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Objective To explore the association of synovial fluid vasoactive intestinal peptide levels with cartilage damage,radiological changes and symptomatic severity in patients with ankle post-traumatic osteoarthritis. Methods 74 patients with ankle traumatic osteoarthritis undergoing ankle anthroscopic debridement or joint replacement and 69 healthy controls receiving body check were enrolled in the this study. Serum and synovial fluid VIP concentrations were measured by a special radioimmunoassay method. Cartilage degradation biomarker colla-gen type Ⅱ(CTX-II)and inflammatory marker interleukin-6 were determined by enzyme-linked immunosorbent assay (ELISA). The symptomatic and functional severity was evaluated using Teeny & Wiss and AOFAS ankle-hindfoot rating scale. The radiographic progression of PTAOA was identified according to the modified ankle osteoar-thritis Kellgren-Lawrence (K-L) grading system. The mankin score was used for assessing the histopathological severity for cartilage lesions. Receiver operating characteristic (ROC) curve was conducted and the area under curve(AUC)was used to evaluate the diagnostic value of VIP,IL-6 and CTX-II levels for the prediction of the modified K-L grading by comparing with other biomarkers. Results There were no significant differences in serum VIP levels between PTAOA patients and controls. VIP levels in synovial fluid showed a negative correlation with modified ankle K-L grading,Mankin scores,CTX-Ⅱand IL-6. In addition,VIP levels were also positively associated with Teeny&Wiss and AOFAS ankle-hindfoot scores. The AUC area of VIP was similar to CTX-Ⅱat early stage of the disease. Conclusions Synovial fluid VIP levels show an independent and negative correlation with disease severity in patients with PTAOA. Low level of VIP in SF can be used as a potential biomarker for reflecting disease progression.
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Objective To explore the association of synovial fluid vasoactive intestinal peptide levels with cartilage damage,radiological changes and symptomatic severity in patients with ankle post-traumatic osteoarthritis. Methods 74 patients with ankle traumatic osteoarthritis undergoing ankle anthroscopic debridement or joint replacement and 69 healthy controls receiving body check were enrolled in the this study. Serum and synovial fluid VIP concentrations were measured by a special radioimmunoassay method. Cartilage degradation biomarker colla-gen type Ⅱ(CTX-II)and inflammatory marker interleukin-6 were determined by enzyme-linked immunosorbent assay (ELISA). The symptomatic and functional severity was evaluated using Teeny & Wiss and AOFAS ankle-hindfoot rating scale. The radiographic progression of PTAOA was identified according to the modified ankle osteoar-thritis Kellgren-Lawrence (K-L) grading system. The mankin score was used for assessing the histopathological severity for cartilage lesions. Receiver operating characteristic (ROC) curve was conducted and the area under curve(AUC)was used to evaluate the diagnostic value of VIP,IL-6 and CTX-II levels for the prediction of the modified K-L grading by comparing with other biomarkers. Results There were no significant differences in serum VIP levels between PTAOA patients and controls. VIP levels in synovial fluid showed a negative correlation with modified ankle K-L grading,Mankin scores,CTX-Ⅱand IL-6. In addition,VIP levels were also positively associated with Teeny&Wiss and AOFAS ankle-hindfoot scores. The AUC area of VIP was similar to CTX-Ⅱat early stage of the disease. Conclusions Synovial fluid VIP levels show an independent and negative correlation with disease severity in patients with PTAOA. Low level of VIP in SF can be used as a potential biomarker for reflecting disease progression.
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Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.
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Objective To construct,express and identify the anti-idiotypic antibody single chain variable fragment(scFv) against Edwardsiella tarda.Methods By using RT-PCR method,the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody(mAb) 1E11 against Edwardsiella tarda were cloned and joined with a(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH was constructed.It was then cloned into vector plasmid pET-28a,expressed in Escherichia coli BL21(DE3),and confirmed by SDS-PAGE,Western blot and ELISA.Results The recombinant scFv could be expressed in E.coli BL21(DE3) in a fusion protein pattern.The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded.The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv had the binding activity to the antigen.Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21(DE3),providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.
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Objective To investigate the distribution of nerve growth factor(NGF) and epidermal growth factor(EGF) and their colocalization with follicle-stimulating hormone(FSH) in rat submaxilary glands,and to study the effects of FSH on the secretion of NGF and EGF by submandibular gland tissue in vitro.Methods Immunohistochemical colocalization method was used in the experiment.Submandibular tissues of rats were incubated in vitro and different concentration of FSH was added.Enzyme link immunoassay was used to measure EGF and NGF in supernatant.Results The serous glandular cells,granular convoluted epithelial cells and all other duct epithelial cells in the submandibular gland showed FSH,NGF or EGF positive immunoreactivity.The positive substance was distributed in the cytoplasm with negative nuclei.Both FSH and NGF or EGF positive products were co-located in rat submandibular gland.When the concentration of FSH was more than 10-5-10-6 IU/L,the secretion of EGF and NGF lessened with the decreased concentration of FSH;when the concentration of FSH was less than 10-5-10-6 IU/L,the secretion of EGF and NGF increased with the decreased concentration of FSH.Conclusion FSH has the same bidirectional regulation effects on EGF and NGF in vitro.FSH may regulate the function of endocrine of rat submandibular gland.
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Objective In this study, we investigated the distribution of Peroxiredoxin II mRNA in follicles at all stages in mouse ovaries and mouse secondary oocytes(MII eggs) basing on our previous researches, to provide the morphological basis for exploring the effect of Peroxiredoxin II on oogenesis and oocyte maturation in the mouse. Methods To observe the distribution of Peroxiredoxin II mRNA in follicles at all stages in ovaries and the localization of Peroxiredoxin II mRNA in Germinal-Vesicle intact oocytes(GV oocytes) and MII eggs by in situ Hybridization. Results In situ hybridization of ovary section revealed that the signals for Peroxiredoxin II mRNA were undetectable in oocytes of primordial follicles, and moderate signals for Peroxiredoxin II mRNA were observed in oocytes of primary follicles. Moreover, strong signals for Peroxiredoxin II mRNA were evident in antral follicles. The signals for Peroxiredoxin II mRNA also existed in GV oocytes and MII eggs in vitro. The hybrid signals were stronger in GV oocytes than in MII eggs. In addition, the weak but consistent signals for Peroxiredoxin II mRNA were detected in follicular cells from primordial follicles to large antral follicles. Peroxiredoxin II mRNA was located in cytoplasm of oocytes and follicular cells, but not in nuclei.Conclusion These results suggested that Peroxiredoxin II might be involved in the regulation of oogenesis and oocyte matruation in the mouse.;
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Objective To investigate the distribution of folliclestimulating hormone (FSH),luteinizing hormone (LH) and colocalization with gonadotropin releasing hormone receptor (GnRHR) in rat submaxilary glands. Methods Distribution of FSH, LH and colocalization with GnRHR consecutive sections of rat submaxilary glands were investigated by immunohistochemical colocalization methods. Results FSH and LH immunoreactivity were observed in the epithelial cells of serous acinus, secretory tubes, excretory ducts and granular convoluted tubule. The immunoreactive materials were brown and distributed in the cytoplasma with negative nucleolus. The results of immunohistochemical colocalization showed not only FSH but also GnRHR immunoreactivity in the same structure of two adjacent section. The distribution of the positive substance of FSH and GnRHR were similar to each other. The most of showing GnRHR immunoreactivity cells were detected LH immunoreactivity in the same structure of two adjacene section and the others were immunonegative. The GnRNR immunoreactive materials were distributed in the cytoplasma with negative nucleolus.Conclusion The epithelial cell of serous acinus, secretory tubes, excretory ducts and granular convoluted tubule of rat submaxilary glands may be synthesized and secreted FSH and LH. These cells with FSH and LH positive immunoreaction of rat submaxilary glands may be regulated by Gonadotropin releasing hormone (GnRH) through autocrine or paracrine.;
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Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.
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Objedctive To study the mechanism of GnRH agalogus(Alarelin)on[ Ca2 + ]; mobilization in stomach smooth muscle cells (SSMc)of rats. Methods Cells were cutured and loaded with Fluo-3-AM. [Ca2 + ]; was measured by nuomeent intensity(FL) in each cell with confocal micoopy. Results (1 ) In Hanks solution. l0-7, l0-6, l0 - 5mol L- 1 Alarelin can elevate[ Ca2 + ], its peak - resting values reached 6. 00 ?0. 50 .9. 23 ?0. 62. l8. 97 ?2. 42 respectively, which indicate that the level of [Ca2+ ]; act in an deede- pendent and time - dependent. (2) thntredly. when Alarelin was 10 4mol?Ｌ－１, its peak - resting value only reached 6. 32 ?0 .67, conpared with Alarelin l0 5m.l L－1 which was sigificantly lower(P 0. 05 ). (5 )When pertreatment with Lacidipine in Hanks solution, the effect of Alarelin l0 5mol L-1.as partly inhibited(P
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Objective To study the effects of DHPs calcium antagonist Mn9202 and lacidipine on 5-HT、TH and 5-HTR of rat stomach fundus smooth muscle cells. Methods It's effects was investigated by using immunocytochemistry and image analysis of stom- ach fundus smooth muscle cells in culture and compared with cyprohetadine(antagonist of 5-HT). Results 5-HT、TH and 5-HTR im- munoreactive substances were existed in SFSMC. The results of image analysis indicated that the content of 5-HT. TH and 5-HTR were significantly lower when using Mn9202.lacidipine and cyprohetadine. Conclusion The results suggest that the rat stomach fundus smooth muscle cells possess 5-HT autocrine function, and DHPs calcium antagonist reduce the number of 5-HT receptor in the rat stomach fundus smooth muscle cells though the reduction of 5-HT sythesis, this results conform with cyprohetadine (antagonist of 5 - HT).
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Dopamine is one of the bioactive amines which regulate many physiological functions. In order to study its localization and quantification in tissues with immunohistochemistry and radioimmunoassay, authors immunised New Zealand rabbits with dopamine-BSA (bovine serum albumin) complex and succeeded in the production of the specific antiserum. The antiserum was tested on routine paraffin sections of Bouin's fixed guinea-pig digestive tract with the ABC technique. The titre of the antiserum was 1:1000-1:2000. Results of substitution, absorption and cross-absorption tests proved that the positive staining obtained with the antiserum demonstrated the specific immunoreaction between the antibody to dopamine and the antigen in the tissues.
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The distribution and morphology of 5-HT immunoreactive endocrine cells in the gastrointestinal tract of 5 adult rats were studied by the immunohistochemicael PAP method with nickel-intensified DAB on paraffin sections of intestine rolls. The density of 5-HT immunoreactive endocrine cells in the gastrointestinal tract of the rat is highest in the pylorus, duodenum and colon and moderate in the jejunum, ileum, caecum and rectum and lowest in the body of the stomach. The 5-HT immunoreactive endocrine cells are various in shape. Some of them have several processes extending between other epithelial cells. The basal portion of some endocrine cells have processes with 5-HT positive substance accumulating in their ends. The processes of the basal portion of some endocrine cells extend into lamina propria through the basal membrane. The 5-HT positive substance of many endocrine cells can be found to extend to luminal surface of the crypt or intestinal tract. These results indicate that the 5-HT immunoreactive endocrine cells can release 5-HT by both endocrine and exocrine ways.
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Distribution and morphology of argyrophil and argentaffin cells in small intestine of 11 rats were studied by means of Huang's method of argyrophil reaction and Singh's method of argentaffin reaction on paraffin sections of intestine rolls. The results are as follows:1. The density of argyrophil and argentaffin cells in rat small intestine is the highest in the duodenum and progressively decreases from jejunum to ileum.2. The staining intensity of argyrophil and argentaffin cells is lowest in the basal portion of crypts and progressively increases from crypts to villus. Intensely stained argyrophil and argentaffin cells in the villus tip were observed. The basal portion of the argyrophil cells has cytoplasmic processes extending to connective tissue of the lamina propria and the argyrophil granules are released to lamina propria along these processes. Argyrophil granules can usually be found to extend to the luminal surface of these cells; occasionally they were observed extracellularly in the gland cavity, suggesting that argyrophil and argentaffin cells may have both endocrine and exocrine functions.3. Some argyrophil cells can be found in connective tissue of the lamina propria. The cells are irregular in shape and possesses processes. There are argyrophil granules in perikaryon and the processes and occasionally outside the cells. The argyrophil cells in the lamina propria are the same as those among epithelial cells in shape, argyrophil property and density of the granules. It is possible that these cells belong to endocrine cells.
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Objective To study the effect of FSH on the secretion of gastrin in the rat stomach,offering experimental evidence for reproductive endocrine hormone regulating digestive function. Methods The FSH was directly injected into the stomach of rats to observe the change of density of gastrin immunoreactive positive cells in the stomach by immunohistochemical SABC method and the gastrin level in circulating blood and gastric liquid by ELISA. Results Compared with the control group,which was injected with saline into the stomach,the density of immunoreactive positive cells was significantly increased in the stomach with FSH treatment group(P
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Objective To investigate the expression of TNF-related apoptosis-inducing ligand(TRAIL) and TNF-related apoptosis-inducing ligand receptor(TRAILR) in thyroid carcinoma. Methods Expression and distribution of TRAIL and TRAILR in thyroid tissues from 13 patients with papillary thyroid carcinoma, 3 patients with follicular thyroid carcinoma and 5 normal necrospy subjects were studied by immunohistochemical methods. Results TRAIL and all of TRAILR immunoreactivity was observed in both thyroid tissues from papillary thyroid carcinoma and follicular thyroid carcinoma. TRAILR4 was expressed highly in all thyroid carcinoma tissues but weakly in normal thyroid tissues.Conclusion TRAIL and all of TRAILR were present in thyroid tissues of papillary thyroid carcinoma and follicular thyroid carcinoma. Expression of TRAIL, TRAILR1 and TRAILR2 by thyrocytes in thyroid carcinoma may induce their apoptosis through autocrine or paracrine. Thyroid carcinoma cells were sensitive to TRAIL-inducing apoptosis despite expression of both decoy receptors. [
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Localization and quantitation of GnRH, somatostatin (SS), and ?-endorphin (?-EP) in human placentalvilla were studied using immunogold-silver staining method. GnRH and SS immunoreactive positive substance existed in the cytotrophoblasts of many placental villi and in syncytiotrophoblasts of few placental villi, ?-EP immunoreactive positive substance localized in syncytiotrophoblasts of many placental villi and in cytotrophoblasts of few placental villi. These results suggest that the cytotrophoblasts and syncytiotrophoblasts are able to synthesize these three kinds of regulatory peptides, however, SS and GnRH may be synthesized mainly in the cytotrophoblasts, ?-EP mainly be synthesized in the syncytiotrophoblasts. The amount of GnRH and ?-EP in placenta villi show obvious change with progress of pregnancy. This change show a negative relationship between the GnRH and ?-EP, which suggested that there may be a functional reciprocal inhibition between the GnRH and ?-EP.
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Distribution of GnRH immunoreactive cells and nerves in gastro entro-pancreatic system of rats was studied by using immunogold-silver staining. The GnRH immunoreactive epithelial cells can be seen in the stomach, small intestine, large intestine and pancreas. The apical portion of these GnRH postive epithelial cells extended to the luminal surface of surface epithelium and glands, and they belong to the open type endocrine cells. The GnRH immunoreactive nerve cells can be found in submucous plexus, myenteric plexus and serosa of the stomach and small intestine. The GnRH postive nerve fibers can be seen in lamina propria and submucosa of the small intestine. These results suggest that the GnRH may play an important role in the regulation of digestion.
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Objective To detect the existence of gonadotropin-releasing hormone receptors(GnRH-R) in rat liver,and to supply morphological evidence for studying the functional significance of the GnRH in hepatocyte. Methods Immunohistochemical SABC method and in situ hybridization with a digoxigenin labeled probe were used to study the localization of GnRH-R in the livers of five rats. Results The rat hepatocytes were found with the GnRH-R immunoreactivity and GnRHR mRNA hybridization signals.The GnRH-R immunoreactive substance was distributed in the cytoplasm of all positive cells,with immuno-negative nuclei.The GnRHR mRNA hybridization signals were also detected in the cytoplasm of all positive cells but not in the nuclei.Hepatocytes in different parts of liver lobules showed different intensity of GnRH-R immunoreactivity and GnRH-R mRNA hybridization signals.Conclusion The rat hepatocyte may express GnRH-R.The hepatocytes in different parts in liver lobules exhibit different levels of GnRH-R expression.
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Objective To observe the localization and distribution of follicle stimulating hormone receptor(FSHR) in the rat submandibular gland and stomach. Methods Immunohistochemical SABC method was used in the experiment. Results The serous glandular cells,granular convoluted epithelial cells and all other duct epithelial cells in the submandibular gland and the parietal cells of gastric gland showed FSHR positive immunoreactivity.The positive substance was distributed in the cytoplasm with negative nuclei.Conclusion The function of rat submandibular gland and gastric gland might be regulated by follicle stimulating hormone(FSH) through FSHR.
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Objective To study the effect of Peroxiredoxin Ⅱ on development of mouse preimplantation embryos in vitro and to investigate the possible action of Peroxiredoxin Ⅱ on preimplantation embryonic development. Methods One-cell embryos collected from the oviduct of the superovulated mice were cultured in microdrops of medium for 48?h.The effect of peroxiredoxin Ⅱ antibody on the development of embryo in vitro was observed,and the percentage of embryos developing to 2-and 4-cell stage of embryos was used as evaluation criteria.With redox-sensitive fluorescence probe 2′,7′-dichlorodihydroflurescin diacete(DCFH-DA),reactive oxygen species(ROS) induced by Peroxiredoxin Ⅱ antibody was monitored in mouse embryos after 24?h culture by laser confocal scanning microscopy. Results The embryonic development rate from 2-cell stage to 4-cell stage was decreased significantly by Peroxiredoxin Ⅱ antibody in the dilution of 1∶100 and 1∶200,but Peroxiredoxin Ⅱ antibody had no inhibitive effect on development from 1-cell to 2-cell stage.Moreover,Peroxiredoxin Ⅱ antibody in the dilution of 1∶200 induced increase in the production of reactive oxygen species in mouse embryos from 24?h culture of one-cell embryo.Conclusion Peroxiredoxin Ⅱ antibody induced the generation of “2-cell block” by increasing the production of reactive oxygen species(ROS) in mouse embryos.These results also indicated that Peroxiredoxin Ⅱ may reduce or eliminate ROS in preimplantation embryos and promote the development of preimplantation embryos.;