hMPV 00-1 strain(AF371337) in the GenBank, and amplify hMPV genomeby RT-PCR. The PCR-products were cloned to T vector and sequenced, the genomic nucleotide sequences were analyzed with the programs Clustal W/X, DNASTAR and MEGA4. 1. Results The cloned strainhMPVgz01 genome is 13 327 bp in length, the genome contains eight open reading frames in the order 3-N-P-M-F-M2-SH-G-L-5. The genomic sequences of hMPVgz01 strain are compared with those of hMPV in GenBank, revealed that the homology with hMPV group A ranges between 92%-97%, homology with group B is 81%, and with avian metapneumovirus group C is 71%, the highest homology is with BJ1887 strain of genogroup A2b. The N, F, G genes of hMPVgz01 strain are compared with those corresponding genes of hMPV subgroups A1, A2, B1, B2, revealed that the highest homology is also with genogroup A2b. Conclusion The complete nucleotide sequence of hMPVgz01 strain isolated from Guangzhou in China is 13 327 bp in length, GenBank accession No. is GQ153651. Comparison of the genomic sequence and three genes of hMPVgz01 strain with those corresponding sequences of hMPV show the highest homology is with genogroup A2b. Sequence and phylogenetic analysis of the hMPVgz01 strain revegled that this isolate belongs to genogroupA2b.

BACKGROUND: Differentiation of bone marrow mesenchymal stem calls(BMSCs) is associated with their microenvironment. After acute myocardial infarction (AMI), the necrosis of cardiomyocytes caused activation of complement, generation of free radical, and secretion of various cell factors. As a result, the ingredients of patients' serum also changed, so does the changes will influence the differentiation of BMSCs into cardiomyocytes? And what influence it will be? OBJECTIVE: To investigative effects of AMI rat serum on the differentiation of BMSCs into cardiomyocytes. DESIGN, TIME AND SETTING: The in vitro controlled cytology experiment was performed at the Laboratory of Center for Stem cells and Tissue Engineering, Sun Yat-sen University from September in 2005 to June in 2006. MATERIALS: SPF Sprague Dawley rats, weighing 50-80 g, aged 3-4 weeks, were used for culture of BMSCs, and SpragueDawley rats, weighing 200-300 g, aged 6-8 weeks, were used for making AMI models. METHODS: BMSCs were isolated and cultured with the adherent method. After making AMI rat models by deligating anterior descending of the left coronary artery, the serum were collected and centrifuged from AMI and normal rats. Then the passage 3 of BMSCs were divided into six groups, non-induction group, 5-azacytidine (5-aza) group, 5-aza plus serum from AMI rat group, 5-aza plus serum from normal rat group, serum from AMI rat group, and serum from normal rat group. MAIN OUTCOME MEASURES: Morphology changes in rat BMSCs after induction; Expression of cardiac troponin T of BMSCs after induction; Expression of GATA-4 and desmin mRNA of BMSCs after induction. RESULTS: After being induced by 5-aza and two kinds of serum, some cells became elongated and thinner. Two to three weeks after induction, some cells had a ball-like or rod-like appearance, and connection were formed between adjacent cells. It showed that some BMSC have differentiated into cardiac like cells. The expression of cardiac troponin T, GATA-4 and desmin were positive in cells differentiated from BMSCs. The troponin T expression in control group and simple serum induction group were negative, but GATA-4 and desmin expressed weakly. CONCLUSION: Serum from AMI rat cannot induce BMSCs to differentiate into cardiomyocytes alone, but it promotes BMSCs induced by 5-aza differentiating into cardiomyocytes and facilitates the differentiated calls into mature.

BACKGROUND:Granulocyte colony-stimulating factor (G-CSF) can strongly mobilize bone marrow hematopoietic stem cells (HSCs). It has been proved that G-CSF has the ability to mobilize both HSCs and mesenchymal stem cells (MSCs).OBJECTIVE:To investigate the therapeutic effect of G-CSF in mobilizing autologous bone marrow stem cells entering cerebral infarction zone on ischemic cerebral infarction in rats.DESIGN:A randomized grouping design, animal experiment.SETYING: Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University.MATERIALS: This experiment was carried out at the Animal Experimental Department of Sun Yat-sen University (North District) and Center for Stem Cell Biology and Tissue Engineering of Sun Yat-sen University from September 2004 to January 2005. Totally 200 male Wistar rats were chosen and randomly divided into autologous bone marrow stem cells transplantation group and control group, with 100 rats in each group.METHODS:Rats of two groups were made cerebral infarction models by line occlusion. Transplantation group introduced intraperitoneal injection of 60 μg/kg G-CSF one hour after operation. The control group introduced intraperitoneal injection of saline of the same dosage at the same time. ①All rats were weighed before operation and 24 hours, 48 hours, one week after operation to evaluate body mass loss rate. They were also given neurological grading. Grading criteria: Grade 0 is normal. Grade Ⅰ is that the right forelimb bends. Grade Ⅱ is that the right forelimb grasped weakly when the tail is lifted. Grade Ⅲ is that the rat has no directivity in automatic action and circumrotates to right when the tail is lifted. Grade Ⅳ is that the rat circumrotates to right in automatic action. ②15 rats in each group were selected. 24 hours, 48 hours, one week after operation, we opened the skulls, took out the brain and used 2,3,5-Triphenyltetrazoluim Chloride (TTC) staining to measure infarction volume, hematoxylin-eosin(HE) staining to observe the pathological change , and immunohistochemistry to detect the infiltration of CD34+ cells.MAIN OUTCOME MEASURES:Body mass loss rate, neurological grade,infarction volume, pathological change and infiltration of CD34+ cells.RESULTS: Totally 180 of 200 rats were successfully made cerebral infarction model. 48 rats died in seven days after operation. As a result, 132 rat models were alive and 120 rats were randomly selected for data analysis. ①Measurement of body mass and neurological grading: There was no significant difference in body mass loss rate between two groups 24 hours and 48 hours after operation (P ＜ 0.05);one week after operation, body mass loss rate was significantly lower in transplantation group [(10.5±8.2)%]than in control group [(17.8±7.1)%] (P ＜ 0.05). There was no significant difference in neurology grade between two groups. ②Infarction volume:Infarction volume and the percent of infarction volume in the whole brain in control group were all higher than those in the transplantation group,with significant difference [ (251.69±52.77) mm3 vs(145.72±28.05)mm3,(17.00±2.69)% vs (9.90±1.62)% ,P ＜ 0.01]. ③Pathological change: 24 hours after operation, the brain tissue of two groups got classical pathological change of cerebral ischemia infarction. There were some mono-nucleus cells infiltrating in transplantation group while none in control group. 48 hours after operation, most nerve cells disappeared and the glial cells were degenerated. There were many mono-nucleus cells infiltrating in transplantation group while a few in control group. One week after operation, tissues in the infarction zone were liquescent with many monocaryons and lymphocytes infiltrating around them in control group. In transplantation group, part of the infarction zone was plerosised through proliferation of newly born capillaries and glial cells and inflammatory cells were not evident. ④Immunohistochemistry: CD34+ mono-nucleus cells were detected in the ischemic territory in transplantation group 24 hours after operation while none in the brain of other side and control group. There were CD34+ mono-nucleus cells and pyramidate cells with mutations in transplantation group 48 hours after operation while none in the brain of other side and control group.CONCLUSION:The stem cell transplantation in situ therapy, which employs self-marrow stem cells mobilized by G-CSF can relieve the ischemic degree and reduce the infarction volume.

Objective:To investigate the effect of HSPC 238 overexpression or low expression on the regulation of the target protein ( HMOX1, MT2A, UBB, RPS27a ) and the regulation pathways.Methods: To construct the interference vector and overexpression vector of HSPC238 respectively,transfected the HEPG2 cell lines by the liposome method,detect the expression of mRNA and protein of the HSPC 238 and the target proteins by the RT-PCR and Western blot , further to transfer the overexpression plasmids of the target proteins into the HEPG 2 cell lines which had been transfected with interference vector and overexpression vector of HSPC238,the experimental group cell lines were treated with proteasome inhibitor MG 132,to purify the target proteins with nickel column,do immunoblotting with HSPC238 to detect the accumulation situation of the target proteins.Results: The HMOX1,MT2A, RPS27a were downregulated obviously when the HSPC 238 was interfered exogenous;and the MT2A,UBB,RPS27a were up-regulated after the HSPC238 overexpressed.The overexpression plasmid of target proteins were transfected into the HEPG 2 cell lines which have been transfected with interference vector and overexpression vector of HSPC 238 ,compared with the control group ,the target protein band in the experimental group was significantly increased after treatment with the proteasome inhibitor MG 132.Conclusion:The HSPC238 overexpression can upregulate the expression of MT 2A,UBB and RPS27a,and interfering HSPC238 can downregulate the expression of HMOX1,MT2A and RPS27a;the HSPC238 target protein may play a regulatory role through the UPP pathway;the HSPC238 may play a regulatory role on the target proteins through the UPP pathway.

Objective To evaluate the therapeutic effect and application value of laparoscopy combined with holmium laser in the treatment of intrahepatic and extrahepatic bile duct stones.Methods Retrospectively analyzed the clinical data of 22 cases with intrahepatic and extrahepatic bile duct stones who treated with laparoscopy choledo-choscopy combined with holmium laser lithotripsy(the observation group),and 21 cases who treated with laparoscopy combined with choledochoscopic lithotomy(the control group).The clinical curative effect was compared between the two groups.Results In the observation group,the residual stones completely removed in 21 cases(95.4%),the mean operation time was (130 ±13)min,the intraoperative bleeding volume was (85 ±20)mL,the average hospitali-zation time was (8.3 ±2.0)days,without conversion to open surgery,no incision infection,1 case had residual stones after operation,through T tube sinus tract choledochoscopy took a stone again.In the control group,took net calculi in 13 cases(61.9%),the average operation time was (135 ±20)min,the intraoperative hemorrhage volume was (70 ± 15)mL,the average hospitalization time was (14.5 ±2)days,6 cases converted to laparotomy,incision infection in 2 cases,8 cases had residual stones after operation,through T tube sinus combined with choledochoscopy again take stone,which 2 secondary mirror lithotomy in 6 cases,3 cases of 2 stones.The differences of the two groups in stone from the net rate,conversion rate,average hospitalization time were statistically significant(χ2 =2.203,2.897,t =4.293,P =0.033,0.006,0.017).The differences of mean operation time,intraoperative hemorrhage had were not statistically significant(t =1.176,1.629,P =0.246,0.111).Conclusion It is safe,effective and minimally invasive for the treatment of intrahepatic and extrahepatic bile duct stones with holmium laser combined with endoscopic holmi-um laser.

Objective:To construct a bait vector for HSPC238, and to screen the target proteins which interact with the HSPC238.Methods:Gene synthesis method was used to synthetic gene HSPC238, then connected with the pGBKT7 vector after digesting by the sfiIA and sfiIB,to obtain the bait plasmid pGBKT7-HSPC238,then transferred into the yeast strains AH109 with the empty plasmid pGBKT7after sequencing,to observe its self-activating effect in the nutrient deficiencies medium,and further to screen the target proteins which interact with HSPC238 from the human fetal liver cDNA library.Results:The bait vector pGBKT7-HSPC238 was successfully constructed,and it had no self-activating effect through the phenotypic screening,after the yeast two-hybrid technology with literature analysis,we preliminary screened and found that the ribosomal protein L5(RPL5) may be the one of the target proteins which interacted with HSPC238 from the human fetal liver cDNA library.Conclusion: We successfully constructed the bait plasmid vector PGBKT7-HSPC238,and after the yeast two-hybrid technology with literature analysis,we preliminary screened and found that the ribosomal protein L5( RPL5) may be the one of the target proteins which interacted with HSPC238.

Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.

Objective: To investigate the expression of HSPC238 in cervical intraepithelial neoplasia and cervical cancer.Methods:We collected 76 cases of cervical cancer,105 cases of CIN and 28 cases of normal cervical epithelial.Then we inves-tigated the expression of HSPC238 by using immunohistochemistry and compared the significant differences between them.Results:There was no significant difference in the expression of HSPC238 between the cervical cancer and normal cervical epithelial ( Z=-0.242,P>0.05).However,there was significant difference between the cervical intraepithelial neoplasia and normal cervical epithelial (χ2=19.159,P<0.01) and the expression of HSPC238 was correlated with the grades of CIN.The expression of HSPC238 decreased when the grade of CIN was increasing.( rs=-0.327,P<0.01 ).Conclusion:The low expression of HSPC238 might be correlated with the development of cervical neoplasia.

Objective To evaluate the efficacy and safety of retroperitoneal laparoscopic pyelolithotomy ( RLP) combined with holmium laser lithotripsy under flexible cystoscopy in the treatment of complicated nephrolithiasis. Methods The retrospective analysis was made on the clinical data of 37 patients who underwent RLP and holmium laser lithotripsy under flexible cystoscopy for complicated nephrolithiasis from January 2013 to January 2014. The clinic parameters involved basic data of patients,operational time,blood loss,post-operative hospital stay,the status of stone-free,perioperative complications,and the follow-up data of patients were observed. Results No patient was converted to open surgery. The mean stone size was (2. 8 ± 0. 9) cm in diameter,operational time was (89 ± 24) min,blood loss was (21. 3 ± 7. 7) mL,post-operative hospital stay was (6. 8 ± 1. 7) d,the stone removal rate in one session was 94. 6%. One case occurred urinary leakage,1 case occurred fever after operation,who were all recovered through conservative treatment. All cases were followed up at the sixth months after operation. Conclusion RLP combined with holmium laser lithotripsy under flexible cystoscopy is effective and safe for the treatment of com-plicated nephrolithiasis.