Objective To explore the biological functions of retinoic acid-inducible gene-I(RIG-I) in vivo through phenotype analysis of RIG-I knockout mice. Methods The gene expression of RIG-Ⅰ in various tissues of mice was examined with Northern blotting and semi-quantitative RT-PCR.The phenotypes observed included body weight measurement,differential count of peripheral blood cells,metabolic parameters measurement and histopathologic examination. ResultsRIG-Ⅰ expressed in various tissues of mice with different levels.No gross developmental abnormalities and expected maturation arrest in granulocytic differentiation were observed in RIG-Ⅰ knockout mice.However,RIG-Ⅰ knockout mice exhibited an unexpected increase in the ratios of neutrophiles to lymphocytes in peripheral blood and increased susceptibility to bacteria infection. Conclusion RIG-Ⅰ may play an important role in immune regulation in mice.

Objective To investigate the effect of 3-oxo-C12-HSL on autophagy in mouse alveolar macrophages MH-S cells. Methods MH-S cells were treated with culture supernatants of the mutant and wild type Pseudomonas aeruginosa(PA) strains of LasI gene(3-oxo-C12-HSL synthetic gene) and chemically synthesized 3-oxo-C12-HSL signaling molecules. GFP puncta was observed by laser confocal fluorescence microscopy and the ratio of LC3Ⅱ/LC3Ⅰ was detected by Western blot to detect the formation of autophagic.Autophagic flux was also detected by mo-nitoring the degradation of p62 and the change of chloroquine to LC3Ⅱ/LC3Ⅰratio. Results The supernatant of the culture medium of the wild type PA strain increased the GFP puncta of the MH-S cells(P<0.05) and the ra-tio of LC3Ⅱ/LC3Ⅰ(P<0.01),The mutant PA strain of LasI gene could not cause the above changes related to autophagy. The chemically synthesized 3-oxo-C12-HSL signal molecules could increase the number of autophagic bodies and the expression of LC3Ⅱ (P<0.01). Autophagic substrate p62 was degraded by 3-oxo-C12-HSL. Chloroquine, a lysosomal inhibitor, enhanced LC3Ⅱaccumulation caused by 3-oxo-C12-HSL (P<0.05,P<0.01).Conclusions 3-oxo-C12-HSL increases the level of autophagy in MH-S cells.

OBJECTIVE: To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells. METHODS: After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays. RESULTS: After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis. CONCLUSION The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.
Animals ; Apoptosis ; physiology ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Mice ; Myoblasts ; cytology ; Myocytes, Cardiac ; cytology ; Oligonucleotides, Antisense ; Phosphoproteins ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVE: To determine the characteristics of a novel gene Mip5 (GenBank accession number AY553870) and its expression under physiological and pathological conditions. METHODS: The characteristics of Mip5 were analyzed by bioinformatic programs including BLAST, spidey, psort, ClustalW and so on. RT-PCR was performed to detect Mip5 expression. RESULTS Bioinformatic analysis showed that Mip5 gene lied in the 13th chromosome and contained 8 exons and 7 introns, its open reading frame contained 909 bp and its protein production was 302 amino acid residues including 6 kelth domains. Under normal conditions, MIP5 expressed abundantly in the heart, brain and kidney, but its expression could not be detected in the liver and muscle. Expression of Mip5 gene was increased significantly after ischemia-reperfusion compared with the sham groups, and reached its peak at 3 h and recovered at 12 h after the reperfusion. Conclusion Mip5 gene is a novel gene containing a putative open reading frame of 302 amino acids residues and may play an important role in rat cardiomyocytes suffering ischemia processing.
Amino Acid Sequence ; Animals ; Base Sequence ; Chromosomes, Human, Pair 13 ; genetics ; DNA, Complementary ; genetics ; Humans ; Male ; Molecular Sequence Data ; Myocardial Ischemia ; genetics ; Myocardial Reperfusion Injury ; genetics ; Open Reading Frames ; genetics ; Rats

OBJECTIVE: To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1. METHODS: A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines. RESULTS: The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts. CONCLUSION The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.
Animals ; Antigens, Polyomavirus Transforming ; pharmacology ; Cell Line ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; Female ; Fibroblasts ; cytology ; Heat Shock Transcription Factors ; Male ; Mice ; Mice, Knockout ; Transcription Factors ; genetics

OBJECTIVE: To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages. METHODS: Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis. RESULTS: After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation. CONCLUSION HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.
Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; pharmacology ; Heat Shock Transcription Factors ; Heat-Shock Response ; Macrophages ; cytology ; Mice ; Rats ; Transcription Factors ; pharmacology ; Transfection

Carcinoma, Non-Small-Cell Lung ; diagnosis ; metabolism ; China ; Consensus ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; diagnosis ; metabolism ; Oncogene Proteins, Fusion ; metabolism ; Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; metabolism

OBJECTIVETo analyze the clinical significance of corrected serum calcium(CSCa) in patients with multiple myeloma.

METHODSThe serum calcium levels of 320 patients with initial multiple myeloma were measured and corrected by serum albumin and its levels measured simultaneously. The differences of serum calcium levels were analyzed before and after the correction by serum albumin.

RESULTSThere was a significant difference between serum calcium and CSCa in MM patients (2.34±0.15 vs 2.6±0.17 mmol/L). The constituent ratio of patients with hypercalcemia was from 11.3% to 23.1% after correction, the MM patients with hypocalcemia was decreased from 42.8% to 7.8% after correction, and the patients with normal calcium level were increased. There was a significant difference between serum calcium level and CSCa in I, II, III stages of MM patients respectively(P<0.05). In the 320 patients, the incidence of anemia was 80%, renal failure was 20.9%, and myeloma bone disease was 68.8%. Calcium concentration in both anemia and renal insufficiency was higher than the normal group, and the difference was more significant after correction. In 220 cases of MM receiving chemotherapy, the median progression-free survival (PFS) was 15 months, and overall survival(OS) time was 20 months. The PFS and OS time of the patients with hypercalcemia were shortened, and the difference was very significant after correction(P<0.01).

CONCLUSIONCorrected serum calcium can more sensitively to reflect the diseases serious extent, thus indicating prognosis has better effect.

OBJECTIVETo investigate the effect of RYBP gene on sensitivity of HL-60 cells to chemotherapy drugs by using RNA interference.

METHODSPlasmid expressing RYBP specific shRNA was constructed and then was used to establish the RYBP knockdown stable HL-60 cell line. Q-PCR and Western blot were used to confirm the efficacy of RYBP gene silencing at mRNA and protein level respectively; then the DNA ladder and Annexin V labeled flow cytometry were used to detect cell apoptosis; CCK-8 was used detect the sensitivity of HL-60 cells to the chemotherapeutic drug cytarabine or daunorubicin.

RESULTSThe lentiviral-RYBP-shRNA vector was succesfully and effectively inhibit the expression of RYBP at mRNA and protein in HL-60 cells. It was found that without chemotherapy drug treatment the apoptosis rate of RYBP shRNA group was lower than that of the empty vector control group (NC group). When treated with cytarabine, the apoptosis rate and inhibitive rate of RYBP shRNA group were lower than those of NC group. Besides, when treated with daunorubicin, the apoptosis rate of RYBP shRNA group was lower than that of NC group, while the inhibitive rate had no significant difference.

CONCLUSIONSRYBP gene silencing can inhibitive the apoptosis of HL-60 cells and significantly reduce the sensitivity to cytarabine, but this gene silencing can't affect the sensitivity to daunorubicin.

Apoptosis ; Genetic Vectors ; HL-60 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering

OBJECTIVETo summarize the clinical characteristics of peripheral blood, immune phenotypes, fusion genes and cytogenetics of patients with t(8;21) acute myeloid leukemia(AML) through the retrospective analysis of 586 patients with t(8;21) AML from 15 blood disease research centers in Northern area of China.

METHODSThe factors affecting prognosis of patients with t(8;21) AML were investigated by using univariate and multivariate COX regression.

RESULTSThe immune type of t(8;21) AML patients was mainly with HLA-DR, CD117, CD34, MPO, CD38, CD13and CD33(>95%), part of them with CD19and CD56; the most common accompanied mutation of t(8;21) AML patients was C-KIT mutation (37.8%); in addition to t(8;21) ectopic, the most common chromosomal abnormality was sex chromosome deletions (38.9%). The univariate analysis revealed a significant survival superiority of OS and PFS in t(8;21) AML patients of WBC≤3.5×10/L without C-KIT mutation, the newly diagnosed ones achieved HSCT(P<0.05), only survival superiority on OS in t(8;21) AML patients with extramedullary infiltration and CD19 positive; the results of multivariate analysis showed a significant survival superiority on OS and PFS in t(8;21) AML patients with WBC≤3.5×10/L(P<0.05).

CONCLUSIONThe clinical features of t(8;21) AML patients in China are similar to those in other countries, WBC≤3.5×10/L is a good prognostic factor while the C-KIT mutation is a poor one in t(8;21) AML patients.