Objective To construct a recombinant lentivirus containing human beta defensins -3 ( hBD3 ) , connective tissue growth factor gene (CTGF) and enhanced green fluorescent protein (EGFP), and to detect its translation in rabbit bone marrow mesenchymal stem cells (BMSC).Methods The lentivirus containing hBD3, CTGF and EGFP genes was constructed in vitro.The titer of lentivirus was tested with end-paint dilution assay .Rabbit BMSCs were transfected with recombinant virus.The best value of multiplicity of infection (MOI) was tested.The expression condition, transfection efficacy and genetic stability of the target genes were evaluated by using fluorescence microscopy and flow cytometry . Western blotting was used to detect the expression of the target protein .Results Recombinant lentivirus vectors: Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained . The titer of the recombinant lentiviruses was 3.21 ×108, 5.80 ×108, and 1.16 ×109, respectively.The best MOI value to transfect BMSCs was 150. The transfection efficacy of these lentivirus vectors was high , reaching 79.72%as assessed by flow cytometry , and it could be stably inherited .Western blotting displayed that target protein expression was successful .Conclusion The construction of recombinant lentiviruses carrying hBD3 and CTGF genes is successful and can be effectively transfected into BMSCs .

Objective: To investigate the effects of PRX-2 gene on phenotype changes in epidermal stem cells differentiating into sweat gland cells. Methods: Epidermal stem cells and sweat gland cells separated and cultured from healthy foreskin and adult full-thick skin respectively, were identified by immunofluorescence staining. Lentiviral vector-mediated overexpression and knockdown of PRX-2 gene in epidermal stem cells were performed respectively, with empty vector-mediated epidermal stem cells as a control group. Overexpression、blank control and knowdown group′s PRX-2 expressions in gene and protein levels were detected using RT-PCR and Western blot technology. The ESCs of each group were co-cultured with sweat gland cells through transwell plate, and the expressions of CEA and β1 integrin in epidermal stem cells were determined by flow cytometry before and after co-culturing. Results: Epidermal stem cells and sweat gland cells were in line with their respective specific antigens. Before co-cultured, epidermal stem cells highly expressed β1 integrin (98.69±0.67)%, hardly expressed CEA (6.20±3.15)%. After co-cultured, β1 integrin expression levels were showed as knockdown group (19.30±0.53)% blank control group (51.20±0.79)%> overexpress group (45.91±0.93)%. There had significant differences between those of each two groups. Conclusions PRX-2 gene can inhibit the phenotypic change of Epidermal Stem Cells differentiating into Sweat Gland Cells and improve the ability to maintain their own specific antigens.

Tissue-engineered skin plays an important role in clinical applications,and even the rapid development of science and technology promotes the research about it.Choosing an appropriate animal model for wound repair is the prerequisite for the objective evaluation of the object of study.In this paper,the research progress of animal models of wound repair was introduced from several aspects,such as selection of experimental animals,making of wound models,skin-related cells and materials,wound healing evaluation indexes,etc.,hoping to provide reference for later research work.

Objective To evaluate the short-term prediction of high-sensitivity cardiac troponin T (hs-cTnT) and other cardiovascular risk biomarkers in patients undergoing maintenance hemodialysis (MHD).Methods We conducted a cohort survey in 296 consecutive MHD patients whose clinical data were retrospectively analyzed.Before MHD,hs-cTnT and other relative cardiovascular biomarkers were detected.The end point (all-cause death) and time of occurring were recorded in the next 13 months.The differences between survival and all-cause death were analyzed by t-test,Mann-Whitney test and x2 test.The best two percentile cutoff point was calculated by X-tile and the survival rate was calculated by Kaplan-Meier Logistic regression analysis was applied to analyze the odd ratio between high risk and non-high risk hs-cTnT group.Non-high risk group was divided into intermediate risk and low risk group based on the 99th percentile of hs-cTnT in healthy population,to further evaluate its short-term prediction value for MHD patients.The short-term significance of hs-cTnT was proved to be independently associated with all-cause death by Logistic regression analysis.Results The mean value of serum hs-cTnT in survival group was 0.05 (0.03～0.07) ng/mL,while in the death group it was 0.07 (0.04～0.14) ng/mL,which had statistical significance (P =0.027).The best two percentile cutoff of hs-cTnT in MHD patients was 0.1 ng/mL.The survival rate in high risk group (hs-cTnT＞0.1 ng/mL) is lower than it in non-high risk group (hs-cTnT≤0.1 ng/mL) (76.67％ vs.96.62％,P ＜0.05).The odd ratios for high risk group and non-high risk group was 7.288 (P＜ 0.001).Moreover,further grouping the non-high risk group by hs-cTnT =0.014 ng/mL,intermediate risk group (hs-cTnT＞0.014 ng/mL) group has lower survival rate than low risk group (hs-cTnT≤0.014ng/mL),while there wasn't any death case occurred in the low risk group.Conclusions Hs-cTnT is an independent risk factor to all-cause death.Thus hs-cTnT can be a strong indicator of short-term prediction and prognostic evaluation.

Objective To probe the periodontal ligament regeneration following the implantation of bone marrow mesenchymal cells ( BMSCs ) sheet-collagen membrane-BMSCs sheet sandwich complex.Methods BMSCs cell sheet-collagen membrane-BMSCs sheet complexes were compounded on the root surface of teeth of Beagle dogs.All the dogs were killed on 4 and 12 weeks after implantation.Periodontal ligament regeneration was observed by radiological means, HE staining and Sirus-red staining.Results Compared with collagen membrane group and blank control group, there was a clearly periodontal ligament like tissue and Sharpey′s like fibres formation in test group only.Conclustion Cell sheet-collagen membrane-cell sheet sandwich complex can effectively improve the periodontal ligament regeneration.

Objective To determine the effect of platelet-derived growth factor-C (PDGF-C) on biological characters of human hair dermal papilla cells cultured in vitro.Methods The human dermal papilla cells(HDPCs) were isolated from human hair skin obtained from rhytidectomy procedure and then cultured in vitro.Cell counting and CCK-8 assay were used to detect the effects of PDGF-C (0,10,20,30 and 40ng/ml) on the proliferation of HDPCs at 0,1 d,2d,3d,4d,5d,6d.Under the optimal concentration,flow cytometry was used to detect cell phases.Transwell assay and cell scratch test were performed to detect cell migration.Alkaline Phosphatase Activity Assay kit was used to detect the inductive activity of HDPCs.Results PDGF-C significantly induced the proliferation of HDPCs.PDGF-C of 30ng/mL promoted the HDPCs proliferation at a summit and increased the percentage of the cells arrested at S phase (P ＜ 0.05).PDGF-C also increased the migration populations of cultured HDPCs.The cell number in lower side of the transwell insert membrane of 30ng/ml PDGF-C treated group was (361.3 ± 24.95)while the control was (246.8 ± 7.525),showing significant difference (P ＜ 0.05).The alkaline phosphate activity of cultured HDPCs was increased comparing to the control group,the difference was significant (P ＜ 0.05).Conclusions PDGF-C can promote the proliferation,migration and inductive activity of cultured human dermal papilla cells,which might be beneficial to promote the cultivation of human dermal papilla cells in vitro.

Objective To determine the effect of platelet-derived growth factor-C (PDGF-C) on biological characters of human hair dermal papilla cells cultured in vitro.Methods The human dermal papilla cells(HDPCs) were isolated from human hair skin obtained from rhytidectomy procedure and then cultured in vitro.Cell counting and CCK-8 assay were used to detect the effects of PDGF-C (0,10,20,30 and 40ng/ml) on the proliferation of HDPCs at 0,1 d,2d,3d,4d,5d,6d.Under the optimal concentration,flow cytometry was used to detect cell phases.Transwell assay and cell scratch test were performed to detect cell migration.Alkaline Phosphatase Activity Assay kit was used to detect the inductive activity of HDPCs.Results PDGF-C significantly induced the proliferation of HDPCs.PDGF-C of 30ng/mL promoted the HDPCs proliferation at a summit and increased the percentage of the cells arrested at S phase (P ＜ 0.05).PDGF-C also increased the migration populations of cultured HDPCs.The cell number in lower side of the transwell insert membrane of 30ng/ml PDGF-C treated group was (361.3 ± 24.95)while the control was (246.8 ± 7.525),showing significant difference (P ＜ 0.05).The alkaline phosphate activity of cultured HDPCs was increased comparing to the control group,the difference was significant (P ＜ 0.05).Conclusions PDGF-C can promote the proliferation,migration and inductive activity of cultured human dermal papilla cells,which might be beneficial to promote the cultivation of human dermal papilla cells in vitro.

BACKGROUND:With the aging population, the incidence of lumbar degenerative disease was apparently increased, but how to treatment of degenerative lumbar disease remains controversial.
OBJECTIVE:To compare clinical and radiographic results of minimaly invasive posterior lumbar interbody fusion and open posterior lumbar interbody fusion for single-segment degenerative lumbar disease.
METHODS: We retrospectively analyzed the clinical data of 97 patients with single-segment degenerative lumbar disease, who were treated in the Huishan District People’s Hospital of Wuxi City from July 2006 to July 2012. These patients were divided into minimal group (minimaly invasive posterior lumbar interbody fusion;n=51) and open group (open posterior lumbar interbody fusion;n=46). These data were compared between the two groups, including operative time, blood loss (intraoperative blood volume+postoperative drainage volume), total blood transfusion, postoperative back pain (visual analogue scale), length of hospital stay, bed time, perioperative complications, clinical function (Oswestry disability index), and radiographic results.
RESULTS AND CONCLUSION:Al of 97 patients were folowed up. The duration of folow-up was 28-78 months and 27-76 months in minimal group and open group, respectively. There was no significant difference between the minimal group and open group in term of folowed-up time (P=0.981). Operative time, blood loss, total blood transfusion, bed time, length of hospital stay and visual analogue scale score during final folow-up were significantly lower in the minimal group than in the open group (P < 0.05). However, there was no significant difference between the two groups in Oswestry disability index during final folow-up, the rate of screw malposition, the rate of Cage shift, loss value of intervertebral height and the rate of interbody fusion (P > 0.05). These results indicate that for the single-segment degenerative lumbar disease, the use of minimaly invasive posterior lumbar interbody fusion or open posterior lumbar interbody fusion can obtain satisfactory clinical function, but the minimaly invasive posterior lumbar interbody fusion has the advantages of a less trauma, shorter length of hospital stay and bed stay, and lighter back pain.

BACKGROUND:Studies have shown that chitosan and other natural polysaccharides have heparin-like anticoagulant function after sulfonated modification. Sulfonated chitosan has good anticoagulant property because the sulfonate group formed by sulfonated chitosan is similar with the active group of heparin. OBJECTIVE: To prepare the anticoagulant chitosan nanoparticles and to detect its morphology, physical and chemical properties and biological security. METHODS: Chitosan nanoparticles were synthesized by emulsion-chemical cross link. Sulfonated chitosan nanoparticles were synthesized by sulfonation reaction. Its morphology was described by transmission electron microscope. The peak-value change of its specific groups was observed by infrared spectroscopy. (1) Coagulation experiment: Heparin, chitosan nanoparticles and 10, 30 and 50 mg of sulfonated chitosan nanoparticles were added into the blood of Spraque-Dawley rats. The coagulation indicators were detected. (2) Hemolysis experiment: deionized water, physiological saline and 10, 30, 50 g/L sulfonated chitosan nanoparticles extracts were added into 2% red blood cel suspension of rabbits. The hemolysis rate was detected. (3) Cytotoxicity experiments: DMEM medium containing fetal bovine serum and 10, 30, 50 g/L sulfonated chitosan nanoparticle extracts were used to culture human umbilical vein endothelial cels. Cel relative growth rate and toxicity grading were detected after 72 hours. RESULTS AND CONCLUSION: Scanning electron microscopy showed that sulfonated chitosan nanoparticles had good morphology, with a diameter of 50 nm. Infrared spectroscopy showed that the sulfonated replacement occurred.In vitro coagulation experiments showed that sulfonated chitosan nanoparticles had significant anticoagulant effects in a dose-dependent manner. Sulfonated chitosan nanoparticles meet the national safety standard for hemolysis rate of less than 5%, non-induced hemolysis property. Cytotoxicity assays showed that sulfonated chitosan nanoparticles extracts had no significant cytotoxicity, and its biological safety was in line with the national standards.

Objective To investigate the relationship between the coagulation system status and the pulmonary hemorrhage in children with severe hand,foot and mouth disease(HFMD)and approach the clinical significance of early detection of coagulation function. Methods By prospective case design method,89 cases with HFMD admitted to Department of Critical Care Medicine of Hebei Provincial Children Hospital from July 2010 to July 2012 were enrolled. The children were divided into severe group(46 cases)and critical group(43 cases)according to the severity of disease,and the children in critical group were subdivided into survivor group(26 cases)and non-survivor group (17 cases). Forty-four healthy children with the same age and in the same period were served as healthy control group. The blood of children was collected immediately after admission for determination of blood routine, prothrombin time(PT),thrombin time(TT),activated partial thrombin time(APTT),fibrinogen(Fg),and D-dimer (DD). Results There were no significant differences in PT,TT,APTT and Fg among severe group,critical group and health control group(all P>0.05). The blood platelets count(PLT)in severe group and critical group was significantly lower than that in health control group(×109/L:245±130,237±156 vs. 389±120),while the DD was significantly higher than that in healthy control group(mg/L:0.34±0.67,0.41±0.08 vs. 0.24±0.13),and the DD in critical group was obviously higher than that in severe group(all P<0.05). The mortality rate in critical group was 39.5%,and there were no significant differences in PT,APTT,Fg,TT and PLT between survivor group and non-survivor group(all P>0.05),but the DD in non-survivor group was significantly lower than that in survivor group(mg/L:0.60±0.09 vs. 0.12±0.09,P<0.05). Conclusions In children with severe or critical HFMD, the coagulation factor and blood platelet were in a state of mobilization,mild consumption state with the existence of fibrinolytic inhibition,but without systemic bleeding tendency,therefore it is in a compensatory stage of disseminated intravascular coagulation(DIC),not the mechanism of pulmonary hemorrhage. The monitor of DD has its clinical significance in evaluations of the disease situation and its prognosis.