Phylogeography of the endangered "eyed" turtles (genus Sacalia) and the discovery of a lineage derived from natural interspecific hybridization.

The herpetofauna of the Indomalayan bioregion of Asia suffers from severe habitat loss, unsustainable harvesting, and lack of research and conservation. Here, we investigated the range-wide phylogeography of the endangered "eyed" turtles (genus Sacalia, including the Beale's Eyed Turtle S. bealei and the Four-eyed Turtle S. quadriocellata) and discovered a natural interspecific hybrid turtle population in China. Based on phylogeny of the mitochondrial Cytochrome b gene of 101 samples in this study and public data, three major clades and six subclades were identified: S. bealei (SBE) in eastern-southern China, east S. quadriocellata in South China (northern east [SQUen] and southern east [SQUes] subclades), and west S. quadriocellata mainly in Vietnam (northern west [SQUwn], central west [SQUwc], and southern west [SQUws] subclades). We sequenced 16 nuclear DNA loci of 87 samples from SBE, SQUen, SQUes, and SQUwn subclades. Population genetic clustering analysis suggested a structure similar to the mitochondrial phylogeny, where most samples were classified into four genetic clusters corresponding to the four mtDNA subclades. However, a proportion of samples carrying SQUen mtDNA haplotypes formed an additional distinct cluster SHY. Those samples are found in the contact zone of the two species bearing mosaic and intermediate morphological characteristics. We detected an admixed ancestry in SHY from SBE and SQUen that conformed to an intrapopulation breeding scenario for at least hundreds of generations after the initial hybrid event, leading to a conclusion that SHY is a distinct and near-panmictic population derived from natural interspecific hybridization. In addition, SQUes (Hainan Island endemic) is of special concern due to significant isolation and low genetic diversity. We suggest that seven evolutionarily significant units should be recognized to facilitate appropriate conservation actions. These findings also highlight the urgent need for further herpetological research and conservation in this region.

Discovery of first active breeding den of Chinese mountain cat ( Felis bieti).

In mid-September 2018, during a field survey in Chiat'ung, Sanjiangyuan (Three-River-Source) Region, Tibetan Plateau, China, we discovered the first active breeding den of the Chinese mountain cat (Felis bieti), inhabited by one adult female and two kittens. Based on fieldwork over the following months, five breeding dens were discovered, and 33 sightings were recorded. In addition, at least five individuals were confirmed to inhabit this overlooked region, and much previously unknown information concerning this cat species and its ecology was revealed for the first time.

AP-1 and NF-κB transcriptionally regulate interleukin-8 in EA.Hy926 cells under shear stress.

Cardiovascular and cerebrovascular diseases remain the leading cause of death in the world. AS (atherosclerosis) is not only an inflammatory disease in which chemokines play the main role but also a disorder that is related to blood SS (shear stress). We have investigated the action of IL-8 (interleukin-8) mRNA expression in human endothelial cells line-EA.Hy926 under SS at different intensities and duration. Expression increases with time in an intensity dependent manner. With regard to the transcriptional mechanism involved, transient transfection of the human wild-type IL-8 promoter (-162/+44)/luciferase reporter plasmid, or site mutation of one of the binding sites [AP-1 (activator protein 1) or NF-κB (nuclear factor κB)] in the IL-8 promoter region was investigated. Both AP-1 and NF-κB were essential for SS-activated transcription, with the cells responding to NF-κB activation within minutes. After stimulated at low SS (4.20 dyne/cm2) for 30 min, the P65 subunit was translocated from the cytoplasm to nucleus for at least 60 min, while the cytoplasmic level of IκB (inhibitory κB) gradually decreased. The combined activation of NF-κB and AP-1 are the upstream regulators of low SS-induced IL-8 production in EA.Hy926 cells, which subsequently trigger an inflammatory reaction in endothelium.

Significant evidence of association between polymorphisms in ZNF533, environmental factors, and nonsyndromic orofacial clefts in the Western Han Chinese population.

The etiology of nonsyndromic orofacial clefts (NSOC) has been considered "complex" or "multifactorial." Etiologic heterogeneity induces disparities in the results among different populations. The zinc finger protein 533 (ZNF533) and several environmental factors have been revealed to be associated with NSOC in several populations. We investigated three single-nucleotide polymorphisms (SNPs) and 10 environmental factors in 211 case-parent trios and 188 control individuals in the Western Han Chinese population to confirm the relationship between ZNF533, environmental factors, and the etiology of NSOC in the Western Han Chinese population. The transmission disequilibrium test, case-control analysis, multiple logistic regression, log-linear model, and conditional logistic regression were tested to confirm the contribution of the ZNF533 gene and environmental factors to the etiology of NSOC. Strong statistically significant evidence of association was found between the rs6757845 and rs1139 markers and NSOC. The haplotype G-G for rs6757845-rs1139 showed significant overtransmission among cleft lip with or without cleft palate (CL/P) trios and among cleft palate only trios. Additional 11 and 5 haplotypes were significantly overtransmitted and undertransmitted among CL/P and among cleft palate only trios, respectively. Maternal disease, use of medication, and passive smoking during the first trimester of pregnancy may increase the risk of NSOC. Maternal folic acid supplementation during the first trimester of pregnancy showed a protective effect on the etiology of NSOC. Genotype-environment interaction test showed a significant evidence of interaction effects between the genotypes at rs6757845 and maternal passive smoking during the first trimester among CL/P trios. These results confirm the effects of the ZNF533 gene and environmental factors on the etiology of NSOC.

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration.

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.

A comparative study of fluctuating asymmetry in Chinese families with nonsyndromic cleft palate.

OBJECTIVE: To compare the asymmetry displayed by Chinese patients with nonsyndromic cleft palate (NSCP), their unaffected parents, and a control population. METHOD: With rigorous inclusion criteria, a total number of 675 individuals with NSCP, 675 parental pairs of these patients, and 650 control individuals were involved in this case-control study. Size-adjusted fluctuating asymmetry (FA) scores were calculated by data on 10 variables. Analysis of variance was used for a three-way comparison of patients/gender-matched parents/gender-matched controls. RESULTS: A significant increase in FA for ear length (p<.05) was noted in NSCP patients when compared with their gender-matched parents. A significant increase in FA for ear length and palpebral fissure width (p<.05) was observed in NSCP patients when compared with the gender-matched control population. A significant increase in FA for palpebral fissure width (p<.05) was detected in parents of NSCP patients when compared with a gender-matched control population. CONCLUSION: Our results indicated that, when compared with a gender-matched control population, patients with NSCP show significantly increased FA in both ear length and palpebral fissure width, but the parents of patients with NSCP show significantly increased FA only in palpebral fissure width. In general, these characteristics seem to be more distinct in male individuals.

[1H-NMR-based metabonomics analysis of plasma from osteoporotic rats induced by ovariectomy].

OBJECTIVE: To investigate the application of metabonomics in research of osteoporosis, through detecting change of the endogenous metabolites in plasma from osteoporotic rats by ovariectomy. METHODS: Six old-months female SD rats were randomly divided into sham and OVX group. Fifth month after ovariectomy, plasma were collected from both groups, respectively. The metabolic profiles were investigated using 1H-NMR spectroscopy of plsama combined with pattern recognition techniques, including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). RESULTS: The PCA PLS-DA plots of the plasma samples presented marked clustering between sham group and OVX group. Compared to sham group, the level of low molecular metabolites such as lactate, acetone and ethonal were higer, glucose, choline/phosphatidylcholine (Cho/PC), alanine (Ala), high density lipoprotein/low density lipoprotein (HDL/LDL), very low density lipoprotein/low density lipoprotein, fatty acid and glucose were lower. CONCLUSION: Obvious changes in metabonomics of plasma from osteoporotic rats were observed.

The parental craniofacial morphological features in Chinese patients with sporadic nonsyndromic cleft lip with or without palate.

OBJECTIVE: To evaluate the parental craniofacial morphology in Chinese patients with sporadic nonsyndromic cleft lip with or without palate. METHODS: A total of 98 parental pairs of nonsyndromic unilateral incomplete cleft lip children, 207 parental pairs of nonsyndromic complete cleft lip and palate children, and 206 normal persons from Sichuan University were involved in this study. A conventional cephalometric analysis was used to measure angles, linear distances, and their ratios. Two-sample Student's t tests and a multivariate discriminant analysis were applied to the data. RESULTS: Data indicate that the unaffected parents of nonsyndromic cleft lip children had on average significantly more acute cranial base angle (Angle N-S-Ba) and larger nasal width (NC-NC') (p < .01). The healthy parents of nonsyndromic cleft lip and palate children consistently displayed a more acute cranial base angle (Angle N-S-Ba), shorter palatal length (A- PNS) and maxillary length (PNS-ANS), a more obtuse gonial angle (Angle Me-Go-Ar), and a larger y-axis length (S-Gn) and nasal width (NC-NC') (p < .01). CONCLUSIONS: All these results indicate that the healthy parents of patients with nonsyndromic cleft lip with or without palate show distinct characteristics in craniofacial morphology. These parental craniofacial features are more obvious in patients with cleft lip with palate than those with cleft lip only. In general, the characteristics seem to be more distinct in the fathers than in the mothers of cleft patients.

Osteogenic effects of D+beta-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) on osteoblasts and bone marrow stromal cells of intact and prednisone-treated rats.

AIM: Previous studies have shown that D(+)beta-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) has anabolic effects on prednisone (GC)-induced osteoporosis in rats. The current study aims to investigate the molecular mechanism of SAA's impact on osteogenesis and adipogenesis in bone marrow stromal cells in intact and GC-treated rats. METHODS: For in vitro study, newborn rat calvaria osteoblasts (rOBs) and rat bone marrow stromal cells (rMSCs) were isolated, identified and cultured with SAA at different concentrations to evaluate SAA's influence on osteogenesis and adipogenesis. In addition, 3-month-old Sprague-Dawley (SD) male rats were treated with distilled water, prednisone alone (3.0 mgxkg(-1)xd(-1)) or prednisone (3.0 mgxkg(-1)xd(-1)) and SAA (25 mgxkg(-1)xd(-1)) for 45 d. At the end point, the different groups of rMSCs were isolated by density-gradient centrifugation and cultured. RESULTS: (1) At 0.1-10.0 mg/L, SAA increased ALP activity, type I collagen (Coll-I) mRNA and OPG mRNA expression and stimulated nodule mineralization of rOBs. SAA (0.5 mg/L) also significantly increased the ALP activity of rMSCs without a need for osteogenesis-inducing medium. At 5.0 mg/L, SAA decreased the number of adipocytes with less lipid droplet formation from the rMSCs, which typically undergo adipocyte induction. (2) Coll-I expression was markedly decreased, whereas lipoprotein lipase (LPL) mRNA expression increased by 98% when compared with the first generation of rMSCs in GC-treated rats. The SAA-treated rats demonstrated an over 2-fold increase in Coll-I expression when compared with intact rats and further showed a significant decrease in LPL expression when compared with GC-treated rats. When rMSCs were co-cultured with SAA (0.5 mg/L) in vitro, SAA did not affect Coll-I and LPL gene expression in intact rats but significantly increased Coll-I and decreased LPL gene expression in GC-treated rats. CONCLUSION: SAA protected bone from GC-induced bone marrow impairment by stimulating osteogenesis and depressing adipogenesis in bone marrow stromal cells both in vivo and in vitro. The data indicated that aqueous extract of Salvia miltiorrhiza, which include SAA, may serve as an active anabolic agent and a useful therapeutic strategy for the treatment of GC-associated osteoporosis.

Cyclic stretching promotes collagen synthesis and affects F-actin distribution in rat mesenchymal stem cells.

Recent studies have shown effects of mechanical environment on bone marrow mesenchymal stem cells (BMSC). In order to examine how BMSC and their cytoskeleton respond to mechanical stimulation, we investigated their collagen synthesis and F-actin expression. Rat BMSC were harvested from adult rats and cultured to passage 4. Then the cells were seeded onto a silicone membrane loaded with an uniaxial cyclic stretching (10%, 1 Hz) during 3, 6, 12, 24 and 36 h. The levels of collagen type I and III before and after stretching were analyzed by immunocytochemistry, and the F-actin in cytoplasm was observed by confocal microscopy. Immunocytochemistry results showed that the stretching enhanced the synthesis of collagen types I and III in BMSC after 24 h stimulation. However, a decrease in fluorescence density of F-actin was observed after the stretching in a time dependent manner. In addition, the F-actin filaments seemed much thinner than those of static cells. These results indicated that the cyclic stretching favored the synthesis of collagen types I and III, but decreased the amount of F-actin in the BMSC.

Effect of uniaxial stretching on rat bone mesenchymal stem cell: orientation and expressions of collagen types I and III and tenascin-C.

Bone marrow mesenchymal stem cells (BMSC) have the potential to differentiate into a variety of cell types like osteoblasts, chondroblasts, adipocytes, etc. It is well known that mechanical forces regulate the biological function of cells. The aim of this study was to investigate the effect of uniaxial stretching on the orientation and biological functions of BMSC. Rat BMSCs were harvested from femoral and tibial bone marrow by density gradient centrifugation. Cells from passages 1-6 were characterized by flow cytometry using monoclonal antibodies. The recovered cells were stably positive for the markers CD90 and CD44 and negative for CD34 and CD45. A cyclic 10% uniaxial stretching at 1Hz was applied on rat BMSC for different time-courses. The length, width, and orientation of the cells were subsequently determined. Expression of collagen types I and III and tenascin-C mRNAs was measured by real-time RT-PCR, and the synthesis of these receptors was determined by radioimmunoassay. Results showed that uniaxial stretching lengthened and rearranged the cells. Compared with control groups, expression of collagen types I and III mRNAs was up-regulated after 12-h of stretching, while significant increase in synthesis of the two collagen protein types was not observed until after 24-h stretching. The expression of tenascin-C mRNA was significantly increased after a 24-h stretching. These data suggest that cyclic stretching promotes the synthesis of collagen types I and III and tenascin-C by the rat BMSC.

Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching.

Adult bone-marrow-derived mesenchymal stroma cells (BMSC) seem to be a potential cell source for tissue engineering of the ligament. The objective of this work was to study the time-related changes in mRNA expression and protein levels of collagen types I and III and of tenascin-C in BMSC under co-culture with fibroblasts or under a uniaxial cyclic condition. Rat BMSC harvested from the femur and tibial bone marrow were co-cultured with ligament fibroblasts or stimulated by cyclic 10% uniaxial stretching at 1 Hz. Image analysis showed significant cell loss in stretched BMSC, particularly in the directions close to the stretching direction. However, these BMSC displayed an equivalent growth rate to that of non-stretched cells. Real-time reverse transcription/polymerase chain reaction revealed that the mRNA expression of collagen types I and III and of tenascin-C by BMSC was significantly up-regulated by co-culture and cyclic stretching. Radioimmunoassay results confirmed the effects of these stimulations, showing increases in the level of these proteins. Thus, BMSC might be useful as a cell source for the tissue engineering of ligament.

Preparation and endothelialization of decellularised vascular scaffold for tissue-engineered blood vessel.

The proliferation of cells on the decellularised tissues fixed by chemical crosslinking agent is retarded for cytotoxicity of crosslinked tissues. To overcome this disadvantage, we prepared the decellularised vascular scaffold through fixing the porcine thoracic arteries with 40 mL/L ethylene glycol diglycidyl ether (EGDE), and reduced the cytotoxicity of this scaffold by treating it with lysine and coating it with type I collagen, finally endothelialized it in vitro. The EGDE-fixed porcine thoracic arteries were examined morphologically. The fixation index determination and the biomechanics test were also performed. Human umbilical vein endothelial cells (HUVECs) were seeded on the type I collagen-coated surface of different modified vascular tissues (fixed with glutaraldehyde or EGDE or EGDE + lysine), and the growths of HUVECs on the specimens were demonstrated by means of MTT test. Finally, HUVECs were seeded on the luminal surface of the modified porcine vascular scaffolds which were respectively treated in the same manner described above, and then cultured for 7 days. On the seventh day, the HUVECs on the specimens were examined by means of light microscopy, scanning electron microscopy and transmission electron microscopy (TEM). The antigenicity of the vascular tissues can be diminished by EGDE through getting rid of cell in the vascular tissues or reducing the level of free amino groups in the vascular tissues. In this study, it was also found that the EGDE-fixed porcine vascular tissues appeared similar to the native porcine vascular tissues in color and mechanical properties. After treated by 2% lysine and coated with type I collagen, the EGDE-fixed porcine vascular tissues were characterized by low cytotoxicity and good cytocompatible. The HUVECs can proliferate well on the modified vascular tissues, and easily make it endothelialized. The results showed that the modified porcine vascular scaffolds should be a promising material for fabricating scaffold of tissue-engineered blood vessel.

[Induction of drug resistance in Tca8113 cell line by exposing to chemotherapy drug].

OBJECTIVE: To study the drug resistance changes in Tca8113 cell lines by exposing to carboplatin. METHODS: The concentration of carboplatin added to Tca8113 cells was increased gradually and continually, which was to induce the carhoplatin-resistance in Tca8113 cells. The sensibility to drugs of the cells was analyzed by MTT method. Immunocytochemistry and RT-PCR were utilized to examine the expression of multidrug resistance proteins and genes. RESULTS: After exposing to carboplatin, the Tca8113/CBP cells had higher drug-resistance to CBP, MTX, PYM, VCR and higher expression of MRP, GST-pi than Tca8113 cells. CONCLUSION: Multidrug resistance of Tca8113/CBP is associated with over expression of MRP, GST-pi and MDR. Tca8113/CBP can provide an ideal model for multidrug resistance research.

[1,25 (OH), dihydroxyvitamin D3 induces the formation of osteoclasts from mononuclear cells of rat marrow].

OBJECTIVE: To investigate the effect of 1,25 (OH)2 dihydroxyvitamin D3 on the generation of osteoclasts from mononuclear cells of adult rats. METHODS: With density gradient centrifugation, the mononuclear cells were isolated from rat bone marrow and cultured in the alpha-MEM with 10(-8) mol/L 1,25 (OH)2 dihydroxyvitamin D3. The induced cells were fixed and stained with tartrate-resistant acid phosphatase (TRAP). The bone resorption pits were examined by scanning electron microscope, and the morphology of osteoclasts were examined with inverted phase contrast microscope. RESULTS: The TRAP+ multinucleated cells could be observed on the seventh culturing day of mononuclear cell group. The number of TRAP+ multinucleated cells went up to a peak on the fourteenth culturing day, and then began to decrease on the twenty-first culturing day. The number of osteoclasts induced from mononuclear cell group was more than that from bone marrow group (P < 0.05) when it was during the fourteenth culturing day to the twenty-third culturing day. The number of bone resorption pits in the mononuclear cell group was much more than that in the bone marrow group (P < 0.05) when it was in period from the tenth culturing day to the twenty-third culturing day. The number of osteoclasts induced from mononuclear cell group could keep the peak value of lasting 7 days, but that induced from bone marrow group could only last its peak value for 3 days. CONCLUSION: The method with 1,25(OH)2 dihydroxyvitamin D3 inducing the formation of osteoclasts from the marrow mononuclear cells is better than that from the whole bone marrow.

Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells.

Variations in the matrix metalloproteinase (MMP)-9 gene are related to the presence and severity of atherosclerosis. The aim of this study was to determine the signaling pathways of MMP-9 in endothelial cells subjected to low fluid shear stress. We found that low fluid shear stress significantly increased MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and phosphorylation of MAPK in cultured human umbilical vein endothelial cells (HUVECs). Inhibition of NF-kappaB resulted in remarkable downregulation of stress-induced MMP-9 expression. Pretreatment of HUVECs with inhibitors of p38 mitogen-activating protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) also led to significant suppression of stress-induced MMP-9 expression and NF-kappaB DNA-binding activity. Similarly, addition of integrins inhibitor to HUVECs suppressed the stress-induced MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and the phosphorylation of p38 MAPK, ERK1/2. Our findings demonstrated that the shear stress-induced MMP-9 expression involved integrins-p38 MAPK or ERK1/2-NF-kappaB signaling pathways.

[Bone marrow stromal cells co-modified with BMP-2 and bFGF gene].

AIM: To establish modified bone marrow stromal cells(BMSCs) which can express BMP-2 and bFGF stably. METHODS: BMP-2 and bFGF gene were amplified by RT-PCR, and then cloned into the expression vector pcDNA3.0. After being confirmed by DNA sequencing, pcDNA3.0-BMP-2 and pcDNA3.0-bFGF were co-transfected into rat BMSCs with Lipofectamine 2000 reagent. The expression of BMP-2 and bFGF gene in rat BMSCs was detected by RT-PCR, Western blot, immunohistochemical staining and ELISA. RESULTS: BMP-2 and bFGF gene were cloned, and their sequences were identical with those in GenBank. The expression plasmids, pcDNA3.0-BMP-2 and pcDNA3.0-bFGF, were constructed and co-transfected into rMSCs successfully. RT-PCR showed the mass transcription of BMP-2 and bFGF mRNA in transfected BMSCs. Western blot, immunohistochemical staining and ELISA confirmed the expression of BMP-2 and bFGF genes in transfected cells and in the supernatant. CONCLUSION: We have constructed the optimal rat BMSCs which can be used in bone tissue engineering.

[Characteristics of porcine thoracic arteries fixed with polyepoxy compound].

OBJECTIVE: To investigate the characteristics of porcine thoracic arteries fixed with ethylene glycol diglycidyl ether (EX-810) and to provide the proper scaffold materials for tissue-engineered blood vessel. METHODS: The porcine thoracic arteries were respectively treated with 40 ml/L EX-810 and 6.25 g/L glutaraldehyde, and then they were examined with naked-eye, light microscope and scanning electron microscope. The fixation index determination, the amino acid analysis and the biomechanics test were also performed. RESULTS: The antigenicity of vascular tissues can be diminished by EX-810 through getting rid of cell in the vascular tissues or reducing the level of free amino groups in the vascular tissues. The structural integrity of vascular tissues can be preserved after treatment with EX-810. It was also found that the EX-810-fixed porcine vascular tissues appeared more similar to the natural vascular tissues in color and mechanical properties, and were more pliable than the glutaraldehyde-fixed tissues. CONCLUSION: The EX-810-fixed porcine thoracic arteries with low cytotoxicity and low antigenicity showed favorable characteristic similar to those of natural vessel, and it should be a promising material for fabricating scaffold of tissue-engineered blood vessel.

[Transcriptional activation of interleukin-8 gene induced by shear stress in human umbilical vein endothelial cells].

OBJECTIVE: To examine the transcriptional activation of IL-8 gene induced by 4.2 dyne/cm2 shear stress in human vascular endothelial cells. METHODS: The one-step RT-PCR was used for detection of IL-8 mRNAs expression in human umbilical vein endothelial cells (HUVECs) after shear stress for 0.5, 1, 2 hours. To construct IL-8 green fluorescent protein reporter gene plasmid pEGFP1-IL8USCS. The endothelial cells were transfected with the pEGFP1-IL8USCS, and stimulated with 4.2 dyne/cm2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by flow cytometry. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining in HUVECs stimulated by shear stress for 0.5, 1, 1.5, 2 hours. Western blotting was used to examine kappaB phosphorylation and degradation after shear stress for 10, 20, 30, 60 minutes respectively. RESULTS: The results of RT-PCR showed that low laminar shear stress could induce IL-8 mRNA expression in HUVECs, its increased effect was time-dependent. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in the pEGFP1-IL8USCS-transfected cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that flow shear stress could induce nuclear translocation of NF-kappaB. Flow shear stress could induce IkappaB phosphorylation and degradation in HUVECs detected by Western blotting. CONCLUSION: These experiments suggested that the NF-kappaB signaling pathway would probably be involved in flow shear stress-induced IL-8 gene transcriptional activation, It may be involved in the development of atherosclerosis.