Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF‐β1, was compared by quantitative real‐time RT‐PCR. The expression of proliferation cell nuclear antigen (PCNA) and α‐smooth muscle actin (α‐SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF‐β1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher α‐SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:374–383, 2008     

Platelet rich plasma promotes skeletal muscle cell migration in association with up‐regulation of FAK,paxillin, and F‐Actin formation

Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague–Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell‐substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F‐actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small‐interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP‐mediated promotion of cell migration. Dose‐dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up‐regulated dose‐dependently. F‐actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up‐regulation of proteins expression of paxillin and FAK as well as increasing F‐actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2506–2512, 2017.

     

GLUD1调控EMT促进结直肠癌细胞侵袭迁移的实验研究

【摘要】 目的 观察GLUD1对结直肠癌细胞侵袭和迁移能力的影响,探讨其可能的分子机制。方法〓采用慢病毒转染下调GLUD1的表达,Transwell侵袭实验检测shGLUD1对肠癌细胞Lovo和SW480侵袭能力的影响,划痕实验检测shGLUD1对Lovo和SW480迁移能力的影响,Western blot检测GLUD1和AG490对E-cadherin、Vimentin、ZEB1、STAT3及pSTAT3表达的影响。结果〓慢病毒转染shGLUD1可显著下调GLUD1在结肠癌细胞中的表达;下调GLUD1的表达可抑制结肠癌细胞Lovo和SW480的侵袭迁移能力;GLUD1可促进STAT3磷酸化,以及上调Vimentin、ZEB1和下调E-cadherin的表达;而AG490处理则可抑制STAT3的磷酸化,上调E-cadherin和下调Vimentin、ZEB1的表达。结论〓GLUD1可通过活化STAT3信号调控上皮间质转化(EMT),进而促进结肠癌细胞的侵袭迁移能力。     

Inhibition of alpha-smooth muscle actin expression in an in vitro wound healing model by certain antibiotics.

OBJECTIVE: This study assesses the effects of antimicrobials on wound healing in an in vitro model of chicken flexor tendons in a collagen gel matrix. Two equidistant tendons were bathed in a culture medium for 28 days as fibroblasts (fb) grew from the tendon ends into the collagen gel and migrated toward each other until gap closure. Five groups of 10 paired tendons each included the control and the study groups, which received oxacillin (Ox), clindamycin (Cl), chloramphenicol (Chl), or tetracycline (Tet) in the culture medium to assess their effects on gap closure rate, fb migration, and myofibroblast alpha-smooth muscle (alpha-SM) actin expression. RESULTS: Gap closure, by day 27, was 98.5% in the controls compared with 97%, 92%, 89.5%, 21.75% in the Tet, Cl, Ox, and Chl groups. Chl retarded gap closure (p < 0.05). Fb migration was similar for all groups. In the control and Ox groups, myofibroblast expressed actin at day 5. By day 7, fb cells were clearly visible in the control, Ox, and Cl groups, whereas, only light actin was present in the Chl and Tet groups. Actin band densities for the Cl, Ox, Tet, and Chl groups were 78.4%, 62.5%, 61.7% and 26.1%, respectively, of the control group. CONCLUSION: These studies suggest that one reason certain antimicrobials impair wound healing, is due to myofibroblast inhibition of alpha-SM actin.     

Expression of insulin-like growth factor binding proteins in healing tendon lesions.

he treatment of overuse tendon injuries with exogenous growth factors such as insulin-like growth factor-I (IGF-I) may facilitate an improved return to sustained athletic function. The biological effects of IGF-I are exerted under the control of a complex of IGF receptors, binding proteins, and proteases. This IGF system includes a family of six structurally related high-affinity IGF binding proteins (IGFBPs) that protect IGF-I from local proteases and restrict access of IGF-I to its receptor. This study describes the expression of the IGFBPs in flexor tendon after acute injury and during healing over time. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8, or 24 weeks following injury. Gene expression was quantitated by fluorescent real-time PCR, and protein expression was evaluated by Western ligand blot (WLB). Message for IGFBPs 2 to 6 was expressed in both normal and healing tendon. No IGFBP-1 mRNA was detected in equine tendon. Message expression for IGFBP-2, -3, and -4 increased following injury, whereas message expression for IGFBP-5 and -6 decreased. Protein expression for IGFBP-2, -3, and -4 was detected by WLB in normal tendon and showed a marked increase following injury. Protein for IGFBP-5 and -6 was not detectable by WLB in normal or healing tendon. The results of this study document the IGFBP response of flexor tendons to injury and healing, which provides information necessary for the design of protocols that may enhance tendon healing through manipulation of IGF-I ligand and binding protein levels.     

Genetic expression for type I procollagen in the early stages of flexor tendon healing.

To determine the precise mechanism by which contact tendon healing occurs at the cellular level, the production of pro alpha (I) collagen messenger RNA (mRNA) produced by fibroblasts of healing intrasynovial flexor tendons was determined by an in situ hybridization technique. The repair site and the proximal and distal tendon stumps of repaired tendons treated with early controlled passive mobilization were fixed and buffered in formalin, 3, 7, 10, and 17 days after repair. A complimentary DNA (cDNA) probe corresponding to alpha (I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. Rising levels of procollagen mRNA, indicating progressively increasing levels of synthetic collagen activity, were detected in the healing tendons through 10 days. A moderate decrease in procollagen mRNA was seen at 17 days. Genetic expression for procollagen mRNA was localized specifically to the epitenon cells on the tendon surface overlying the repair site and to cells in the gap between the tendon stumps. No detectable expression was noted in endotenon fibroblasts. The finding of high levels of expression for procollagen type I mRNA in the surface layer of healing tendons demonstrates that cells intrinsic to tendon epitenon contribute the greatest quantity of native tendon collagen to the repair site during these important early intervals after tendon suture.     

Ex vivo static tensile loading inhibits MMP-1 expression in rat tail tendon cells through a cytoskeletally based mechanotransduction mechanism.

To determine the effect of various degrees of ex vivo static tensile loading on the expression of collagenase (MMP-1) in tendon cells, rat tail tendons were statically loaded in tension at 0.16, 0.77, 1.38 or 2.6 MPa for 24 h. Northern blot analysis was used to assay for mRNA expression of MMP-1 in freshly harvested, 24 h load deprived, and 24 h statically loaded tendons. Western blot analysis was used to assay for pro-MMP-1 and MMP-1 protein expression in fresh and 24 h load deprived tendons. Freshly harvested rat tail tendons demonstrated no evidence of MMP-1 mRNA expression and no evidence of the pro-MMP-1 or MMP-1 protein. Ex vivo load deprivation for 24 h resulted in a marked increase in the mRNA expression of MMP-1 which coincided with a marked increase of both pro-MMP-1 and MMP-1 protein expression. When tendons were subjected to ex vivo static tensile loading during the 24 h culture period, a significant inhibition of this upregulation of MMP-1 mRNA expression was found with increasing load (p<0.05). A strong (r2=0.78) and significant (p<0.001) inverse correlation existed between the level of static tensile load and the expression of MMP-1. Disruption of the actin cytoskeleton with cytochalasin D abolished the inhibitory effect of ex vivo static tensile loading on MMP-1 expression. The results of this study suggest that up-regulation of MMP-1 expression in tendon cells ex vivo can be inhibited by static tensile loading, presumably through a cytoskeletally based mechanotransduction pathway.     

JAK2激酶抑制剂AG490对胃癌细胞侵袭的影响

目的　观察JAK2激酶抑制剂AG4 90对胃癌细胞侵袭以及对基质金属蛋白酶类(MMPs)、组织型基质金属蛋白酶抑制物 (TIMPs)表达的影响 ,探讨STAT3信号转导通路在胃癌侵袭转移调控中的作用。方法　应用JAK2激酶抑制剂AG4 90处理胃癌BGC 82 3细胞 ,Matrigel肿瘤体外侵袭模型检测肿瘤细胞侵袭 ;Westernblot检测STAT3、MMP 2、MMP 9蛋白表达及活性 ;RT PCR检测MMPs、TIMPsmRNA的表达。结果　AG4 90可抑制JAK2 /STAT3信号转导通路活化及胃癌BGC 82 3细胞侵袭 ,在此过程中AG4 90在mRNA和蛋白水平抑制MMP 2和MMP 9表达 ,同时上调TIMP 1、TIMP 2mRNA的表达。结论　STAT3信号转导通路参与胃癌细胞侵袭调控 ,阻断STAT3通路活化可以抑制胃癌细胞侵袭 ,这一作用可能是通过抑制MMP 2和MMP 9表达以及上调TIMP 1和TIMP 2表达实现的。     

In vitro alterations in cytoskeletal tensional homeostasis control gene expression in tendon cells.

An in vitro collagen gel system was used to determine the effect of alterations in cytoskeletal tensional homeostasis on gene expression in tendon cells. Collagen gel matrices, seeded with rat tail tendon cells, underwent cytochalasin D and gel contraction treatments designed to alter the internal cytoskeletal homeostasis of the cells. Gels were examined for cytoskeletal organization using a rhodamine phalloidin stain for actin. The effect of altered cytoskeletal organization on mRNA expression of a catabolic (interstitial collagenase) and anabolic (alpha1(I) collagen) gene was examined using northern blot analysis. Tendon cells in adhered gels demonstrated a highly organized cytoskeleton and showed evidence of alpha1(I) collagen mRNA expression but no evidence of collagenase mRNA expression. Treatment of the attached gel with cytochalasin D disrupted the cytoskeletal organization and resulted in the up-regulation of collagenase mRNA and the inhibition of alpha1(I) collagen mRNA expression. Release of the gels resulted in a cell mediated gel contraction, an immediate loss of cytoskeletal organization, and an mRNA expression pattern similar to that seen with cytochalasin D treatment. Isometric contraction of the gel on itself or around a 3-point traction device resulted in an mRNA expression pattern similar to the adhered gel. Gene expression in the contracted gels could be reversed through chemical cytoskeletal disruption or removal of the traction device which permitted further gel contraction. The results of the study suggest that tendon cells can establish an internal cytoskeletal tension through interactions with their local extracellular environment. Alterations in this tension appear to control the expression of both catabolic and anabolic genes in a reciprocal manner.     

The presence of smooth muscle actin in fibroblasts in the torn human rotator cuff.

The rotator cuff frequently sustains athletic and occupational injury, often resulting in chronic pain and disability. However, despite the high incidence of such shoulder problems, the pathophysiology of rotator cuff injury and healing has not yet been fully elucidated. The notable finding of this study was the presence of a contractile actin isoform, alpha-smooth muscle actin (SMA), in nonvascular cells in all of the seven torn human rotator cuff specimens evaluated immunohistochemically. Up to 95% of cells in any one region, and over 95% of elongated cells found in association with crimped collagen, contained SMA. Most of the cells staining positive for SMA in these sections had morphological features of the fibroblast, though a small number were chondrocyte-like. Treatment of cells growing out from human rotator cuff explants with TGF-beta1 significantly increased the amount of SMA evaluated by Western blot analysis. PDGF-BB and IFN-gamma had no effect on the cell content of SMA. This is the first documentation of the presence of SMA-positive cells in the human rotator cuff tendon. SMA has been found in a number of other healing connective tissues including skin, ligament, meniscus, cartilage, and other types of tendon. Of importance are previous findings that SMA-positive cells can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. The results of the present study thus suggest that SMA-containing cells could contribute to the retraction of the torn ends of a ruptured rotator cuff and play an important role in healing.     

RNA激活技术上调p21WAF1/CIP1基因表达对胆囊癌细胞增殖、侵袭与迁移能力的影响

目的利用RNA激活技术上调人胆囊癌细胞(GBC-SD)中p21基因的表达,观察其对GBS-SD细胞体外增殖、侵袭和迁移能力的影响。方法将与p21基因启动子DNA序列互补的双链RNA分子(dsRNA)转染入人胆囊癌细胞中,采用RT-PCR法和Western blot分别检测p21基因mRNA及蛋白的表达情况;MTT法检测细胞增殖活性;Transwell小室法检测RNAa后细胞侵袭、迁移能力的变化。结果 dsRNA转染GBC-SD细胞72h后能显著上调p21基因mRNA和蛋白的表达,与空白组和对照组比较,转染dsp21后,GBC-SD细胞的增殖活性明显受到抑制,细胞侵袭及迁移能力明显下降。结论 RNAa技术能有效上调p21基因的表达并抑制细胞的增殖活性,降低其侵袭及迁移能力,为胆囊癌疾病的基因治疗提供依据。