OBJECTIVETo study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.

METHODSRT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.

RESULTS1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.

CONCLUSIONSoluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Endothelial Cells ; cytology ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Retroviridae ; genetics ; Sarcoma 180 ; metabolism ; pathology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics ; physiology

OBJECTIVETo report clinical and laboratory features of 4 cases of myeloid neoplasm with t (5;12) (q33;p13).

METHODSCytogenetic examination of bone marrow cells obtained from patients was performed by 24 h culture method. R banding technical was used for karyotype analysis. PDGFRβ gene rearrangement was detected by FISH using dual color break apart PDGFRβ probe. ETV6-PDGFRβ fusion genes were detected by multiple-reverse transcription polymerase chain reaction (RT-PCR). Direct sequencing analysis was performed on the PCR products in case 1. Immunophenotype analysis was carried out by flow cytometry. Four cases were treated with imatinib (IM) and followed up.

RESULTSThe diagnoses included 3 MPN and 1 AML-M2. The t (5;12) (q33;p13) was a primary abnormality in 3 cases of MPN and a secondary abnormality in 1 case of AML-M2. PDGFRβ gene rearrangement and ETV6-PDGFRβ fusion genes were detected by FISH and multiple-RT-PCR in 4 cases, respectively. The immunophenotypical analysis of leukemia cells showed positive for CD13, CD33 and CD34. Two cases obtained MMR after the treatment of IM, one case complete hematologic and complete cytogenetic response. ETV6-PDGFRβ was negative detected by multiple-RT-PCR after the treatment of IM, but relapsed and died soon in case 4.

CONCLUSIONSThe t (5;12) myeloid neoplasm was a subtype with unique features. The t (5;12) maybe a primary chromosome abnormality in MPN and a secondary in AML. MPN with t (5;12) could benefit from IM, but not for AML. Dual-FISH was a reliable tool for detecting PDGFRβ rearrangement.

Chromosome Banding ; Gene Rearrangement ; Hematologic Neoplasms ; genetics ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Karyotyping ; Myeloproliferative Disorders ; genetics ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-ets ; genetics ; Receptor, Platelet-Derived Growth Factor beta ; genetics ; Remission Induction ; Repressor Proteins ; genetics ; Translocation, Genetic

Objective To investigate the effect of Immutol on inducing the immune tolerance of cardiac grafts in rat models. Methods A rat model of heterotopic abdominal heart transplantation was established. The recipient rats were divided into 5 groups: blank control group (n=6); dimethyl sulfoxide (DMSO) group (n=6), in which DMSO was administered until the cardiac graft arrest; Immutol group (n=6), in which Immutol was administered until the cardiac graft arrest; ciclosporin (CsA) group (n=10), in which CsA was administered for 20 d; combined group (n=13), in which Immutol was given for 60 d combined with CsA for 20 d. The survival time and pathological changes of cardiac grafts in each group were observed. The contents of serum interleukin (IL)-10 and interferon (IFN)-γ were detected. The expression levels of indoleamine 2, 3-dioxygenase (IDO) and fibrinogen-like protein 2(Fgl2) messenger RNA(mRNA) in heart tissues of rats in each group were measured. Results In the combined group, the cardiac grafts survived for >180 d and immune tolerance was induced. The pathological score of cardiac grafts in the combined group was significantly lower than that in the CsA group at postoperative 39 d (P < 0.05). The levels of serum IL-10 and IFN-γ in the combined group were significantly higher than those in the CsA group at 9 d and 39 d after operation (both P < 0.05). The content of serum IL-10 and IFN-γ in the combined group were gradually increased over time. At postoperative 39 d, the expression levels of IDO and Fgl2 mRNA in the combined group were significantly higher than those in the CsA group (both P < 0.05). The expression level of IDO mRNA in the combined group tended to gradually elevate after operation. In the combined group, the expression level of Fgl2 mRNA at postoperative 180 d was significantly higher than those at 9 d and 39 d after operation (both P < 0.05). Conclusions Combined administration of Immutol and CsA can effectively inhibit the incidence of acute rejection, and maintain the long-term survival of the cardiac grafts and induce immune tolerance after drug withdrawal.

Objective To investigate the relationship between immune tolerance and the changes of helper T cell (Th), regulatory T cell (Treg) cytokines, related signaling pathway proteins during immune tolerance process in rat models of liver transplantation. Methods The orthotopic liver transplantation rat models were established by double-cuff technique. All rats were divided into 3 groups. In the operative control group (n=6), sham operation was performed without liver transplantation. In the short-term group (n=10), the rats survived for 10 d after liver transplantation. In the immune tolerance group (n=10), the rats survived for 100 d after operation and the function of the transplanted liver was restored to normal. The expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), Th1 cytokines [interferon (IFN)-γ, interleukin (IL)-2 and tumor necrosis factor (TNF)-α], Th2 cytokines (IL-4, IL-5, IL-6 and IL-13), Th17 cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A], Treg cytokines [IL-10, transforming growth factor (TGF)-β and IL-12p] were quantitatively measured. The serum sample of rats in each group was detected by protein chip analysis. Results Compared with the operative control group, the AST level in the short-term group was significantly down-regulated, whereas the ALT level was significantly up-regulated (both P < 0.05). However, the AST and ALT levels did not significantly differ between the immune tolerance group and operative control group (both P > 0.05). In the liver tissues of rats in each group, the expression levels of Th1 cytokines IFN-γ and IL-2 in the short-term group were significantly higher than those in the operative control group (both P < 0.05). The expression level of Th2 cytokine IL-4 in the immune tolerance group was significantly lower than that in the operative control group (P < 0.05). The expression levels of Th2 cytokines IL-5, IL-6 and IL-13 in the short-term group were significantly lower than those in the operative control group (all P < 0.05). The expression level of IL-17A in the immune tolerance group was significantly higher than that in the operative control group (P < 0.05). In the immune tolerance group, the expression levels of IL-10and IL-12p were significantly higher than those in the operative control group (both P < 0.05). The expression level of TGF-β in the short-term group was significantly higher than that in the operative control group (P < 0.05). Compared with the operative control group, the expression levels of intercellular adhesion molecule (ICAM)-1, pro-platelet basic protein (Ppbp), Neuropilin-2, Notch-2 protein in the short-term group were significantly up-regulated (all P < 0.05). The expression levels of CXC chemokine ligand 17 (CXCL17), ICAM-1 and Neuroleptin-2 protein were markedly up-regulated (all P < 0.05), whereas that of B7-1 protein was significantly down-regulated (P < 0.05) in the immune tolerance group. Conclusions Treg cytokines (IL-10, TGF-β and IL-12p), IL-6, IL-17 and trans-membrane signaling pathway molecules (ICAM-1, Neuropilin-2, B7-1 proteins) play a pivotal role in the natural immune tolerance process of rat models of liver transplantation.

Objective:We proposed a Mingdao immune score system(MISS)to evaluate recipient's immune status after liver transplantation.Methods:From January 2017 to June 2019, retrospective analysis was conducted for 89 recipients of liver transplantation. Age/gender-matched 385 healthy controls(HC)were selected. The percentages of 30 lymphocyte subgroups of patients and HC were measured by flow cytometry. The score of each individual was calculated with our proposed MISS method. And drug concentrations and relevant clinical data were collected.Results:The normal MISS value of a healthy person was 0 score according to our criterion. In this study, the value of MISS for HC was distributed in a nearly normal fashion(-0.73±4.02). When the data from patients at different timepoints were compared, the MISS value started with -1.21±7.42 pre-operation, then declined sharply down to -8.95±8.05 at 1 month and jumped to -4.50±7.80 at 3 months. Afterward it stabilized at -4.18±7.83 between 3~12 months post-operation and finally reached -2.00±5.51 at 1 year ( P<0.05). Patients with acute rejection had higher MISS values than those without acute rejection, ( P<0.05). No significant correlation existed between blood drug concentrations and MISS values ( P>0.05). Conclusions:Our proposed MISS method may reflect the whole immune status. It is useful to manage the application of immunosuppressants in conjunctions with blood drug concentrations and liver graft function.

To analyze the genetic characterization of epidemic rubella virus strains isolated in Liaoning from 2007-2012, a total of 145 rubella virus strains were isolated using Vero/Slam cell line from the patients' throat swabs during rubella outbreaks and sporadics cases in Liaoning Province from 2007 to 2012. Fragments of 945 nucleotides containing 1E gene from 145 rubella virus isolates were amplified by RT-PCR, the PCR products were sequenced and analyzed. Based on the 739 nucleotides of 1E gene, the phylogenetic trees were constructed with 32 WHO rubella reference strains of 13 genotypes downloaded from GenBank and 145 rubella virus strains. The results showed that the 145 rubella virus strains in 2007 -2012 belonged to genotype 1E, nucleotide acids and amino acids similarities were 97.2%-100.0% and 97.6%-100.0%, respectively. Compared to the 1E reference strains(Rvi/ Dezhou.CHN/02, RVi/MYS/01), the nucleotide acids and amino acids similarities were 96.6%-99.2% and 98.2%-100.0%, respectively except for one amino acid change (Val246-Ala246) of RVi/Shenyang. Liaoning. CHN/13.11/13, and Asp262-Asn262 of RVi/Shenyang. Liaoning. CHN/13.11/4 and RVi/Liaoyang. Liaoning. CHN/26. 11/2. there had no change found in the important antigenic epitope sites, the hemagglutination inhibition and neutralization epitopes of the other rubella viruses. All the 145 strains isolated had the same amino acid change (Leu338--Phe338) in E1 protein. These findings suggested that genotype 1E of rubella virus was the predominant genotype in Liaoning province. the rubella prevailed in recent six years was mainly caused by rubella viruses genotype 1E with multi-transmission routes.
Amino Acid Sequence ; China ; epidemiology ; Epidemics ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Rubella ; epidemiology ; virology ; Rubella virus ; classification ; genetics ; isolation & purification ; Sequence Alignment ; Viral Envelope Proteins ; chemistry ; genetics