2.Relation between alternatively spliced tissue factor and tumor
Linyan ZHAO ; Bo JIANG ; Ri ZHANG
Journal of International Oncology 2015;(5):361-363
Serine/ aginine rich(SR)protein,SR protein kinase and other enzymes participate in the composition of alternatively spliced tissue factor(asTF). Recently researchers have found that this protein takes a part in tumor angiogenesis,tumor progression,metastasis and so on,so the detection of its clinical content will be very significant.
3.Observation and nursing of adverse reactions in chronic myelocytic leukemia patients receiving imatinib therapy
Ling LU ; Sufang ZHAO ; Ri ZHANG
Chinese Journal of Practical Nursing 2013;29(19):33-35
Objective To discuss the observation and nursing of adverse reactions in chronic myelocytic leukemia patients receiving imatinib therapy.Methods Adverse reactions were observed and recorded in 193 chronic phase myelocytic leukemia patients who received imatinib therapy,and corresponding treatment and nursing were given to them.Results Among 193 patients,more than 60% of patients had adverse reactions,of which,54% of patients showed gastrointestinal adverse reactions including nausea,vomiting,anepithymia and diarrhea; 22% of them had muscle and bone pain; 7% had rash; 65% got edema.After proper treatment and nursing,all adverse effects obtained satisfactory remission.Condusions During the treatment course of chronic myelocytic leukemia patients using imatinib,careful observation of any possible adverse reactions,and giving corresponding treatment and nursing can facilitate good compliance and longterm remission of patients.
4. Mechanism analysis of Salviae Miltiorrhiza and Ligustrazine Hydrochloride Injection based on network pharmacology
Chinese Traditional and Herbal Drugs 2018;49(11):2606-2613
Objective To investigate the efficacy and mechanism of Salviae Miltiorrhiza and Ligustrazine Hydrochloride Injection (SMLHI). Methods The targets of chemical components of SMLHI were predicted and the compounds-targets (C-T) network was constructed. The key targets were screened through the topology analysis of the C-T network. Also, protein-protein interaction (PPI) network was established, and the Gene ontology and KEGG pathway was enriched and analyzed. The pharmacological action mechanism of SMLHI was predicted, and the mechanism was preliminarily verified by pretreating with SMLHI on the MARK expression of cerebral ischemia rats. Results Fifteen main compounds in SMLHI act on 94 targets and PRSS1, PTGS2, F2, and PTGS1 were key targets in the C-T network. There were 71 targets in the PPI network including several key nodes such as SRC, MAPK-1, MMP-9, MAPK-14, PTGS2, BCL-2, and so on. All the targets were enriched in 26 GO items and six KEGG pathways. Conclusion Results in this study preliminarily verified the action of SMLHI on cerebral infarction and diabetic peripheral neuropathy, thus laying a solid foundation for further study on the mechanism of action.
6. Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia
Otgonbat A ; Ming Feng Zhao ; Wu Ri Mao
Health Laboratory 2016;5(1):5-11
Background:Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN), since the first description of CNL in 1920, more than 150 cases have reported in the literature. The World Health Organization (WHO) recognizes CNL, as a MPN and, for the frst time, provides recognized criteria to permit the operational classifcation of this poorly defned disease. Until recently, the molecular pathogenesis of CNL was unknown and the diagnosis was based on morphological aspects, clinical criteria and exclusion of known genetic entities like the Philadelphia translocation indicative of CML, or JAK2 mutations indicative of MPNs. Recent discovery of highfrequency granulocyte-colony stimulating factor receptor (CSF3R) mutations in CNL identifes a new major diagnostic criterion, and lend more specificity to the WHO diagnostic criteria for CNL, which are variably applied in routine clinical practice. In 2013 Maxson et al., and Pardanani and colleagues identified granulocyte-colony stimulating factor 3 receptor (CSF3R) mutations in 8 of 9 (89%), and in 13 of 13 (100%) patients with CNL, respectively. CSF3R mutations fall into 2 classes: nonsense or frameshift mutations that lead to premature truncation of the cytoplasmic tail of the receptor (truncation mutations) and point mutations in the extracellular domain of CSF3R (membrane proximal mutations). These nonsense or frameshift mutations truncate the cytoplasmic tail of CSF3R, impair its internalization,and alter its interactions with proteins such as SHP-1/2 and SOCS family members. These structural and functional alterations are thought to perturb the capacity of CSF3R to regulate granulocyte differentiation and to increase granulocytic proliferative capacity. Thetwo types of CSF3R mutations may have differential susceptibility to classes of tyrosine kinase inhibitors,with CSF3R truncation mutations showing activation of SRC family–TNK2 kinase signaling and sensitivity to dasatinib, and CSF3Rmembrane proximal mutations strongly activate the JAK/signal transducer and activator of transcription pathway and are sensitive to JAK kinase inhibitors such as ruxolitinib.The most common CSF3R mutation in CNL is themembrane proximal mutation: T618I. On September 2012 we got a case (a 67-years-old Chinese man), which had fulflled the WHO diagnostic criteria for CNL with a novel mutation site of colony stimulation factor 3 receptor (CSF3R). In our case was identifed a membrane proximal mutations CSF3RT618I and also a unreported novel mutation site of CSF3R-H54A in the CD34+ and CD15+ cell fractionsby sorting bone marrow samples (BD FACSAria™ III; BD Biosciences) using a PCR-based DNA pyrosequencing method. Thus, we sought to determine CSF3R-FL, CSF3R-T618I, CSF3R-H54A mutations have some correlation with molecular pathogenesis of CNL.Objective:To determine CSF3R-FL, CSF3R-T618I, CSF3RH54A mutations that have some signifcance on the molecular pathogenesis of CNL.Materials/Methods:1. Plasmid construction. Plasmids were constructed by PCR amplifcation of the insert, restriction digestion and ligation using standard molecular biology methods, briefly: the host vectors pLV-EF1α-EYFP-N, pLP-2, pVSV-G, pLP-1 gag pol were purifed with Omegabiotek maxi prep kit and digested by restriction enzymes (ECO RI, NOTI). The linearized vectors were purified from agarose gel using a AxyPrep TM DNA Gel Extraction Kit and the concentration of the samples was estimated on an agarose gel stained with ethidium bromide.The inserts (CSF3R-FL, CSF3R-T618I, CSF3R- H54A) were generated by PCR, the sequences of the primer-pairs used and the conditions of the PCR reactions. The amplifed DNA fragments were purifed from agarose gels using AxyPrep TM DNA Gel Extraction kits and digested by ECO RI, NOTI was used to linearize the acceptor vector. Enzymatic reactions in the case of the i) insert: 100–3000 ng purifed PCR product was digested by appropriate amount of enzyme and 4 µl of 10x reaction buffer in 40 µl of fnal volume for overnight at the appropriate temperature; ii) vector: 2000–4000 ng plasmid DNA was digested by appropriate amount of enzyme and 2 µl of 10x reaction buffer in 20 µl of fnal volume for 4 hours at the appropriate temperature. When necessary Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia Otgonbat Altangerel1, Ming Feng Zhao2, Wu Ri Mao21Department of Internal Medicine, Division of Hematology, Mongolian National University of Medical Sciences, Mongolia 2Department of Hematology, Tianjin First Central Hospital, First Central Clinical College of Tianjin Medical University,P.R. China11 the digested fragments were purifed again and the concentrations of the inserts were estimated on agarose gels, as described above. A 1:3 vector: insert molar ratio was used for the ligation reactions. Chemically competent DH5α Escherichia coli bacteria were transformed with the products of the ligation reactions and were grown on Luria Bertrani (LB) agar plates containing the required antibiotic, such as ampicillin (Sigma). A day later single colonies were picked from the plate, inoculated into and grown overnight in LB medium containing with ampicillin. Plasmids were purifed from the overnight cultures as above and tested by restriction mapping for the presence of the insert. Selected clones were sequenced by Sanger sequencing.2. Lentiviral packaging system we used 3 main components, such as the lentiviral expression vector(Plasmid DNA of CSF3R-FL, CSF3R-T618I), the lentiviral packaging plasmids (pLP-1, pLP-2 plasmids that encode for gag, pol, and rev from the HIV or FIV genome and pVSV-G), 293TNN producer cells. We seed 1X105 293TNN cells per 10 cm2 culture plate in 2-3 ml of culture medium containing DMEM medium supplemented with 4 mM L-glutamine, 4.5 g/l glucose, and fetal bovine serum (10%) without antibiotics. Grow for 18-24 hours at 37 °C with 5% CO 2 so that the cell density reaches ~60 - 80% confluency at the time of transfection. We used a GFP as a positive control, to confrm transfection experiment was successful. Then we collected the cell culture supernatant, which is containing infectious pseudoviral particles.3. Transduction of Pseudotyped Viral Particles into the primary cell of mouse. In the fnal step we have used the Mouse Colony Forming Unit Assay using MethoCultTM to assess the effects of CNL-associated oncogenes on the morphology and number of primary murine cells derived bone marrow. For this purpose cells are transduced with either control, which is without viral construct or a construct expressing the oncogene of interest (CSF3R-FL, CSF3R-T618I).Results:1. On the Plasmid construction step we successfully extracted and purifcated of recombinant plasmids ofCSF3R-FL and CSF3R-T618I cloning, but we still didexperiment to obtain the recombinant plasmid CSF3R- H54A cloning.2. After 24 hours of transfection 293TNN Cells with Packaging Plasmids and the Expression Construct, cells we visualized with green fluorescence protein under the fluorescence microscope.3. The both two of CSF3Rcloning were capable of transforming murine colony forming cells. After transforming, CFU-GM colonies were counted manually by light microscopy seven days after plating. We found that the membrane proximal mutation (T618I) transformed CFU-GM colonies number was more than the full length non-mutants (CSF3RFL), which indicates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.Conclusions:1. The establishment of the plasmid reconstruction, lentiviral packaging system and Mouse Colony Forming Unit Assay were done successfully.2. We confrmed the transformation capacity of the CSF3R mutations, especially CSF3R-T618I was higher than CSF3R-FL. This result demonstrates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.3. Further studies will be continued and are needed to prove the effects of the novel mutation site CSF3RH54A on the transduced murine bone marrow progenitor cells by using CFC assay
7.Improvement of rat pulmonary microvascular endothelial cell primary culture methods
Ge, MENG ; Jian, ZHAO ; Xin-Huai, L(U) ; Ri-Gao, DING
Bulletin of The Academy of Military Medical Sciences 2009;33(6):567-569
Objective:To improve the primary culture method of rat pulmonary microvascular endothelial cells(PMVECs) and obtain purified PMVECs.Methods:The modified tissue block pasted culture method was used to isolate and culture Wistar rat PMVECs. The morphous of cultured cells were observed by microscopy. The cultured cells were identified by detecting factor Ⅷ related antigen and binding isolectin B4. Results and Conclusion:The morphous of cultured primary PMVECs in vitro showed short fusiform shape or polygon, and the monolayer of cultured cells displayed the shape of pavingstone. But the morphous changed followed the transfer of culture and the change of culture condition. The cultured cells had characterization of binding isolectin B4 and negative immunocytochemical staining for factor Ⅷ related antigen. The cultured PMVECs have good growth state and purity,and can be subcultringed stably.The observation of cell morphous integrating with immunocytochemical staining is a reasonable identification method for PMVECs.
8.Isolation, purification, primary culture and identification of alveolar type Ⅱ epithelial cell from rats
Ge, MENG ; Jian, ZHAO ; Xin-huai, L(U) ; Ri-gao, DING
Bulletin of The Academy of Military Medical Sciences 2010;34(1):34-36
Objective To establish a method of isolation, purification, primary culture and identification of alveolar type Ⅱ epithelial cells(AT-Ⅱ).Methods The AT-Ⅱs were isolated from Wistar rats by trypsin,purified by differential centrifugation, erythrocyte spallation, differential adherence and immune adherence, and identified by observing the morphology of cultured cells under the inverted phase and tannic acid staining. Results and Conclusion The cultured primary AT-Ⅱs in vitro presented single or island form growth, and their shapes were round or elliptical. A great deal of fine particles showed sharp contrast, and were observed in intracytoplasm. The cell nuclei were clear. They were positive for tannic acid staining.The primary culture AT-Ⅱs obtained from improved isolation and purification have good growth state and purity, and are suitable for research in vitro.
9.Clinical features of mtDNA A3243G mutation in adult patients
Sheng YAO ; Ri-Liang ZHENG ; Hong-Yan BI ; Zhao-Xia WANG ; Yun YUAN ;
Chinese Journal of Neurology 2000;0(04):-
Objective To analysis the clinical manifestations of mtDNA A3243G mutation in adulthood.Methods The clinical features were investigated in 36 cases (28 patients from 5 families with the mutation and 8 sporadic cases),in whom mtDNA A3243G mutation was confirmed genetically in 23 cases (15 cases from 5 mutation families and 8 sporadic cases).Cranium radiology was performed in 14 cases.Muscal biopsies were performed in l0 cases.Results Among 28 cases in the 5 family,there were 9 cases (32.1%) with stroke like episodes,17 cases (60.7%) with diabetic mellitus and 16 cases (57.1%) with deafness.Such symptoms usually combined with each other and rarely existed alone. Cardiomyopathy and renal failure were uncommon.In the 23 cases with mtDNA A3243G mutation,14 cases (61.0%) had mitochondria] myopathy,encephalopathy,lactic acidosis,and stroke-like episodes (MELAS),mostly presenting cognitive abnormalities,dysarthria or aphasia and headache,3 cases (13.0%) were asymptomatic carriers,2 cases (8.7%) had autonomic dysfunction,2 cases (8.7%) had diabetic mellitus with or without nerve deafness,1 case (4.3%) had diabetic mellitus with infertilitas and cardiomyopathy,respectively.Cranial radiological images revealed the changes more commonly in the temporal and occipital lobes and less frequently in the frontal lobes.Ragged red fibers were confirmed in 9 of 10 cases with muscle biopsies.The proportion of mutant mtDNA A3243C was not significantly different between MEALS (28.75%?13.69%) and non-MELAS (25.08%?11.54%).Conclusions mtDNA A3243G mutation mainly results in the lesions in the central nerve system,pancreatic island and acoustic nerve in adulthood.Heart and kidney are less frequently involved.Cognitive abnormalities,aphasia and headache are the major symptoms of adult MELAS.Families have with more than 1 patient with diabetic mellitus and deafness,indicating that the mutation is other than MELAS mutation.We should pay more attention to the non-MELAS symptoms in the families with mtDNA A3243G mutation.
10.Epidemic situation analysis of human brucellosis in Inner Mongolia during 1952 to 2007
Dong-ri, PIAO ; Lan-yu, LI ; Hong-yan, ZHAO ; Bu-yun, CUI
Chinese Journal of Endemiology 2009;28(4):420-423
Objective To explore the possible factors associated with twice human brucellosis epidemics in Inner Mongolia during 1952 to 2007 to provide scientific tactics for prevention and control brucellosis. Methods Surveillance data and literature about human brucellosis during 1952 to 2007 in Inner Mongolia was collected, descriptive analysis of human brucellosis incidence on distribution in the regions and among occupations was carried out during 1952 to 2007. Results In Inner Mongolia, the first epidemic of human brucellosis peak appeared in the early 1960s, spreading to 12 regions, at an incidence of 55.28/100 000 in 1961, 72.9% of the Brucella infected people were herdsman;another epidemic peak seriously hit middle and eastern regions after 2000, the incidence being 38.44/100 000 in 2005;51.9% and 28.7% of the new brucellosis cases were respectively peasant and herdsman. Conclusions In Inner Mongolia, animal husbandry industry has been rapid developed since the early 1990's, resulting frequent livestock trade without quarantine, at the same time the public health system doesn't match the development, so the epidemic situation of brucellosisbecomes more and more serious after mid-90's, and has reached the peak during 2004 and 2007.