Objective To observe the analgesic effect of COX-2 inhibitors combined with PCA in treatment of the pain after total knee arthroplasty(TKA)and explore novai method for pain control after TKA. Methods This study was,a double blind,controlled,randomized,placebo-controlled,parallelgroup and single center study and obtained the consent of the Medical Ethics Committee of Chongqing Medical University.Inclusion criteria:the patients with severe trauma or degenerative knee,at age of 58-75 vears.ASA Ⅰ or Ⅱ, tolerance of the intravenous anesthesia.The same solvent formula of PCA was routinely prepared based on body weight after TKA.Intravenous injection of 40 mg Parecoxib plus 2 ml normal saline or injection of only 2 ml normal saline was done when closing the incision.which was recorded and prescribed by the researcher A.The same reagent waft,used continually every other 12 hours till48 hours.Celecoxib or placebo capsules was not usod orally at 72 hours until two weeks later.The knee ioint function was observed preoperatively and at days 3,7 and 15 after TKA. Results The study was completed in 60 patients including 30 patients in the group treated with Parecoxib and Celecoxib sequential therapy and 30 patients in placebo-control group.There was no statistical difference in aspects of general condition in both groups(P>0.05).There was no statistical difference in aspect of preoperative knee score between two groups(P>0.05).The analysis showed statistical difference in the knee function score at days 3.7 and 15 after operation(P<0.05 or0.01).Conclusions The COX2 inhibitors combined with PCA has definite analgesic effect on the pain after TKA,which positively correlates with early modified HSS evaluation of TKA.

OBJECTIVETo construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA).

METHODSThe RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector. The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing. A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1, pHelper 1.0, and pHelper 2.0. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into rat mesenchymal stem cells was calculated.

RESULTSPCR and DNA sequencing demonstrated that the constructed lentivirus vector of L-smNRSE/RE-1 produced RE-1/NRSE dsRNA.The titer of the concentrated virus was 4x108 TU/m1. The virus was stably transfected into rat mesenchymal stem cells, and the infection efficiency reached 100% when the multiplicity of infection was 80.

CONCLUSIONThe lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.

Animals ; Bone Marrow Cells ; Cells, Cultured ; Genetic Vectors ; Lentivirus ; genetics ; Mesenchymal Stromal Cells ; Plasmids ; genetics ; RNA, Double-Stranded ; genetics ; Rats ; Repressor Proteins ; genetics ; Silencer Elements, Transcriptional ; genetics ; Transfection

OBJECTIVETo establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs).

METHODShAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry.

RESULTSThe cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45.

CONCLUSIONSThis study establishes a potential method for isolation of hAMSCs from human amnion,in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Amnion ; cytology ; Cell Culture Techniques ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo analyze the change of the neuronal restricted silencing factor (NRSF) gene as well as the NRSF regulation genes in beta-mercaptoethanol induction of the marrow mesenchymal stem cells (MSCs) to neurons, and to discuss the function of NRSF in neural induction of the MSCs and the mechanism of the differentiation from MSCs to neurons.

METHODWe used beta-mercaptoethanol, serum-free DMEM, and dimethyl sulfoxide to induce rat MSCs to differentiate to neurons, and then analyzed the changes of the expressions of NRSF gene and NRSF-regulated genes through real-time PCR.

RESULTSThe rat MSCs were successfully induced to differentiate into neuron-like cells. The induced neuron marker, neuron-specific enolase, was positive. Real-time PCR showed that the expression of NRSF gene remarkably declined. The expressions of neurotrophic tyrosine kinase receptor, type 3, synaptosomal-associated protein 25, L1 cell adhesion molecular,neuronal pentraxin receptor in the NRSF-regulated genes also increased at varied extents.

CONCLUSIONSThe differentiation from MSCs to neurons is relevant with the decline of NRSF expression and the increase of the expressions of NRSF-regulated genes. The NRSF may be the key gene during the differentiation from MSCs to neurons.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Phosphopyruvate Hydratase ; genetics ; metabolism ; Rats ; Rats, Wistar ; Repressor Proteins ; metabolism ; physiology ; Synaptosomal-Associated Protein 25 ; genetics ; metabolism

Cell reprogramming is a progress in which the memory of a mature cell is erased and then the cell develops novel phenotype and function; ultimately, the fate of the cell changes. Cell reprogramming usually occurs at genes expression levels that no genomic DNA sequence change will be involved. By changing the programs of the genetic expressions of cells in terms of space and time, cell reprogramming alters the differentiation of cells and thus produces the required cells. Further research on cells reprogramming will elucidate the mechanisms that govern the cell development, and thus provides more information of the sources of seed cells used for regeneration medicine. More cells differentiated from many terminally differentiated cells will be obtained, which is extremely important for the understanding of molecular differentiation and for the development of cell replacement therapy. This article summarizes the classification, influencing factors, approaches and latest advances of cells reprogramming.
Animals ; Cell Dedifferentiation ; genetics ; Cell Differentiation ; genetics ; Cellular Reprogramming ; Gene Expression ; Humans ; Nuclear Transfer Techniques

OBJECTIVETo observe the effect of the human amniotic membrane (HAM) loaded with human amniotic mesenchymal stem cells (hAMSCs) on the skin wounds of SD rats.

METHODSThe amniotic epithelial cells were removed by trypsin digestion, hAMSCs were loaded onto HAM and then covered on rats' skin defects. The wound healing was observed by HE staining and immunohistochemistry, and the results were compared with the amniotic membrane group and blank control group.

RESULTSThe average wound healing time was (18.3 +/- 0.9) d in the HAM load with hAMSCs group, which was significantly faster than those in the blank control group [(26.4 +/- 0.7) d, P < 0.01] and the amniotic membrane group [(21.5 +/- 1.2) d, P < 0.05]. After 11 d and 14 d, the wound healing rates in the HAM load with hAMSCs group were (81.5 +/- 7.2)% and (94.3 +/- 3.6)%, respectively, which were significantly higher than those in the blank control group [(48.5 +/- 3.2)% and (74.3 +/- 4.3 )%] and the amniotic membrane group [(68.5 +/- 4.5)% and (86.8 +/- 4.8)%] (all P < 0.01). Skin biopsy/HE staining confirmed that the quality of wound healing in the HAM load with hAMSCs group was significantly better than in the amniotic membrane group and the blank control group. Immunohistochemical staining showed that the number of CK19-positive epidermal stem cells in the HAM load with hAMSCs group (48.2 +/- 3.2) was significantly larger than those in the amniotic membrane group (37.7 +/- 3.1) (P < 0.05) and the blank control group (29.6 +/- 2.4) (P < 0.01). Furthermore, the vascular endothelial growth factor expression (64.5 +/- 4.5) in the HAM load with hAMSCs group was also significantly higher than those in the amniotic membrane group (52.6 +/- 3.8) (P < 0.05) and the blank control group (40.7 +/- 3.1) (P < 0.01).

CONCLUSIONHAM loaded with hAMSCs may promote the repair of skin wounds by promoting the regeneration of epidermal stem cells and capillaries.

Amnion ; cytology ; Animals ; Cells, Cultured ; Disease Models, Animal ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Rats ; Rats, Sprague-Dawley ; Skin ; injuries ; physiopathology ; Wound Healing

OBJECTIVETo explore the mechanism via which the epidermal growth factor (EGF) affects the migration of human amnion-derived mesenchymal stem cells (hAMSCs).

METHODSIn vitro cultured hAMSCs were divided into control (untreated), EGF group, inhibitor AG1478 + EGF group, inhibitor LY294002 + EGF group, and inhibitor U0126 + EGF group. The migration ability of hAMSCs in each group was measured using Transwell chamber. The expressions of phosphorylated EGFR (P-EGFR), phosphorylated AKT (P-AKT), and phosphorylated ERK1/2 (P-ERK1/2) as well as the expressions of metalloproteinase (MMP) -2 and MMP-9 were detected using Western blot analysis. The differentially expressed genes in the culture solutions in EGF groups and control group were analyzed with RNA-Seq technique.

RESULTSCells in EGF group had significantly stronger migration ability than in control group (P = 0.0361), inhibitor AG1478 + EGF group (P = 0.0113), inhibitor LY294002 + EGF group (P = 0.0169), and inhibitor U0126 + EGF group (P = 0.0293). EGF increased the phosphorylation levels of EGFR, AKT and ERK, and increased the expression of MMP-2. However, the increased expressions of P-AKT and P-ERK could be suppressed by AG1478 and LY294002. As shown by GO functional enrichment analysis and KEGG pathway analysis, EGF increased the transcription of genes, which were mainly involved in transcriptional regulation, protein modification, and apoptosis inhibition. Genes that were involved in the MARK pathway included DUSP5, IL1B, DUSP6, NGF, and HSPA2.

CONCLUSIONEGF-induced migration of hAMSCs may be mediated by the signaling pathways of PI3K and ERK, which needs MMP-2 expression and the co-expression of genes involved in transcriptional regulation, protein modification, and apoptosis inhibition.

Amnion ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; physiology ; Proto-Oncogene Proteins c-akt ; metabolism

Objective To label human amniotic epithelial cells(hAECs) by using PEI-FeOnanoparticles. Methods The PEI-FeOnanoparticles were characterized by using transmission electron microscopy and dynamic light scattering. The primary cultured hAECs were labeled with the nanoparticles,and the labeling efficiency was evaluated by Prussian blue staining. The cell survival rate and viability were tested by using placenta blue staining and CCK-8 assay,respectively. Results The PEI-FeOnanoparticles were compact spheres with an average particle size of 13 nm,a hydrodynamic radius of 17.56 nm,and a zeta potential of+34.5 mV. The labeling efficiency of the nanoparticles on hAECs reached 91% when the concentrations were greater than 20 μg/ml. When the concentrations of nanoparticles were at 50 μg/ml(t=16.37,P<0.0001;t=10.39,P<0.0001) and 100 μg/ml(t=29.89,P<0.0001;t=16.86,P<0.0001),the cell survival rates and cell viabilities were significantly reduced versus controls. Conclusion The PEI-FeOnanoparticles can be used for labeling hAECs without obvious cytotoxicity at its working concentration.