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Objective To comparatively study the characteristics of 3 kinds of culture substrates of human odontogenic induced pluripotent stem cells(iPSCs).Methods The human odontogenic iPSCs were cultured by 3 kinds of substrates:mouse embryonic fibroblasts(MEF),matrigel and recombinant human vitronectin(VTN-N).The iPSCs growth situation was compared among three groups.Results The preparation time of these 3 kinds of substrates was 14,3,1 hlespectively,and,the difference was statistically significant (P＜0.05).The iPSCs reprogramming time was (30± 1.6),(26 ± 2.1),(27 ± 1.4) d,lespectively,wht that in the MEF group significantly higer than in other two groups (P＜0.05).The reprogramming efficiencies were 0.3 ％ ± 0.03 ％,0.56 ％ ± 0.08 ％,0.7 ％ ± 0.02 ％ respectively (P＜ 0.05).Three kinds of substrate could better support iPSCs growth and make them to maintain un-differentiation status.Conclusion with no heterologous animal components,and the adrantaga of simple pleparation,oonfrollable standard and shorter gramming time is easy to prepare,the standard is controllable and the reprogramming time is shorter,which is an ideal substrate for supporting iPSCs growth.
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Objective To analyze radiation induced alterations of vitronectin and collagen expressions in fibroblasts at different times post-irradiation,so as to evaluate the potential to apply vitronectin as a biomarker of radiation-induced lung fibrosis.Methods The human fibroblast cells WI-38 and IMR-90 were irradiated with 137Cs γ-rays at doses of 0 (control),4,6,8,10 and 12 Gy,respectively.The cells and its supernatant were collected at 6,12,24,36,48 and 60 h post-irradiation.The expressions of vitronectin and collagen Ⅰ and Ⅲ were analyzed by Western blot,PCR and ELISA.Results After irradiation,the expressions of vitronectin and collagen Ⅰ and Ⅲ were positively correlated (r=0.40-0.79,P＜0.05) and were all significantly higher than that in control group (t =3.04-25.45,P ＜0.05) and reached the highest expression levels at 48 h after 8-10 Gy of irradiation (t =2.92-18.86,P ＜ 0.05).Analyses of Real-time PCR and ELISA assay showed that expressions of vitronectin mRNA and its protein level in the cell lysis were significantly increased by radiation (F =27.09-42.62,P ＜ 0.05).Conclusions The expressions of vitronectin in cellular supernatant and its mRNA may be a potential biomarker of radiation-induced fibrosis,and 48 h after 8 Gy irradiation may be an optimum condition of measurement.
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Objective To investigate the expression of vitronectin (VN) in placental basal plate and its relationship with the pathogenesis of severe preeclampsia.Methods From March 2010 to December 2011,17 patients with early-onset severe preeclampsia who delivered in the Second Hospital of Jilin University were recruited as the early-onset severe preeclampsia group; and 16 women were recruited as the late-onset severe preeclampsia group.Meanwhile,15 healthy pregnant women who delivered before 34 weeks were defined as the early control group (termination of pregnancy was carried out because of fetal heart malformations),and 15 healthy pregnant women delivered after 34 weeks were defined as the late control group.Imnunohistochemistry and semi-quantitative reverse transcription (RT)-PCR were used to investigate the expression of VN protein and mRNA in the placental infarct center and its surrounding tissue of placental basal plate.The levels of serum prothrombin time (PT),part thromboplastin time (APTT) and fibrinogen (FIb) were detected and the international normalized ratio (INR) was calculated.The correlation of abnormal coagulation markers and VN expression levels in the early-onset severe preeclampsia group and the early control group was studied.Results (l) VN protein was detected in all placental basal plate of the four groups.It was highly expressed in the necrotic tissue of placental infarct center and weakly expressed in the tissue far from the infarcted area.(2) The mean levels of VN protein expression in placental basal plate of the early-onset severe preeclampsia group,the late-onset severe preeclampsia group,the late control group and the early control group were 0.152 ± 0.019,0.113 ± 0.023,0.095 ± 0.014 and 0.055 ± 0.010,respectively.And the differences between the groups were statistically significant (P ＜ 0.01).The VN protein expression in placental infarct center,infarct edge,peri-infarct tissue and tissue far from the infarcted area gradually reduced,and the differences were statistically significant (P ＜ 0.01).Compared with the same areas of each group,the differences were not statistically significant (P ＞ 0.05).(3) VN mRNA were detectable in infarct center,infarct edge,per-infarct tissue and tissue far from the infarcted area of placental basal plate.In the early-onset severe preeclampsia group and the early control group,it was statistically higher in infarct center than in tissue far from the infarcted area (P ＜ 0.05).(4) PT of the early-onset severe preeclampsia group was (9.45 ± 0.63) s,significantly shorter than that of the early control group [(9.88 ± 0.17) s,P ＜ 0.05].While there was no statistically significant difference in APTT,FIB and INR among the four groups (P ＞ 0.05).(5) In the early-onset severe preeclampsia group,VN expression level and PT were significantly negative correlated (r =-0.612,P ＜0.05) ; while in the early control group there was no correlation (r =0.489,P ＞ 0.05).Conclusion VN was highly expressed in placental basal plate of the early-onset severe preeclampsia group,which caused the imbalance of coagulation and fibrinolysis system and the pathogenesis of severe preeclampsia.
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Background Vitronectin is a glycoprotein that has a variety of functions.Its expression was markedly higher in the retina of oxygen induced mice,which was confirmed in our animal model,and also increased in human umbilical vein endothelial cells (人 UVECs) that were cultured in high glucose.However,there was no evidence that showed vitronectin was involved in retinal neovascularization.Objective This study was to observe the influence of vitronectin on cytoskeleton remodeling,cell migration and blood vessel formation in 人 UVECs conditioned by high glucose.Methods 人 UVECs were cultured in high glucose and the expression of vitronectin was knocked down using RNA interference technology.The experiments were divided into the high glucose group (人 UVECs were conditioned with DMEM medium that contained 50 mmol/L glucose),negative interference group (人 UVECs were transfected with control siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose) and positive interference group (HUVEC were transfected with vitronectin siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose).The protein expression of vitronectin was measured by Western blot,and the microfilament cytoskeleton of 人 UVECs was examined by immunofluorescence cytochemical staining followed by fluorescence microscopy.Cell migration ability in a scratch wound assay and blood vessel formation ability in a matrigel assay of 人 UVECs were evaluated.The general differences were analysed by One-Way ANOVA ;further contrasts of the two groups were analysed by the LSD-t test.Results The differences in vitronectin expression of the three groups were not obvious at 0 hour (F=1.064,P＞0.05).After 24 hours,vitronectin expression was highest in the high glucose group,lower in the negative interference group,and the lowest in the positive interference group,and the differences were significant (F =15.519,P＜0.05).After 48 hours,vitronectin expression of the three groups displayed the same pattern,and the differences were also significant (F=37.521,P＜0.05).Immunofluorescence showed that the cytoskeleton structure was most obvious in the high glucose group,moderate in the negative interference group,and was the least obvious in the positive interference group,after both 24 hours and 48 hours.In the scratch wound assay,the cell migration ability of the high glucose group was the highest,lower in the negative interference group,and the lowest in the positive interference group after 24 hours,and the differences were significant (F=90.685,P＜0.05).After 48 hours,the cell migration abilities of the three groups displayed the same pattern,and the differences were also significant (F=67.880,P＜0.05).In the matrigel assay,after 6 hours,the number of blood vessels formed in the high glucose group was more than that in the negative interference group,and the least amount was found in the positive interference group.The differences among of them were significant (F =86.653,P＜0.05).The number of blood vessel formed in the positive interference group was also the lowest after 12 hours,and the differences were also significant (F=18.992,P＜0.05).Conclusions Vitronectin can bring about cytoskeleton remodeling,increase in cell migration,and enhancement of blood vessel formation in 人 UVECs conditioned in high glucose.It may be one of the important influence factors of diabetic retinopathy.
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El objetivo del presente trabajo fue desarrollar un péptido híbrido, que presente una secuencia capaz de quelatar al 99mTc y otra con afinidad por el receptor de la vitronectina, que permita la detección in vivo de tumores malignos. El marcaje del péptido PICIC3 con 99mTc se realizó de forma directa. Se estudió la estabilidad del complejo en exceso de L-cisteína y en plasma, la unión a proteínas plasmáticas, el coeficiente de partición en el sistema NaCl 0.9 por ciento:n-octanol, la carga del complejo mediante electroforesis y la afinidad por el receptor de la vitronectina se valoró a partir de un ensayo de saturación con membranas de células B16-F10. Se determinó la biodistribución en ratones C57BL/6 con injertos de melanoma B16-F10. Conclusiones: El péptido desarrollado mostró una afinidad satisfactoria por el receptor de la vitronectina y que permitió la detección in vivo de los melanomas múridos del tipo B16-F10 en los ratones injertados.
The aim of the present work was to develop a hybrid peptide, with a sequence for the chelation of 99mTc and other with affinity for the vitronectine receptor, to allow in vivo detection of malignant tumors. 99mTc-labeling of peptide PICIC3 was directly performed. The stability in presence of L-cysteine excess and plasma of the complex, its binding to plasma proteins, the partition coefficient in NaCl 0.9 percent:n-octanol, the charge of the chelate by electrophoresis and the peptide affinity for the vitronectine receptor by a saturation assay using membranes of B16-F10 cells, were studied. Biodistribution in C57BL/6 mice injerted with melanoma B16-F10 was assessed. Conclusions: Developed peptide showed a satisfactory affinity for the vitronectine receptor and allowed in vivo detection of murine melanomas in mice with allografts.
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Animals , Mice , /metabolism , Melanoma, Experimental , Melanoma, Experimental/metabolism , Peptides, Cyclic/pharmacokinetics , Technetium/pharmacokinetics , Tissue Distribution , Drug Stability , Time Factors , Isotope Labeling/methods , Peptides, Cyclic/metabolism , Technetium/metabolismABSTRACT
Purification of a lectin from Bothrops jararacussu venom (BjcuL) was carried out using agarose-D-galactose affinity gel. MALDI-TOF gave a major signal at m/z 32028, suggesting the presence of a dimmer composed of two identical subunits. Divalent cations were required for the lectin activity, as complete absence of such ions reduced hemagglutination. BjcuL was more effective at neutral pH and showed total loss of activity at pH values below 4.0 and above 9.0. Its agglutinating activity remained stable at 25°C until 60min, but increased when at 35°C for at least 15min. Adhesion assays to extracellular matrix (ECM) glycoproteins showed that the biotinylated lectin (0.039-5.0µg/100µl) was capable of binding to fibronectin and vitronectin in a dose-dependent manner. The binding was partially inhibited in the presence of D-galactose. BjcuL (1.25-10µg/30µl) potential was investigated for leukocyte rolling and adhesion to endothelial cells in living microvessels using intravital microscopy, which showed that it induced a dose-dependent increase in rolling and adherence of leukocytes, acting directly on endothelial cells of postcapillary venules. The specific association between lectins and their ligands, either on the cell surface or on the ECM, is related to a variety of biological processes. The complementary characterization of BjcuL, shown here, is useful to further understand the venom effects and as a background for future investigation for therapeutic strategies.
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Both fibronectin and vitronectin bind to Pneumocystis carinii (P. carinii) and mediate the attachment of the organisms to respiratory epithelial cells. Surfactant A and D play a role in the interaction between P. carinii and host cells. In this study we examined the expression of fibronectin, vitronectin, surfactant-A and D in the interaction between P. carinii and alveolar epithelial cells by immunohistochemistry and pre-embedding immunoelectron microscopy. The experimental rat model of P. carinii pneumonia was induced by administration of low protein diet (8%) and drinking water containing dexamethasone (2 mg/liter) for 6 to 8 weeks. The primary antibodies for light and electron microscopic immunohistochemistries were monoclonal antibodies including fibronectin (1:100) and vitronectin (1:100), and polyclonal antibodies including surfactant A (1:50) and D (1:50), respectively. Light microscopic immunohistochemistry for the fibronectin, vitronectin, surfactant-A and D showed strong expressions on the P. carinii and surface linings of type I alveolar epithelial cells. The electron microscopic immunohistochemistry of the fibronectin and vitronectin showed a strong immunoexpression along the surface pellicles and tubular extensions of P. carinii trophozoites, and surface membranes of the type I epithelial cells. The surfactant-A and D proteins showed a strong expression on the pellicles of P. carinii and surface membranes of the type I epithelial cells, but a weak expression on the free-floating surfactant materials. In conclusions, the trophozoites of P. carinii were mostly attached to type I epithelial cells. The fibronectin, vitronectin, surfactant-A and D were strongly expressed, and played an enhancing role in the binding between the P. carinii organisms and the type I alveolar epithelial cells.
Subject(s)
Antibodies , Antibodies, Monoclonal , Dexamethasone , Diet, Protein-Restricted , Drinking Water , Epithelial Cells , Fibronectins , Immunohistochemistry , Membranes , Microscopy, Immunoelectron , Models, Animal , Pneumocystis carinii , Pneumocystis , Pneumonia , Pneumonia, Pneumocystis , Trophozoites , VitronectinABSTRACT
Objective To investigate the function of integrin ?v?3 receptor in bone marrow micrometastasis in breast cancer. Methods Flow cytometry was used to examine the expression of integrin ?v?3 receptor in breast cancer cell lines MCF7, MDA-MB-231 and MDA-MB-435s. Functional activation of integrin ?v?3 was assayed by cell adhesion, cell migration in Boyden Chamber and by confocal microscopy. The role of LM609 in bone marrow micrometastasis in athymic mice was detected by quantitative real-time RT-PCR. Results Integrin ?v?3 expression in cellular membrane was positive in MDA-MB-231 and MDA-MB-435s cell lines and negative in MCF-7 cell line. Cell adhesion and migration was attenuated significantly by LM609 in MDA-MB-231 and MDA-MB435s cell lines. Treated by LM609, the number of tumor cells significantly decreased in high integrin ?v?3 expression tumor in nude mice model(P