AIM:To evaluate the morphological and functional changes of retinas induced by treatment of tunicamycin with optical coherence tomography (OCT) in rats.METHODS:Totally 60 SD rats were randomly divided into 3 groups (20 in each group), 0.5mg/kg (in low dose group), 1.5mg/kg (in high dose group) tunicamycin were injected into vitreous cavity and saline (9g/L NaCl) were injected in the same dose as a control group.Changes of retinas were observed by OCT on the 1,7 and 14d after treatment of tunicamycin.Then the rats were sacrificed, retinas were taken out and embedded by the paraffin, tissue sections and the HE staining were performed.RESULTS:OCT results suggested that tunicamycin played damage effects on retinal morphology and structure which appeared a time-and dose-dependent.Fundus photography results suggested that 2wk after tunicamycin treatments, with the gradually changing of tunicamycin concentration, peripheral retinal and macular region became pale color gradually, edema occurred in optic disk, retinal vessels appeared thinner in the high dose group, optic nerve came out atrophy.Fluorescein angiography confirmed that tunicamycin injection in vitreous cavity 2wk later, retinal vessels injury occurred, resulted in leaking of intravascular contrast agent from peripheral to the central part of the retinas.Electrophysiological data showed that retinal electrogram occurred disorder induced by tunicamycin, such as the amplitude of a wave, b wave decreased gradually, even closed to zero, which was very different from control significantly (P<0.05).HE staining of paraffin sections showed that retina injuries induced by tunicamycin were in dose-time dependent, which was consistent with the results of OCT.CONCLUSION: Clinical retinal diseases could be simulated by retinal damage animal model induced by tunicamycin treatment.OCT detection offered real-time images of the retinal cross-section, which provided a helpful non-invasive method for detecting and evaluating the retinal damages.

OBJECTIVETo investigate the effect of total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21(Ras) and proliferating cell nuclear antigen (PCNA) gene in erythroleukemia cell line K562.

METHODSThe effect of total flavonoids of Hedysarum polybotry on K562 cell line survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay. The time- and dose-dependent manner was also observed. The cell cycle and apoptosis were analyzed with flow cytometry (FCM). The immunocytochemistry method was applied to quantitatively analyze the effects of flavonoids of Hedysarum polybotry on changes p21(Ras) and PCNA gene expressions.

RESULTSFlavonoids of Hedysarum polybotry (20-100 μg/mL) significantly inhibited the proliferation of K562 cells in a time- and dose-dependent manner. After K562 cells were cultured for 48 h, total flavonoids of Hedysarum polybotry had no significant effect on the apoptosis of K562 cells but showed significantly inhibition (P<0.01), indicating that total flavonoids of Hedysarum polybotry could induce K562 cells arrested at G(0)/G(1) and G(2)/M phases. Compared with the control group, p21(Ras) and PCNA gene expressions were decreased significantly in K562 cells treated with total flavonoids of Hedysarum polybotry (40 and 80 μg/mL, respectively) for 48 h.

CONCLUSIONThe inhibitory effect on proliferation of K562 cells was observed in the groups treated with flavonoids of Hedysarum polybotry, which might be related to cells arresting.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; drug therapy ; genetics ; pathology ; Oncogene Protein p21(ras) ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ranunculaceae ; chemistry

Objective To investigate the changes of Th1/Th2 cells and related cytokine levels in chronic hepatitis B (CHB) fibrosis.Methods Forty-six patients with CHB fibrosis underwent liver biopsy during March and October,2011.According to the stage of fibrosis,the patients were divided into S0-1 group (n =15),S2-3 group (n =20) and S4 group (n =11).Ten healthy subjects served as controls.The frequencies of circulating Th1,Th2 cells were detected by flow cytometry.The expressions of interferon-γ (IFN-γ) and interleukin 4 (IL-4) mRNA in peripheral blood mononuclear cells (PBMCs) were detected by real-time quantitative PCR.The serum IFN-γand IL-4 concentrations were determined by enzyme-linked immunosorbent assays.Intrahepatic expressions of IFN-γ and IL-4 were detected by immunohistochemical staining.Differences between groups were analyzed using non-parametric Kruskal-Wallis H test,followed by Mann-Whitney U test for multiple comparisons.Logistic regression was used for multivariate analysis.Results With the degree of liver fibrosis exacerbations,the peripheral Th1/Th2 cells frequencies ratio,IFN-γ/IL-4 mRNA ratio in PBMCs,serum IFN-γ/IL-4 ratio and intrahepatic IFN-γ/IL-4 ratio were declined (x2 =36.259,40.822,26.321 and 31.852,respectively,all P ＜ 0.05).Serum and intrahepatic IFNγ/IL-4 ratio were negatively associated with the stage of liver fibrosis (r =-0.616 and-0.531,P ＜0.01).Logistic regression analysis showed that AST,PT and the serum IFNγ/IL-4 ratio were the risk factors for significant liver fibrosis (S2-4) (OR =5.933,95％ CI:1.324-26.586,P =0.02; OR =12.866,95％CI:1.746-94.788,P =0.01; OR=4.755,95％CI:1.034-21.862,P =0.04).Conclusions The CHB patients has imbalanced Th1/Th2 ratio.With the degree of liver fibrosis exacerbations,Th1/Th2 cytokines drift into Th2 lymphocyte sub-cluster,which suggests that Th1/Th2 imbalance may be involved in the pathogenesis of CHB fibrosis.

OBJECTIVETo determine the correlation between DNA load of human papillomavirus (HPV) and recurrence of condyloma acuminata (CA).

METHODSThe HPV6/11 and HPV16/18 DNA load of 31 cases of primary CA and 32 cases of recurrent CA were detected by real-time fluorogenic quantitative PCR.

RESULTSAmong the 63 CA patients, 62 cases were HPV6/11 DNA positive. The positive rate was 98.4%. The ranges of HPV6/11 DNA load in primary and recurrent CA were 1.4x10(3)-6.7x10(7) copies/ml and 1.2x10(4)-3.6x10(8) copies/ml respectively. Of 62 cases with HPV6/11 DNA positive, 7 cases were HPV16/18 DNA positive (11.3%). The ranges of HPV16/18 DNA load in primary and recurrent CA were 1.9x10(3)-1.6x10(4) copies/ml and 1.4x10(5)-1.7x10(7) copies/ml respectively. The HPV6/11 and HPV16/18 DNA load in recurrent CA were higher than in primary CA (P < 0.05). The DNA load of HPV6/11 was positively correlated with times of recurrence and course of disease (r=0.37 and 0.30 respectively).

CONCLUSIONCertain correlation exists between DNA load of HPV and recurrence of CA.

Adult ; Condylomata Acuminata ; virology ; DNA, Viral ; analysis ; Female ; Humans ; Male ; Middle Aged ; Papillomaviridae ; genetics ; isolation & purification ; Papillomavirus Infections ; virology ; Recurrence ; Sequence Analysis, DNA ; Viral Load

OBJECTIVEThe present study aimed to clone and express three fragments of genomic RNA derived from SARS associated coronavirus (SARS-CoV) S1 domain and to study its immunogenicity.

METHODSThe S1 domain gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pQE-30. Three fragments (40-751, 746-1344 and 746-2001 bp) derived from S1 domain produced after the recombinant plasmid (pQE-30/S1) was digested by restriction endonucleases. The three fragments were cloned into pQE-30 and expressed in M15 strains of Escherichia coli. The expression products, designated S1a, S1b and S1c respectively, were purified by Ni affinity chromatography. The immunogenicity was analyzed by Western Blot and ELISA using serologically confirmed sera from SARS patients and the sera from healthy donors was used as control at the same assay.

RESULTSThree recombinant plasmids (pQE-30/S1a, pQE-30/S1b, pQE-30/S1c) were constructed.Fusion proteins with relative molecular mass of 26,700, 22,500 and 46,000 dalton were successfully expressed with amounts of 35%, 35% and 30% of total cell protein and purified by Ni affinity chromatography, respectively. Western Blot and ELISA analysis showed that the S1c protein could be specifically recognized by the sera from SARS patients.

CONCLUSIONThe recombinant S1c protein was a good immunogen and has the potential to be used as a vaccine against SARS-CoV infection.

Antibodies, Viral ; blood ; Antigens, Surface ; genetics ; immunology ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; SARS Virus ; genetics ; immunology ; metabolism ; Severe Acute Respiratory Syndrome ; blood ; virology ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.

METHODSH5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.

RESULTSThe recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.

CONCLUSIONThe recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.

Animals ; Baculoviridae ; genetics ; Cell Line ; Erythrocytes ; cytology ; immunology ; Genetic Vectors ; genetics ; Guinea Pigs ; Hemagglutination Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Spodoptera ; Transfection

OBJECTIVETo prepare and characterize monoclonal antibodies (mAbs) against the recombinant nucleocapsid (N) protein of 3 human coronaviruses SARS-CoV, 229E and OC43 and study the antigenic relationship between the 3 N proteins.

METHODSBALB/c mice were immunized with the recombinant N proteins of SARS-CoV, 229E and OC43 to obtain the mAbs by means of hybridoma. Screening and identification of the mAbs were performed using indirect enzyme-linked immunosorbent assay (ELISA), Western blotting and indirect immunofluorescence assay. Cross-reactivity between the N proteins of the 3 coronaviruses was analyzed with the prepared mAbs.

RESULTSThe mAbs against the recombinant N proteins of SARS-CoV, 229E and OC43 were obtained, which reacted specifically with the corresponding viral N protein as shown by indirect ELISA, Western blotting and indirect immunofluorescence assay. No cross-reactivity was found between the 3 N proteins.

CONCLUSIONThe prepared mAbs against the recombinant N proteins may provide valuable assistance in studying antigenic relationships of N proteins between the 3 human coronaviruses.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Coronavirus 229E, Human ; genetics ; immunology ; Coronavirus OC43, Human ; genetics ; immunology ; Cross Reactions ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology

This study was aimed to investigate the effect of clostridium difficile toxin A (Tcd A) on proliferation of K562 cells and its mechanism. The proliferative activity of K562 cells exposed to Tcd A was tested by MTT assay; cell cycle distribution and mitochondrial membrane potential were analyzed by flow cytometry; the protein expression of cytochrome C and DNA fragmentation were observed by immunohistochemistry staining and agarose gel electrophoresis respectively. The results indicated that Tcd A inhibited proliferation of K562 cells in a time-and concentration-dependent manner. Cells were arrested at G(0)/G(1) phase. Peak of apoptosis appeared. The protein expression of cytochrome C increased as compared with control group (p < 0.05). Agarose gel electrophoresis of DNA from K562 treated with Tcd A revealed a "ladder" pattern. It is concluded that clostridium difficile toxin A can inhibit proliferation and induce apoptosis of K562 cells. The mechanism may be in relation to decrease of mitochondrial membrane potential and the release of cytochrome C from mitochondria matrix.
Apoptosis ; drug effects ; Bacterial Toxins ; pharmacology ; Cell Proliferation ; drug effects ; Enterotoxins ; pharmacology ; Humans ; K562 Cells

This study was purposed to investigate the growth inhibition and apoptosis-inducing effect of Clostridium difficile toxin A (TcdA) on the leukemia cell line K562. The proliferative activity of K562 cells exposed to Tcd A was tested by MTT assay, cell apoptosis was detected by flow cytometry; immunocytochemistry and colorimetric assay were employed to detect the protein expressions of BCL-2/BAX and the activity of Caspase-3, respectively. The results indicated that the proliferation of K562 cells was inhibited in a time-and dose-dependent manner after exposure to Tcd A for 24, 48 and 72 hours, the cells displayed the typical apoptotic, morphological changes, the expression of BCL-2 protein was down-regulated but the expression of BAX protein was signficantly increased, compared with control group (p < 0.05). In addition, caspase-3 was activated in a concentration-dependent manner. It is concluded that Tcd A inhibits cell growth of K562 by inducing apoptosis, and the up-regulation of BAX protein and activation of caspase-3 may play important roles in these processes.
Apoptosis ; drug effects ; Bacterial Toxins ; pharmacology ; Caspase 3 ; metabolism ; Enterotoxins ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

Objective:To discuss the management principles and skills for treatment of intractable ureterostenosis under ureteroscope.Methods:Our management experience on 19 patients with intractable ureteral stenosis was retrospectively analyzed.The 19 cases included urological TB-caused multiple ureteral stenosis,oncothlipsis to ureters from intestinal tract or gynecology,restenosis 3 months to 12 years after pelviureteric junction plasty,operative site stenosis after ureterolithotomy. double ureter back flow accompanied by stenosis,ureter imperforation after renal parenchyma lithotomy without placing double"J",ureter imperforation 3 months after extracorporeal shock-wave lithotripsy due to ureterolith,tubal bladder stoma stenosis after renal transplantation,restenosis after tubal bladder stoma due to distal ureterostenosis,and so on.All the patients were treated under ureteroscope.The management methods included:the Wolf 8/9.8 CH12?and Wolf 6/7.6 CH5?ureteroscope was used as a dilator to dilate the stenoses:balloon expanding under ureteroscope was used to dilate the stenoses;the ureter pliers was used to expand the stenoses to different directions;the cold knife was used to open the stenoses;if the diameter of stenoses were smaller than the that of the ureteroscopes,F4.5 or F3 double"J"tubes were inserted guided by a wire under ureteroscope; and 2 or 3 weeks later,a larger tube or two tubes were introduced into the stenoses already dilated partly by the former tube. Results:Ureteroscopic method failed in treating 2 patients in our group and succeeded in treating all the other patients.The outcomes of patient were fine during 9 months to 3 years'follow-up.Conclusion:It is difficult to treat patients with intractable ureterostenoses.With good experience in manipulation of ureteroscope,the flexible application of several techniques according to the different conditions of different patients can guarantee successful treatment in most patients.