蛋白质印迹法中免染技术和总蛋白定量方法的研究进展

当含有蛋白质的SDS-PAGE胶浸泡在三氯乙酸或氯仿中时,经三氯化合物的作用,使得蛋白质中的色氨酸经紫外光照射会发出可见光,因而可在SDS-PAGE凝胶上实现对蛋白质条带的定位与定性。免染技术即是基于此原理而发明的。基于免染技术,我们可以检测到待测样品中的总蛋白条带的光密度,进而利用目的蛋白条带光密度与总蛋白条带光密度的比值对目的蛋白进行定量。本文就蛋白质印迹法技术中的免染技术和总蛋白定量方法的原理和优势进行简要综述。

蛋白质免染系统的应用、常见问题及改进

聚丙烯酰胺凝胶电泳是蛋白质分离的关键方式,随着蛋白质印迹法的应用,常被用作生物医学等方面分析的下游工具。近年来,近红外荧光材料膜的运用以及3D打印等新兴技术逐渐应用于组织工程中,对蛋白质免染系统提出了更高的定量要求。本文阐述了免染系统的原理,操作和分析中存在的问题及改进方法,并对免染技术的发展进行了展望,以期为实现标准免染技术对蛋白质的准确计量分析提供基础和思路。

DYNAMIC ANALYSIS OF A TYPE OF FINANCIAL RISK CONTAGION MODEL INVOLVING IMMUNITY PERIOD AND SELF-RESCUE

In this paper,we study the dynamics of a Susceptible-Exposed-Infectious-Recovered(SEIR)nancial risk contagion model with time delay.Using stability theory and Hopf bifurcation theory,equilibria stability and Hopf bifurcation are analyzed in detail.Based on the epidemic model,we improve it by taking prior prevention and self-rescue into consideration,conclude pre-ventive intensity and self-rescue capabilities e ect the number of infections.At the same time,the analytical conditions for Hopf bifurcation are obtained,and the relevant results are veri ed by numerical simulations.

原液着色粘胶纤维的开发生产及应用

本文介绍了原液着色粘胶纤维的生产方法，重点结合颜料的选择对着色颜料性质、在丝条内的呈色机理进行阐述，并对纺前注射生产过程中可能出现的色泽差异进行分析，对原液着色粘胶纤维的应用范围及前景进行了说明。

IN SITU IMAGING OF BREAST CANCER CELLS USING GREEN SEMICONDUCTOR QUANTUM DOTS

The breast cancer is the most common cause of cancer death in women. To establish an early stage in situ imaging of breast cancer cells, green quantum dots （QDs） are used as a fluorescent signal generator. The QDs based imaging of breast cancer cells involves anti-HER2/neu antibody for labeling the over expressed HER2 on the surface of breast cancer cells. The complete assay involves breast cancer cells, biotin labeled antibody and streptavidin conjugated QDs. The breast cancer cells are grown in culture plates and exposed to the biotin labeled antibodies, and then exposed to streptavidin labeled QDs to utilize the strong and stable biotin-streptavidin interaction. Fluorescent images of the complete assay for breast cancer cells are evaluated on a microscope with a UV light source. Results show that the breast cancer cells in the complete assay are used as fluorescent cells with brighter signals compared with those labeled by the organic dye using similar parameters and the same number of cells.

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells

Objective To investigate whether hypertonic saline （HS） can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. Methods Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat （1 day）. The astrocytes were randomly divided into five groups： isotonic （IS） and HS groups, astrocytes were incubated by IS and HS （320 mosM NaCl） medium, respectively, for 1, 3, 5, 10 or 15 rain; carbenoxolone （CBX） ＋IS and CBX＋HS groups, astrocytes were pre-treated with CBX （100 mmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2＋＋HS group, astrocytes were pre-incubated with Ca2＋ （1 000 μmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein （GFAP） and anti-glutamate. The C6 cells were divided into four groups： IS, HS, CBX＋IS and CBX＋HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry （FCM）. Results （1） Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. （2） The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 rain （P 〈 0.01 vs the 5 min of HS group）. In CBX＋HS group, the glutamate intensity was higher than that in CBX＋IS and HS groups. （3） The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min （P 〈 0.01 vs 1 min and 3 min）. On the contrary, the medium glutamate concentrations in the CBX＋HS or Ca2＋＋HS group were significant lower than that in the HS group （P 〈 0.01）. （4） FCM showed HS and CBX＋HS induced glutamate increase in C6 cells. Conclusion HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Effects of Heavy Metal and Pollutants on the Non-special Immunity of the Shrimp and Crab

This paper attempted to review and evaluate existing information about the effects of heavy metal, ammonia-N, nitrite and the organic pollutants on the non-special immune response of the shrimp and crab, which providing theory for improving the self-immunity of shrimp and crab by meliorating cultural environment. In addition, it could provide information for further study on this field.

Optimization of Parameters of Exogenous Gene Mediated by Liposome to Transfect Yak Mammary Epithelial Cells in Vitro

[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...

Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma

AIM: To investigate the expression of programmed death (PD)-1,PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC).METHODS: Liver biopsies and HCC specimens from patients were collected and histologically examined.The expression of PD-1,PD-L1,and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining.The association between the expression level of PD-1,PD-L1,and PD-L2 and clinical and pathological variables was analyzed statistically.RESULTS: Expression of PD-1 was found in liverinfiltrating lymphocytes.In contrast,PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells.The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756,P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001,P = 0.018).PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051,P = 0.000;1.05 ± 1.099 vs 4.29 ± 3.885,P = 0.004;1.80 ± 1.473 vs 3.81 ± 3.400,P = 0.020).CONCLUSION: PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B.PD-1 and PD-Ls may play a role in immune evasion of tumors.

Effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B virus infection

AIM： To investigate if the nucleoside analogue lamivudine （LAM）, a potent inhibitor of HBV replication, could restore the function of dendritic cells derived from patients with chronic hepatitis B （CHB） in an Asian population.METHODS： Dendritic cells （DCs） derived from mononuclearcytes of patients with chronic HBV infection were cultured in the presence of IL-4, granulocytemacrophage colony-stimulating factors （GM-CSF） and gradient concentrations of LAM （0-2 mmol/L）. Cell morphology was observed under light microscopy. Cell surface molecules, including HI_A-DR, CD80, CD83, and CDla, were analyzed with flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were assayed by ELISA. T cell proliferation was assayed by methyl thiazoM tetrazolium （MTT）.RESULTS： The expression of CDlα on DC treated with 0.5 mmol/L LAM （LAM-DC 0.5 mmol/L） was significantly higher than that of DC untreated with LAM （54.1 ± 4.21 vs 33.57 ± 3.14, P 〈 0.05）, and so was the expression of CD83 （20.24 ± 2.51 vs 12.83 ± 2.12, P 〈 0.05） as well as the expression of HLA-DR （74.5 ± 5.16 vs 52.8 ± 2.51, P 〈 0.05）. Compared with control group, LAM-DC group （0.5 mmol/L） secreted significantly more IL-12 （910 ± 91.5 vs 268 ± 34.3 pg/mL, P 〈 0.05）, had lower levels of IL-6 in the culture supernatant （28 ± 2.6 vs 55 ± 7.36 pg/mL, P 〈 0.05）, markedly enhanced the stimulatory capacity in the allogeneic mixed leukocyte reaction （MLR） （1.87 ± 0.6 vs 1.24 ± 0.51, P 〈 0.05）.CONCLUSION： The lower expression of phenotypic molecules and impaired allogeneic mixed lymphocyte reaction function of dendritic cells derived from patients with HBV infection could be restored in vitro by incubation with LAM.

Immunogenetic phenotypes in inflammatory bowel disease

The currently accepted etiopathogenic hypothesis suggests that the chronic intestinal inflammation and related systemic manifestations characteristic of inflammatory bowel disease （IBD） are due to an overly aggressive or pathologic immune response to resident luminal bacterial constituents. Predisposing factors are genetic dysregulation of mucosal immune responses and/ or barrier function, with onset triggered by environmental stimuli. These factors and their interactions may also be important determinants of disease phenotype and disease progression. The emergence of immunogenetic phenotypes lends support to the proposed hypothesis that susceptibility genes regulate distinct immune processes, driven by luminal antigens, expressed as specific immune phenotypes which in turn influence clinical phenotypes in IBD patient.

Three cases of retroperitoneal schwannoma diagnosed by EUS-FNA

Schwannomas are peripheral nerve tumors that are typically solitary and benign.Their diagnosis is largely based on surgically resected specimens.Recently,a number of case reports have indicated that retroperitoneal schwannomas could be diagnosed with endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA).We report the diagnosis of three cases of schwannoma using EUS-FNA.Subjects were two males and one female,ages 22,40,and 46 years,respectively,all of whom were symptom-free.Imaging findings showed well-circumscribed round tumors.However,as the tumors could not be diagnosed using these findings alone,EUS-FNA was performed.Hematoxylin-eosin staining of the resulting tissue fragments revealed bland spindle cells with nuclear palisading.There was no disparity in nuclear sizes.Immunostaining revealed S-100 protein positivity and all cases were diagnosed as schwannomas.Ki-67 indexes were 3%-15%,2%-3%,and 3%,respectively.No case showed any signs of malignancy.As most schwannomas are benign tumors and seldom become malignant,we observed these patients without therapy.All tumors demonstrated no enlargement and no change in characteristics.Schwannomas are almost always benign and can be observed following diagnosis by EUS-FNA.

Nuclear β-catenin expression as a prognostic factor in advanced colorectal carcinoma

AIM： To investigate the changing pattern of β-catenin expression and its prognostic value in advanced colorectal cancer （CRC）. METHODS： Archival tumor samples were analyzed for β-catenin using immunohistochemistry （IHC） in 95 patients with advanced CRC. RESULTS： Membranous β-catenin expression was found in the normal colorectal epithelium. Almost 100% of CRC cases showed membranous and cytoplasmic expression, and 55 （58%） cases showed nuclear expression. In univariate （Kaplan-Meier） survival analysis, only the nuclear index （NI） was a significant predictor of disease free survival （DFS） （P = 0.023; n = 35）, with a NI above the median associated with longer DFS （34.2 too） than those with a NI below the median （15.5 too） （P = 0.045, ANOVA）. The other indices were not significant predictors of DFS, and none of the three tested indices （for membranous, cytoplasmic, or nuclear expression） predicted diseasespecific survival （DSS）. However, when dichotomized as positive or negative nuclear expression, the former was a significant predictor of more favorable DFS （P = 0.041） and DSS （P = 0.046）. CONCLUSION： Nuclear β-catenin expression provides additional information in predicting patient outcome in advanced CRC.

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T

AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.

Expression of cytokeratins in Helicobacter pylori-associated chronic gastritis of adult patients infected with cagA+strains:An immunohistochemical study

AIM： To investigate the expression of different cytokeratins （CKs） in gastric epithelium of adult patients with chronic gastritis infected with Helicobacter pylori （H pylon） cagA ＋ strains. METHODS： The expression of CK 7, 8, 18, 19 and 20 was studied immunohistochemically in antral gastric biopsies of 84 patients. All the CKs were immunostained in cagA＋Hpylori gastritis （57 cases）, non-Hpylori gastritis （17 cases） and normal gastric mucosa （10 cases）. RESULTS： In cagA＋ H pylori gastritis, CK8 was expressed comparably to the normal antral mucosa from surface epithelium to deep glands. Distribution of CK18 and CK 19 was unchanged, i.e. transmucosal, but intensity of the expression was different in foveolar region in comparison to normal gastric mucosa. Cytokeratin 18 immunoreactivity was significantly higher in the foveolar epithelium of H pylori-positive gastritis compared to both Hpylori-negative gastritis and controls. On the contrary, decrease in CK19 immunoreactivity occurred in foveolar epithelium of H pylori-positive gastritis. In both normal and inflamed antral mucosa without Hpyloriinfection, CK20 was expressed stronglyl moderately and homogenously in surface epithelium and upper foveolar region, but in H pylod -induced gastritis significant decrease of expression in foveolar region was noted. Generally, in both normal antral mucosa and H pylori-negative gastritis, expression of CK7 was not observed, while in about half cagA＋ H pylori-infected patients, moderate focal CK7 immunoreactivity of the neck and coiled gland areas was registered, especially in areas with more severe inflammatory infiltrate. CONCLUSION： Alterations in expression of CK 7, 18, 19 and 20 together with normal expression of CK8 occur in antral mucosa of H pylori-associated chronic gastritis in adult patients infected with cagA＋ strains. Alterations in different cytokeratins expression might contribute to weakening of epithelial tight junctions observed in H pylori-infected gastric mucosa.

AtFKBP53 is a histone chaperone required for repression of ribosomal RNA gene expression in Arabidopsis

Chromatin structure is important for controlling gene expression, but mechanisms underlying chromatin remodel- ing are not fully understood. Here we report that an FKBP （FK506 binding protein） type immunophilin, AtFKBP53, possesses histone chaperone activity and is required for repressing ribosomal gene expression in Arabidopsis. The At- FKBP53 protein is a multidomain FKBP with a typical peptidylprolyl isomerase （PPIase） domain and several highly charged domains. Using nucleosome assembly assays, we showed that AtFKBP53 has histone chaperone activity and the charged acidic domains are sufficient for the activity. We show that AtFKBP53 interacts with histone H3 through the acidic domains, whereas the PPIase domain is dispensable for histone chaperone activity or histone binding. Ri- bosomal RNA gene （18S rDNA） is overexpressed when AtFKBP53 activity is reduced or eliminated in Arabidopsis plants. Chromatin immunoprecipitation assay showed that AtFKBP53 is associated with the 18S rDNA gene chro- matin, implicating that AtFKBP53 represses rRNA genes at the chromatin level. This study identifies a new histone chaperone in plants that functions in chromatin remodeling and regulation of transcription.

Hepatitis B and C virus co-infections in human immunodeficiency virus positive North Indian patients

AIM： To determine the prevalence of hepatitis B and C virus infections in human immunodeficiency virus （HIV） -positive patients at a tertiary care hospital in New Delhi, India. METHODS： Serum samples from 451 HIV positive patients were analyzed for HBsAg and HCV antibodies during three years （Jan 2003-Dec 2005）. The control group comprised of apparently healthy bone-marrow and renal donors. RESULTS： The study population comprised essentially of heterosexually transmitted HIV infection. The prevalence Fate of HBsAg in this population was 5.3% as compared to 1.4% in apparently healthy donors （P 〈 0.001）. Though prevalence of HCV co-infection （2.43%） was lower than HBV in this group of HIV positive patients, the prevalence was significantly higher （P 〈 0.05） than controls （0.7%）. Triple infection of HIV, HBV and HCV was not detected in any patient. CONCLUSION： Our study shows a significantly high prevalence of hepatitis virus infections in HIV infected patients. Hepatitis viruses in HIV may lead to faster progression to liver cirrhosis and a higher risk of antiretroviral therapy induced hepatotoxicity. Therefore, it would be advisable to detect hepatitis virus coinfections in these patients at the earliest.

T cell immunopathogenesis and immunotherapeutic strategies for chronic hepatitis B virus infection

Hepatitis B is caused by the host immune response and T cells play a major role in the immunopathogenesis. More importantly,T cells not only destroy hepatocytes infected by hepatitis B virus(HBV),but also control HBV replication or eradicate HBV in a noncytolytic manner.Therefore,analysis of T cell immune response during acute and chronic HBV infection is important to develop a strategy for successful viral control,which could lead to immunotherapy for terminating persistent HBV infection.There have been many attempts at immunotherapy for chronic HBV infection,and some have shown promising results.High viral load has been shown to suppress antiviral immune responses and immunoinhibitory signals have been recently elucidated, therefore,viral suppression by nucleos(t)ide analogs, stimulation of antiviral immune response,and suppression of the immunoinhibitory signals must be combined to achieve desirable antiviral effects.