Objective To construct mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) gene eukaryotic expressing plasmid pcDNA3-GM-CSF, to transfect the recombinant into erythroleukemia cell line FBL-3, and identify their biological activity.Methods GM-CSF gene eukaryotic expressing plasmid was constructed by subclone and recombinant was transfected into FBL-3 cells by electroporation. After screening by G418 and cloning by limiting dilution,we obtained positive cell clones(FBL-3-GM-CSF). PCR and RT-PCR were used to identify the integration and stable expression of GM-SF gene in FBL-3-GM-CSF cells. The biological activity was confirmed by the hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. Results Mouse GM-CSF cDNA was amplified from the prokaryotic expressing plasmid PET-30a(+)-GM-CSF by PCR firstly and BamH Ⅰ and EcoRⅠrestriction sites were introduced. The inserted fragment was cut by BamH Ⅰ and EcoR Ⅰ digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleared with appropriate endoneucleases and sequenced. The findings showed that the orientation of the insert was correct, while no rearrangement or mutation was found. PCR and RT-PCR assay showed that GM-CSF gene had integrated into FBL-3-GM-CSF cells and stably expressed. The hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay demonstrated that the cultured supernatant of FBL-3-GM-CSF cells of expressing GM-CSF should obviously stimulate proliferation of murine marrow mononuclear cells, and could stimulate hematopoietic progenitor cell colony formation. The number of colony formation was 54.67?4.83. The rate of colony formation was 0.547 %.Conclusions GM-CSF gene eukaryotic expressing plasmid is constructed successfully. A cell clone, which can express stably GM-CSF gene and possess biological activity,is obtained. Our studies have founded the base for the preparation of GM-CSF gene-modified vaccine of tumor cell and the study of feasibility of immune therapy of leukemia.

OBJECTIVETo study pharmacokinetic effect of Aikeqing Granule (AG) by different medication ways on zidovudine (AZT) in highly active antiretroviral therapy ( HAART) of rats.

METHODSTotally 36 rats were administered with corresponding medications by gastrogavage, group I [HAART: AZT 31.5 mg/kg +3TC 31.5 mg/kg + Efavirenz (EFV) 63.0 mg/kg], group II (HAART+AG525 mg/kg), group III (HAART and AG 525 mg/kg after a 2-h interval). Drug concentrations of AZT were determined by high performance liquid chromatography-mass spectroscopy (HPLC-MS) before HAART, and at 0.5, 1, 2, 3, 4, 6, 8, 10, 12 h after HAART, respectively. Pharmacokinetic parameters [such as t1/2, Tmax, Cmax, AUCo-t, plasma clearance rate (CL)] were calculated by DAS2.0 Software.

RESULTSThe-equation of linear regression of AZT was good, with the precision, coefficient of recovery, and stability definitely confirmed. AUC in group II and III was larger than that of group I. There was no statistical difference in t1/2, Tmax, Cmax, AUC0-12 h, or AUC0-∞ among groups (P > 0.05).

CONCLUSIONAG combined HAART could enhance the Cmax of AZT.

Animals ; Antiretroviral Therapy, Highly Active ; Benzoxazines ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; pharmacokinetics ; pharmacology ; Mass Spectrometry ; Rats ; Zidovudine ; pharmacokinetics ; pharmacology

Objective An experiment study on the femoral head plastic operation to prevent and treat aseptic necrosis of the femoral head. Methods 60 Chinese white rabbits were equally random divided into 4 groups. The four groups except A group were made a globose defect and filled with 95% alcohol tampon for 30 minutes. The B group was natural repair group. The C group was filled with bone cement after being made defect. The D group was filled with bone cement added barium sulfate agent after being made defect. After 12 weeks, all rabbits in four groups were killed. The specimens were random divided into two parts. The articular cartilage was observed and measured immediately. The other specimen was determined with histological examination and extreme anti-pressure rigidity test . Result The femoral heads of bone cement group and the femoral heads of contrast agent bone cement group kept their outline all the time through the articular cartilage observation. After 12 weeks the cartilage thickness of natural repair group (511.74 ± 69.00) was thinner than the other three groups [ control group ( A group ) (511.74 ± 69 00 );Bone cement group ( C group) (468. 36 ± 82. 99 ); Bone cement group contrast agent ( D group ) ( 515.61 ±64. 65 ) ], and it had significant difference ( P < 0. 05, P < 0. 01 ). The rigidity of natural repair group [ (676. 57 ±50. 92) N/mm] had significant difference with that of the other three groups[ A group( 884. 66±52. 29)N/mm;C group(1178.40 ± 170.45) N/mm;D group(928.60 ± 104.42) N/mm] ( P <0.05, P<0. 01 ). Conclusions Femoral head plastic operation was simple and it could release pain. And femoral head outline could be kept integrity. It would be a new operation which was used to treat young patients in Ficat Ⅱ～Ⅲ period to postpone or substitute total hip replacement.

Objective To investigate the role of directly constitutive activation of p38 mitogen-activated protein kinases(p38MAPKs)signaling in ?-globin gene expression and fetal hemoglobin(HbF)induction,and provide direct data for the relationship between phosphorylation of p38 and erythroid differentiation of human K562 erythroleukemia cells.Methods The human K562 erythroleukemia cells were transfected with pCDNA 3.1-MKK3(Glu)and pCDNA 3.1-MKK3(Ala)recombinant plasmids by lipofectamineTM 2000.Then,the stable cell lines overexpressing constitutively active p38 and constitutively inhibitive p38 activation were established by the addition of G418 to select single cell G418-resistant clones and identification with reverse transcriptase-polymerase chain reaction(RT-RCR)and Western blot assays,named K562-MKK3(Glu)and K562-MKK3(Ala)cells,respectively.Furthermore,the direct effects of constitutively active p38 on the ?-globin gene expression and HbF induction were analyzed by RT-PCR and benzidine staining,respectively.Results The results of RT-PCR and Western blot showed that there were no evident changes in the mRNA and protein levels of p38 for various cell models,but compared with K562,K562-vect,and K562-MKK3(Ala)cells,the phosphorylation of p38 and expression of ?-globin levels in K562-MKK3(Glu)cells were significantly up-regulated.The results of benzidine staining displayed that the mean percentages of positive cells stained by benzidine in K562,K562-vect,K562-MKK3(Ala),K562-MKK3(Glu)cells,and K562-MKK3(Glu)cells treated with SB203580 were(3.2?1.4)%,(3.7?1.2)%,(2.8?0.9)%,(32.6?5.3)%,and(7.8? 2.3)%(q = 7.56 P

1)which represented 340 genes and 171 down-regulated(SLR

Objective: To learn the concentration of target genes of severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) and the influencing factors in the confirmed cases with coronavirus disease 2019 ( COVID-19 ) in Ningbo. Methods: Demographic information and clinical diagnosis of COVID-19 cases reported in Ningbo from January 21 to February 20, 2020 were collected through China Disease Control and Prevention Information System. The Ct values of ORF1ab and N of the cases were collected through Ningbo Center for Disease Control and Prevention and designated hospitals, and analyzed in terms of gender, age, severity, clinical classification, source of case, sampling time and specimen type. Results: There were 157 confirmed COVID-19 cases, including 56 males ( 35.67% ) and 101 females ( 64.33% ). Sixty-seven cases ( 42.68% ) aged between 40 and 60 years old. The Ct values of ORF1ab and N in females were 29.96±5.28 and 30.38±4.90, which were higher than 27.56±4.94 and 28.03±4.88 in males ( P<0.05 ). The Ct values of ORF1ab and N were the lowest when sampled at 1-7 days after onset ( P<0.05 ), which were 27.84±4.80 and 28.35±4.65. The Ct values of ORF1ab ( rs=0.288, P=0.001 ) and N ( rs=0.296, P=0.001 ) were positively correlated with sampling time. The Ct values of ORF1ab in pharyngeal swab and sputum were 29.19±4.85 and 28.74±6.40, the Ct values of N in pharyngeal swab and sputum were 29.61±4.60 and 29.22±6.10, both without significant differences between different samples ( P>0.05 ). Conclusions Sampling time has a great influence on the Ct values of ORF1ab and N of SARS-CoV-2.

Objective: To investigate the status of electronic cigarette use among adolescents in Ningxia Hui Autonomous Region, and to provide evidence for tobacco control in adolescents. Methods: Based on the 2019 National Youth Tobacco Epidemic Monitoring Program, multistage proportional sampling method was used to select middle school students from Ningxia Hui Autonomous Region. A questionnaire revised by Chinese CDC was used to collect the general information, the cognition and use of electronic cigarettes, and the access to advertising of electronic cigarettes and related products. Results: Totally 9 019 questionnaires were distributed, 8 401 valid ones were recovered, and the response rate was 93.2%. The rates of electronic cigarette use and attempt among students were 4.3% and 13.4%. The rates of electronic cigarette use and attempt in male students were 7.7% and 22.9%, which were higher than that in female students (0.8% and 3.8%, P<0.05) . The rates of electronic cigarette use and attempt varied in different schools ( P<0.05 ), which were higher in vocational high school students ( 11.5% and 26.8% ). Among 246 students who used electronic cigarettes, 30.1% did not thought electronic cigarettes contained nicotine, while 60.2% did not know whether electronic cigarettes contain nicotine. In the past 30 days, 27.0% of the students had seen the advertisements of electronic cigarettes and related products, mainly through TV, store, supermarket, convenience store, grocery store, electronic cigarette experience store or retail store. Conclusions The rates of electronic cigarette use and attempt among adolescents in Ningxia Hui Autonomous Region are 4.3% and 13.4%. Boys and vocational high school students have higher rates. Students generally know electronic cigarette and have more access to it.

OBJECTIVEThe objective of this article is to summarize the development of evaluation and treatment of posterior malleolus fracture (PMF).

DATA SOURCESData used in this review were mainly from English literature of PubMed data base. Study selection Articles were included in this review if they were related to the PMF or trimalleolar fracture.

RESULTSNo consensus was found regarding what sizes of posterior malleolus fragments would lead to ankle instability thus affecting prognosis and should be fixed. X-ray measurement is unreliable, while CT scan is widely recommended and it can recognize the occult posterior malleolus fractures associated with tibia shaft fractures, which are always undetected previously. Direct posterior malleolus fixation is suitable to stabilize syndesmotic injury. The basic and clinical researches support direct reduction and buttress plate fixation of posterior malleolus fracture through the posterolateral approach. Operative indications and timing of weight bearing are still in discussion.

CONCLUSIONSKnowing whether ankle instability occurs and the proper methods to diagnose, evaluate, and operate can help manage the fracture. Further biomechanical research on ankle stability and clinical study to compare various treatment methods are required.

Ankle Injuries ; surgery ; Fractures, Bone ; surgery ; Humans ; Orthopedic Procedures ; methods ; Tibial Fractures ; surgery

OBJECTIVETo investigate the influence of early enteral nutrition with synbiotics on the plasma endotoxin level, the nutritional state, the inflammatory response and the incidence of infectious complications in severely burned patients.

METHODSRandomized double blind and control method was employed im the study. Forty severely burned patients were randomly divided into A and B groups with 20 in each group. The patients in group A received early enteral nutrition with synbiotics including four kinds of lactic acid bacteria and four kinds of fibers, while those in group B received early enteral nutrition with synbiotics including only four kinds of fibers. The patients with 80% to 280% coefficient unit burned surface(UBS) were further divided into A1 (n = 10) and B1 (n = 11) groups. The plasma endotoxin level in group A and B was determined dynamically on the 1st, 3rd, 7th, 10th, 14th, and 21st postburn days (PBD), and its abnormal rate in both groups was statistically analyzed in correlation with the normal endotoxin level. meanwhile, the mortality, the incidence of infectious complications and the blood bacterial culture results were compared between the two groups. The plasma levels of IL-1, IL-6 and prognostic inflammatory nutrition index (PINI) were also determined at the above time points.

RESULTSThe plasma endotoxin level in group A (37.9 +/- 5.4) ng/L was evidently lower than that in group B (59.1 +/- 7.9) ng/L (P < 0.05) on 10 PBD. The abnormal rate of plasma endotoxin in group A (36.7%) was evidently lower than that (49.2%) in group B (P < 0.05). Blood culture was positive in 3 patients in group A, and 5 in group B. There was no obvious difference in the incidence of infectious complication between the two groups. Two patients died in group A and 1 group B. There was no obvious difference in plasma IL-1 level between A1 and B1 groups at different time points. The plasma IL-6 level in A1 group in 10th and 14th PBD was evidently lower than that in B1 group (P < 0.05). The PINI in A1 group on the 10 PBD was remarkably lower than that in B1 group.

CONCLUSIONEarly enteral nutrition with synbiotics was helpful in decreasing inflammatory stress response and lowering the plasma endotoxin level. Enteral supplementation of synbiotics might be beneficial to the controlling of burn infection.

Adult ; Burns ; therapy ; Dietary Fiber ; administration & dosage ; therapeutic use ; Double-Blind Method ; Endotoxins ; blood ; Enteral Nutrition ; methods ; Female ; Humans ; Intestinal Mucosa ; drug effects ; Male ; Probiotics ; administration & dosage ; therapeutic use

OBJECTIVETo study long-term behavioral and ultrastructural alterations in a hypoxic-ischemic brain damage (HIBD) model of neonatal rats.

METHODSSixty seven-day-old Sprague-Dawley rats were randomly subjected to unilateral carotid artery ligation followed by hypoxic exposure (HIBD group) or sham operation (n=30 each). A battery of behavioral tests, including Morris water maze test and sensorimotor tests, were performed at a postnatal age of 5 weeks. Nissl staining was used for counting neurons. Transmission electron microscopy was used for observing synapse structures and measuring the thickness of the postsynaptic density area and the length of the postsynaptic active area. The correlations of histological changes with the results of behavioral tests were evaluated.

RESULTSThe HIBD group showed a significantly longer escape latency (P<0.05) and a lower frequency of original platform crossing (P<0.05) in the Morris water maze test compared with the sham operation group. The sensorimotor function test showed that the sensorimotor function in the HIBD group was worse than in the sham operation group. Nissl staining showed that the number of neurons in the HIBD group was significantly reduced (P<0.01) compared with the sham operation group. Transmission electron microscopy showed that synapses were significantly reduced in number, and that the thickness of the postsynaptic density area and the length of the postsynaptic active area were reduced in the HIBD group. The thickness of the postsynaptic density area was negatively correlated with escape latency in the Morris water maze test (r=-0.861, P<0.01), and also negatively correlated with the total score of sensorimotor function tests (r=-0.758, P<0.05) in the HIBD group.

CONCLUSIONSHypoxia ischemia can lead to neuron loss and ultrastructure damage, resulting in long-term deficit of behavioral functions in neonatal rats.

Animals ; Animals, Newborn ; Brain ; pathology ; ultrastructure ; Female ; Hypoxia-Ischemia, Brain ; pathology ; psychology ; Male ; Maze Learning ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Reaction Time