Objective:To investigate the effect of EBV protein on the production of Immunoglobulin(Ig)by cultured B cells.Methods:The purified human umbilical blood B cells were treated respectively with UV and Heat-inactivated EBV and cultured in complete IMDM. CD5, CD3, CD4 and CD8 cells were measured by flow cytometric analysis. IgG and IgM in the supernatant were detected by ELISA. At the mean time, IgG and IgM in the supernatant of cultured EBV-transformed B lymphocytes were detected by ELISA,and compared with that of UV-inactivated EBV group at the same period.Results:In UV-inactivated EBV group,CD3, CD4 and CD8 T cells couldn’t be detected,CD5 + cells occupied about 42% of all detected cells at the 14th day; and at the 28th day, 47%.The IgG A value of the 10th、18th、22th and 26th day in UV-inactivated EBV group have a significant difference compared with that in the control group ( P

ObjectiveTo evaluate the clinical application value of a kind of rapid urine microalbumin( mALB )screening test.Methods Six hundred and thirteen samples for urine routine detection from Affiliated Fuxing hospital,Capital Medical University were included from December 2006 to February 2007.mALB of all urine samples was detected with H-800 automated urine analyzer based on a dry chemical qualitative screening test.To validate the screening effect of the dry chemical screening test,all of the positive samples and a part of negative samples were determined by a mALB quantitative test as confirmation by lmmage-800 automated specific protein analyzer.Comparison of mALB quantitative level between screening positive group and negative group was carried out by 2 independent samples rank sum test.Comparison between enumeration data was carried out by Kappa test for consistency.Results One hundred and fourteen positive samples out of 613 were detected by screening method ( 18.6％ ).Out of the 114 positive samples by screening test,seventy-one samples were confirmed positive by quantitative test (mALB ＞ 13.3 mg/L).Out of 76 negative sample by screening test,eight samples were confirmed positive by quantitative test.The mALB level of the screening positive group and negative group was 17.70 ( 9.19 -32.90) mg/L and 4.55(2.38-7.60) mg/L,respectively,and the mALB level of the screening positive group was much higher than that of the negative group ( Z =- 8.644,P ＜ 0.01 ).According to the quantitative test,the sensitivity and specificity of the mALB screening method were 89.9％ (71/79) and 61.3％ (68/111 ),respectively.ConclusionsThe mALB rapid screening test in the research has a certain screening value.But the test method should be further perfected to improve its sensitivity for its certain omission rate.

Objective To study the potential therapeutic effect of potassium antimonyl tartrate (PAT) on human colon cancer in transplanted tumor nude mice models. MethodsSixty transplanted animal models were constructed with colon cancer cell line SW480 injected in nude mice. Nude mice were then random divided into 4 groups ( n = 15) :Normal saline group, 5-Fu group and different dose of PAT groups [ (20 mg/( Kg · d) ,40 mg/( Kg · d) ]. The volume of mass was measured every 3 days. After final-administration for 24 hours, immunohistochemical staining was used to detect the expression of PCNA in colon cancer cells. ResultsAfter the use of PAT, the growth of mass slowed down. PCNA levels [ (63. 63 ±8. 88)％ ,(59. 13 ±6. 15)％ ,(33. 38 ± 12. 76)％ ] in SW480 cells was reduced by PAT( P <0. 05, P <0. 01 ). ConclusionPAT potentially inhibited the growth of colon cell lines and induced apoptosis of SW480 colon cancer cells.

AIM:To observe the expression and tissue localization of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta 1 ( TGF-β1 ) in the rat acute cerebral ischemia model.METHODS:Male Wistar rats were used to establish acute cerebral ischemia model by a suturing method.The rats were divided into normal control group, sham group and ischemia 6 h, 12 h, 1 d, 2 d, 6 d and 14 d groups.The rat cerebral cortex and hippocampus of the brain were collected at different time points.The mRNA and protein levels of MMP-9 and TGF-β1 in the brain tissues were detec-ted by real-time PCR and in situ histochemistry staining, respectively.The levels of MMP-9 and TGF-β1 in the plasma were also measured by ELISA.RESULTS:The results of real-time PCR showed that the mRNA levels of MMP-9 began to in-crease 6 h after acute ischemia and reached to a peak 2 d after acute ischemia.Similarly, the mRNA level of TGF-β1 began to rise 12 h after acute ischemia and reached to the highest level 6 d after acute ischemia.Compared with the sham rats, the mRNA levels of MMP-9 and TGF-β1 in the rat brains that collected at ischemic time of 12 h, 1 d, 2 d, 6 d and 14 d were significantly increased.Moreover, results of in situ histochemical staining showed that the expression of MMP-9 was detected at cerebral cortex and hippocampus 1 d after acute cerebral ischemia.Further studies showed that MMP-9 dyeing of the rat cerebral cortex was most obvious 2 d after the acute cerebral ischemia.Similarly, the rat cortex and hippocampus began to express TGF-β1 2 d after acute ischemia and TGF-β1 staining at rat cerebral cortex was most obvious 6 d after the acute cerebral ischemia.In addition, ELISA showed that the increase in MMP-9 and TGF-β1 was detected in the plasma 12 h after ischemia.Compared with the sham rats, the level of these 2 factors significantly upregulated since 1 d after ischemia. CONCLUSION:The brain tissue itself contributes to the upregulation of MMP-9 and TGF-β1 post acute cerebral ischemia, which shed light on the related research in the field.

Objective To investigate the mRNA and protein expressions of nine integrin subunits in human keloid-derived mesenchymal stem cells (KD-MSCs).Methods Cultured KD-MSCs and normal skin-derived MSCs (NS-MSCs) served as the experiment group and control group respectively.Real-time quantitative PCR and Western blot were performed to measure the mRNA and protein expressions of nine integrin subunits in the two groups respectively.Statistical analysis was carried out by t test.Results Real-time quantitative PCR showed no significant difference in the mRNA expression level of any of the integrin units α2,α3,α5,αV,α10,α11 or β1 between KD-MSCs and NS-MSCs (all P ＞ 0.05).The mRNA expression level of integrin α8 was decreased,while that of integrin β3 was significantly increased in KD-MSCs compared with NS-MSCs (both P ＜ 0.01).Western blot revealed that the changes in protein expression levels of integrin units α8 and β3 were consistent with those in their mRNA expression levels in both KDMSCs and NS-MSCs (both P ＜ 0.01).Conclusions Integrin units α8 and β3 may be involved in the occurrence and development of keloid,and the receptors made up of them may play important roles in the pathogenesis of keloid.

OBJECTIVE:To discuss clinical efficacy and safety of alteplase combined with butylphthalide to the patients with acute ischemic stroke. METHODS:98 patients with acute ischemic stroke in our hospital were selected and divided into ob-servation group and control group according to random number table,with 49 patients in each group. Control group was addition-ally given Butylphthalide and sodium chloride injection 100 ml,ivgtt,bid,on the basis of routine treatment as controlling blood glucose,blood pressure,etc.;observation group additionally received Alteplase for injection 5 mg added into NS 10 ml,iv+Al-teplase for injection 45 mg added into NS 100 ml,ivgtt,qd,on the basis of control group. Both groups were treated for 2 weeks. Clinical efficacies of 2 groups were compared as well as cerebral infarction area,NIHSS score,ability score of daily liv-ing,the levels of IL-6,IL-8,IL-10,CRP,24 h urine protein,Scr and creatinine clearance rate before and after treatment. The occurrence of ADR was also recorded. RESULTS:1 patient of observation group and 2 patients of control group withdrew from the study due to severe hemorrhage. Total effective rate of observation group(95.83%)was significantly higher than that of con-trol group(80.85%),with statistical significance(P<0.05). Before treatment,there was no statistical significance in above in-dexes between 2 groups (P>0.05). The cerebral infarction area and NIHSS score were reduced significantly in observation group after treatment,ability score of daily living were increased significantly in observation group after treatment,and the im-provement of observation group was significantly better than control group,with statistical significance (P<0.05). 24 h urine protein and Scr of observation group was significantly lower than those of control group,with statistical significance(P<0.05). the level of IL-6 in observation group 1 week after treatment and the levels of IL-6,IL-8,IL-10 and CRP 2 weeks after treat-ment were significantly lower than in control group,with statistical significance(P<0.05). There was no statistical significance in the incidence of ADR between 2 groups (P>0.05). CONCLUSIONS:Alteplase combined with butylphthalide show signifi-cant therapeutic efficacy,can effectively reduce the level of serum inflammatory factors,control brain tissue ischemia and cere-bral infarction area,and improve neurologic function and pro-tect renal function in patients with acute ischemic stroke.

Objective To design and testify a novel strategy for acquiring mimetic epitope mapping by screening for a phage random peptide library using polyclonal anti keratin autoantibodies (AK auto Ab). Methods AK auto Ab were isolated and purified from pooled human sera by keratin affinity column in which keratin had been linked with CNBr Sepharose 4B,then biotinylated by the biotin ester. A 15 mer phage random peptide library was biopanned for 3 cycles and positive clones were identified by ELISA,competition assay and DNA sequencing. ResultsBy sequence comparison 23 positive clones were selected randomly and three epitopes were confirmed. Among the three epitopes SLSPMPTTNRR was the dominant epitope. The phages carrying positive clones reacted with AK auto Ab specifically and keratin could prevent interaction between AK auto Ab and positive phages. Conclusion The designed strategy is successfully applied in acquiring epitopes of polyclonal autoantibodies to keratin, which could provide a new approach for the discovery of epitope mapping which binds to natural autoantibodies.

Translational medicine is a subject on translating basic research results to clinical applications and pathophysiology is a bridge course combining basic and clinical medicine.Results were good by applying concept of translational medicine in pathophysiology teaching and detailed measures included carrying out case analyses,adding clinical pathophysiology course,developing comprehensive experiments and opening laboratories,etc.

Objective To investigate UV- or heat-inactivated Epstein-Barr virus(EBV)stimulating the production of anti-keratin autoantibody(AK auto Ab)in cultured human umbilical cord blood B cells. Methods Mononuclear cells were isolated routinely from umbilical cord blood, in which monocytes, NK cells and cytotoxicity T cells were eliminated by L-leucine methyl ester method, and T cells were removed by sheep red blood cells(SRBCs)treated with 2-amino ethyl-isothiouronium bromide (AET). The purified B cells were treated with UV- or heat-inactivated EBV respectively and then cultured in complete IMDM. CD5, CD3, CD4 and CD8 cells were detected by flow cytometry. IgG and IgM of AK auto Ab were measured by ELISA in the supernatant which came from the B cells treated by UV-inactivated EBV or EBV-transformed B cells respectively. Results In UV-inactivated EBV group CD5+B cells accounted for 43% and 47% of all cells detected on the 14th and 28th day, respectively. No CD3, CD4 and CD8 cells were detected during this period. In UV-inactivated EBV group the AK auto Ab of IgG and IgM increased significantly on the 18th and 26th day, respectively (P 0.05). On the 40th day the AK auto Ab of IgG and IgM were significantly higher in EBV-transformed B cell group than those in UV-inactivated EBV group. Conclusions UV-inactivated EBV is able to induce AK auto Ab production but heat-inactivated EBV does not, which suggests that EBV protein might be the effective agent in inducing the production of AK auto Ab.

Objective To prepare natural anti-keratin IgM monoclonal antibody. Methods Spleen cells of BALB/c mice raised in specific pathogen free conditions were directly fused with Sp2/0 myeloma cells. The hybridoma supernatants were tested by ELISA using pre-extracted keratin. The natural IgM obtained was further identified by immunochemistry and immunoblot methods. Results The cell fusion rate was about 60% without pre-immunization. About 14% supernatants reacted with the keratin antigen. Three hybridoma strains secreting natural IgM monoclonal antibody against keratin were obtained. The immunochemistry results showed that the natural anti-keratin IgM was able to bind to epidermis, sebaceous gland, hair follicule, and muscle tissues. Conclusion B lymphocytes in normal BALB/c mice spleen can produce natural antibody against kerain.