Mechanical properties and cytoskeletal protein changes after anln deficiency / 中国组织工程研究

BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported. OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells. METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy. RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.

Biological behaviors of fibroblasts on the surface of TiO2 nanotubes with different diameters after annealing treatment / 中国组织工程研究

BACKGROUND: Previous studies have shown that titanium nanotubes with different diameters affect the adhesion and growth of cells on the implant surface. OBJECTIVE: To investigate the effects of annealing treatment on the biological behaviors of fibroblasts on the surface of TiO2 nanotubes with different diameters. METHODS: TiO2 nanotubes were prepared by polishing pure titanium samples through anodizing at 5 V and 20 V respectively, and then annealed. The pure titanium samples were divided into six groups: P (polishing titanium), NT5 (TiO2 nanotubes prepared at 5 V), NT20 (TiO2 nanotubes prepared at 20 V), A-P (annealing treatment and polishing titanium), A-NT5 (TiO2 nanotubes prepared at 5V and annealed) and A-NT20 (TiO2 nanotubes formed at 20 V and annealed). Field emission scanning electron microscopy was used to obsesrve the surface topography. Fibroblasts were inoculated on the surface of each group of samples. After 60 and 120 minutes of culture, the number of cells adhering to titanium sample surface was counted using cell nucleus staining method. On day 1 of culture, cell morphology was observed using scanning electron microscopy. On days 1, 3 and 5 of culture, cell proliferation was detected by MTT method. On day 3 of culture, the secretion of collagen fiber was detected by picro-sirius red staining method. RESULTS AND CONCLUSION: (1) The annealing treatment had no significant effect on the morphology and diameter of the nanotubes. (2) The number of adherent cells in the NT5 and NT20 groups was significantly lower than that in the P group. The annealing treatment increased the number of fibroblasts on the surface of polished pure titanium and decreased the number of fibroblasts in the NT5 and NT20 groups. (3) Annealing enhanced the viability of fibroblasts on the surface of polished pure titanium and decreased the viability of cells on the surface of nanotubes prepared at 5 and 20 V. (4) Cell viability in the NT5 and NT20 groups was lower than that in the P group. Annealing treatment increased the viability of cells on the surface of polished pure titanium and decreased the viability of cells on the surface of nanotubes prepared at 5 and 20 V. (5) The level of collagen on the surface of nanotubes in the NT5 and NT20 groups was higher than that in the P group. Annealing treatment increased the level of collagen on the surface of polished pure titanium and decreased the level of collagen in the NT5 and NT20 groups. These findings suggest that TiO2 nanotubes inhibit the adhesion, spreading and proliferation of fibroblasts to different degrees; annealing treatment can enhance this inhibitory effect; TiO2 nanotubes enhance collagen secretion by fibroblasts to different degrees, and annealing treatment inhibits the enhancement.

The effects of diameter-controlled titanium nanotubes on the proliferation, streching and collagen secretion of fibroblasts / 实用口腔医学杂志

Objective:To study the effects of Ti-TiO2 nanotubes with different diameters on the proliferation,stretching and collagen secretion of fibroblasts.Methods:TiO2 nanotubes formed at 1,5,10 and 20 V potentials served as the experimental samples and polished pure titanium served as the control.Fibroblasts was cultivated on the surface of the various samples.MTT assay was used to examine the cell proliferation.The surface morphology of the cells was observed with SEM.Collagen secrection was tested by sirius red/bitter acid staining.Results:The nanotubes prepared by 1,5,10 and 20 V were with the diameter of 15,30,50 and 100 nm respectively.At day 1,3 and 5,the cell proliferation on polished pure titanium surface was more than that on the nanotubes surfaces at the same time;at day 5,cell proliferation on 100 nm nanotubes was significantly more than that on the other nanotube surfaces (P ＜ 0.01).At day 3,fibroblasts at polished pure titanium surface stretched as typical long spindle form,while with obvious pseudopodium at nanotube surfaces.The collagen secretion of fibroblasts was highest at 100 nm nanotubes (P ＜ 0.01).Conclusion:100 nm nanotubes may have minimal negative effect on fibroblast proliferation and the greatest positive effect on the collagen secretion.

Effects of different substrate stiffness on adhesion, spreading and migration of human hepatocellular carcinoma cells / 医用生物力学

Objective To investigate the effects of substrate stiffness on the adhesion, spreading and migration of hepatocellular carcinoma cells as well as the regulation of cytoskeleton assembly and integrinβ1 expression, and to explore the role of substrate mechanical properties in the metastasis of hepatocellular carcinoma cells. Methods The polyarcylamide gel with different stiffness was achieved by varying the relative ratio of acrylamide to bis acrylamide. The substrate surface was cross linked with extracellular matrix molecules for cell adhesion. The adhesion, spreading and migration of hepatocellular carcinoma cells on substrates with different stiffness were recorded by phase contrast microscope and made quantitative analysis by Image J software. The cytoskeleton assembly on substrates with different stiffness was detected by immunofluorences assay, and the expression of integrinβ1on different substrates was measured by flow cytometer. Results The rigid substrate enhanced the adhesion and spreading of hepatocellular carcinoma cells in shortened time. Neither the soft (1.1 kPa) nor over rigid (glass) substrate facilitated the migration of hepatocellular carcinoma cells, and the maximum migration velocity was found on the substrate with moderate stiffness（10.7 kPa）. The rigid substrate could promote cytoskeleton assembly and integrinβ1 expression. Conclusions The effects of substrate stiffness on adhesion, spreading and migration of hepatocellular carcinoma cells are regulated by the cytoskeleton assembly and integrin expression.

Caracterização funcional das isoformas de splicing do gene ADAM23 / Functional characterization of ADAM23 gene splicing isoforms

A ADAM23 é uma glicoproteína transmembrana pertencente à família ADAM (A Disintegrin and Metalloprotease) que apresenta a estrutura protéica típica dos membros desta família, mas não possui atividade de metaloprotease. O gene ADAM23 apresenta três isoformas de splicing, α, ß e γ, que codificam proteínas com porções C-terminais distintas. As isoformas α e ß codificam proteínas com domínios transmembranas diferentes, enquanto γ provavelmente consiste em uma isoforma secretada ou citoplasmática de ADAM23. Foi demonstrado que o gene ADAM23 está epigeneticamente silenciado em tumores de mama de estágios mais avançados e que seu silenciamento está associado a um maior risco de desenvolvimento de metástases e a um pior prognóstico. Recentemente, foi descrito que a proteína ADAM23 interage diretamente com a integrina αVß3 na linhagem tumoral de mama MDA-MB-435, sendo capaz de modular seu estado conformacional, controlando sua ativação. Utilizando RNAi, observou-se que o silenciamento completo do gene ADAM23 (i.e., as três isoformas) aumenta os níveis de αVß3 em conformação ativa na superfície das células MDA-MB-435, promovendo um incremento de sua capacidade migratória e adesiva. No presente trabalho, avaliamos por reações de amplificação em tempo real o perfil de expressão das três isoformas de splicing do gene ADAM23 em cinco tecidos normais (mama, cólon, cérebro, próstata e pâncreas) e em doze linhagens tumorais derivadas destes tecidos. Observamos diferenças nos níveis de expressão das isoformas em todas as amostras avaliadas, tanto dentro de uma determinada amostra, como quando comparamos tecidos normais entre si ou com linhagens tumorais. A isoforma γ é a mais expressa em todos os tecidos normais (exceto em cérebro) e em todas as linhagens tumorais. Em tecido normal de mama e de próstata e nas doze linhagens tumorais, ADAM23α é a segunda isoforma mais expressa, sendo ß a menos expressa. Constatamos também que a fração representada por cada isoforma, em relação à expressão total do gene ADAM23, está alterada nas linhagens tumorais, em comparação aos tecidos normais correspondentes. Com o intuito de elucidar a função das isoformas de ADAM23 separadamente, utilizamos shRNAs (short hairpin RNAs) para reduzir a expressão de cada isoforma de modo individual e específico na linhagem tumoral MDA-MB-435, e avaliamos seu efeito na proliferação, na morfologia, na adesão e no espraiamento celular. Verificamos que a redução da expressão da isoforma γ aumentou significativamente a taxa de proliferação das células MDA-MB-435 cultivadas em modelo tridimensional. Demonstramos também que ADAM23γ participa da regulação da morfologia e da capacidade de espraiamento das células MDA-MB-435 em condições padrão de cultivo (i.e., meio de cultura completo e placas não-sensibilizadas com substratos) e em componentes específicos da matriz extracelular, como fibronectina, colágeno I e matrigel. A isoforma α também está envolvida no controle da morfologia e do espraiamento da linhagem MDA-MB-435, porém, de modo distinto da isoforma γ. Já ADAM23ß não interfere na morfologia das células MDA-MB-435 e tem efeito marginal no espraiamento celular apenas em condições padrão de cultivo. Em conjunto, nossos resultados demonstram que as isoformas de ADAM23 são diferencialmente expressas em tecidos normais e tumorais, e exercem funções biológicas distintas

ADAM23 is a transmembrane glycoprotein that belongs to the ADAM (A Disintegrin and Metalloprotease) family of proteins and exhibits the typical protein structure of the family members, but it doesn't have metalloprotease activity. The ADAM23 gene has three splicing isoforms, α, ß and γ, that code for proteins with different C-terminal regions. Isoforms α and ß code for proteins with different transmembrane domains, while γ probably constitute a secreted or cytoplasmatic isoform of ADAM23. It has been demonstrated that the ADAM23 gene is epigenetically silenced in advanced stage breast tumors and that its silencing is associated with a higher risk of developing metastases and with a worse prognosis. Recently, it was described that ADAM23 protein interacts directly with αVß3 integrin in the breast tumor cell line MDA-MB-435, modulating its conformational state and controlling its activation. Using RNAi, it was observed that the complete silencing of ADAM23 gene (the three isoforms) raises the levels of αVß3 in its active conformation in the surface of MDA-MB-435 cells, promoting an increase in its migratory and adhesive capacity. In the present work, we evaluated by real time PCR the expression pattern of the three splicing isoforms of ADAM23 gene in five normal tissues (breast, colon, brain, prostate and pancreas) and in twelve tumor cell lines derived from these tissues. We observed differences in the expression levels of the three isoforms in all samples, either within a specific sample or comparing normal tissues among them or with tumor cell lines. Isoform γ has the highest expression in all normal tissues (except for brain) and in all tumor cell lines evaluated. In breast and prostate normal tissues and in all tumor cell lines, ADAM23α is the second most expressed isoform, while ß is the less expressed. We also noticed that the ratio represented by each isoform, relative to the total expression of ADAM23 gene, is altered in the tumor cell lines, compared to the corresponding normal tissues. With the aim to elucidate the function of ADAM23 isoforms separately, we used shRNAs (short hairpin RNAs) to reduce the expression of each isoform specifically in the MDA-MB-435 tumor cell line, and studied its effects in proliferation, morphology, adhesion and cell spreading. We observed that the reduced expression of isoform γ significantly increased the proliferation rate of MDA-MB-435 cells cultivated in tridimensional system. Also, we demonstrated that ADAM23γ participates in the regulation of cell morphology and spreading of MDA-MB-435 cells, both in standard culture conditions (cell culture media with fetal serum and in plates not sensitized with substrates) and in specific components of extracellular matrix, such as fibronectin, collagen type I and matrigel. Isoform α is also involved in the control of morphology and spreading of MDA-MB-435 cell line, although in a distinct manner from isoform γ. ADAM23ß doesn't interfere in the morphology of MDA-MB-435 cells and plays a discrete role in cell spreading only under standard culture conditions. Together, our results demonstrate that ADAM23 isoforms are differently expressed in normal and tumoral tissue, and play distinct biological roles

Spreading dynamics of β2 integrin expressed human neutrophils onto ICAM-1-immobilized substrate / 医用生物力学

Objective To elucidate the spreading dynamics of β2 integrin expressed human neutrophils (PMNs) on ICAM-1-immobilized substrate. Methods The fraction of PMN spreading on the substrate pre coated by 10, 20, or 100 μg/mL intercellular adhesive molecule-1 (ICAM-1) was quantified when that on 2% human serum albumin (HSA) immobilized or that on blank substrate was served as control. The site density of β2 integrin expressing on PMNs was determined using flow cytometry and the regulation of β2 integrin subunits was defined using the fraction of PMN spreading on 100 μg/mL ICAM-1 substrate by blocking CD11a or CD11b subunit of β2 integrin. Results PMN spreading was presented on ICAM-1-immobilized substrate but absent on 2% HSA-immobilized substrate, supporting the specificity of β2 integrin induced spreading. Time course of neutrophil spreading on ICAM-1 substrate was density dependent of both ICAM-1 and β2 integrin molecules. The fraction of PMN spreading was reduced significantly when the expression of CD11b subunit was blocked. Conclusions PMN spreading was mediated specifically by β2 integrin-ICAM-1 interactions and determined by the expression of β2 integrin and ICAM-1, in which CD11b subunit played a dominate role.

CD2AP depletion impairs the adhesion and spreading function of podocyte / 中华肾脏病杂志

Objective To study the effects of CD2-associated protein (CD2AP) on podocyte adhesion and extension ability and to explore its possible mechanism. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and serambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen IV coated plates. The relative cell adhesion and cell area were examined 90 min later. Apoptotic rates of CD2AP siRNA transfected podoeytes and different PAN concentrations incubated podoeytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confoeal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative ceil adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[(41.72±6.07)% vs (64.46±8.53)%, P<0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [(5.73±0.61)% vs (3.26±0.45)%, P<0.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability (P<0.05). Nevertheless, no significant difference was found in cell body spreading (P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level was conspicuously descended to some degree (P<0.05). Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may he partly responsible for the decline of cell adhesion and spreading.