Apoptosis of Endothelial Cells Induced by Anti-Platelet Integrin β3 Antibody / 中国实验血液学杂志

OBJECTIVE@#To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin β3 antibodies in vitro.@*METHODS@#The serum from 36 chronic ITP patients were collected, flow cytometry and monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to collect antiplatelet integrin β3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin β3 antibodies, the cell damage was detected by Lactate dehydrogenase (LDH) assay, cell apoptosis was detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR (RT-qPCR), and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot. HUVEC were treated by PS combined with Akt activator SC79, the cells damage were detected by LDH assay, apoptosis of the cells were detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by RT-qPCR.@*RESULTS@#Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin β3 antibodies were collected. After HUVEC was treated by PS, the viability of LDH was significant increased（P<0.05）, so as for the apoptosis of the cells（P<0.05）, the expression of gene and protein of Bax was increased up-regulated（P<0.05）, the protein expression of pAkt was down-regulated（P<0.05）. Comparing with HUVEC cultured with PS alone, the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced（P<0.05）, so as for the apoptosis of the cells（P<0.05）, and gene expression of Bax was significantly decreased（P<0.05）.@*CONCLUSION@#Anti-integrin β3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin β3 antibodies to HUVEC was effectively reversed by SC79.

Megacariopoyesis humana in vitro: determinación de la concentración óptima de trombopoyetina / In vitro human megacaryocytopoiesis: determination of thrombopoietin optimal concentration / Megacariópoiesis humana in vitro: determinação da concentração ótima da trombopoietina

El estudio de la megacariopoyesis humana se ha visto obstaculizado por la relativa escasez de megacariocitos en la médula ósea (0,05-0,2 % de las células medulares), lo que ha llevado a la optimización de protocolos de expansión in vitro a partir de precursores de diversos orígenes (cordón umbilical, médula ósea y sangre periférica con o sin movilización previa). Los cultivos celulares a partir de precursores han permitido la producción y el estudio tanto de megacariocitos así como de proplaquetas y plaquetas Sin embargo, la producción in vitro óptima de megacariocitos que culminen todos los estadios de diferenciación es un reto aún no resuelto. En este trabajo reportamos los hallazgos concernientes a la determinación de las condiciones y concentraciones de trombopoyetina para lograr una óptima relación entre la cantidad de trombopoyetina empleada y el porcentaje y grado de diferenciación megacariocítica en muestras obtenidas de cinco donantes alogénicos aceptados para trasplante de médula ósea.

The study of human megakaryocytopoiesis has been hampered by the relative scarcity of megakaryocytes in bone marrow (0.05-0.2 % of medullary cells), which has led to the optimization of protocols of in vitro expansion of precursors from diverse sources (umbilical cord, bone marrow and peripheral blood with or without previous mobilization). Cell cultures from different precursors have allowed the production and study of megakaryocytes as well as proplatelets and platelets. However, the in vitro production of megakaryocytes that culminate all stages of differentiation is a challenge that has not yet been resolved. In this work we report the findings related to the determination of thrombopoietin treatment conditions and concentrations to achieve an optimal relationship between the amount of thrombopoietin and the percentage and degree of megakaryocytic differentiation in five allogeneic donors that were accepted for bone marrow transplantation.

O estudo da megacariopoiese humana tem sido dificultado pela relativa escassez de megacariócitos na medula óssea (0,05-0,2 % das células medulares), o que levou à otimização dos protocolos de expansão in vitro a partir de precursores de diversas origens (cordão umbilical, medula óssea e sangue periférico com ou sem mobilização prévia). Culturas de células a partir de precursores permitiram a produção e o estudo tanto de megacariócitos e de proplaquetas e plaquetas. No entanto, a produção ótima in vitro de megacariócitos que culminam em todas as fases de diferenciação é um desafio ainda não resolvido. Neste trabalho, relatamos as descobertas relativas à determinação das condições e concentrações de trombopoietina para obter uma relação ótima entre a quantidade de trombopoietina usada e a taxa e o grau de diferenciação megacariocítica em amostras obtidas de cinco doadores alogênicos aceitos para transplante de medula óssea.

Effect of Amino Acid Motifs in Integrin β3 Cytoplasmic Tail on αⅡbβ3-Mediated Cell function in 293T cell models / 中国实验血液学杂志

OBJECTIVE@#To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function.@*METHODS@#293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested.@*RESULTS@#293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen.@*CONCLUSION@#To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.

Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and ß3 integrin receptors / Disminución de la infección por rotavirus en células MA104 por la unión de proteínas de extractos probióticos a receptores celulares integrina ß3 y Hsc70 / Diminuição da infecção por rotavírus em células MA104 pela união de proteínas de extratos probióticos a receptores integrina ß3 e Hsc70

Abstract Probiotic bacteria are microorganisms beneficial to human health, useful to improving biological conditions. Thanks to probiotic bacteria the symptoms of viral infections can be alleviated. Different mechanisms whereby probiotic bacteria exert they antiviral effect have been proposed. The aim of this study was to determine whether probiotic bacteria extracts bind to receptors of host cells susceptible of rotavirus (RV) infection. To accomplish this objective, four probiotic bacterial strains of Lactobacillus spp. and Bifidobacterium spp. were tested. Probiotic extracts were obtained after bacterial growth, cell lysis and centrifugation. Obtained probiotic extracts were used in assays to interfere with adhesion and penetration of a RV strain in the mammal cell line MA104. Furthermore, the interaction between probiotic extracts and MA104 cell receptors was evaluated by co-immunoprecipitation assays using anti-β3-integrins and anti-Hsc70 antibodies. All four probiotic, protein-rich, extracts reduced RV infections in MA104 cells, suggesting a successful antiviral activity mediated by these probiotic extracts. All probiotic extracts significantly exerted their antiviral activity by interfering with RV adhesion on MA104 cell receptors, with proteins in probiotic extracts competitively interacting with cell surface receptors necessary to RV infection. Co-immunoprecipitation assay results showed that proteins in probiotic extracts were able to bind to β3-integrinsand Hsc70, which are two cellular receptors required to viral infection. The most significant contribution of this study is an insight into the mechanisms of probiotic antiviral activity, thus expanding current probiotics fundamental knowledge.

Resumen Las bacterias probióticas son microorganismos con efectos positivos en la salud humana, gracias a las bacterias probióticas los síntomas de infecciones virales pueden mitigarse. Al respecto, varios mecanismos antivirales de las bacterias probióticas han sido propuestos. El propósito de este estudio fue determinar, de manera experimental, si extractos de bacterias probióticas reducen la infección rotavírica al interferir con la unión entre el rotavirus y sus receptores celulares blanco. Extractos de cuatro cepas probióticas de Lactobacillus spp. y Bifidobacterium spp. fueron obtenidos a partir de cultivos bacterianos lisados y centrifugados. Cada uno de los extractos fue usado en experimentos para determinar si estos interfieren con la adhesión y penetración del rotavirus en células de mamífero MA104. Además, la interacción entre extractos probióticos y receptores de las células MA104 fue evaluada con ensayos de co-inmunoprecipitación, usando anticuerpos anti-integrina β3 y anti-Hsc70. Se observó que los cuatro extractos probióticos, ricos en proteínas, redujeron significativamente la infección de rotavirus en las células MA104. También se estableció que la que la actividad antiviral de los extractos probióticos es mediada por la interacción competitiva de sus proteínas con los receptores integrina β3 y Hsc70 de las células MA104, necesarios para iniciar la infección por rotavirus. Estos hallazgos constituyen un aporte al conocimiento de los mecanismos básicos de acción antiviral de las bacterias probióticas.

Resumo Bactérias probióticas são microrganismos com efeitos positivos na saúde humana, úteis na melhora de certas condições biológicas. Gracas a bactérias probióticas os sintomas de uma infecção viral podem ser aliviados. Diferentes mecanismos pelos quais as bactérias probióticas exercem seus efeitos antivirales têm sido propostos. O objetivo de este estudo foi determinar se extratos de bactérias probióticas reduzem a infecção de rotavírus (RV) ao interferir com a união entre o RV e seus receptores celulares alvo. Quatro cepas probióticas de Lactobacillus spp. e Bifidobacterium spp. foram testadas. Os extratos probióticos foram obtidos após o crescimento bacteriano, lise celular e centrifugação. Os extratos probióticos obtidos foram utilizados em ensaios para determinar se interferem com a adesão e penetração de uma cepa de RV em células de mamífero MA104. Adicionalmente, a interação entre os extratos probióticos e os receptores das células MA104 foi avaliada por ensaios de co-imunoprecipitação usando anticorpos anti-integrina β3 e anti- Hsc70. Os quatro extratos probióticos, ricos em proteínas, reduziram as infecções por RV em células MA104, sugerindo uma atividade antiviral mediada por estes extratos. Todos os extratos interferiram na adesão do RV aos receptores de células MA104, sendo que as proteínas presentes nos extratos mostraram uma interação competitiva com os receptores integrina β3 e Hsc70 das células MA104, necessários para iniciar a infecção por RV. Estes resultados contribuem para o conhecimento dos mecanismos básicos de ação antiviral de bactérias probióticas.

Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.</p><p><b>METHODS</b>The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.</p><p><b>RESULTS</b>DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.</p><p><b>CONCLUSION</b>Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.</p>

Significant correlation of angiotensin converting enzyme and glycoprotein IIIa genes polymorphisms with unexplained recurrent pregnancy loss in north of Iran

Background: Spontaneous abortion is considered as the most complex problem during pregnancy. Thrombophilia is resumed as a cause of recurrent pregnancy loss [RPL]. Glycoprotein IIIa [GPIIIa] gene is involved in thrombosis and abortion. Angiotensin converting enzyme [ACE] converts angiotensin I to angiotensin II and is involved in thrombosis. The most common polymorphism in this gene is the insertion/deletion [I/D]

Objective: In this study, we analyzed the association between ACE I/D and GPIIIa c.98C >T polymorphisms in women with unexplained RPL from the north of Iran

Materials and Methods: Sample population consisted of 100 women with unexplained RPL and 100 controls. The ACE I/D and GPIIIa c.98C>T polymorphisms were genotyped by TETRA-ARMS PCR. The association between genotypes frequency and RPL were analyzed using ?P2P and exact fisher tests. Associated risk with double genotype combinations was also investigated by binary logistic regression

Results: There was significant association between ACE DD genotype and RPL [OR=2.04; 95% CI=0.94-4.44; p=0.036]. ACE D Allele was also significantly associated with the RPL [OR=1.59; 95% CI=1.05-2.41; p=0.013]. No significant association was observed between GPIIIa c.98C>T polymorphism and RPL

Conclusion: ACE I/D polymorphism may probably be a prognostic factor in female family members of women with the history of recurrent abortion

Study of in vitro expression of human platelet ITGB3 gene nonsense mutation c.1476G>A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.</p><p><b>METHODS</b>An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.</p><p><b>RESULTS</b>The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.</p><p><b>CONCLUSION</b>The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.</p>

Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction.</p><p><b>METHODS</b>A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested.</p><p><b>RESULTS</b>myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation.</p><p><b>CONCLUSION</b>The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.</p>

Effect of the Integrin β3 Cytoplasmic NITY Motif on α II bβ3-Mediated Cell Functions in CHO Cell Model / 中国实验血液学杂志

<p><b>UNLABELLED</b>OBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.</p><p><b>METHODS</b>Stable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3ΔNITY (β3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions.</p><p><b>RESULTS</b>CHO-αIIbβ3, CHO-αIIbβ3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbβ3 had the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbβ3. The β3ΔNITY mutation substantially reduced kindlin-2 association.</p><p><b>CONCLUSION</b>Deletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.</p>

Methylglyoxal inhibits human umbilical vein cell migration in vitro by down-regulating integrinβ3 / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of methylglyoxal on endothelia cell migration.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVECs) were stimulated by serial concentrations of methylglyoxal (MGO, 0, 25, 50, 100 and 200 µmol/L) for 24 h, and the cell migration was assessed by scratch wound and Transwell assay. The expression of integrin β3 in the treated cells was examined by immunoblotting, and the effect of an anti-β3 antibody, LM609, on cell migration was investigated.</p><p><b>RESULTS</b>Methylglyoxal significantly inhibited HUVEC migration in a concentration-dependent manner (P<0.05). Methylglyoxal decreased the expression of integrin β3 in a time- and concentration-dependent manner (P<0.05). LM609 also significantly inhibited HUVEC migration (P<0.05).</p><p><b>CONCLUSION</b>Methylglyoxal inhibits HUVEC migration in vitro by down-regulating integrin β3 expression.</p>

Analysis of Human Platelet Antigen-1 System Alloantibodies Using Recombinant GPIIIa Fragments Coupled to Luminex Beads / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect platelet anti-HPA-1a and -1b antibodies using recombinant GPIIIa fragments coupled to Luminex beads.</p><p><b>METHODS</b>The sensitivity of 2 techniques, monoclonal antibody specific immobilization of platelet antigen (MAIPA) and Luminex bead assay, was compared using 12 twofold-serial dilutions (from neat to 1 in 2048) of an anti-HPA-1a WHO international standard. The specificity of Luminex assay to identify anti-HPA-1a and -1b antibodies was assessed using 8 negative or positive controls and 36 blinded samples provided by WHO Platelet Workshop.</p><p><b>RESULTS</b>The sensitivity of MAIPA and Luminex bead assay to detect anti-HPA-1a was dilution 1/64 (i.e. 1.56 IU/ml) and far more than dilution 1/2048 (i.e. 0.049 IU/mL), respectively. The Luminex bead assay could specifically identify negative and positive controls of anti-HPA-1a and -1b. All results of 36 blinded samples by Luminex assay were accordant to reference results except one sample which contained high concentration antithetical antibody and resulted in false positive of anti-HPA-1b. Cross-reactivity was also not observed with the samples containing HLA, ABO or other platelet antibodies.</p><p><b>CONCLUSION</b>The Luminex beads coupled with recombinant GPIIIa fragments can be used to detect HPA-1 system antibodies with sufficient sensitivity and specificity, that is suitable for the detection of platelet alloantibodies in clinical alloimmune thrombocytopenia.</p>

Overexpressions of Vimentin and Integrins in Human Metastatic Spine Tumors

OBJECTIVE: To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. METHODS: Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins alphav, beta1, and beta3 was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Immunohistochemical staining showed that vimentin and integrin alphav was broadly expressed in all tissues examined. By contrast, integrin beta1 was weakly expressed only in 38.4% (5/13) of tissues. Integrin beta3 was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin alphav in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin beta1 was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin beta3 was variable. CONCLUSION: Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin alphav and beta1 in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.

Assessment of alpha4, alphav, beta1 and beta3 integrins expression throughout the implantation window phase in endometrium of a mouse model of polycystic ovarian syndromes

Endometrial integrin expression changes might be a reason for implantation failure in polycystic ovarian syndromes [PCOS]. Assessment of integrin genes and proteins expression upon endometrium in the PCOS experimental mouse model was the main goal of this study. 30 NMRI female mice were equally divided into control, experimental [PCOS; received estradiol valerate [40 mg/kg]] and sham group [received; olive oil]. After 8 weeks, each group was hyper stimulated by 7 IU PMSG and then, after 48hrs, 7 IU HCG was injected. Vaginal plaque was checked. After 5 days, Progesterone and estradiol levels and endometrial tissues were investigated to evaluate of alpha4, alphav, beta1 and beta3 integrins gene and protein by qPCR method and immunohistochemistry, respectively. Tissue samples were assessed and showed that level of progesterone was significantly decreased in PCOS group. Results of molecular part in the amount of alphav, beta3, beta1 and alpha4 gene expressions showed a great difference in beta3 and alphav genes expressions between experimental groups, alphav, beta3, alpha4 and beta1 proteins in the endometrial stroma in the control group were expressed, but they were not detected in PCOS group. According to the results, integrins had different expression patterns in different areas of the endometrium; such as epithelial and stromal. It seems that in PCOS, this pattern has changed and the results might have a great influence on implantation failure. Therefore, this study suggests that a great attention to this problem may be essential in patients who are involved

Expression of alpha4, alphav, beta1 and beta3 integrins during the implantation window on blastocyst of a mouse model of polycystic ovarian syndromes

It has been hypothesized that blastocyst integrin expression changes can affect the spontaneous miscarriage in polycystic ovarian syndromes [PCOS]. In this study, the profile of integrin genes and proteins was investigated on blastocyst of the PCOS experimental mouse model. 30 NMRI female mice were equally divided into 3 groups: control, experimental [PCOS that was injected estradiol valerate [40 mg/kg]]. After 8 weeks, each group was hyper stimulated by PMSG and HCG. Vaginal plaque was checked, and mice were investigated 5 days after the test. Progesterone and estradiol levels were determined; alpha4, alpha v, beta 1 and beta 3 integrin genes and protein of blastocysts were examined by real time PCR method and immunohistochemistry, respectively. Estradiol level was significantly increased [p

The Sysmex XN-2000 Hematology Autoanalyzer Provides a Highly Accurate Platelet Count than the Former Sysmex XE-2100 System Based on Comparison with the CD41/CD61 Immunoplatelet Reference Method of Flow Cytometry

No abstract available.

Expression heterogeneity research of ITGB3 and BCL-2 in lung adenocarcinoma tissue and adenocarcinoma cell line

OBJECTIVE@#To analyze expression heterogeneity of Integrin beta 3 (ITGB3) and B-cell lymphoma 2 (BCL-2) in lung adenocarcinoma tissue and adenocarcinoma cell line and further provide theoretical direction for molecular biological research of lung adenocarcinoma.@*METHODS@#Tissue microarray was used to observe relation among expression, heterogeneitpy and clinical characteristics of ITGB3 and BCL-2 in lung cancer.@*RESULTS@#ITGB3 and BCL-2 increased significantly in A549 cells in CAFs group withβ-actin as control; the expression level of BCL-2 also increased in ITGB3 transfected cells with GFP plasmid transfected A549 cells as control; immunohistochemistry staining showed that positive rates of ITGB3, ITGB1 and BCL-2 in normal lung tissues were 0, the positive rates in lung adenocarcinoma were 7.04%, 84.51% and 4.23%, respectively; in the results of immunohistochemistry staining, the expression of Girdin protein in lung adenocarcinoma was homogeneous, however protein expression of ITGB3, ITGB1 and BCL-2 showed different patterns in the same location with significant heterogeneity; majority of ITGB3, ITGB1 or BCL-2 positive tissue showed heterogeneity that expression in trailing edge was higher than that of trailing edge in lung adenocarcinoma tissue, the patients with BCL-2 heterogeneity showed higher lymph node metastasis ratio and lower clinical stage (P0.05).@*CONCLUSIONS@#Expression of ITGB3 and BCL-2 in lung adenocarcinoma and adenocarcinoma cell line showed heterogeneity that expression in trailing edge was higher than that of trailing edge, which may play an important role in promoting tumor lymph node metastasis and vascular invasion, and provides a new research direction for exploration of lung adenocarcinoma metastasis mechanism.

Synergistic effect of platelet-derived growth factor-BB and transforming growth factor-beta1, on expression of integrin beta3 in periodontal membrane of rat orthodontic tooth / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the synergistic effect of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-BB (PDGF-BB) on the expression of integrin beta3, in periodontal membrane of rat orthodontic tooth.</p><p><b>METHODS</b>An orthodontic tooth movement model was established. Up to 32 experimental rats were randomly divided into four groups according to a random number table. The four groups were injected with 1% PBS, TGF-beta1 (5 ng), PDGF-BB (10 ng), and combined TGF-beta1 (5 ng) and PDGF-BB (10 ng) in the buccal submucosal, respectively. The volume injected in each group was 0.1 mL. The animals were then sacrificed on the 10th day. The left maxillary first molar and periodontal tissue were taken. Different expressions of integrin beta3 were detected in periodontal tissues through immunohistochemistry. Mean optical density (OD) values of the positive fields were examined. The data obtained were analyzed through ANOVA. The data followed normal distribution, and were compared via t-test.</p><p><b>RESULTS</b>Compared with the control groups, the expression of integrin beta3 was higher in the experimental groupin tension sides (P < 0.01). Significant differences in tension sides between the single-injection groups and the combined group were observed (P < 0.01). Compared with the control groups, the expression of integrin beta3 was higher in the experimental group in compression sides (P < 0.05). In addition, there was no significant differences in compression sides between the single-injection groups and the combined group (P > 0.05).</p><p><b>CONCLUSION</b>In terms of local regulatory factors, TGF-beta1 combined with PDGF-BB enhance the expression of integrin beta3 in the periodontal membrane and accelerate periodontal remodeling. The synergistic effect of the two growth factors is better than the single growth factor.</p>

The Effects of Pueraria lobata on Osteoclast Differentiation and Bone Resorption / 체질인류학회지

Previous researches have proved that Pueraria lobata up-regulates bone mineral contents and bone mineral density in bone-loss model, ovariectomized mice and orchidectomized rats. However, the precise effects and mechanisms of Pueraria lobata on osteoclast differentiation and bone resorbing activity of mature osteoclasts still remains unknown. Therefore, we investigated the effect and mechanism of Pueraria lobata on receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony stimulation factor (M-CSF)-induced osteoclast differentiation in bone marrow macro-phages (BMMs). First of all, we treated BMMs derived from mice with various concentrations of Pueraria lobata in order to perform screening by tartrate-resistant acid phosphatase (TRAP) staining. Also, we conducted western blotting and RT-PCR for the purpose of verifying the treatment mechanism of Pueraria lobata and lastly, we used hydroxyapatite-coated plate to evaluate the effects of Pueraria lobata on bone resorbing activity of mature osteoclasts. As a result, Pueraria lobata has inhibitory effect on phosphorylation of p38, Akt, c-Jun N-terminal kinase (JNK), and IkappaB which are essential early signaling pathway of osteoclastogenesis. Also, the inactivation of nuclear factor of activated T cells (NFAT)c1, and c-Fos which is caused by Pueraria lobata is followed by the suppression effects of Pueraria lobata on osteoclast-related various genes, osteoclast-associated receptor (OSCAR), TRAP, Integrin beta3, osteoclast stimulatory transmembrane protein (OC-STAMP), and dendritic cell-specific transmembrane protein (DC-STAMP). Particularly, Pueraria lobata blocks the formation of pit area on hydroxyapatite-coated plate in a dose-dependent manner as well as the mRNA expression of Cathepsin K, which is associated with bone resorbing activity. These results demonstrate the molecular mechanism relating to anti-osteoclastogenesis effect of Pueraria lobata as well as the inhibitory effect of Pueraria lobata on mature osteoclast formation and bone resorbing activity.

Transgenic mouse models of the truncated platelet integrin β3 cytoplasmic tail established by stem cell transplantation / 中国实验血液学杂志

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.

Effects of Er'zhi Tiangui Granule () on sequential expressions of integrin β 3 and its ligand osteopontin in mouse endometrium during controlled ovarian hyperstimulation / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Er'zhi Tiangui Granule (, ETG) on sequential expressions of integrinβ3 and its ligand osteopontin in the mouse endometrium during controlled ovarian hyperstimulation (COH) and implantation period.</p><p><b>METHODS</b>Seventy-five Mature female Kunming mice were randomly divided into 3 groups, a normal control group, a model group, and a treatment group administrated with ETG for 10 days, 25 in each group. After mated with male mice, every 5 mice were sacrified in each group at the 0, 2nd, 4th, 6th, and 8th days to take their endometrium. In-situ hybridization was used to detect the expressions of integrinβ3 and osteopontin in the endometrium.</p><p><b>RESULTS</b>mRNA expressions of integrinβ3 and osteopontin in the endometrium during implantation period showed similar time sequence rules in the treatment group to those in the normal control group; the peak values of them were a little lower in the treatment group than the normal control without significant differences. In the model group, integrinβ3 mRNA expression was higher at the 2nd day, obviously lower at the 4th and 6th days, and insignificantly lower at the 8th day; and osteopontin expression was remarkably lower at the 4th, 6th, and 8th days, compared with the normal control and the treatment groups (P<0.05, P<0.01).</p><p><b>CONCLUSIONS</b>COH might influence the sequential expressions of integrinβ3 and its ligand osteopontin, bring forward the integrinβ3 expression peak, impact on the cooperation of integrinβ3 and osteopontin, so as to damage the endometrial receptivity. ETG could regulate the sequential expressions of integrinβ3 and its ligand osteopontin to improve the mouse endometrial receptivity during COH.</p>