Raw264.7 cells were incubated with receptor activator of NF-kappa B ligand (RANKL) and α-melanocyte stimulating hormone(α-MSH) for6 d.The amount of osteoclast cells were counted by tartrate resistant acid phosphatase staining and the acid phosphatase activity was assayed.The expressions of 5 melanocortin receptors (MCR) in Raw264.7 cells were determined by RT-PCR.The results showed that the number of osteoclasts in RANKL +α-MSH group was significantly increased compared with RANKL group (P ＜ 0.05),but there was no osteoclast formation in α-MSH group.Compared with control group and α-MSH group,the acid phosphatase activities were significantly increased in RANKL group and α-MSH+RANKL group (P＜0.05).All five MCRs were expressed in the Raw264.7 cells shown by RT-PCR.These results suggest that α-MSH may promote osteoclasts formation through RANK signaling pathway.

AIM: To evaluate the efficacy of combined therapy for amblyopia in children by making use of pattern visual evoked potential ( P-VEP) game.
METHODS: This was a prospective case control study. These asthenopic children were divided into two groups. The control group ( 66 eyes of 49 patients ): occlusive therapy with glasses, cover, precision work, red light treatment and so on, later the stereo vision training was added. The experimental group (72 eyes of 52 patients):conventional methods mentioned above with P - VEP games.
RESULTS: The total effective rate and cure rate of experimental group in 6mo were higher than those of control group. The overall effective rate was 94. 4% in the experimental group and 83. 3% in the control group. There was a statistically significant difference between them (P<0. 05).
CONCLUSION: The comprehensive therapy by making use of P-VEP game is an individualized effective new way in treating amblyopia.

Animals ; Ascorbic Acid ; therapeutic use ; Coxsackievirus Infections ; drug therapy ; mortality ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; mortality ; virology ; Superoxide Dismutase ; blood ; therapeutic use ; Survival Rate

OBJECTIVETo observe the effect of Chinese drugs for strengthening Pi, harmonizing Wei, and dispersing blood stasis (CDSPHWDBS) on the expression of gastric mucosal heat shock protein 70 (HSP70) in chronic atrophic gastritis (CAG) patients.

METHODSA total of 100 CAG patients were assigned to the control group and the treatment group by random digit table, 50 in each group. Patients in the control group took Folic Acid Tablet 10 mg each time, 3 times per day. Those in the treatment group took CDSPHWDBS, 100 mL each time, once per day. The treatment course was 6 months for all. Clinical symptoms and signs, endoscopic and histopathological changes were observed before and after treatment in the two groups. The expression of gastric mucosal HSP70 in CAG patients was determined using SP immunohistochemistry. Data were collected by HPIAS-1 000 pathological graphic analysis system, and its expression semi-quantitatively analyzed.

RESULTSThe total effective rate of clinical Chinese medical symptoms and signs was 88. 0% (44/50 cases) in the treatment group and 56. 0% (28/50 cases) in the control group, with significant difference between the two groups (P <0. 01). The improvement rate of endoscopic manifestations such as congestion and edema, erosion, bile regurgitation, pale gastric mucosa, exposed blood vessels, particles proliferation in the treatment group were superior to those in the control group (P <0. 05). The total effective rate of atrophy was 80. 0% (40/50 cases) in the treatment group and 54. 0% (27/50 cases) in the control group, with significant difference between the two groups (P<0. 01). The effective rate of intestinal metaplasia was 75. 0% (12/16 cases) in the treatment group and 33.3% (5/15 cases) in the control group, with significant difference between the two groups (P < 0. 05). The optical density value of gastric mucosal HSP70 was significantly elevated in the two groups after treatment (both P <0. 05). It was higher in the treatment group than in the control group after treatment with significant difference (P <0. 01).

CONCLUSIONCDSPHWDBS had obvious effect in treatment of CAG and could improve pathological changes of precancerous lesions possibly by promoting the expression of gastric mucosal HSP70 in CAG patients.

Drugs, Chinese Herbal ; therapeutic use ; Gastric Mucosa ; metabolism ; Gastritis, Atrophic ; drug therapy ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Immunohistochemistry ; Medicine, East Asian Traditional

Objective To construct the recombinant eukaryotic expression vector pRNAT-U6,1- siEdg4 which curries small interfering RNA(siRNA)of Edg4 and observe the silencing effect of Edg4 gene targeted siRNA in ovarian cancer cell line SKOV3.Methods The Edg4 gene-targeted hairpin siRNA sequence was designed according to the Edg4 sequence in Genbank,and the two complementary oligo nucleotide strands were synthesized and annealed and inserted into the pRNAT-U6.1 plasmid to build a recombinant Edg4 siRNA eukaryotic expression vector,which was sequenced and identified to contain the correct Edg4 siRNA sequence.The human ovarian carcinoma cell lines SKOV3 were transfeeted with the vector using lipofeetamine method.The efficiency of transfecting cells was observed with fluorescent microscope and the mRNA expression level of Edg4 gene was detected by real time quantitative PCR.The LPA levels in cell supernatants were detected using a biochemical method.And the apoptosis of SKOV3 cells induced by the vector was evaluated by flow cytometry.Results The recombinant eukaryotic expression vector was confirmed to contain correct Edg4 siRNA sequence by PCR and sequencing.After transfection large amounts of green fluorescence were seen in plasma and nuclei of SKOV3 cells and the positive cell rates were 64%.The expression level of Edg4 mRNA in transfeeted SKOV3 cell line was significantly decreased (0.05?0.01 vs 0.29?0.04,P

This study was purposed to investigate the sensitivity and specificity of conventional cytogenetics (CC), nested-reverse transcriptase polymerase chain reaction (nested-RT-PCR) and dual-color/dual-fusion fluorescence in situ hybridization (D-FISH) technique in monitoring the tumor load of chronic myeloid leukemia (CML) during treatment with transplantation. CC, nested-RT-PCR and interphase D-FISH were simultaneously carried out to detect the tumor load of 7 CML patients during treatment with non-myeloablative allogeneic stem cell transplantation (allo-NSCT). 40 specimens from 7 CML patients before and after allo-NSCT were analyzed. The results showed that 29 specimens were Ph (+) with different positive ratio and 3 specimens with lower cells were not analyzed by CC. 36 specimens were bcr/abl mRNA (+) by RT-PCR. 4 specimens from case 1 at 12, 18, 26 and 38 months after allo-NSCT were Ph (-) and bcr/abl mRNA (-), 4 Ph (-) bcr/abl (+) specimens containing 2 from case 1 at 9 and 10 months after allo-NSCT, 1 from case 2 at 15 months after allo-NSCT, 1 from case 3 at 12 months after allo-NSCT showed 5.4%, 0%, 16.5% and 1.5% bcr/abl (+) cells by FISH. 3 specimens with lower cells containing 2 from case 5 at 20 and 60 days after allo-NSCT and 1 from case 7 at 40 days after allo-NSCT were analyzed by FISH and showed 55.0%, 27.5% and 73.5% bcr/abl (+) cells. The Ph (-) bcr/abl (-) specimen from case 1 at 12 months post-allo-NSCT showed 0% bcr/abl (+) cells by FISH. It is concluded that CC can be used as a basic tool to monitor the change of tumor load in CML during treatment. When specimen with lower cells can not be analyzed by CC in early period after allo-NSCT, or result of CC can not evaluate precisely dynamic change of tumor load and when tumor load in treated patient are lower to Ph (-) by CC while bcr/abl mRNA (+) by RT-PCR, FISH must be used to detect precisely tumor load and monitor dynamic change of it. More sensitive RT-PCR is used to monitor tumor load when it is lower to bcr/abl (-) by FISH during treatment.
Adult ; Cytogenetic Analysis ; methods ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; therapy ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Stem Cell Transplantation ; Tumor Burden

Baculoviridae ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; chemistry ; classification ; genetics ; metabolism

OBJECTIVETo explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of abnormal numbers of chromosome 8 in patients with hematologic malignancies.

METHODSConventional cytogenetics(CC) and interphase FISH using chromosome 8 centromere specific probe were simultaneously carried out to detect the abnormal numbers of chromosome 8 in eight acute myeloid leukemia cases with CC unveiled abnormal numbers of chromosome 8, ten chronic myeloid leukemia cases in accelerated phase or blast crisis, and three normal individuals.

RESULTSNine cases that displayed trisomy 8 by means of CC were confirmed by FISH. Among them, Case 5 only displayed diploidy 8, trisomy 8 and tetrasomy 8 by CC, at the same time, FISH confirmed the presence of trisomy 8 and tetrasomy 8 and also revealed a low percentage of a pentasomy 8 clone. Case 3 and Case 17 had each only one cell with trisomy 8 by means of CC, and this could not determine whether they had the trisomy 8 clone, yet FISH confirmed the existence of trisomy 8 clone. Case 9 did not display trisomy 8 by CC, but FISH revealed the existence of trisomy 8 clone. In the other cases, the percentages of trisomy 8 cells determined by FISH were close to or significantly lower than those by CC, but for Case 16 where the percentage of trisomy 8 cells by FISH was significantly higher than that by CC.

CONCLUSIONInterphase FISH is a useful method for the detection of abnormal numbers of chromosome 8, especially in the patients with normal or unsure karyotype or with less and bad metaphases. It is an important complement to CC.

Acute Disease ; Adolescent ; Adult ; Chromosome Aberrations ; Chromosomes, Human, Pair 8 ; genetics ; Female ; Hematologic Neoplasms ; diagnosis ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; Leukemia, Myeloid ; diagnosis ; genetics ; Male ; Middle Aged ; Reproducibility of Results

This study was purposed to characterize the first case of acute promyelocitic leukemia (AML-M(3a)) with t(15;17), trisomy 8 and tetrasomy 8, and explore its characteristics of morphology, cytogenetics, molecular biology, immunology and clinical features. Morphological changes of peripheral blood and bone marrow smears were observed under microscope. Chromosome specimen was prepared by 24 h short-term culture of bone marrow cell, RHG-banding technique was used for karyotypic analysis. PML-RARa fusion gene transcript was detected by nested-reverse transcription-polymerase chain reaction (nested RT-PCR). Interphase fluorescence in situ hybridization (FISH) using chromosome 8 centromere specific probe were carried out to detect abnormal numbers of chromosome 8. Immunophenotypic analysis was performed by flow cytometry. The results showed that peripheral blood smear revealed 65% promyelocyte, and bone marrow aspirate was hypercellular with 72.4% promyelocyte, moderately basophilic cytoplasm with numerous azurophilic granules. Karyotype analysis demonstrated 48, XY, +8, +8, t(15;17)(q22;q12) [16]/47, XY, +8, t(15;17)(q22;q12) [3]/46, XY, t(15;17)(q22;q12) [1]. RT-PCR assay revealed PML-RARa fusion gene transcript (+). FISH showed that the percentages of cells exhibiting 1, 2, 3, 4, 5, 6 green fluorescence signals were 0.5, 7, 19, 55, 18 and 0.5, respectively. This confirmed the presence of tetrasomy 8 and trisomy 8 and also revealed a low percentage of a pentasomy 8 clone. Immunophenotypes of the blasts displayed that CD13 (96.2%), CD33 (55.9%), CYMPO (93.5%) were positive. All the lymphoid markers tested were negative. The patient survival time was just 10 days. It is concluded that tetrasomy 8 is secondary cytogenetic event after t(15;17) in this case. It may be a consequence of clonal evolution of trisomy 8. t(15;17) AML-M(3) with tetrasomy 8 heralds a poor prognosis.
Chromosomes, Human, Pair 15 ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 8 ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Promyelocytic, Acute ; genetics ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; Oncogene Proteins, Fusion ; genetics ; RNA, Messenger ; analysis ; Translocation, Genetic ; Trisomy

AIM:To observe the concentration of RBP4 and IL-6 in vitreous of proliferative diabetic retinopathy (PDR). METHODS: A total of 65 patients (66 eyes) were enrolled in Department of Ophthalmology, Renmin Hospital of Wuhan University from February 2017 to July 2017 with the informed consent. The patients were divided into PDR group (23 cases) and NPDR group (16 cases). Twenty- six patients without diabetic mellitus (DM) served as control group. The demography was matched among the groups, but the course of DM, the blood glucose level and the HbA1c level were elevated in the PDR group and the NPDR group (all P<0.05). Vitreous samples were collected during the procedure of vitrectomy. RBP4,IL-6,TNF-α concentrations in vitreous specimens were detected by ELISA. The differences of vitreous RBP4, IL-6 and TNF-α in various groups were statistically analyzed by ANOVA, respectively. The correlations between RBP4 and IL - 6, TNF - α were calculated by Pearson correlation analysis. RESULTS:The concentration of RBP4 in PDR group,the NPDR group and control group were 13.68士2.66, 11 03士1 12,10.45士1.17μ g/Ml, and the concentration of IL-6 were 56.0士10.27, 20.92士5.77, 10.26士1.91pg/Ml. RBP4 and IL- 6 concentrations were elevated in PDR group compared with NPDR group and control group, with significant difference among three groups (F = 12. 135, 161.167; P < 0. 01). IL - 6 concentrations in vitreous increased in the NPDR group in comparison with control group(P<0.05). RBP4 concentrations had no significant difference between the NPDR group and the group(P>0 05). Pearson correlation coefficient was significant positive between RBP4 concentration and IL - 6 concentration(r=0.606,P=0.001). CONCLUSION: RBP4 is probability involved in the inflammation pathogenesis of PDR. These results indicate that RBP4 could be a new target for the diagnosis and treatment of PDR.