The purpose of this paper is to investigate the probable molecular mechanism in mechano-transduction of the regulation of integrins and the effects of cyclic biaxial mechanical strain on proliferation and synthetic function in the osteoblasts isolated from 3-month-old female Sprague-Dawley (SD) rats. The osteoblasts were cultured in F-12 medium contained with 10% fetal bovine serum(FBS) and grown to subconfluency in Flexercell type I dishes in a humidified incubator with 5% CO2 and 95% air at 37 degrees C. Mechanical strains were applied to the cells for periods of 30 min, 2, 4 and 8 hours every day, lasting 2 days. The amplitude of mechanical strain applied to the cells were 400, 1,000 and 4,000 mu strain respectively, at a frequency of one hertz(1 Hz). Unstrained cells were used as control. The expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts and proliferation activity of osteoblasts were studied with Flow Cytometry(FCM). The content of osteocalcin, carboxyterminal propeptide of type-I procollage(PICP), total protein secreted by osteoblastes were detected with the isotope labelling method. The results showed that there are actual expressions of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts without mechanical strain and that the expression of integrins beta 1 is highest. The mechanical strain increased the expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts, but the strain-related up-regulation of expression of integrins alpha 2, beta 1, beta 3 are different in various amplitude and different duration of mechanical stains. The up-regulation of expression of integrins beta 3 is most sensitive to mechanical strain. The up-regulation of expression of integrins alpha 2, beta 1, beta 3 is higher at 4,000 mu strain than at 400, 1,000 mu strain. The mechanical strain can elevate the proliferation activity and the synthetic function of osteoblast at 400, 1,000 mu strain. However, the mechanical strain increased significant the proliferation in the osteoblasts and suppressed obviously the synthetic function in the osteoblasts. In the present study, the reaction of the osteoblasts in 3 month-old rat to the mechanical stimulation suggested that 1) expressions of integrins alpha 2, beta 1, beta 3 were increased in a amplitude of strain-dependent manner; 2) the changes of expression of integrins alpha 2, beta 1, beta 3 relate close to the changes of the proliferation and synthetic function of the osteoblasts. Low amplitude of strain can increase the proliferation and the synthetic function of the osteoblasts along with up-regulation of expression of integrins alpha 2, beta 1, beta 3; while higher amplitude of strain elevated significantly the proliferation of osteoblasts and suppressed obviously the synthetic function of the osteoblasts along with up-regulation of expression of integrins alpha 2, beta 1, beta 3. The amplitude of 4,000 mu strain is an optimal amplitude as stimulus for up-regulation of expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts and increase the proliferation activity, but decrease the synthetic function of osteoblasts in the present study. Accordingly it indicates that integrins have a important role in regulation of signal transduction pathway in osteoblasts as a result of mechanical strain.
Animals ; Cell Division ; Cells, Cultured ; Female ; Integrin alpha2 ; biosynthesis ; Integrin beta1 ; biosynthesis ; Integrin beta3 ; biosynthesis ; Mechanics ; Osteoblasts ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley

BACKGROUNDTo express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin.

METHODSThe human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay.

RESULTSThe alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells.

CONCLUSIONSThe results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Animals ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Hantavirus ; Integrin beta3 ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Virus ; Vero Cells ; metabolism

BACKGROUNDGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China.

METHODSA three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping.

RESULTSThe proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately.

CONCLUSIONSThe alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

Asian Continental Ancestry Group ; Child ; Female ; Flow Cytometry ; Heterozygote ; Humans ; Integrin alpha2 ; genetics ; Integrin beta3 ; genetics ; Mutation ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction ; Thrombasthenia ; genetics ; metabolism ; pathology

No abstract available.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Automation ; Blood Platelets/*cytology/metabolism ; Child ; *Flow Cytometry ; Humans ; Integrin beta3/*metabolism ; Middle Aged ; Platelet Count/*instrumentation ; Receptors, IgG/*metabolism ; Young Adult

OBJECTIVETo study the association of integrin alpha-2 (ITGA2) gene C807T, integrin beta-3 (ITGB3) gene T176C polymorphisms with ischemic stroke and the effect of the polymorphisms on plasma lipid and lipoprotein levels.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to detect the integrin genotypes in 265 patients with ischemic stroke and 280 healthy controls. The plasma lipid and lipoprotein levels were measured by routine method.

RESULTSPlasma total cholesterol (TC), triacylglycerol (TG) and low density lipoprotein-cholesterol (LDL-C) in the patients with ischemic stroke were significantly higher than those in the controls (P< 0.05). The distributions of the ITGB3 gene T176C polymorphism were not different between the ischemic stroke group and control group, but the ITGA2 gene C807T polymorphism was significantly different. The relative risk suffering from ischemic stroke of the T allele carrier was 1.455 times as that of the C allele carrier (OR=1.455, 95%CI: 1.134-1.866). The level of plasma lipid in the T allele carriers was significantly higher than that in the C allele carriers (P< 0.05).

CONCLUSIONThe ITGA2 gene C807T polymorphism was associated with ischemic stroke, the 807 T allele may be a genetic risk factor for ischemic stroke. The ITGA2 gene C807T polymorphism may affect ischemic stroke through plasma lipid and lipoprotein levels.

Brain Ischemia ; blood ; genetics ; metabolism ; Cholesterol, LDL ; genetics ; metabolism ; Female ; Genetic Predisposition to Disease ; Humans ; Integrin alpha2 ; genetics ; metabolism ; Integrin beta3 ; genetics ; metabolism ; Lipid Metabolism ; genetics ; Lipids ; blood ; Male ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

OBJECTIVETo observe the effects of Bushen Zhuyun Recipe (BZR) on protein expressions of estrogen receptor (ER), progesterone receptor (PR) and integrin alpha5 and beta3 in endometrium of rats at the implantation stage, for exploring the possible mechanism of the recipe in treating luteal phase defect (LPD) infertility.

METHODSFemale SD rats were randomly divided into 6 groups, the blank group, the model group, the WM group treated by Western medicine, and the three BZR groups treated by low-, middle- and high-dose BZR respectively. Rats were made to pregnancy and sacrificed at the implantation stage, their middle segment of uterus, about 1 cm in length was gotten for detecting the protein expressions by Western blot. Results The protein expressions of endometrial ER and PR were significantly higher, while those of integrin alpha5 and beta3 were significantly lower than those of the control group (P < 0.05). The protein expressions of endometrial ER and PR were significantly lower, but those of integrin alpha5 and integrin beta3 were higher in rats treated by middle- and high- dose BZR than those in model rats (P < 0.05).

CONCLUSIONBZR can raise the receptivity of rats' endometrium through down-regulating the expressions of ER, PR and increasing the protein expression of integrin alpha5 and beta3 in endometrium and thus to enhance the pregnant rate.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; physiology ; Endometrium ; drug effects ; metabolism ; Female ; Integrin alphaV ; metabolism ; Integrin beta3 ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

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.
Amino Acid Motifs ; Animals ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; HEK293 Cells ; Humans ; Integrin beta3 ; genetics ; metabolism ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo study the effects and underlying mechanisms of Zhuyun Recipe (ZR) on the endometrial receptivity in ovarian stimulation (OS) and blastocyst implantation dysfunction (BID) mice.

METHODSTotally 200 normal female Kunming mice were randomly divided into 6 groups, i. e., the control group (Group A), the OS group (Group B), the OS + ZR group (Group C), the BID group (Group D), the BID + ZR group (Group E), and the ZR group (Group F). The pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) were intraperitoneally injected to mice in Group B. Mifepristone was subcutaneously injected to mice in Group D at 9:00 am on the 4th gestation day. Corresponding medications were given to mice in Group C, E, and F at 1.5 mL/100 g by gastrogavage at 8:00 am from the first to the 4th gestation day. Eight uterus samples were collected at 9:00 pm on the 4th gestation day and fixed. The expression levels of leukemia inhibitory factor (LIF) and integrin beta3 were detected using immunohistochemical assay. The pregnant mice were sacrificed at 9:30 pm on the 8th gestation day, and their uterus were taken out. The number of blastocysts was counted.

RESULTSCompared with Group A, the pregnant rate was 6.67% (1/15 cases) in Group B and 18.75% (3/16 cases) in Group D, the mean OD value of LIF was 0. 18 +/- 0.02 in Group B and 0.23 +/- 0.02 in Group D, and the mean OD value of integrin beta3 was 0.20 +/- 0.05 in Group B and 0.19 +/- 0. 02 in Group D, showing statistical difference (P < 0.01). The pregnant rate was 54.55% (12/22 cases) in Group C and 65. 22% (15/23 cases) in Group E, the mean OD value of LIF was 0.37 +/- 0. 09 in Group C and 0.39 +/- 0.02 in Group E, and the mean OD value of integrin beta3 was 0.34 +/- 0.04 in Group C and 0.38 +/- 0.08 in Group E, showing statistical difference when compared with those of Group B and Group D respectively (P < 0.05).

CONCLUSIONSOS and BID had negative effects on the endometrial receptivity and hindered the blastocyst implantation. ZR could improve the uterine receptivity and elevate the pregnant rate by up-regulating the expressions of endometrial LIF and integrin beta3.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; physiology ; Female ; Integrin beta3 ; metabolism ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred Strains ; Ovulation Induction ; Pregnancy

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.
Animals ; Blood Platelets ; metabolism ; Genetic Vectors ; Hematopoietic Stem Cell Transplantation ; Integrin beta3 ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Retroviridae ; genetics

OBJECTIVETo explore the effects of methylglyoxal on endothelia cell migration.

METHODSHuman 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.

RESULTSMethylglyoxal 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).

CONCLUSIONMethylglyoxal inhibits HUVEC migration in vitro by down-regulating integrin β3 expression.

Cell Movement ; drug effects ; Cells, Cultured ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Integrin beta3 ; metabolism ; Pyruvaldehyde ; pharmacology