BACKGROUND: Uncontrolled-rate freezing in -80 ℃ refrigerator is convenient, while controlled-rate freezing in -196 ℃ liquid nitrogen is reliable and long-term, the combination of the two can simplify the process and has been successfully used in clinics. OBJECTIVE: To explore the influences of different cryoprotectants by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen on the cryopreservation of hemopoietic stem cells. METHODS: The experiments were divided into four groups: 10% dimethyl sulfoxide (DMSO) group, 5% DMSO combined with 3% hydroxyethyl starch group, 5% DMSO combined with 0.25 mol/L trehalose group, 5% DMSO combined with 3% hydroxyethyl starch and 0.25 mol/L trehalose group. Peripheral hemopoietic stem cells were cryopreserved by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen. The ultrastructural changes were examined by transmission electron microscopy, the expressions of Annexin-V, PI and Caspase-3 were detected by flow cytometry. RESULTS AND CONCLUSION: There was no significant difference in survival rate, apoptotic rate and necrotic rates of the cryopreserved cells in the four groups (P > 0.05). The ultrastructural changes had no significant difference under the transmission electron microscopy. The viability was more than 90% in frozen-thawed mononuclear cell colonies, and the apoptosis was roughly 50% in the frozen-thawed CD45+ cell population, which contained many mature cells. Of hemopoietic stem cells, early stage cells have greater resistance to damage of cryopreservation than late stage cells. It is concluded that the addition of hydroxyethyl starch or trehalose into DMSO exhibits no synergistic protective effect on the cryopreservation of hemopoietic stem cells.

The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on proliferation of vascular smooth muscle cells (VSMCs) stimulated by advanced glycation end products (AGE) and neointima hyperplasia after rat carotid arterial injury. Rat aortic VSMCs were cultured and treated with rhIL-10 or AGE respectively, and then co-treated with rhIL-10 and AGE. Proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytomertry. Sprague-Dawley rats were treated with recombinant human IL-10 (rhIL-10) for 3 d after carotid arteries injury. The ratio of neointima to media area at the site of arterial injury was measured 28 d after balloon injury. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control, AGE stimulated VSMCs proliferation. rhIL-10 alone had no effect on VSMCs growth. With AGE stimulation, rhIL-10, at dose as low as 10 ng/ml, inhibited VSMCs growth (P<0.05). The cell number in G(0)/G(1) phase of AGE and rhIL-10 co-treatment group was higher than that of AGE treatment alone (P<0.01) by flow cytometry analysis. Compared with the control group of neointima hyperplasia in rats, the ratio of neointima to media area of recombinant human IL-10 group was reduced by 45% (P<0.01). The p44/42 MAPK activity was significantly enhanced by AGE. The AGE effects were opposed by rhIL-10. The anti-inflammatory cytokine rhIL-10 inhibits AGE-induced VSMCs proliferation. Recombinant human IL-10 also inhibited neointima hyperplasia after carotid artery injury in rats. The results suggest the possibility that recombinant human IL-10, as a potential therapeutic approach, prevents neointimal hyperplasia.
Animals ; Aorta, Thoracic ; cytology ; Atherosclerosis ; physiopathology ; Carotid Artery Injuries ; pathology ; physiopathology ; Carotid Intima-Media Thickness ; Cell Proliferation ; drug effects ; Cells, Cultured ; Glycation End Products, Advanced ; antagonists & inhibitors ; pharmacology ; Hyperplasia ; prevention & control ; Interleukin-10 ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; Neointima ; drug therapy ; prevention & control ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Tunica Intima ; pathology

Vessel injury provokes a release in proinflammatory cytokines that influence vascular smooth muscle cell (VSMC) proliferation. The purposes of this study was to determine the effects of recombinant human interleukin-10 (rhIL-10) on rat vascular smooth muscle cell proliferation and the activity of p44/p42 mitogen-activated protein kinase (MAPK) promoted by tumor necrosis factor-alpha (TNF-alpha). Rat aortic VSMCs were cultured and treated with rhIL-10 or TNF-alpha respectively, and then cotreated with rhIL-10 and TNF-alpha. The proliferation of VSMCs was quantified by colormetric assay. Cell cycle analysis was performed by flow cytometry. The p44/42 MAPK activity was evaluated by the immunoblotting technique using anti-p44/42 phospho-MAPK antibody. Compared to control group, TNF-alpha stimulated significantly VSMC proliferation in TNF-alpha group. rhIL-10 alone had no effect on VSMC growth, but significantly inhibited VSMC proliferation induced by TNF-alpha at a dose of 10 ng/ml. The cell number in G(0)/G(1) phase of TNF-alpha and rhIL-10 co-treatment group was higher than that of TNF- alpha group (P<0.01) by flow cytometry analysis. The p44/42 MAPK activity was significantly enhanced by TNF-alpha and the TNF-alpha effect was opposed by rhIL-10. It is suggested that rhIL-10 can inhibit TNF-alpha induced VSMC proliferation and phosphorylation of p44/42 MAPK.
Animals ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Interleukin-10 ; pharmacology ; Male ; Mitogen-Activated Protein Kinases ; biosynthesis ; Muscle, Smooth, Vascular ; cytology ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To observe the effects of aerobic training and aerobic combined with resistance training on motor function, like muscle strength,cardiopulmonary endurance and so on,in patients with chronic kidney disease(CKD). Methods From July,2015 to August,2016,60 patients with CKD were randomly divided into control group(A,n=20), aerobic training group(B,n=20)and aerobic combined with resistance training group(C,n=20).Group B per-formed cycle ergometer at 50% peak oxygen uptake(VO2peak)for 30 minutes a time,and group C performed one section of Thera-Band resistance training based on group B, three times a week for twelve weeks.All patients were evaluated with one repetition maximum-upper limb (1 RM-U), one repetition maximum-lower limb (1 RM-L),Cardiopulmonary Exercise Test(CPET),Arm Curl Test(ACT),30-second Chair Stand(CS-30),Six-Min-ute Walk Test(6MWT),and estimated gomerular filtration rate(eGFR)and serum creatinine(sCr)were calculat-ed and recorded before and after training. Results There was no significant difference in all indexes among three groups before training(F<1.841,P>0.05).After training,all indexes improved in groups B and C(t>2.162,P<0.05),and were better in groups B and C than in group A(t>2.132, P<0.05).After training, 1 RM-U, 1 RM-L, VO2peak,ACT, CS-30 and 6MWT were better in group C than in group B(t>2.081,P<0.05). Conclusion Aerobic training could improve the motor function of patients with CKD,and it is more effective combined with resistance training.

OBJECTIVETo explore a new method for finite element modeling to achieve material property assignment based on in situ CT gray value in simulated osteotomies for deformities.

METHODSA CT scan dataset of the lower limb of a patient with extorsion deformity was obtained for three-dimensional reconstruction using Mimics software and preparing a solid model. In the CAD software, the parameters for osteotomy simulation were defined including the navigation axis, rotation angle and reference plane. The tibia model was imported to the FEA pre-processing software for meshing procedure and then exported to Mimics. All the segments of the tibia meshed model were assigned uneven material properties based on the relationship between CT gray values and material properties in the Mimics software. Finally, all the segments of the tibia model, reference axis and reference plane were assembled in the pre-processing software to form a full finite element model of a corrected tibia, which was submitted to resolver for biomechanical analysis.

RESULTSThe tibia model established using our modeling method had inhomogeneous material properties based on CT gray values, and was available for finite element analysis for the simulation of osteotomy.

CONCLUSIONSThe proposed finite element modeling method, which retains the accuracy of the material property assignment based on CT gray value, can solve the reposition problem commonly seen in modeling via the routine method of property assignment and provides an efficient, flexible and accurate computational biomechanical analysis method for orthopedic surgery.

Finite Element Analysis ; Humans ; Models, Anatomic ; Osteotomy ; Software ; Tibia ; pathology ; Tomography, X-Ray Computed

BACKGROUND:The use of normal hyaline cartilage to repair large areas of full-thickness knee cartilage defect has been a hot topic recently; however, a follow-up study with a relative large number of patients is required. OBJECTIVE:To make a preliminary study concerning the methods and therapeutic effects of tissue-engineered cartilage (TEC) implantation for treating large-area full-thickness knee cartilage defects. METHODS:Twenty-one patients (23 knees) diagnosed with cartilage defect of the knee joint (Outbridge III-IV) were enrolled. The area of the cartilage defect was 3.5-11.2 cm2. All of the patients were given TEC treatment. Postoperative functional exercise of the knee joint was carried out in these patients as planned. We regularly reviewed the knee MRI and calculated visual analog scale score, International Knee Documentation Committee (IKDC) score, and Lysholm score. RESULTS AND CONCLUSION:All the patients were followed up for 3 to 12 months. Postoperatively knee pain relieved obviously, and the visual analog scale score was significantly declined compared with the preoperation (P<0.05). All the patients manifested painless 1 year after surgery. The 1-year postoperative MRI showed that the injured cartilage grew well. The thickness and MRI signal of the graft was the same as the normal cartilage, and the bone healed completely. The IKDC and Lysholm scores were significantly improved at 3, 6, 12 months after the surgery, and the difference was statistically significant before and after the surgery (P<0.05). Overall, TEC is an improved technique of chondrocyte implantation, which is an effective and safe method for cartilage defect repair.

Objective To investigate isolation of pathogens from bile and clinical characteristics of patients with hepatobiliary diseases.Methods Bacterial culture result of bile and related clinical data of patients with hepatobiliary diseases in a hospital were collected and analyzed by retrospective survey.Results A total of 406 bile specimens from patients with hepatobiliary diseases were collected,the positive rate of culture was 64.53％.Of 262 positive specimens,62.21％ (n =163),32.83％ (n =86),and 4.96％ (n =13) were isolated single pathogen,2 kinds of pathogens,and 3 kinds of pathogens respectively.374 pathogenic strains were isolated,242 (64.71％),131 (35.02 ％),and 1 (0.27 ％) were gram-negative bacteria,gram-positive bacteria,and fungus respectively.Patients with cirrhosis of liver,history of hepatobiliary surgery,and cholelithiasis had higher isolation rates of pathogens from bile than control group(all P＜0.05),isolation rates of pathogens from bile in patients with cholelithiasis of different sites were varied;but there was no significant differences among patients of different age,gender,and whether or not with hepatobiliary tumors(all P＞0.05).There were no statistical difference in constitute of pathogenic species from bile between patients with and without gallstones,as well as with and without history of hepatobiliary surgery(both P＞0.05);while constitute of pathogenic species from bile between patients with and without cirrhosis of liver was statistically different(x2 =14.058,P =0.001).Conclusion Pathogens from bile in patients with hepatobiliary diseases are mainly Enterobacteriaceae and Enterococcus spp.which caused single infection.Positive culture rate of pathogens from bile is higher in patients with cholelithiasis,history of hepatobiliary surgery,and liver cirrhosis.

OBJECTIVETo investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.

METHODSPrimers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.

RESULTSThe enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.

CONCLUSIONNeither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.

Blotting, Western ; Cell Line ; Humans ; Mitochondria ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; SUMO-1 Protein ; metabolism ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism

Deep vein thrombosis(DVT)is a common complication after surgery for lower limb fracture.It has the features of high morbidity,high disability rate and high mortality.At present,the measures for clinical prevention and treatment of post-operative DVT in lower limb fracture mainly include perioperative nursing,intervention with medical auxiliary instruments,western medicine prevention and treatment,traditional Chinese medicine(TCM)intervention,and patients'self-cooperation.The patients'self-cooperation is the basis for the smooth implementation of other measures for prevention and treatment,and the patients'active cooperation is the premise of achieving the efficacy of prevention and treatment.Perioperative nursing is helpful for the patients to understand the risk factors of postoperative DVT and the possible risks after the occurrence of DVT,guides the patients to choose the food,assists the patients to do postoperative exercises,improves the level of patients'hemorheological indexes,and reduce the incidence of postoperative DVT.Medical devices are helpful for assisting patients to do postoperative rehabilitation exercises,improving the levels of hemodynamic indicators,promoting patients'rehabilitation and reducing the incidence of postoperative DVT.Western medicines such as low molecular weight heparin,Rivaroxaban,Enoxaparin and other anticoagulant drugs can reduce the aggregation of coagulation factors and blood viscosity,and reduce the incidence of postoperative DVT.TCM interventions mainly include oral administration of Chinese medicine and external treatment such as acupuncture,moxibustion and massage.Oral administration of Chinese medicine is helpful for improving blood flow status.Acupuncture,moxibustion and massage are beneficial to the activation of the function of zang-fu organs,and can stimulate the healthy qi to improve the qi-blood state of the whole body.Each method of prevention and treatment has its advantages and disadvantages.In clinical application,reasonable prevention and treatment methods should be selected according to the specific conditions and individual conditions of the patients.TCM intervention of DVT can be performed in patients with lower limb fracture before and after surgery,and has the advantages of low cost and definite efficacy,which is worthy of continuous research and inheritance and innovation.

Objective To explore the antimicrobial resistance of carbapenem-resistant Klebsiella pneumoniae(CRKP)isolated from blood and the related risk factors for infection in patients.Methods Clinical data of 383 KP-infected patients from whose blood Klebsiella pneumoniae(KP)were isolated during hospitalization period in a hos-pital from January 2018 to December 2021 were retrospectively analyzed.Patients were divided into CRKP group(n=114)and non-CRKP group(n=269)based on antimicrobial resistance.According to the prognosis,114 patients in the CRKP group were subdivided into the death group(n=30)and the survival group(n=84).General informa-tion,underlying diseases,antimicrobial use,and infection outcomes of two groups of patients were compared,and risk factors for infection and death after infection were analyzed.Results The resistance rates of KP to tigecycline and compound sulfamethoxazole showed upward trends,with statistically significant differences(both P=0.008).The CRKP group had higher resistance rates to amikacin,aztreonam,compound sulfamethoxazole,ciprofloxacin,cefepime,cefoperazone/sulbactam,piperacillin/tazobactam,tigecycline,ceftazidime,tobramycin,and levofloxacin,as well as higher in-hospital mortality than the non-CRKP group,with statistically significant differences(all P＜0.05).Acute pancreatitis prior to infection(OR=16.564,P＜0.001),hypoalbuminemia(OR=8.588,P＜0.001),stay in in-tensive care unit prior to infection(OR=2.733,P=0.017),blood transfusion(OR=3.968,P=0.001),broncho-scopy(OR=5.194,P=0.014),surgery within 30 days prior to infection(OR=2.603,P=0.010),and treatment with carbapenems(OR=2.663,P=0.011)were independent risk factors for the development of CRKP blood-stream infection(BSI).Cardiac insufficiency before infection(OR=11.094,P=0.001),combined with pulmonary infection(OR=20.801,P=0.010),septic shock(OR=9.783,P=0.002),disturbance of consciousness(OR=11.648,P=0.001),and receiving glucocorticoid treatment(OR=5.333,P=0.018)were independent risk factors for mortality in patients with CRKP BSI.Conclusion The resistance rate of KP from BSI to tigecycline and com-pound sulfamethoxazole presents upward trend.Underlying diseases,invasive procedures,and carbapenem treat-ment are closely related to CRKP BSI.Cardiac insufficiency,pulmonary infection,septic shock,disturbance of con-sciousness,and glucocorticoid treatment can lead to death of patients with CRKP BSI.