90%inasinglestep,andtheproteinyieldwas2.5mg/L.ConclusionPurifiedChsp60kDantigenisobtained,andtheantigenmightbeappliedtodetectantibodyinpatientsinfectedwithChlamydialtrachomatis,andwillcontributetostudytheroleofChsp60inimmunopathogenesis.

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Objective To observe insulin resistance in first-degree relatives of patients with Graves disease. Methods All subjects in control group and experiment group including first-degree relatives of GD patients underwent oral glucose tolerance tests (OGTT) and insulin releasing tests then the degree of insulin resistance was analyzed. Results Blood glucose at each point of OGTT, insulin level and insulin resistance index 1 (HOMA-IR) of experiment group were higher than those in control group, while insulin activity index (IAI) and HOMA-βwere significantly lower than those in control group. Conclusion Patients insulin resistance could be found among first-degree relatives of GD patients.

Objective: To prepare a blood deficient model and study the effect of Tangkuei Blood Supplementing Decoction on hemopoiesis in this model. Methods: This model was made in mice by i.p. an accumulate doses of 60mg/kg acetylphenylhydrazine (APH) and 160mg/kg cyclophosphamidum (CY). The RBCs, WBCs, reticulocytes and bone marrow nucleated cells (BMNC) were counted, the micro structure of bone marrow was observed. The swimming time, the body temperature and the plasma cAMP, cGMP levels were measured. The effects of Tangkuei Blood Supplementing Decoction by p.o. administration on promoting the hemopioetic function were observed with the model mice. Results: Tangkuei Blood Supplementing Decoction could remarkably increase RBC, WBC, BMNC, improve the proportion of reticulolytes in peripheral blood and the micro struture of bone marrow, prolony the swimming time, raise the body temperature and the specific value of cAMP/cGMP. Conclusion: The model exhibits the main features of blood deficiency, and can be used as one of models of blood deficiency. Tangkuei Blood Supplementing Decoction can obviously improve the dual deficiention of qi and blood of model mice by supplementing qi and blood.

Objective To investigate The Th1/Th2 and Tc1/Tc2 polarization in the peripheral blood of first degree relatives of pemphigus vulgaris(PV) and healthy control individuals, and to approach the mechanism of the Dsg3-specific autoimmunity in PV. Methods The peripheral blood mononuclear cells (PBMC) from first degree relatives and healthy control was stimulated for72 h with Dsg3 and without Dsg3. Th1/Th2, Tc1/Tc2 was assessed by four-color flow cytometry. Results The mean frequency of Dsg3-spe-cific Th2 cells for PV antibody positive first degree relatives was 10.13%±3.72%, compared with stimula-tion without antigen 7.28%±3.58%, the difference was significant (P<0.05). The percentage Dsg3-spe-cific Th2 was markedly higher in the PV antibody positive first degree relatives group than that in the control group(10.13%±3.72% vs 6.10%±2.82%, P<0.05) , Tc2 was markedly higher also (20.01%± 10.43% v514.91%±8.06%, 20.01%±10.43% vs 9.58%±5.49%, P<0.05). Conclusion When Dsg3 stimulated PBMC were used to stimulate autologous T cells an increased amount of Th2 and Tc2 was observed, it is implied that the imbalance of Th1/Th2, Tc1/Tc2 might play an important role in the initia-tion of PV.

Objective To identify the peptide that have strong ability binding to HCMV-UL149 encoded protein,and to analyze the characteristics of the amino acid sequence of UL149-binding peptides.Methods Expressed UL149 proteins of three genotypes were used to screen the binding peptide in the random peptide display library,then the encoding sequence of binding peptides in the selected clones were sequenced.The amino acid sequences of the binding peptides were analyzed for their homology,and were com pared with those of the known protein in protein banks.Results The homologous amino acid sequence W/A/F/V-D/E-D/E-G-W/F/I/L were found within the binding peptides selected by proteins of all the three UL149 genotypes proteins,and no difference between three groups was found.The alignment with amino acid sequences of the known proteins in protein banks showed that the binding peptides of UL149 putative protein have homologous amino acid sequences with immunoglobulin heavy chain variable region(IgHV),the serine/threonine protein kinases,compliment factor H,zinc finger protein,MHC Ⅰ molecule,eukaryotic translation initiation factor,nuclear factor and so on.Conclusion The UL149 encoding proteins have binding ability to proteins mentioned above,and might interfere with the immunity responds to HCMV infection through multiple mechanisms.

Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.

Objective To observe the therapeutic effects of recombinant human granulocyte colony stimulating factor(rhG-CSF)on CCl4 induced chronic liver injury.Methods Male BALB/C mice were randomly allocated into treatment and control groups.The mice model were established by injection with daily for 7 days,while the control mice were received the same volumes of saline.The mice were sacrificed to get weight,liver mass and spleen mass.The count of CD34+ cells and Thy-1+ cells were analyzed by flow cytometry and immunohistochemical staining,respectively.Results The ratio of liver/spleen was 15.94±1.20 and 10.52±0.66 on day 8 and 15 in treatment group,respectively,while those were 7.14±1.68 and 8.31±1.71 in control group,respectively(all P value＜0.05).But there was no significant difference in body weight and liver mass between two groups(P＞0.05)The concentration of album in treatment group was raised rapidly on day 15.The concentrations of alanine aminotransferase (ALT),aspartate aminotransferase(AST),hyaluronic acid(HA)and laminin(LN)on day 30 were significantly lower in treatment group compared to control group(P＜0.05).There was significant difference in score of liver fibrosis on day 30 between two groups(treatment group:5.49±2.16,control:8.74±1.86,P＜0.05).The number of CD34+ cell and Thy-1+ in treatment group(on day 8:9.54±2.24 and 5.10±1.25 and on day 15:8.18±1.93 and 7.53±1.39,respectively)were higher than those in control group(on day 8:5.40±0.99 and 3.25±0.75;on 15 days:4.46±0.77 and 3.35±0.86,all P value＜0.05).Conclusion The rhG-CSF may improve the reparation of chronic liver injury,and may provide a novel method in treatment of liver fibrosis.

Objective To evaluate the bone repair capacity ofpolylactic acid-polyglycolic acid copolymer (PLGA)/collagen type Ⅰ (CoI) microspheres combined with BMSCs after being injected in intertrochanteric bone defect of osteoporotic female rats.Methods Prepared PLGA microspheres.The microspheres were coated with Col.BMSCs of the third passage were cultured with PLGA/CoI microspheres.Forty 3-month-old female SD rats were ovariectomized to establish osteoporotic animal models.The osteoporotic rats were randomly divided into 5 groups,including SHAM group,PBS group,Cell group,MS group and Cell+ MS group.There were 8 rats in each group.Different material was injected into the intertrochanteric bone defect site which was made with electric drill.Four rats of each group were sacrificed at 1 month and 3 months post-operation.The fenora were taken to measure the intertrochanteric bone mineral density (BMD) with DEXA and evaluate trabecular stucture with Micro CT.Results After 7 days of coculture,BMSCs seeded on PLGA/CoI microspheres had nice adherance and proliferation.There was no difference of BMC and BMD among all groups at 1 month post-operation.Tb.Th of Cell+MS group was higher than that of PBS group and MS group at 1 month post-operation.％Tb.Ar of Cell+MS group was higher than that of Cell group and MS group at 1 month post-operation.Tb.Sp of Cell+MS group had a tendence to decrease compared with other groups but there was no statistical difference at 1 month post-operation.After 3 months of operation,the BMC of Cell+MS group had a tendence to increase compared with that of PBS group and MS group but showed no statistical difference.BMD and Tb.Th of Cell+MS group was higher than those of other groups.％Tb.Ar of Cell+MS group was higher than that of SHAM group and PBS group.Tb.Sp of Cell+MS group had a tendence to reduce compared with other groups but showed no statistical difference.Conclusion The bone defect of osteoporotic site can be repaired 1 month after the injection of the PLGA/CoI microspheres combined with BMSCs.The trabecular reconstruction and bone quality of osteoporotic site can be improved 3 months after the injection.