cyclin-d1 and Burns

Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs).. Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model.. After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly.. These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.

Topics: Animals; Bone Marrow Cells; Burns; Cell Differentiation; Cell Line; Cell- and Tissue-Based Therapy; Cellular Reprogramming Techniques; CRISPR-Cas Systems; Cyclin D1; Disease Models, Animal; Doxycycline; Ectodysplasins; Gene Editing; Hedgehog Proteins; HEK293 Cells; Humans; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; RNA, Guide, Kinetoplastida; Sweat Glands

Epidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism.. Human ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo.. RhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo.. This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; ADP Ribose Transferases; Animals; Botulinum Toxins; Burns; Carbazoles; Cell Proliferation; Cells, Cultured; Cyclin D1; DNA Replication; Epidermal Cells; Epithelial Cells; Humans; Indole Alkaloids; Male; Mice; Mice, Inbred C57BL; Mutation, Missense; Primary Cell Culture; Protein Kinase C; Random Allocation; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Signal Transduction; Stem Cells; Transfection; Wound Healing

Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Burns; Capillary Permeability; Caspase 3; Cell Line; Cyclin D1; Cytochromes c; Electric Impedance; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation; Genes, bcl-2; Intracellular Signaling Peptides and Proteins; Male; Membrane Potential, Mitochondrial; Microvessels; Mitochondrial Proteins; Propofol; Rats, Sprague-Dawley; Reactive Oxygen Species; Serum; Signal Transduction

To explore the influence of different nutritional support routes on the intestinal mucosal epithelial cell cycle in burned rats.. Sixty-six Wistar rats inflicted with 30% TBSA III degree burns on the back were employed as the model and were randomly divided into enteral feeding group (EF) and intravenously parenteral nutrition group (PN). Equal volume of nutritional support fluid containing predetermined equal amount of calories and nitrogen was applied via feeding or intravenously infusion through external jugular vein. The indices were observed on 6, 12, 24, 48 and 72 postburn hours (PBHs) with the reference to those in 6 normal rats. The intestinal epithelial cell cycle in jejunal and ileal mucous membrane was analyzed by flow cytometry. Western blotting method was employed in the examination of the expression of cyclin D1, E and that of cyclin dependent kinase (CDK)2 and CDK4.. (1) lntestinal mucosal epithelial G0/G1 ratio in jejunum in EF group was significantly lower than that in PN group at 72 PBHs (P < 0.05). While the ratio in ileum in EF was obviously higher than that in PN groups at 6, 12, 48 and 72 PBHs (P < 0.05). (2) The cell percentage of S phase in EF group was evidently higher than that in PN group (P < 0.05 - 0.01) at 48 and 72 PBHs. (3) Intestinal mucosal cyclin D1 expression increased significantly in EF group at 24 PBHs and in PN group at 48 PBHs (P < 0.05) and which in EF group was obviously higher than that in PN group at 72 PBHs (P < 0.05). (4) The expression of the intestinal mucosal cyclin E in EF group at 72 PBHs was evidently higher than the control value and that in PN group (P < 0.05). (5) The expression of CDK2 exhibited no obvious difference among PN,EF and control group (P < 0.05). The CDK4 expression in EF group increased obviously at 72 PBHs (P < 0.05).. Early postburn enteral feeding was beneficial to the progression of intestinal mucosal epithelial cell cycle and to the repairing and renovation of injured intestinal mucosal membrane. Cyclin and CDK might be important in the modulation of the intestinal mucosal epithelial cell cycle.

Topics: Animals; Burns; CDC2-CDC28 Kinases; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Disease Models, Animal; Enteral Nutrition; Female; G1 Phase; Intestinal Mucosa; Male; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Rats, Wistar; Resting Phase, Cell Cycle; S Phase