ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.

BACKGROUNDLarge-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap.

METHODSA fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks.

RESULTSImmunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal.

CONCLUSIONSFlow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.

BACKGROUND: Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. METHODS: A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. RESULTS: Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. CONCLUSIONS Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.
Animals ; Bioreactors ; Groin ; Male ; Perfusion ; RNA ; analysis ; Rats ; Rats, Inbred Lew ; Surgical Flaps ; Tissue Engineering

AIM:To understand the clinic effect of intravitreal anti-vascular endothelial growth factor(VEGF) drugs injection in the treatment of fundus disease in the real-world study (RWS).METHODS:The clinical cases treated with anti-VEGF drugs in our department from September 2012 to June 2015 were enrolled in this study.Retrospective investigation was reviewed to the kinds of diseases, frequency, usage, efficacy, adverse reaction, and the effects on visual acuity, fundus and macular thickness which were treated with intravitreal anti-VEGF drugs injection.RESULTS:In 305 patients (340 eyes) treated with anti-VEGF drugs, 53 patients (60 eyes, 17.6%) were wet age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV) 16 cases (18 eyes, 5.3%), diabetic macular edema (DME) 120 cases (134 eyes, 39.4%), branch retinal vein occlusion (BRVO) secondary macular edema 61 cases (68 eyes, 20.0%), central retinal vein occlusion (CRVO) secondary macular edema 29 cases (32 eyes, 9.4%), idiopathic choroidal neovascularization (ICNV) 16 cases (18 eyes, 5.3%), high myopia with choroid neovascularization 4 cases (4 eyes, 1.2%), neovascular glaucoma 4 cases (4 eyes, 1.2%), retinal angiomatous proliferation (RAP) 1 cases (1 eyes, 0.2%) and optic papillary neovascularization 1 cases (1 eyes, 0.2%).The minimum age was 16 years old, and the maximum age 90 years old.There were 247 cases (275 eyes, 80.9%) were treated with intravitreal ranibizumab injection, 58 cases (65 eyes, 19.1%) intravitreal conbercept injection.The time number of all patients accepted anti-VEGF drugs treatment was 465, with an average of 1.7 times per eye.Which, the 3 + PRN treatment method in 98 patients (109 eyes, 32.1%), 1 + PRN treatment in 207 patients (231 eyes, 67.9%).69 cases (77 eyes, 22.6%) were used alone to receive anti-VEGF drugs therapy, 10 cases (11 eyes, 3.2%) combined with intravitreal triamcinolone injection(TA), 35 cases (39 eyes, 11.5%) combined with vitrectomy, 26 cases (29 eyes, 8.5%) combined with photodynamic treatment (PDT), 165 cases (184 eyes, 54.1%) combined with simple laser treatment.After anti-VEGF drug treatment, majority of patients' the best corrected visual acuity (BCVA), fundus and central macular thickness(CMT) were significantly improved, compared with the pre-treatment, the difference is significant (P<0.05).So that anti-VEGF drugs can effectively improve visual function and ocular fundus for fundus diseses.There were no serious adverse reactions except 3 patients appearling skin redness, itching, rash, 1 patient low low-grade fever and 1 patient acute cerebral infarction during the treatment.CONCLUSION:Intravitreal anti-VEGF drugs injection can significantly improve the visual function and ocular fundus for patients with fundus diseases, but there are still some adverse events, which should be attached great importance to medical workers.

BACKGROUNDMany studies on periostin have focused on its role in tumors and vascular reconstruction. However, the effect of periostin on stem cell function remains unclear. The aim of this study was to enhance vitality in adipose-derived stem cells (ADSCs), the effect of periostin on the function of ADSCs was observed.

METHODSHuman ADSCs (hADSCs) were isolated from human adipose tissue by collagenase I digestion and collected in multi-periods for in vitro culture. CD29, CD34, CD44, CD45 and CD105 were detected by flow cytometry. In addition, directed differentiation of hADSCs was induced using adipogenic, osteogenic and chondrogenic induction mediums. The induced morphological changes were observed using oil red O, Alizarin red and alcian blue staining. Periostin was administered to hADSCs in an acidic environment. The treatments of cells were divided into three groups: a periostin group (P); an acidic control group (A); a normal group (N). Then the resulting cell proliferation and migration were detected using a Cell Counting Kit-8 (CCK-8) and a transwell chamber assay, respectively.

RESULTSThe detection rates of CD29, CD44, CD105, CD34 and CD45 were 98.89%, 93.73%, 86.99%, 0.19% and 0.16%. The specific staining of cells was positive after induction culture. The mean absorbance of the cells in group P and A at 12 hours were 16.67% and 22.22% greater than group N, respectively (P < 0.01). The mean absorbance of cells from group P was 20.00% greater than that of group A at 48 hours (P < 0.05). The mean number of migratory cells per visual field in group A was 50.38% lower than that in group N (P < 0.05). The migratory cell number in group P was 119.98% greater than that in group A (P < 0.05).

CONCLUSIONSThe acidic environment impacted hADSC proliferation and inhibited cell migration. However, periostin was able to promote the proliferation and migration of hADSCs despite the acidic environment.

Adipose Tissue ; cytology ; Adult ; Antigens, Surface ; analysis ; Cell Adhesion Molecules ; pharmacology ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Humans ; Stem Cells ; drug effects ; physiology

OBJECTIVECutaneous wound is a common health problem of humans. Loropetalum chinens, a medicinal plant, is widely used to treat wounds among the people. The research aims to observe whether L. chinens can promote the rats' wounds healing process, isolate the extracts primarily and commit the wound healing selection, which provide work basis for wound healing research of L. chinens.

METHODFirst we analyzed the possible components with HC-MS/MS, then committed our wound healing experiments for L. chinens in the rat incision wound model and excision wound model, which are commonly used worldwide. After that, we carried on the preliminary isolation of the L. chinens and we screened the heal-promoting effects of the isolations in incision wound model.

RESULTL. chinens significantly accelerates the wound healing of rat's skin, shortens the healing period, enhances the healing intensity and promotes the cell proliferation and blood vessels formation around the wounds. The isolations, which are petroleum ether layer, ethyl acetate layer and n-butyl alcohol layer, exert heal-promoting effects. It indicates that the possible morphon that promotes wound healing may exist in these three components, with small polar.

CONCLUSIONSL. chinens possesses strong wound healing promoting effects, and the active constituent, with small polar, exists in petroleum ether layer, ethyl acetate layer and n-butyl alcohol layer, and we should focus on these three layers when carrying on further studies.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; Hamamelidaceae ; chemistry ; Humans ; Male ; Phytotherapy ; Rats ; Rats, Wistar ; Skin ; drug effects ; injuries ; physiopathology ; Skin Diseases ; drug therapy ; physiopathology ; Wound Healing ; drug effects

3'-Deoxyadenosine, so-called cordycepin, is a bioactive component of the fungus Cordyceps militaris. It has been known to exhibit multiple-biological effects including: modulation of immune response, inhibition of tumor growth, hypotensive and vasorelaxation activities, and promoting secretion of adrenal hormone. To investigate its lipid-lowering effect, hyperlipidemic hamsters and rats fed by high-fat diet were both administered orally with cordycepin extracted from Cordyceps militaris for four weeks. The levels of lipids in hamsters and rats were measured enzymatically before and after the administration of cordycepin (12.5, 25 and 50 mg x kg(-1)). The results suggested that levels of serum total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C) increased markedly in the two animal models by feeding high-fat diet. Meanwhile, cordycepin reduced levels of serum TC, TG, LDL-C, VLDL-C as well as LDL-C/HDL-C (high density lipoprotein cholesterol) and TC/HDL-C ratios. In concert with these effects, an increase in lipoprotein lipase (LPL) and hepatic lipase (HL) activity afforded by cordycepin was considered to contribute to the regulation on lipid profiles. Furthermore, no toxicity of cordycepin was observed by intragastric administration at the maximal tolerant dose in ICR mice for 14 days. The exact lipid-lowering effect of cordycepin needs further investigation.
Animals ; Cholesterol ; blood ; Cholesterol, LDL ; blood ; Cholesterol, VLDL ; blood ; Cordyceps ; chemistry ; Cricetinae ; Deoxyadenosines ; adverse effects ; isolation & purification ; pharmacology ; Hyperlipidemias ; blood ; Hypolipidemic Agents ; isolation & purification ; pharmacology ; Lipase ; blood ; Lipids ; blood ; Lipoprotein Lipase ; blood ; Male ; Mesocricetus ; Mice ; Mice, Inbred ICR ; Rats ; Rats, Wistar ; Triglycerides ; blood

OBJECTIVETo explore the pathogenesis mechanism of hypertrophic scar (HS) and the effective means for its clinical treatment, the difference of the gene expressions between HS and normal skin was compared.

METHODSThe differentially expressed genes between HS and normal skin were obtained by mining PubMed. The dysregulated genes in HS were analyzed by a series of bioinformatics methods, including protein-protein interaction networks, pathways, Gene Ontology and functional annotation clustering analysis.

RESULTSA total of 55 dysregulated genes in HS was identified (46 up-regulated genes and 9 down-regulated genes). Fifty-one genes were found to encode proteins with interaction network, including up-regulated genes TGFB1, FN1, JUN, COL1A1, CTGF, VEGFA, FOS, COL3A1, IGF1, IL4, PELO, SMAD2, TIMP1, PCNA, and ITGA4 and down-regulated genes ITGB1 and DCN as the central nodes for this network. The dysregulated genes in HS involved in a variety of biological pathways, such as focal adhesion formation, integrin signal transduction, and tumor formation. Furthermore, the dysregulated genes in HS played the important roles in biological processes of cell surface receptor linked signal transduction, tissue development, cell proliferation and apoptosis, and macromolecule biosynthetic process, as well as in molecular function of calcium ion binding, double-stranded DNA binding, heparin binding, promoter binding and MAP kinase activity. The results of functional annotation clustering analysis revealed that the dysregulated genes in HS involved in epidermis development, angiogenesis, and apoptosis.

CONCLUSIONSuch key genes as TGFB1, FN1, and JUN, along with the pathways, biological processes and molecular functions involving epidermis development, angiogenesis, and extracellular matrix-integrin-focal adhesion signal transduction may play the important roles in the development of HS. The investigations of the dysregulated genes in HS could provide the new targets for clinical treatment.

Cicatrix, Hypertrophic ; genetics ; Cluster Analysis ; Computational Biology ; Data Mining ; Gene Expression ; Gene Expression Profiling ; Gene Regulatory Networks ; Humans

BACKGROUNDPeriostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway.

METHODSA human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.

RESULTSThe transfection efficiency of periostin/pGCsi to U2OS cells was about 70% - 80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F = 564.71, P < 0.001) and 58% (F = 341.51, P < 0.001), respectively. Meantime, the earlier apoptosis value increased by 417% (F = 28.69, P < 0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F = 47.00, P < 0.001), however, that of G0 - G1 phase cells increased by 12% (F = 14.50, P < 0.001). The capability of migration and invasion reduced by 41% (F = 17.79, P < 0.001) and 72% (F = 197.08, P < 0.001), respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-β and vascular endothelial growth factor decreased by 17% (F = 73.99, P < 0.001) and 47% (F = 30.25, P < 0.001), respectively. A tendency of lower focal adhesion kinase (FAK) was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21% (F = 16.81, P < 0.001).

CONCLUSIONSRNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.

Apoptosis ; Bone Neoplasms ; etiology ; pathology ; Cell Adhesion Molecules ; antagonists & inhibitors ; genetics ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alphaVbeta3 ; physiology ; Neoplasm Invasiveness ; Osteosarcoma ; etiology ; pathology ; Phosphorylation ; RNA Interference ; Transfection