Objective To develop a visible detection system prepared by protein microarrays which was based on GoldMag particles-labeled technique and compare the results of detection using the protein which was labeled with GoldMag particles and colloidal gold.Methods Human IgG was printed on the glass slides modified with epoxy groups,and goat anti-human IgG conjugated with GoldMag particles and colloidal gold respectlively was then added on the slide.The glass slides were incubated,and then the black images of microarray spots were produced by immuno-gold-silver staining method and observed by naked eyes and recorded with common flatbed scanner.Results The high signal-to-noise ratio could be obtained when the optimized procedures of GoldMag particles-labeling probe were introduced to the protein chip.The conditions of optimum assay were as follows:the spotting concentration of human IgG was 0.2 mg/ml,the glass slides were incubated at 37 ℃ for 2 hour to immobilize human IgG,and the silver enhancement time was 10 min-15 min.Parallelly,the optimized conditions for colloidal gold were as follows:the spotting concentration of human IgG was 0.1 mg/ml,the slides were incubated at 37 ℃ for 1 hour to immobilize human IgG,and the silver enhancement time was 15 min-20 min.Conclusions GoldMag particles-labeled protein technique is comparable to colloidal gold in applying to protein microarrays.The method is considerably simple and practical,and the protein labeled by GoldMag particles could be quantitative.

Objective To investigate the alteration of the cytoskeleton of airway smooth muscle cells in young asthmatic rats with airway remodeling and the effect of RhoA/ROCK signal pathway.Methods Airway smooth muscle cells (ASMCs) were primary cultured and purified from Sprague-Dawley(SD) rats that were induced by ovalbumin (OVA) inhalation for 8w,then incubated by Pho kinase inhibitor Y27632.Real time quantitative polymerase chain reaction (RT-PCR),Western blot,and immunohistochemistry were used to measure the alteration of F-actin,and α-tubulin in the cytoskeleton of airway smooth muscle.Results (1) The asthma group showed a high average gray value of F-actin in ASMC than control groups,especially 8 weeks;and were significantly down in the group after adding Y27632(P ＜0.01).(2) The intension and intensity of fluorescence signal of α-tubulin in asthma groups in 8 weeks were higher than control greup(P ＜0.01),and were significantly decreased in Y27632 group.(3) A higher expression of α-tubulin protein was shown in the asthma group in 8 weeks relative to control group(P ＜0.01),and was significantly down-regulated in Y27632 group(P ＜0.05).Conclusions Alteration of the cytoskeleton of airway smooth muscle exists in young asthmatic rats and the RhoA/ROCK signal pathway possibly plays a significant role.

BACKGROUND: As a new radiotherapy for malignant tumor, brachytherapy of radioactive seed implantation enables the inter-tissue implantation of radioactive seeds to be applied further with the appearance of seed implantation treatment planning system (TPS), and the gradual consummation of ultrasound and CT-guided precise positioning system.OBJECTIVE: To evaluate the methods, feasibility, safety and efficacy of CT-guided iodine-125 (125I) implantation for malignant tumors.DESIGN: A controlled observation before and after implantation.SETTING: The First People's Hospital of Hefei City.PARTICIPANTS: Twenty-one patients with malignant tumors, who were not suitable for surgical treatments of operation,were selected from the First People's Hospital of Hefei City from January 2004 to March 2005. There were 26 lesions, including 9 lesions of primary unresectable carcinoma and 17 lesions of metastasis tumors.METHODS: Under CT guidance, 125I seeds were implanted into malignant tumor according to TPS, the radioactivity quantum were 22, 26, 30 and 33 MBq per granule. Larger radiation 125I wes suitable for the implantation in the center of a lesion and smaller ones were for the margin of the lesion and the peripheral region of some important organisms such as vessels and nerves. The interval between larger seeds was about 1.5 cm whereas the interval between smaller ones was about 1.0 cm.MAIN OUTCOME MEASURES: The seed distribution, existence of complication and changes of the size of lesion after implantation were observed.RESULTS: ① The seeds were implanted successfully in all the 21 cases. No complication was observed. The practical distribution of the implanted seeds was basically the same as the scheduled scheme before implantation. All the 21 patients were involved in the analysis of results. ② The number of seeds implanted in one lesion was 5 to 40 (mean 14).Pain relief was obtained in all 10 cases of malignant tumors of bone after implantation. Follow-up CT reexamination demonstrated that 18 lesions were obviously diminished, necrosis was found in 4 lesions and remaining 4 lesions had no significant changes in size. ③ The average sizes of 14 lesions in 11 patients at 5-7 months after implantation were obviously smaller than those before implantation (1.84 cm vs 3.41 cm, t =5.7237, P ＜ 0.001). The average sizes of 12 lesions in 10 patients at 8-10 months after implantation were also obviously smaller than those before implantation (1.96 cm vs 3.43 cm, t =5.577 8, P ＜ 0.002).CONCLUSION: CT-guided 125I implantation is a safe, effective and feasible method for the treatment of malignant tumor.

Objective To observe the serum γ-glutamyltransferase (γ-GGT) levels in patients with chronic hepatitis B virus (HBV) infection in different immune status and investigate their relationship with HBV DNA loads and ALT levels. Methods Blood samples were collected from 191 patients with chronic HBV infection in different immune status, including inactive HBV carrier state (group B,n = 55), immune tolerance phase (group C, n=47), HBeAg-negative CHB (group D, n =17), immune-reactive phase (group E, n=72) and 61 healthy individuals ( group A) for the detection of the serum γ-GGT, ALT level and HBV DNA loads. Results γ-GGT level were obviously higher in groups D and E than those in groups of A, B and C (P<0.01). Correlation analysis showed that the γ-GGT levels were positively correlated with serum ALT , AST levels in HBeAg-negative CHB and immune-reactive phase , but not correlated with HBV DNA loads. Conclusions The levels of γ-GGT are different during different immune status in patients with chronic HBV infection. The increased serum γ-GGT level may be an indicator for patients with chronic HBV infection entering immune active phase. The liver inflammation is the major impact factor to the γ-GGT levels.

Objective To analyze the clinicopathologic characteristics of atypical polypoid adenomyoma (APA) of endometrium,and investigate the special characteristics of cancerous transformation from APA.Methods Fourteen cases of APA were collected in General Hospital of People' s Liberation Army from January 2007 to March 2013.The clinical data,morphologic features,immunohistochemistry and the related literature were reviewed.Results The median age of the 14 patients was 38 years (ranged from 23 to 72 years),only 1 patient was postmenopausal.The most common symptom was irregular vaginal bleeding (4/14),and 4 patients were identified during routine physical examination for infertility.Among 14 cases,4 cases were diagnosed as well differentiated endometrioid adenocarcinoma originating from APA,and their median age was 35 years (ranged from 28 to 41 years); color Doppler flow imaging (CDFI) of ultrasound showed rich blood flow signal.The tumors with cancerous components were obviously larger than the usual APA (mean diameter:4.7 versus 1.8 cm).Histologically,irregular and branched glands were embedded in fibromuscular stroma and the glandular epithelium were atypical hyperplasia in varying degrees.While carcinoma developed in the APA,the sieve,solid and papillary structures were noticeable,and necrosis were common.Conclusions APA is a rare lesion of the uterus.Although the clinical behavior is benign in most cases,there may be possible for some cases developing carcinomas.If the APA mass is more than 4 cm in diameter,and microscopically demonstrates prominent sieve,solid,papillary structures and necrosis,the diagnosis of carcinoma developed from APA can be made.Thorough analysis should be done before the most proper therapeutic regimen is drawn up.

Objective To assess the protective effect of vaccination in routine application on hepatitis B virus (HBV) exposed infants and to clarify whether hepatitis B immunoglobulin (HBIG) administration of pregnant women may reduce the risk of maternal-fetal transmission of HBV. Methods Serum samples of 6398 pregnant women at gestation of 15-20 weeks from 6 urban and 8 rural areas across Jiangsu province were previously tested for serologic markers of HBV by ELISA from July 2002 to August 2004. In this study, infants born to 419 HBV carrier mothers were taken as the study group, while infants born to 453 non-carrier mothers were taken as the control group by stratified random sampling. They were followed-up and screened for HBV markers during October 2009 to March 2010. Information including HBIG administration during pregnancy, HBV vaccination and HBIG administration of the infants were collected. χ2 test or Fisher′s exact method were used to compare the rates and the comparison of the means was by t test. Results The follow-up rates of the study group and control group were 71.12% (298/419) and 72.41% (328/453), respectively. Of the 298 infants born to HBV carrier mothers, 11 (3.7%) were positive for HBsAg, while none of the 328 infants born to non-carrier mothers was HBsAg positive (χ2=12.32, P<0.01). All of the 11 children were born to mothers with both HBsAg and HBeAg positive, and nine of the 11 children were not injected HBIG or not immunized with hepatitis B vaccine within 24 hours after birth, with only one received regular vaccination and detailed information was unknown in one case. The positive rates of anti-HBs in the study group and the control group were 69.46% and 69.21% respectively (χ2=0.01, P=0.95). HBsAg positive rate of the children born to pregnant women treated with HBIG during late pregnancy (n=92) was 2.17% (n=2), whereas that in the children born to women not treated with HBIG (n=197) was 4.57% (χ2=0.98, P=0.51). Conclusions The protective effect of immunoprophylaxis in routine application against perinatal HBV infection in Jiangsu province is good. Efforts are required to emphasize the importance of HBIG administration in infants born to HBV carrier mothers, especially in HBeAg positive mothers within 24 hours after delivery. Treatment of HBsAg positive pregnant women with HBIG in third trimester would not decrease the risk of maternal-fetal transmission of HBV.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

We established a purification system for glutathione-S-transferase (GST) fusion protein using glutathione coupled magnetic particle. Glutathione was coupled covalently to the surface of magnetic particles with isothiocyanate functional groups. Cell lysate, containing the fusion protein, was then incubated with these glutathione coupled magnetic particles at room temperature. Unbound and non-specifically bound proteins were removed by wash steps. Subsequently, the GST-fusion protein was eluted from the magnetic particles by the addition of reduced glutathione. The resulting fusion protein was tested for purity using SDS-PAGE and demonstrated by Western blotting. The concentration of the fusion protein was measured by Bradford method. Both the conditions for incubation and washing were optimized. The results showed that 150 microg glutathione could be bound on 1 mg of particle surface and 10 mg of the glutatione-coupled magnetic particles was suitable for 100 microL lysate, the optimal incubation time for reaction between particles and lysate was 40 min. The magnetic particles could help purify efficiently GST-fusion protein with a yield of around 516 microg fusion protein per 10 mg particles. Magnetic particles can be successfully used in a simple, rapid and reliable method for the purification of GST-fusion proteins.
Glutathione ; chemistry ; Glutathione Transferase ; chemistry ; isolation & purification ; Magnetite Nanoparticles ; chemistry ; Protein Binding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

Objective To investigate the roles and mechanisms of hepatocyte nuclear factor 4α (HNF4α) in chenodeoxycholic acid (CDCA) induced gastric intestinal metaplasia (IM).Methods After the immortalized gastric mucosal epithelial cells GES-1 were stimulated with CDCA at different concentration,the changes of HNF4α,caudal-related homeobox 2 (CDX2) and trefoil factor family 3 (TFF3) expressions at mRNA and protein levels in GES-1 cells and gastric cancer cell lines (AGS,SGC7901 and BGC823) were detected by real time-polymerase chain reaction (RT-PCR) and Western blotting.After GES-1 were transfected with HNF4α short hairpin RNA (shRNA) or control shRNA,and followed by CDCA stimulation,the expressions of HNF4α,CDX2 and TFF3 at protein level were determined by Western blotting.HNF4α was overexpressed in GES-1 cells and SGC7901 cells,and HNF4α was silenced in BGC823 cell line and AGS by lentiviral vector system.The expressions of HNF4α,CDX2 and TFF3 at mRNA and protein levels were tested by RT-PCR and Western blotting.Luciferase reporter assay was used to analyze the regulation role of HNF4α on CDX2.T test was performed for statistical analysis.Results The expressions of HNF4α in GES-1,SGC7901,BGC823 and AGS cells at mRNA level were 1.00 ± 0.12,263.01±10.23,848.01±18.13 and 3 049.86±91.75,respectively.The mRNAlevels of HNF4α in AGS,BGC823 and SGC7901 cells were all higher than that of GES-1 cells,and the differences were statistically significant (t=33.23,46.72 and 25.62,all P＜0.01).The expressions of HNF4α in GES-1,SGC7901,BGC823 and AGS at protein level were consistent with mRNA level.The expressions of CDX2 and TFF3 at protein level of HNF4α shRNA transfected group were lower than those of non-HNF4α shRNA transfected group.In GES-1 cells,the expressions of HNF4α,CDX2 and TFF3 of HNF4α overexpressed group at mRNA level were 16 281.839 ± 1 843.017,6.275 ± 0.137 and 17.310± 1.533,respectively;which were all higher than those of overexpressed control group (1.000 ± 0.048,1.000 ± 0.012 and 1.000±0.108,respectively),and the differences were statistically significant (t =8.83,38.29 and 10.61,all P＜0.01).In AGS cells,the expressions of HNF4α,CDX2 and TFF3 of HNF4α silenced group at mRNA level were 0.021 ± 0.001,0.088 ± 0.007 and 0.074 ± 0.002,respectively,which were lower than those of silenced control group (1.000 ± 0.108,1.000 ± 0.131 and 1.000 ± 0.122),and the differences were statistically significant (t=9.09,6.93 and 7.57,all P＜0.01).In GES-1 overexpressed cells and AGS silenced cells,the expressions of HNF4α,CDX2,TFF3 at protein level were consistent with mRNA level.In double reporter plasmid containing the CDX2 promoter CDX2 1 (-2 000～-1 bp) and CDX2-2 (-1 510～1 bp),after transfected with HNF4α shRNA,the activities were 0.387 ± 0.013 and 0.533 ± 0.040,respectively,which were lower than those of HNF4α shRNA transfected control group (0.605 ± 0.012 and 0.882 ± 0.019),and the differences were statistically significant (t =21.49 and 13.53,both P＜0.01).Conclusion HNF4α may be involved in bile acid induced intestinal metaplasia by upregulating the expression of CDX2.

OBJECTIVETo explore the feasibility of karyotype analysis using cells cultured from fetal bladder centesis samples.

METHODSSamples were derived from fetal bladder centesis for 3 fetuses featuring giant bladder and oligohydramnios. Following in vitro culture, cells were routinely processed and stained for chromosome analysis.

RESULTSFor all 3 cases, cell culture has achieved success. Sufficient metaphase cells were obtained for chromosome counting and karyotype analysis. The karyotypes of the 3 fetuses were respectively 46, XY, 46, XX, t(1;5)(q22;q12)[7]/46, XX[4], and 46, XY.

CONCLUSIONCells cultured from fetal bladder centesis may be used for karyotype analysis following in vitro culturing. This new approach can enable prenatal chromosome analysis for fetuses featuring smaller gestational weeks, giant bladder and oligohydramnios.