OBJECTIVETo investigate the spatial and temporal expression changes of fibronectin and integrin beta1 mRNA during fracture healing.

METHODSUsing in situ RT-PCR technique, the spatial and temporal expression pattern of both fibronectin and integrin beta1 mRNA was detected on paraffined slices in different rabbit mandibular fracture healing phases.

RESULTS(1) The mRNA of integrin beta1 and fibronectin was widely expressed in fractured bone cells. The positive stain existed in cytoplasm and nucleolus. (2) After 7 days of fracture, increaseded integrin beta1 mRNA expression was observed, and it reached maximal levels approximately 14- 30 days post-fracture and returned to normal levels after 60 - 90 days. (3) Fibronectin mRNA was detected on 3 days post-fracture, it reached maximal levels at 7 - 14 days, faint expression was detected at 30 days, undetectable 60 days afterwards.

CONCLUSIONDuring fracture healing, the mRNA expression of integrin beta1 and fibronectin increased locally, FN and Itg beta1 cooperate with each other, it is suggested that they have an important influence on fracture healing.

Animals ; Fibronectins ; Fracture Healing ; Integrin beta1 ; Mandible ; RNA, Messenger ; Rabbits

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

OBJECTIVETo investigate the effect of different kinds of mechanical stress on the mRNA expression of integrin beta1 subunit in cultured human periodontal ligament fibroblasts (hPDLF).

METHODSTo scalp and remove the periodontal ligament attached to the mid-third part of the fresh root of young premolars extracted for the cause of orthodontics. Cultured hPDLF by the method of digesting by I-type collagenase combining with tissue adhering. Then hPDLF was isolated and purified by cells passage. The sixth passage's cells were selected to be loaded. A new cyclic strain loading apparatus. Forcel four point bending device was used for mechanically loading. Cells were loaded by three levels (1000, 2000, 4000 microstrain) of tensional and compressive forces and collected at different times (0, 0.5, 1, 4, 8, 12 h) course after strain loading. The quantity of integrin beta1 mRNA in every group was analyzed by means of quantitative real-time PCR with the special primers of up- and down-regulated genes.

RESULTSDynamic mechanical forces down-regulated the expression of integrin beta1 subunit mRNA in hPDLF and the difference in groups by different magnitude, different kinds, and different time of mechanical forces loading were statistically significant. The stronger stimulated forces, the more down-regulated expression. Compression down-regulated the expression of integrin beta1 subunit mRNA more than tension did.

CONCLUSIONDynamic mechanical forces could regulate the expression of integrin beta1 subunit mRNA. The difference among all the groups by different magnitudes, different kinds, and different time of mechanical forces loading were statistically significant.

Cells, Cultured ; Fibroblasts ; Humans ; Integrin beta1 ; Periodontal Ligament ; RNA, Messenger ; Stress, Mechanical

OBJECTIVETo study the integrin beta1 mRNA changes after orthodontic tooth movement in normal teeth and periodontitis teeth of rats.

METHODSThe OD of positively stained osteoclasts for integrin beta1 mRNA using in situ hybridzation was detected after orthodontic tooth movement in normal teeth and periodontitis teeth groups.

RESULTSIntegrin beta1 mRNA expression were detected on all osteoclasts in tooth movement samples of normal and periodontitis teeth. There were stronger positive signals after given orthodontic force in both of the two groups. But no differences were found after 0.5, 1, 2, 3, 5, 7, 10 days since orthodontic tooth movement. The integrin beta1 mRNA signals in normal tooth movement group were not different from that in periodontitis group.

CONCLUSIONThe integrin beta1 of osteoclasts may play a role in the stability and remodeling of periodontal ligament in orthodontic tooth movement. There were no difference in the OD of integrin beta1 mRNA staining in orthodontic tooth movement between normal teeth group and periodontitis teeth group.

Animals ; Integrin beta1 ; metabolism ; Osteoclasts ; Periodontal Ligament ; Periodontitis ; physiopathology ; RNA, Messenger ; metabolism ; Rats ; Tooth Movement Techniques

OBJECTIVETo study the effect of basic fibroblast growth factor on the mRNA expression of beta1 integrin subunit by periodontal ligament fibroblasts in culture; to discuss the effect of basic fibroblast growth factor in periodontal regeneration.

METHODSHuman periodontal ligament fibroblasts were cultured and stimulated by basic fibroblast growth factor (0.1, 1.0, 10.0 ng x mL(-1)) for 24, 48, 72 h respectively, and then mRNA expression of beta1 integrin subunit was assessed by fluorescent quantitative real-time reverse transcription polymerase chain reaction.

RESULTSBasic fibroblast growth factor enhanced the mRNA expression of beta1 integrin subunit, and there was optimal effect when the concentration of basic fibroblast growth factor was 1.0 ng x mL(-1) at 24, 48, 72 h respectively; the mRNA expression of beta1 integrin subunit at 72 h was higher than that at 24, 48 h.

CONCLUSIONBasic fibroblast growth factor can strengthen human periodontal ligament fibroblasts' adhesion and may be one of important factors which participate in the periodontal regeneration.

Cells, Cultured ; Fibroblast Growth Factor 2 ; Fibroblasts ; Humans ; Integrin beta1 ; Periodontal Ligament ; RNA, Messenger ; Regeneration

OBJECTIVETo investigate the regular pattern of the distribution of skin epidermal stem cells (ESCs) in the different parts of a healthy human body, and to evaluate the feasibility of the identification of ESCs by P63 and CD29 with single and double labeling.

METHODSFull-thickness skin samples from 21 parts (including scalp, dorsum of foot, sole of foot, pubic region, and scrotum) of 5 healthy persons were harvested for the study. Immunohistochemistry method with biotin-streptavidin-horseradish peroxidase (SP) was employed with P63 and CD29 as the first antibody to carry out single and double labeling. The staining results were subjected to image analysis. The distribution of the ESCs in the skin from the above parts was observed and expressed as positive unit (PU) value.

RESULTSIt was found by P63 single labeling and P63 and CD29 double labeling that the PU value in the dorsum of foot was the lowest while that in the scalp was the highest among all the parts of a healthy body. It was also found by CD29 single labeling that the PU value in the dorsum of foot was the lowest [(11.9 +/- 1.5)%] while highest in the scalp [(29.1 +/- 5.0)%]. The PU value in the hairy region of a human body was evidently higher than that in the non-hairy region (P < 0.01), when examined by P63 and CD29 single and double labeling. But there was no difference in the PU values between the trunk and limbs by means of P63 and CD29 single and double labeling (P > 0.05).

CONCLUSIONThere are more ESCs in the skin from the scalp, mons pubis and scrotum than other parts of the body. Single P63 or CD29 labeling exhibits higher sensitivity but lower specificity in the identification of ESCs. While the double labeling method exhibits higher specificity but lower sensitivity. Above all, it seems that the double labeling may be a simple and effective method for the identification of ESCs.

Epithelial Cells ; cytology ; Humans ; Immunohistochemistry ; Integrin beta1 ; Male ; Skin ; cytology ; Stem Cells

Objective To study the characteristics of positive expression of integrin β1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin β1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin β1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin β1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin β1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin β1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin β1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.
Animals ; Brain/metabolism* ; Brain Injuries ; Brain Injuries, Traumatic ; Integrin beta1/metabolism* ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the regulatory effect of Jingang Jiangu pill (see text, JGJG) on expression of integrin in ovariectomized rats.

METHODSFifty ovariectomized 10 months old female rats were randomly divided into 5 groups: Fushanmei group (FSM), Jingang Jiangu pill (see text) group (JGJG), Gusongbao granule group (GSB), Model group (OVX), Sham group. After ovariectomized,the rats were raised in the same environment for 13 weeks. The rats in JGJG group took 0.13 g JGJG pill orally each day for each rat; the rats in GSB group took 0.86 g GSB granule orally each day for each rat; the rats in FSM group took 0.28 mg FSM orally each day for each rat; and the rats in OVX and sham groups took sodium. The treatment duration of rats in above 5 groups was 13 weeks. Bone mineral density (BMD) and the expression of integrin beta1 and alphavbeta3 were detected in each group after the treatment. RESYKTS: The BMD and the expression of integrin beta1 in FSM group, JGJG group and GSB group improved obviously than that of OVX group. There were statistical difference between these groups (P<0.05). The expression of integrin alphavbeta3 of the three treating groups significantly depressed.

CONCLUSIONThe JGJG pill improves BMD and express of integrin beta1, in ovariectomized rats and reduces express of integrin alphavbeta3 through the regulation of the coupling of osteoblasts and osteoclasts.

Animals ; Bone Density ; Disease Models, Animal ; Female ; Integrin alphaVbeta3 ; analysis ; Integrin beta1 ; analysis ; Medicine, Chinese Traditional ; Osteoporosis ; drug therapy ; metabolism ; Ovariectomy ; Rats ; Rats, Wistar

OBJECTIVETo observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action.

METHODSAttachment assay was used to assess the adhesion of wwox-transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells. Integrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane.

RESULTSAttachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours (P<0.01). The level of membranous integrins alpha2 and alpha3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in vector-transfected controls (P<0.05), but there was no association with the level of functioning integrins betal or beta2 in clone cells (P>0.05). The attachment assays were repeated after pre-incubating the cells with integrin alpha2 or alpha3 function-blocking antibodies. These results showed that blocking integrin alpha3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody (P<0.05). In contrast, preincubation with alpha2 blocking antibody had very little effect on fibronectin binding in these cells (P>0.05). FACS analysis showed that membranous integrin alpha3 expression revealed a marked reduction in WWOX-transfected cells than that in vector-transfected cells.

CONCLUSIONWWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.

CD18 Antigens ; metabolism ; Cell Adhesion ; Female ; Fibronectins ; metabolism ; Humans ; Integrin alpha2 ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Oxidoreductases ; genetics ; metabolism ; Protein Binding ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo explore the association between renal transforming growth factor-β1 (TGF-β1) and the expressions of α3 and β1 integrins and observe the effect of irbesartan on their expressions in diabetic rats.

METHODSThirty 8-week-old male Wistar rats were randomly divided into normal control group (n=7), diabetic control group (n=14) and irbesartan group (n=9). Rat models of diabetes were established by a single peritoneal injection of streptozotocin (STZ), and 4 weeks later the rats received irbesartan treatment for 8 weeks. Enzyme-linked immunosorbent assay (ELISA) was used to measure the urinary albumin excretion rate, and PAS staining was utilized to observe the renal pathologies. Immunohistochemistry was performed for semi-quantitative determination of podocyte density, and real-time RT-PCR was used to detect the renal TGF-β1 and α3/β1 integrin mRNA expressions.

RESULTSIn diabetic rats, the expression of renal TGF-β1 mRNA was significantly increased, while α3 and β1 integrin mRNA expressions and podocyte density significantly decreased with increased proteinuria. Irbesartan obviously improved such changes.

CONCLUSIONIn diabetic rats renal TGF-β1 can regulate α3 and β1 integrin mRNA expressions to reduce the number of podocytes, and inhibition of this pathway may be one of the mechanisms of the renal protective effect of irbesartan.

Animals ; Biphenyl Compounds ; pharmacology ; Diabetes Mellitus ; metabolism ; Diabetic Nephropathies ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Kidney ; drug effects ; metabolism ; Male ; Rats ; Rats, Wistar ; Tetrazoles ; pharmacology ; Transforming Growth Factor beta1 ; metabolism