用细胞-ELISA法检测抗CEA抗体

目的 以高表达CEA的人结肠癌细胞系LoVo作为抗原 ,建立检测抗CEA抗体的细胞 ELISA法。方法 将1.0× 10 6 mL浓度 2 0 0 μL的LoVo加入培养板培养 2 4h ,用 0 .0 2 5 %戊二醛固定 ,加入经CEA基因疫苗免疫小鼠血清 ,亲和素 生物素复合物 ,酶标仪检测OD值。结果 建立了ABC CELISA检测抗CEA抗体 ,2套基因疫苗诱导抗CEA抗体效价不同。结论 ABC CELISA法简便 ,敏感度高 ,为研究CEA体液免疫提供了方法。

抗中性粒细胞胞浆抗体对溃疡性结肠炎诊断价值的研究

目的 :建立固定中性粒细胞 - ELISA,分析血清抗中性粒细胞胞浆抗体 (ANCA)对溃疡性结肠炎 (UC)的诊断价值。方法 :采用固定中性粒细胞 - ELISA法检测 2 5份溃疡性结肠炎血清、2 6份细菌性结肠炎血清、1 5份混合类型腹泻血清和 50份正常对照血清中的 ANCA,对各组血清的吸光度 (A值 )进行统计分析。结果 :UC组 ANCA与其它对照组 ANCA之间的差异非常显著(P<0 .0 0 5)。 UC组 A值超过正常对照组 A值 2 s以上病例达 80 % ,诊断特异性达 89%。结论 :UC血清中存在有诊断价值的 ANCA,固定中性粒细胞 - ELISA可作为血清 ANCA检测的重要手段。

Effect of Estrogen and Antiestrogen on Chemotherapeutic Sensitivity of Endometrial Carcinoma Cell Line

Objective: To study the effect of estrogen and tamoxifen on chemotherapeutic sensitivity in ER(+) endometrial carcinoma cells.Methods: DNA fragmentation as the criteria for apoptotic cell death was used to evaluate the value of estrogen, tamoxifen and adriamycin in ER(+) endometrial carcinoma cells. DNA fragmentation was measured with the cell death ELISA.Results: Adriamycin and tamoxifen could induce apoptosis in ER(+) endometrial carcinoma cell. The cell apoptosis level was decreased with the increasing of 17-β-estradiol concentration (P<0.001) and was inversely proportional to 17-β-estradiol concentration (IgM) (P<0.01). The cell apoptosis level was increased with the increasing of tamoxifen concentration (P<0.01) and was also directly proportional to tamoxifen concentration (IgM). Furthermore, the cell apoptosis level was increased significantly after treated with both tamoxifen and adriamycin.Conclusion: Estrogen may block apoptosis induced by adriamycin in ER(+) endometrial carcinoma cell. Tamoxifen can increase the sensitivity of endometrial carcinoma cell to adriamycin. Tamoxifen combined with chemotherapeutic drug may be of significant therapeutic benefit in ER(+) endometrial carcinoma. Key words endometrial carcinoma - estrogen - tamoxifen - adriamycin - cell apoptosis

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.

Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.

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.

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 cagE gene on Helicobacter pylori-associated gastritis and levels of interleukin-8

Objective: To investigate the status of cagE gene of Helicobacter pylori(H. pylori) isolated from patients with various gastrointestinal diseases and its relationship with the pathological inflammation grade and levels of IL 8 in the gastric mucosa and IL 8 secretion in gastric epithelial cells stimulated by H. pylori . Methods: cagE was amplified by polymerase chain reaction (PCR) in 145 clinical isolates. The inflammation grade of gastric mucosa was evaluated pathologically. IL 8 levels of gastric mucosa and IL 8 concentration of the supernatant of the cocultured SGC 7 901 cells and H. pylori was assayed by enzyme linked immunosorbent assay (ELISA). Results: cagE was positive in 79.3% of all the H. pylori strains. The mean score of the cagE positive gastritis in the antrum and corpus was (1.865±0.335) and (1.759±0.310). Meanwhile, the cagE negative grade was (1.689±0.294), (1.608±0.284). There was no significant difference ( P >0 05). The mean levels of IL 8 in cagE positive group in antrum and corpus were (390.6±101.4) pg/mg and (368.6±91.2) pg/mg; and cagE negative group were (328.6±102.8) pg/mg and (332.6±96.7) pg/mg. IL 8 in SGC7901 cells induced by cagE positive and negative group averaged (789.5±146.7) pg/ml and (757.6±136.4) pg/ml. There was still no significant difference( P >0.05). Conclusion: Positive rate of cagE is very high in Chinese patients regardless of the clinical outcome. And there was no direct relationship between cagE gene and the inflammation grade and IL 8 levels of H. pylori infected gastric mucosa and IL 8 secretion in gastric epithelial cells stimulated by H. pylori .

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.

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 （IL-2）-like factor was detected in the supernatant of plant haemoglutinin （PHA）-stimulated lymphocyte culture from peripheral blood,spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-1ike factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes （P〈0.01）. The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2 （rhIL-2） was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor （IL-2R） on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25（IL-2R） was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

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.

SPECIFIC BINDING OF HUMAN BONEMORPHOGENETIC PROTEIN （2A） WITH MOUSEOSTEOBLASTIC CELLS

Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM21. Human BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the surface of mouse osteoblastic cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the surface of MC3T3 cells exist the receptor for hBMP2A.

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus （FMDV）, BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies （mAbs） cell lines against the 3AB protein of foot-and-mouth disease virus （FMDV） were obtained, named C6 and E7 respectively. The mieroneutralization titer was 1：1024 for mAb C6, and 1：512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease （SVD） antigens. The titers in abdomen liquor were 1：5×10^6 for C6 and 1：2×10^6 for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.

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）.

Concentration, distribution and expression of interleukin-5 in human nasal polyp tissues

OBJECTIVES: To study the concentration, distribution and expression of IL-5 in nasal polyp tissues and explore its significance in micro-environment differentiation of eosinophil accumulation. METHODS: The concentration and expression of IL-5 in nasal polyp tissues of 40 patients were determined by ELISA and immunohistochemistry and inferior turbinate mucosa from patients with nasal polyps and healthy volunteers were used as control. RESULTS: IL-5 concentration in polyp tissues was significantly higher than that in turbinate mucosa (P 0.05). IL-5 concentrations in polyp tissues were markedly higher in patients with allergic rhinitis compared with those without (P 0.05). 80.1% of the eosinophils were positive for IL-5 and 90.9% of IL-5 positive cells were eosinophils. Only 3.7% of lymphocytes and neutrophils were positive for IL-5; IL-5 was not detectable in epithelial cells. IL-5 expression in eosinophils of polyp tissues was remarkably stronger than that of the turbinate mucosa (P 0.05). IL-5 expression of eosinophils in polyp tissue was significantly stronger in patients with allergic rhinitis compared with those without (P 0.05). CONCLUSION: IL-5 is the key cytokine in eosinophilic pathologic mechanisms in nasal polyp tissues.

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.