CELISA法的建立及其在免疫导向药物制备过程中的应用

用细胞ＥＬＩＳＡ（ＣＥＬＩＳＡ）法测定了导向药物Ｂ１５９－表阿霉素结合物和Ｂ１５９－阿霉素结合物制备过程中含单抗Ｂ１５９腹水、Ｂ１５９纯化物和结合物的抗体活性。结果表明，ＣＥＬＩＳＡ法较活细胞免疫荧光法有更高的敏感性，是导向药物制备过程中鉴定免疫活性的一种稳定、实用、经济而简单的方法。

Modulation of Lianbizi Injection (Andrographolide ) on Some Immune Functions

Objective: To study the effects of andrographolide on immune functions and the immune mechanism in clinical therapy.Methods: The amounts of IFN-α,IFN-γ, TNF-α, IL-8 in the peripheral blood mononuclear cells (PBMC) culture supernatants dealt with by different concentrations of LianBiZhi (LBZ) injection, the effective component of which is andrographolide, were detected by biological activity test or ELISA in vitro. The effects of LBZ injection on macrophage phagocytotic function and natural killer cells cytotoxicity were examined by means of macrophage to phagocytize cock erythrocyte and measurement of lactate dehydrogenase (LDH) activity released from the damaged cells, respectively.Results: The LBZ injection could not only enhance the phagocytosis activity of peritoneal macrophage from guinea pig to phagocytosis cock erythrocytes, but also augment the cytotoxicity mediated by natural killer cells from PBMCs.Conclusion: Andrographolide is an immunostimulant agent which can modulate both antigen specific and nonspecific immune function by means of its natural killer cells, macrophage and cytokines.

Expression and Immunological Analysis of Capsid Protein Precursor of Swine Vesicular Disease Virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

Effects of transfecting Bcl-2 shRNA and celecoxb on the SGC-7901 cell line

Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000,the rate of cell growth inhibition of all groups was detected by multiply-table tournament(MTT) method,apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa.Results:After siRNA was transfected to SGC-7901 cells,the rate of cell growth inhibition was increased,the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa.Conclusion:The growth of SGC-7901 cells which was transfected siRNA has been inhibited,and the sensitivity to celecoxib has been increased.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 （epitopel）, tandem repeat 200-213 （epitope2 （＋2）） and the combination of two epitopes （epitopel-2） was genetically cloned into the prokaryotic expression vector PPRoExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 （＋2） and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 （＋2）, GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.

Effects of glucose and aldosterone on the proliferation of cardiac fibroblasts

Objective To investigate the effects of glucose and aldosterone on the proliferation of rat cardiac fibroblasts. Methods The neonatal SD rat cardiac fibroblasts （Cfs） were separated by the differential attachment technique. Cfs were incubated in different D- glucose concentrations for 24 hours, with or without aldosterone. DNA synthesis and metabolic activity of the Cfs were measured simultaneously with Brdu incorporation and WST-1 ELISA, respectively. Results Glucose at high concentrations （15 and 25 retool/L） significantly increased the proliferation of Cfs, compared with glucose at low concentration （5.6 retool/L, P〈0.01 ）, while no difference was shown on Cfs proliferation between the two high glucose concentration groups. Addition of aldosterone （10-Tmmol/L） to low- glucose media resulted in significant proliferation compared with those without aldosterone （WST- 1, P〈0.01; Brdu, P-4）.019）. However, when incubated in high glucose media, the stimulation effect of aldosterone for Cfs proliferation disappeared （P〉0.05）. Further analysis suggested that aldosterone have significant interaction on Cfs DNA synthesis （P=0.012）. Conclusions Both glucose and aldosterone could stimulate the proliferation of cultured Cfs. In high glucose concentration, the stimulatory effect for Cfs proliferation may be masked （J Geriatr Cardio12010; 7：36-39）.

Immune Function Changes among HIV+/ARC/AIDS Patientsand Its Relationship to Disease Progression

Objective: To analyze HIV replication and immune functionchanges among Chinese patients with HIV/ARC/AIDS andtheir association with disease progression. Methods: T cell subsets, plasma cytokine concentrations andviral loads from 42 HIV+ individuals, and 13 ARC/AIDSpatients, and 10 controls were analyzed by flow cytometry(FCM), quantitative ELISA and reversetranscription-polymerase chain reaction (RT-PCR),respectively. Results: CD4 cell counts and plasma IL-2 in HIV/AIDSpatients were significantly less than in normal control subjects(P<0.001). The plasma concentrations sIL-2R, TNF-α, andNeopterin increased significantly with decreasing CD4 cellcounts. Plasma IL-2 among AIDS/ARC patients was also lessthan in HIV+ patients (P<0.01). CD4 cell counts, the ratio ofCD4 to CD8 and plasma IL-2 levels decreased significantlywith infection duration. CD4 cell counts declined an averageof 43/ml per year. In contrast, the concentration of plasmasIL-2R, sTNFR-I, and Neopterin increased an average of9.03pg/ml, 8.69pg/ml, 2.11ng/ml per yean respectively.Furthermore, a significant reverse linear correlation wasobserved between CD4 cell count, CD4/CD8 ratio, and CD3,CD4 and CD8 counts with plasma levels of sTNFR-I,Neopterin, and HIV RNA load. A positive linear correlationwas noted between plasma sIL-2R levels and changes ofplasma IL-6 level (P<0.001), IL-10(P<0.001), TNF-α(P<0.001),sTNFR-I (P<0.005), and Neopterin (P<0.002) and betweenIL-6 and TNF-α(P<0.001), Neopterin and IL-10 (P<0.05),sTNFR-I (P<0.001), plasma viral load and sTNFR-I (P<0.001),and Neopterin (P<0.002). Conclusion: These findingr suggest a close relationshipbetween IL-6 and TNF-aimmune activation, HIV replicationand disease progression in primary HIV infections and AIDSpatients. Declining CD4 cells and plasma IL-2, and increasingviral load, sIL-2R, TNF-α, sTNFR-I, and Neopterin might beconsidered as important predictors of the progression of HIVinfection to AIDS.

Effect of Tangnaikang on TGF-β_1-induced transdifferentiation of human renal tubular epithelial HK-2 cells

OBJECTIVE： To explore the function of Tangnai- kang （TNK） in the prevention and treatment of re- nal interstitial fibrosis through transdifferentiation of the human renal tubular epithelial cell line HK-2 induced bytransforming growth factor-β1 （TGF-β1）. METHODS： HK-2 cells cultured in dulbecco＇s modi- fied eagle medium/F12 （1：1） with 10% fetal calf se- rum were divided into six groups： blank control group, TGF-β1 group （TGF-β1 10 ng/mL）, serum con- trol group （TGF-β1 10 ng/mL ＋ 10% serum）, treat- ment group 1 （TGF-β1 10 ng/mL ＋ 5% TNK serum）, treatment group 2 （TGF-β1 10 ng/mL＋10% TNK se-rum）, and treatment group 3 （TGF-β1 10 ng/mL＋ 20% TNK serum）. Cell proliferation was detected by 4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazoliu m bromide assay. Expression of a-smooth muscle ac- tin （a-SMA） and E-cadherin were observed by im- munohistochemical assay. The contents of collagen Ⅰ （Col Ⅰ）, collagen Ⅲ（ColⅢ）, and fibronectin （FN） in the culture medium supernatant were detected by ELISA. RESULTS： E-cadherin was expressed and α-SMA was not expressed in normal HK-2 cells. In HK-2 cells cultured with TGF-β1, α-SMA expression signifi- cantly increased, HK-2 cells significantly proliferat- ed, and secretion of Col Ⅰ, Col Ⅲ, and FN significantly increased compared with the blank control group （all P〈0.05）. In the HK-2 cells cultured with TGF-β1 and TNK serum, the expression of α-SMA signifi- cantly decreased, the expression of E-cadherin sig- nificantly increased, and the cell proliferation and the secretion of Col Ⅰ, Col Ⅲ and FN were significant- ly inhibited compared with the TGF-β1 group （all P〈 0.05. CONCLUSION： TNK can inhibit cell proliferation and reduce secretion of Col Ⅰ, Col Ⅲ, and FN.This in- dicates that TNK can inhibit transdifferentiation of human renal tubular epithelial cells induced by TGF-β1, with the effect of preventing and treating renal interstitial fibrosis.

Variation and significance of NKT cell and its subset in patients with severe multiple injuries

Objective： To observe the variation and significance of natural killer T （NKT） cells in patients with severe multiple injuries. Methods： Peripheral blood was drawn from 30 patients with severe multiple injuries and 20 healthy individuals. NKT cells and the subsets of NKT cells were stained and analyzed on fluorescence activated cell sorter （FACS） using Cellquest software. The level of IL-4 and IFN- γ in blood serum was detected by ELISA. Results： The proportion of NKT cells was significantly increased. CD4^＋ NKT cells was increased （t=-3.11, P〈0.01） and CD4^＋CD8^＋NKT （double negative NKT, DN NKT） cells decreased in patients with severe multiple injuries compared with healthy controls （t=2.99, P〈0.01）. There was a positive correlation between the proportion of NKT cells and injury severity score （ISS） by Spearman correlation analysis （r=0.70, P〈0.01）. The level of IFN-γ was significantly decreased and the level of IL-4 significantly increased in patients with severe multiple injuries. Conclusions： We demonstrate that human NKT cells are increased in trauma patients. Most significantly, there is an association between ISS and NKT cells. The increased CD4^＋NKT cells may contribute to the reduction of Thl cytokine production and the growth of Th2 cytokine production, leading to the suppression of immunity after injury.