Objective To explore a separation and culture method of human adipose-derived stem cells(ADSCs) suitable for the clinical application .Methods The non-enzymatic method and the collagenase digestion method were adopted to isolate and culture the cells from the human adipose tissue in the individuals with liposuction .The characteristics of isolated mesenchymal stem cells were comparatively analyzed .Results The required time in the non-enzymatic method was one third of that in the collagenase di-gestion method and the cellular morphology ,reproductive capacity ,immunophenotype and differentiation potential of the isolated cells were consistent to those isolated by the collagenase digestion method .Conclusion The no-enzymatic method may isolate and culture ADSCs from the adipose tissue in the individual with liposuction ,which is a safety and reliable isolation and culture method of human adipose tissue-derived stem cells suitable for clinical application .

Objective To introduce a new intelligent stomach syringe based on PC,which can deal with the stop of waste pipe automatically and feed back to the control of water speed.Methods On the foundation of analyzing the defects of traditional stomach syringe,the electric circuit of the design was improved.It realized the judgment,analysis and handling of acquired signals in the PC board.Results This machine can display the numerical value of pressure,PH,flux,and give the sound or light alarm.It can work automatically according to the demand or be operated artificially.Conclusion The instrument is automatic,secure and intelligent.Furthermore,it increases the successful rate for rescuing the patients.

Objective To determine the affinities of five oligopeptides specifically binding with DNA binding domain of NF-?B p65 subunit and identify their inhibiting effect on DNA binding activity of NF-?B. Methods By using biosensor the affinities were measured by means of kinetic analysis, and the inhibiting effects were determined by competitive ELISA. Results The results of biosensor showed that all of five oligopeptides really possessed the capability of specific interacting with NF-?B p65 subunit. The affinity constants of these oligopeptides were 2.67?10~ -7 mol/L, 9.02?10~ -6 mol/L, 1.07?10~ -6 mol/L, 8.03?10~ -6 mol/L, 9.83?10~ -7 mol/L respectively. The results of competitive ELISA indicated that five oligopeptides could inhibit NF-?B from binding with ?B motif, and their inhibiting effect depended on their concentration. Conclusion Five oligopeptides that were screened by yeast two-hybrid system method can really interact with p65, and possess the inhibiting effect on DNA binding activity of NF-?B. So it will be possible that these oligopeptides are regarded as model to design and develop novel anti-inflammatory peptide drug targeting NF-?B.

Objective To study the therapeutic effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on acute liver failure ( ALF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS) in rats. Methods The ALF model was obtained through intraperitoneal injection of D-gal(300 mg/kg)and LPS (20μg/kg)in Wister rats. The hUCMSCs were transplanted after intoxication. All rats were divided into four groups, and each group received either hUCMSCs or 0.9% NaCl solution through intraperitoneal or tail-intravenous injection. To evaluate the liver function of each group, the levels of alanine aminotransferase (ALT), total bilirubin (TBil) and serum albumin (Alb) were measured on the day of hUCMSCs transplantation and the following 1, 2, 3, 5 and 7 days. All rats were then sacrificed to examine the liver histology at day 7. Analyses were done by using Fisher's exact test, unpaired t test and Mann-Whitney U test. Results There were no significant differences of survival rates among four groups (Fisher's exact test, both P = 1. 00). The levels of ALT, TBil and Alb in group receiving hUCMSCs intraperitoneally were (804. 9 ± 88. 0) U/L,(17. 4±2. 7) μmol/L and (20. 9±0. 8) g/L, respectively after 2 days of injection, whereas in the corresponding control group, those were (1294. 3± 171. 4) U/L, (32. 3±5. 5) μmol/L and (16. 1±0. 9) g/L, respectively, which indicated that hUCMSCs transplantation significantly improved the liver function (t = 2. 640, P =0.020;t=2.529, P = 0. 025;t= - 3. 833, P = 0. 002). Both of hUCMSCs-transplanted groups showed no significant differences. Liver histological data showed that transplantation of hUSMSCs through either intraperitoneal or tail-intravenous injection alleviated liver damage (U=4. 500, P = 0. 005;U=4. 500, P = 0. 008) and the mitotic index also increased in hUCMSCs-transplanted groups (U=4. 000, P = 0. 005; U=5. 500, P = 0. 013). Conclusions The levels of ALT, TBil and Alb can rapidly normalize in ALF rats after injected with hUCMSCs either intraperitoneally or tail-intravenously. hUCMSCs application raises the mitotic index, enhances hepatocellular regeneration and improves histological status.

OBJECTIVETo detect the expression of cell cycle positive regulators cyclin D(1), cyclin E, CDK(2), CDK(4) and negative regulators p21(cip1), p27(kip1), p16(ink4a) and p15(ink4b) during wound healing in rats.

METHODSOpen wounds of full-thickness skin, diameter 1.8 cm, on rat backs were used as the wound model. Wound tissues were harvested on postwounding days 3, 5, 7, 9, 11, 14, 21 and 30. Ki67 expression in granulation tissue was detected by immunohistochemical assay. The patterns of the expression of cyclin D(1), cyclin E, CDK(2), CDK(4) and p21(cip1), p27(kip1), p16(ink4a), p15(ink4b) were detected by Western blot.

RESULTSCell proliferation in granulation tissue took place predominantly within the first week after injury, with the proliferation peak occurring at postwounding day 5. There were no dramatic variations in the expression of cyclin D(1), CDK(2) and CDK(4) during wound healing. Up-regulated cyclin E was maintained from day 3 to 11 after injury, and then was down-regulated. No expression of p16(ink4a) and p15(ink4b) was found. p21(cip1) was expressed only from day 7 to 14, with peak expression observed on day 9. Constitutive p27(kip1) was expressed throughout wound healing with low levels in the proliferating period of day 3 to 5 and with increased levels in the post-mitotic and remodeling stage. The expression of p21(cip1) and p27(kip1) showed an inverse gradient to that of Ki67.

CONCLUSIONp21(cip1) and p27(kip1) play a supervising role in preventing the hyperproliferative tendency in tissue repair.

Animals ; Cell Cycle ; physiology ; Cell Cycle Proteins ; biosynthesis ; physiology ; Cell Division ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; biosynthesis ; Cyclins ; biosynthesis ; Male ; Rats ; Rats, Wistar ; Skin ; cytology ; metabolism ; Tumor Suppressor Proteins ; biosynthesis ; physiology ; Wound Healing

Objective:To explore the role and potential mechanism of middle layer adhesion γ2 (LAMC2) in the regulation of macrophage polarization in diabetic nephropathy (DKD).Methods:Using GEO database, LAMC2 was identified as the main research object. Mouse mononuclear macrophages RAW264.7 were cultured in vitro and divided into the following 6 groups: Control group (blank control group), HG group (high glucose group), Treated with 30 mmol/L glucose, si-NC group (treated with 30 mmol/L glucose and transfected with negative control), si-LAMC2 group -1# (treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 1), si-LAMC2 group -2# (treated with 30 mmol/L glucose and transfected with SHRnA-LAMC2 construct 1), and SI-LAMC2 group-2# (treated with 30 mmol/L glucose) Group 3# of si-LAMC2 was treated with mmol/L glucose and transfected with shRNA-LAMC2 construct 2, and Group 3# of Si-LAMC2 was treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 3. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) and Western Blot assay were used to detect LAMC2 expression in macrophages, small interfering RNA (siRNA) technique was used to inhibit LAMC2 expression, cell proliferation was evaluated by EdU staining, and cell invasion ability was evaluated by Transwell assay. The expressions of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, Tumor necrosis factor-α (TNF-α), Arg-1, CD163 and IL-10 were detected by qRT-PCR, the expressions of CD86 and CD206 were detected by cellular immunofluorescence staining, and the expressions of key proteins in nuclear factor-κB (NF-κB) and extracellular signal regulated kinase (ERK) signaling pathways were detected by western blot assay.Results:Gene analysis in GEO database showed that the expression of LAMC2 in DKD and M1 macrophages was significantly up-regulated (all P<0.01). Compared with the blank Control group, the mRNA and protein expression of LAMC2 in macrophages of HG group were higher (all P<0.01). EdU staining and transwell statistics showed that, the EdU positive rate in HG and si -NC groups was lower than that in the Control group, and the number of invasive cells was significantly higher than that in the Control group (all P<0.05). The EdU positive rate of the si-LAMC2 group was higher than that of the si-NC group, and the number of invaded cells was significantly lower than that of the si-NC group (all P<0.05). Compared with Control group, the mRNA expression levels of M1 macrophage markers iNOS, IL-12 and TNF-α were higher in HG group and si-NC group (all P<0.01). Compared with the si-NC group, the mRNA expression levels of the above three markers in the si-LAMC2 group were lower (all P<0.01). Compared with the Control group, the mRNA expression levels of M2 macrophage markers Arg-1, CD163 and IL-10 in HG and si-NC groups were significantly decreased (all P<0.01). Compared with the si-NC group, the mRNA expression levels of the above three markers in the si-LAMC2 group were higher (all P<0.01). Compared with the Control group, the fluorescence intensity of CD86 in HG and si-NC groups was significantly higher, and the fluorescence intensity of CD206 was lower. Compared with the si-NC group, the fluorescence intensity of CD86 in the si-LAMC2 group was significantly lower, and CD206 were higher. Compared with the Control group, the expression levels of p-ERK1/2 and p-NF-κBP65 in HG and si-NC groups were significantly higher (all P<0.01). Compared with the si-NC group, the expression levels of two proteins in the si-LAMC2 group were significantly lower (all P<0.01). Conclusions:LAMC2 may play important physiological and pathological functions in diabetic nephropathy by promoting the polarization of macrophage M1 and affecting NF-κB and ERK1/2 signaling pathways.

Objective To provide better emergency and patient services in well-equipped comprehensive hospitals, the organization and wisdom therapeutic strategy are of great importance for the recovery of injured patients from the earthquake zone. Method From 12 May 2008, following the 8.0 Magnitude earthquake in Wenchuan county of Sichuan Province, six Chongqing hospitals with third class in grade A were involved in the rescue of the injured patients with great effort. A total of 533 patients were retreated and followed up from quake zone. All the patients were scored with ISS and AIS system. The profiles of the patients examined, operated and clinical infection investigation were documented. Results Of 533 patients, the number of the patients whose ISS is below 16 is 456 (83.6%), the number between 16 and 25 is 65 (12.2%), and the humor above 25 is 12 (2.3%). The patients were classfled based on their fracture parts as follows: head and neck (n = 42), face (n = 7), chest (n = 114), abdominal and cavitas pelvis (n =81), limb and pelvis (n =314), body surface (n =205), with 180 single fracture site, 139 of them being two combined fracture sites, and 114 of them being above three combined fracture sites. Thirty-two of the patients were suffered from amputation. The number of patients suffered from crushing syndrome reached 21, with 281 surgical operations in hospitals. Seventy-nine patients were suffered from infections including 87.3% of pre-hespital infections. The results from bacteria culture and antibiotic susceptibility showed that the infected bacteria mainly involved in Escherichia coli, Staphylococcus anreus, Staphylococcus haemolyticns, Klebsiella pneumoniae, Baumanii, Aerobacter cloacae, Pseudomonas aeruginosa, C type chain coccus, Bacillus aerogenes capsulatus. The antibiotic susceptibility to diverse bacteria has no obvious changes and exists partial overlapping, and infected patients should be given the treatment of cephalosporin, macrolide antibiotic and so on. Conclusions For the emergency conditions after the catastrophe, the comprehensive hospitals must be prepared to meet large quantities of severe trauma and infection therapy. The scientific selection of antibiotics in the combinative therapy is of great importance to the enhancement of early specific treatment, prevention of severe trauma complications and rehabilitation of patients.

OBJECTIVETo observe the relations among expression of interleukin-2 (IL-2) in spleen lymphocytes, DNA binding activity of nuclear factor of activated T cells (NFAT) and expression of the partly family members C-Fos, C-Jun after trauma.

METHODSA murine closed trauma model was used, animals were sacrificed 6, 12 hours and 1, 4, 7, 10, 14 days, respectively after injury. Spleen lymphocytes were isolated from injured mice and stimulated with concanavalin-A. The culture supernatants were harvested and assayed for IL-2 activity. Total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. Nuclear protein was extracted, and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay (EMSA), the expressions of C-Fos, C-Jun protein determined by Western blot analysis.

RESULTSThe expressions of IL-2 activity and IL-2 mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice, and the most obvious decrease appeared on the 4th day after injury. The DNA binding activity of NFAT decreased gradually and reached the minimum that was only 41% of the control on the 4th day after injury, which was closely associated with the decline of IL-2 activity and IL-2 mRNA. An decrease in the expression of C-Fos on the 1st and 4th day after injury, trauma had no significant effect on the C-Jun expression.

CONCLUSIONSThese results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice; and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.

Animals ; Blotting, Western ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Female ; Interleukin-2 ; metabolism ; Lymphocyte Activation ; physiology ; Male ; Mice ; NFATC Transcription Factors ; Nuclear Proteins ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-jun ; metabolism ; Random Allocation ; T-Lymphocytes ; physiology ; Transcription Factors ; metabolism

OBJECTIVETo investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1).

METHODSMice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis.

RESULTSDNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury.

CONCLUSIONDecreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.

Animals ; Cell Nucleus ; chemistry ; DNA ; metabolism ; DNA-Binding Proteins ; metabolism ; Electrophoretic Mobility Shift Assay ; Female ; Interleukin-2 ; analysis ; genetics ; Male ; Mice ; NFATC Transcription Factors ; Nuclear Proteins ; Proto-Oncogene Proteins c-fos ; analysis ; Proto-Oncogene Proteins c-jun ; analysis ; RNA, Messenger ; analysis ; Transcription Factor AP-1 ; metabolism ; Transcription Factors ; metabolism