Construction of granulocyte-macrophage colony stimulating factor gene eukaryotic expressing plasmid and identification of its biological activity / 实用儿科临床杂志

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.

Dynamic variability of intestinal flora and endotoxin in rat with fulminate hepatic failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic variability of intestinal flora and endotoxins in rats with fulminate hepatic failure.</p><p><b>METHODS</b>Establishing the fulminate hepatic failure models by intraperitoneal injection of Galactosamine. Forty Sprague-Dawley rats were divided into three groups: group A (n=10) were killed at the beginning of the experiment as control; while Group B (n=12) and C (n=18), the fulminate hepatic failure models, were killed 24 and 48 hours respectively after successful induction. Then, the contents of the jejunum, ileum and colon descendents were collected and a quantitative analysis was made about intestinal flora. Meanwhile, the concentrations of endotoxin in portal vein and right ventricle were determined and so were those in contents of ileums and colons.</p><p><b>RESULTS</b>Our experiments showed that the livers of rats in group B were injured most seriously among three groups, and a minor recovery of hepatic function was observed in group C with the decrease of total bile acids (P< 0.05). Analysis on intestinal flora show: the intestinal enterobacteriacea increase and the lactobacillus decrease in group B (P< 0.01 in jejunum and ileum and P<0.05 in colon). The comparisons between group C and B showed that the enterobacteriacea in the former decreased in both jejunum and colon (P< 0.05) while the number of lactobacillus recovered in the jejunum of group C (P<0.05). Quantitative analysis on endotoxins showed that the ileum endotoxin increased in group B (P< 0.05) and in group C, endotoxins in ileum and colons also increased (vs. control, P<0.01); portal endotoxin in group B showed higher level than that in group A and C (P< 0.01).</p><p><b>CONCLUSION</b>The alteration of intestinal flora was observed in fulminate hepatic failure rats. Abnormal intestinal flora might lead to incline of endotoxin in ileum, colon and portal vein, while the recovery of normal intestinal flora would decrease the level of portal endotoxin.</p>

Preliminary comparative study on the effects of early enteral supplementation of synbiotics on severely burned patients / 中华烧伤杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Randomized 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Early 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.</p>

Study of severe hepatitis treated with a hybrid artificial liver support system / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To evaluate the efficacy of a hybrid artificial liver support system in the treatment of chronic severe hepatitis.</p><p><b>METHODS</b>The hybrid artificial liver support system (HALSS) consisted of a bioreactor containing more than 5 x 10(9) porcine hepatocytes and plasma exchange device. 15 patients with chronic severe viral hepatitis were treated with the hybrid system.</p><p><b>RESULTS</b>All the patients experienced a reduction in symptoms, such as fatigue, abdominal distention or ascites. After each treatment serum total bilirubin decreased markedly (from 493.5 micromol/L+-139.8 micromol/L to 250.9 micromol/L+-91.3 micromol/L, t=8.695, P<0.001), while prothrombin activity increased (from 24.5%+-8.4% to 30.6%+-6.3%, t=3.325, P<0.01). There were 11 patients whose progress of hepatocytes necrosis stopped after HALSS treatment, and finally they recovered completely. Four patients died of their worsen conditions. No serious adverse events were noted in the 15 patients.</p><p><b>CONCLUSION</b>HALSS is a reliable hepatic support device for chronic severe hepatitis.</p>

Effect of Sodium Butyrate on Erythroid Lineage by Expressional Profiles Microarray in K562 Cells / 实用儿科临床杂志

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

Relationship between Phosphorylation of p38 and Erythroid Differentiation of Human K562 Erythroleukemia Cells / 实用儿科临床杂志

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