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2.
Article in Chinese | WPRIM | ID: wpr-879176

ABSTRACT

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.


Subject(s)
Boraginaceae/genetics , Cloning, Molecular , Coenzyme A , Coenzyme A Ligases/genetics , Ligases , Phylogeny
3.
Article in Chinese | WPRIM | ID: wpr-879149

ABSTRACT

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.


Subject(s)
Cells, Cultured , Secondary Metabolism , Sorbus , Stress, Physiological
4.
Article in Chinese | WPRIM | ID: wpr-878915

ABSTRACT

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.


Subject(s)
Boraginaceae/genetics , Caffeic Acids , Chromatography, Liquid , Cinnamates , Cloning, Molecular , Depsides , Glycosyltransferases/genetics , Phylogeny , Tandem Mass Spectrometry
5.
Bol. latinoam. Caribe plantas med. aromát ; 19(6): 591-600, 2020. tab, ilus
Article in English | LILACS | ID: biblio-1284301

ABSTRACT

To investigate the influence of Kuntai capsules on the expression level of leukemia inhibitory factor (LIF), insulin-like growth factor-I (IGF-1)and epidermal growth factor (EGF) during the mouse's implantation window of superovulation period and controlled ovarian hyperstimulation period. 90 female mice were randomly divided into six groups in control, superovulation and controlled ovarian hyperstimulation (COH) conditions. The RNA expression of EGF, LIF and IGF-1 in the endometrium on the 4th day of pregnancy was detected, and the relative expression was compared. mRNA expression of these three factors in endometrium was significantly lower in superovulation and COH groups than control group (p<0.001). mRNA expression of these three factors in endometrium remained obviously lower in superovulation plus kuntai capsule group and COH plus kuntai capsule group than control group (p<0.01). mRNA expression of these three factors in endometrium was lower in control group than in the NS plus kuntai capsule group (p<0.05). Kuntai capsule cannot completely reverse the endometrial damages caused by superovulation and COH. Thus Kuntai capsule could partially improve a mouse's endometrial receptivity during the implantation window.


Para investigar la influencia de las cápsulas de Kuntai en el nivel de expresión del factor inhibidor de la leucemia (LIF), el factor de crecimiento similar a la insulina I (IGF-1) y el factor de crecimiento epidérmico (EGF) durante la ventana de implantación del ratón del período de superovulación y la hiperestimulación ovárica controlada período, se dividieron aleatoriamente 90 ratones hembra en seis grupos en condiciones de control, superovulación e hiperestimulación ovárica controlada (COH). Se detectó la expresión de ARN de EGF, LIF e IGF-1en el endometrio al cuarto día de embarazo, y se comparó la expresión relativa. La expresión de ARNm de estos tres factores en el endometrio fue significativamente menor en los grupos de superovulación y COH que en el grupo control (p<0,001). La expresión de ARNm de estos tres factores en el endometrio permaneció más baja en el grupo de cápsulas de superovulación más Kuntai y en el grupo de cápsulas de COH más Kuntai respecto del grupo control (p<0,01). La expresión de ARNm de estos tres factores en el endometrio fue menor en el grupo control que en el grupo de cápsula NS más Kuntai (p<0,05). La cápsula de Kuntai no pudo revertir completamente los daños endometriales causados por la superovulación y la COH. Por lo tanto, se sugiere que la cápsula de Kuntai podría mejorar parcialmente la receptividad endometrial de un ratón durante la ventana de implantación.


Subject(s)
Animals , Female , Mice , Ovulation Induction/methods , Somatomedins/drug effects , Drugs, Chinese Herbal/pharmacology , Epidermal Growth Factor/drug effects , Leukemia Inhibitory Factor/drug effects , Embryo Implantation , Superovulation , Somatomedins/genetics , Somatomedins/metabolism , Capsules , Polymerase Chain Reaction/methods , Electrophoresis , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , Leukemia Inhibitory Factor/genetics , Leukemia Inhibitory Factor/metabolism
6.
Article in Chinese | WPRIM | ID: wpr-828429

ABSTRACT

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.


Subject(s)
Boraginaceae , Genetics , Cytochrome P-450 Enzyme System , Naphthoquinones , Phylogeny , Plant Roots , RNA , RNA Interference
7.
Article in Chinese | WPRIM | ID: wpr-827988

ABSTRACT

This paper summarized the effects of ecological planting on secondary metabolism firstly and pointed out that ecological planting can increase the content of secondary metabolites in plants, especially the content of defensive secondary metabolites. The possible mechanism was analyzed subsequently. Then, we reviewed the induction and utilization of secondary metabolism in the ecological planting of Chinese materia medica from the perspectives of biological control of pests and diseases, promotion of beneficial microorganism accumulation, optimization of mixed planting, regulation of no-tillage and straw cover. In this article, we pointed out that paying close attention to secondary metabolism is the most important feature of ecological planting of Chinese materia medica. Ecological planting can promote the accumulation of secondary metabolites of Chinese materia medica which means can improve the quality of Chinese materia medica, beneficial to the prevention and control of diseases, insects and weeds. Furthermore, lacking of systemic researches,the extensive verifications and systematic in-depth researches on the ecological planting of Chinese materia medica should be carry out urgently.


Subject(s)
Drugs, Chinese Herbal , Materia Medica , Medicine, Chinese Traditional , Plants, Medicinal , Secondary Metabolism
8.
Article in Chinese | WPRIM | ID: wpr-827189

ABSTRACT

OBJECTIVE@#To investigate the influence of conventional CAG regimen and decitabine + decreased dose CAG (D+dCAG) regimen on the clinical efficacy and safety of patients with MDS-RAEB/AML-MRC.@*METHODS@#The clinical data of 67 patients with MDS-RAEB/AML-MRC hospitalized in our hospital from March 2012 to July 2017 were analyzed retrospectively. According to chemotherapecctic regimens, 76 patients were divided into 2 groups: 37 patients treated with conventional CAG regimen were enrolled in control group, 30 patients treated with decitabine + decreased dose CAG regimen were enrolled in D+dCAG group. The complete remission (CR) rate, overall remission rate (ORR), OS and PFS time and incidence of adverse reactions in 2 groups were compared.@*RESULTS@#The CR in D+dCAG group was significantly higher than that in control group (P<0.05). ORR was not significanly different between 2 groups (P>0.05). There was no significant difference in the cumulative OS rate between 2 groups (P>0.05). There was no significant difference in the cumulative OS rate and PFS rate in nonimplantation between 2 groups (P>0.05). The incidence of adverse reactions of hematological system, pulmonary infection, skin and soft tissue infection, agranulocytosic fever and mycotic infection was not significanly different between 2 groups (P>0.05). The duration of granulocyte deficiency and platelet count less than 20×10/L were not significanly different between 2 groups (P>0.05).@*CONCLUSION@#Compared with conventional CAG regimen, decitabine + decreased dose CAG regimen in the treatment of patients with MDS-RAEB/AML-MRC can efficiently improve the remission effects and showed the well overall safety, but can not increase the survival rate.


Subject(s)
Anemia, Refractory, with Excess of Blasts , Antineoplastic Combined Chemotherapy Protocols , Cytarabine , Decitabine , Granulocyte Colony-Stimulating Factor , Humans , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Retrospective Studies , Treatment Outcome
9.
Article in Chinese | WPRIM | ID: wpr-781482

ABSTRACT

OBJECTIVE@#To analyze the correlation of the minimal residual disease level with the prognosis of the AML patients with NPM1 gene mutation positive after chemotherapy.@*METHODS@#The clinical data of 112 newly diagnosed adult AML patients with positive NPM1 gene were collected and retrospectively analyzed. The correlation of the transcripts of NPM1 gene mutation with prognosis of patients was analyzed.@*RESULTS@#In 112 AML patients, the median transcript level of NPM1 gene mutation accounted for 83.68% (5.86%-486.57%), FLT3-ITD mutation positive was found in 44 cases (39.29%), chromosomal abnormalities in 22 cases (19.64%) and complete remission in 96 cases (85.71%). Multivariate logistic regression analysis showed that the initial induction therapy and white blood cell count closely related with complete remission (P<0.05). The median follow-up time was 22 (3-36) months, and the 3-year overall survival rate was 66.07% in 112 patients. Multivariate logistic regression analysis showed that both the high level of minimal residual disease at the initial complete remission and the high level of minimal residual disease after consolidation therapy were the independent risk factors for overall survival (P<0.05).@*CONCLUSION@#In newly diagnosed adult AML patients with NPM1 mutation positive, the early high level of minimal residual disease after chemotherapy closely relates with poor prognosis.

10.
Article | WPRIM | ID: wpr-833825

ABSTRACT

Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.

11.
Chinese Journal of Neurology ; (12): 209-215, 2019.
Article in Chinese | WPRIM | ID: wpr-745915

ABSTRACT

Objective To investigate the relationship between total cerebral small vessel disease (CSVD) burden and intracranial hemorrhage transformation (HT) after intravenous thrombolysis in patients with acute ischemic stroke (AIS).Methods One hundred and fifty-four patients who suffered from ischemic stroke within 4.5 hours of onset and received recombinant tissue plasminogen activator thrombolytic therapy in the emergency green channel of the First Affiliated Hospital of Soochow University from August 2016 to January 2018 were enrolled.HT examined by computed tomography scan within 24 hours after thrombolysis was included.The magnetic resonance imaging examination was performed within 48 hours.The patients were divided into two groups:HT group and control group according to the presence or absence of HT.Periventricular white-matter hyperintensities (WMH) with Fazekas score of 3 or deep WMH with Fasekas score of 2 or 3 was recorded as 1 point,MRI of cerebral microbleeds (CMBs) or lacunar infarction (LI) was recorded as 1 point respectively,and peripheral vascular space (PVS) in basal ganglia graded 2-4 (≥11)was counted 1 point.Single-factor analysis was used to compare total CSVD burden score,baseline data and clinical data between the two groups.Multivariate Logistic regression analysis was performed to explore the relationship between total CSVD burden score and HT.Results The age of the 154 patients was 66.00(59.00,74.25) years,males accounted for 66.9% (103/154),onset to treatment time (OTT) was 174.50 (131.50,200.00) minutes and the NIHSS score before thrombolytic therapy was 6.00 (3.00,10.25).There were 43 cases (27.9%) with moderate to severe WMH,35 cases (22.7%) with CMBs,52 cases (33.8%) with PVS graded 2-4,and 96 cases (62.3%) with LI.There were 21 enrolled patients (13.6%) who suffered from HT.Symptomatic intracranial hemorrhage occurred in nine cases (5.8%).In the multivariate Logistic regression model,the results demonstrated that baseline diastolic pressure (OR=1.072,95%CI 1.027-1.118,P=0.001)and atrial fibrillation (OR=28.564,95%CI 6.217-131.241,P=0.000) were independently associated with HT.After using the mild CSVD burden score as a reference,moderate CSVD burden (OR=0.810,95% CI 0.154-4.257,P=0.804) was not associated with HT after thrombolysis,and severe CSVD burden (OR=8.429,95% CI 1.643-43.227,P=0.011) was independently associated with HT.Conclusions The severity of total CSVD burden in patients with AIS was closely related to HT after thrombolysis.Severe CSVD was an independent risk factor for HT after thrombolysis.

12.
Article in Chinese | WPRIM | ID: wpr-771887

ABSTRACT

OBJECTIVE@#To investigate the regulatory effect of miR-543 on proliferation and apoptosis of human leukemia cell line K562 and its mechanism.@*METHODS@#46 CML patients treated in our hospital from 2018.2-2018.9 was selected and enrolled in CML group, and 30 healthy persons were selected and enrolled in control group at the same time. After Lipofectamine 2000 liposome was used to transfer the miR-543 mimic and negative control (Scramble mimic) to K562 cells, CCK8 assay was used to detect the effect of miR-543 on proliferation of K562 cells; Luciferase reporter assay was used to report the targeting relationship of miR-543 to Wnt gene. Flow cytometry was used to detect the effect of miR-543 on apoptosis of K562 cells; Western blot method was used to detect the Wnt, β-catenin, BCL-2, c-MYC and BAX expression level.@*RESULTS@#The level of miRNA-543 in CML patients was significantly lower than that in healthy controls (P<0.05). The expression level of miR-543 in mimic group was significantly higher than that in blank control group (P<0.05). The Wnt gene expression level in mimic group was significantly lower than that in blank control group (P<0.05). The expression of luciferase of Wnt wild plasmid was decreased by miR-543 mimic (P<0.05), and the luciferase activity of Wnt mutant plasmid was not inhibited by miR-543 mimic after mutation of binding site (P<0.05). There was no significant difference in the proliferation ability of K562 cells between the blank control group and the negative control (Scramble mimic) group (P>0.05). The proliferation level of K562 cells in mimic group was significantly lower than that in blank control group and negative control (Scramble mimic) group (P<0.05). Apoptotic level of K562 cells in miR-543 mimic group was significantly higher than that in blank control group and negative control (Scramble mimic) group (P<0.05). Western blot assay showed that Wnt, β-catenin, BCL-2 and c-MYC protein levels in miR-543 mimic group were significantly lower than those in blank control group and negative control (Scramble mimic) group (P<0.05); BAX protein level in miR-543 mimic group was higher than that in blank control group and negative control (Scramble mimic) group (P<0.05).@*CONCLUSION@#miR-543 can target Wnt protein to inhibit the activity of Wnt signaling pathway, thereby inhibiting the proliferation of leukemia cells and increasing the level of apoptosis.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Humans , K562 Cells , Leukemia , MicroRNAs , Genetics
13.
Article in Chinese | WPRIM | ID: wpr-807186

ABSTRACT

Objective@#To establish a set of rules for autoverification of blood analysis, in order to provide a way to validate autoverification rules for different analytical systems, which can ensure the accuracy of test results as well as shorten turnaround time (TAT) of test reports.@*Methods@#A total of 34 629 EDTA-K2 anticoagulated blood samples were collected from multicenter cooperative units including the First Hospital of Jinlin University during January 2017 to November 2017. These samples included: 3 478 cases in Autoverification Establishment Group, including 288 cases for Delta check rules; 5 362 cases in Autoverification Validation Group, including 2 494 cases for Delta check; 25 789 cases in Clinical Application Trial Group. All these samples were analyzed for blood routine tests using Sysmex XN series automatic blood analyzers.Blood smears, staining and microscopic examination were done for each sample; then the clinical information, instrument parameters, test results and microscopic results were summarized; screening and determination of autoverification conditions including parameters and cutoff values were done using statistical analysis. The autoverification rules were input into Sysmex Laboman software and undergone stage Ⅰ validation using simulated data, and stage Ⅱ validation for post-analytical samples successively. True negative, false negative, true positive, false positive, autoverification pass rate and passing accuracy were calculated. Autoverification rules were applied to autoverification blood routine results and missed detection rates were validated, and also data of autoverification pass rate and TAT were obtained.@*Results@#(1)The selected autoverification conditions and cutoff values included 43 rules involving WBC, RBC, PLT, Delta check and abnormal characteristics. (2)Validation of 3 190 cases in Autoverification Establishment Group showed the false negative rate was 1.94%(62/3 190)(P<0.001), autoverification pass rate was 76.74%, passing accuracy was 97.47%; Validation of 2 868 cases in Autoverification Validation Group, the false negative rate was 3.38%(97/2 868)(P=0.002), autoverification pass rate was 42.26%, passing accuracy was 92.00%; Validation of Delta check on 288 cases in Autoverification Establishment Group and 2 494 cases in Autoverification Validation Group showed the false negative rates were respectively 1.39% and 2.61%(P<0.001). (3)Three hospitals adopted these rules of autoverification for 25 789 blood routine samples, and found that the average TAT of blood routine test reports were shortened by 24min, 32min and 7min respectively, the rate of samples reported within 30min were elevated by 33%, 53% and 7%. The autoverification pass rates were 72%-74%.@*Conclusions@#The application of this set of 43 autoverification rules in blood sample analysis can ensure test quality while shortenTAT and improve work efficiency. It is worth pointing out that for the same analytical systems in this research, validation is necessary before application of this set of rules, and periodic validation is required during application to make necessary adjustment; for different analytical systems, as this research provide a way to establish autoverification rules for blood routine tests.Clinical labs may establish their own suitable autoverification rules on the basis of technological parameters. (Chin J Lab Med, 2018, 41: 601-607)

14.
Article in Chinese | WPRIM | ID: wpr-698660

ABSTRACT

BACKGROUND: Although a large number of related studies have been carried out, there is still a lack of practical methods to amplify hematopoietic stem cells(HSCs)in vitro.Mesenchymal stem cells(MSCs)secrete a variety of cytokines that promote the HSCs proliferation and inhibit their differentiation. These cytokines play an important role in maintaining the hematopoietic microenvironment and regulating HSCs function. OBJECTIVE:To investigate the effect of bone marrow MSCs on the proliferation of HSCs in vitro under different coculture modes. METHODS:Mesenchymal stem cells from the bone marrow of C57BL/6 mice were cultured in vitro using the whole bone marrow adherent culture. CD117+cells (HSCs) were sorted from passage 3 cells by using miniMACS magnetic beads sorting. Then, CD117+cells were co-cultured with MSCs under different coculture models, including single culture of HSCs (control group), Transwell coculture (upper chamber, HSCs; lower chamber, MSCs) and two-dimensional contact coculture (coculturing HSCs and MSCs in 24-well plates). The morphology of HSCs was observed under phase contrast microscope and fluorescence microscope, and the number of active cells of HSCs was counted at 1, 3, 5, and 7 days after coculture. RESULTS AND CONCLUSION: During the coculture of 1-7 days, the number of HSCs in the two groups was increased with culture time (P <0.05). After 3 days of coculture, HSCs in each group was grown into the logarithmic growth phase, and morphological changes in some HSCs were detected at 5 days of coculture. At 7 days of coculture, the viabilities of HSCs in different culture models were ranked as follows: single culture model < Transwell coculture model < two-dimensional contact coculture model (P < 0.05). These findings suggest that MSCs can effectively promote the proliferation of HSCs in vitro,and the promotion effect is increased under contact coculture conditions.

15.
Journal of Experimental Hematology ; (6): 1523-1530, 2018.
Article in Chinese | WPRIM | ID: wpr-689903

ABSTRACT

<p><b>OBJECTIVE</b>To study the effect and mechanism of shh and mesenchymal stem cell(MSC)synergism on the proliferation of hematopoietic stem cells in noninvasive co-culture system in vitro.</p><p><b>METHODS</b>The mesenchymal stem cells were cultured in vitro,CD34 cells were sorted by mini MACS magnetic bead separator,flow cytometry was used to identify the purity of 2 cells. CD34 cells and MSCs were seeded to upper and low of transwell respecibely for non-contact coculture,and add exogenous shh protein for intervenece. The number of MSCs and HSCs,the total amount of RNA,the expression of ki67 and Tie-2 mRNA of HSC,the expression of VEGF and Ang-1 mRNA of MSC were detected for investigating the condition of cell proliferation and the expression of angiogenic factors.</p><p><b>RESULTS</b>The total number of cells,the total amount of RNA and the relative expression of ki67, Tie-2, VEGF and Ang-1 in non-contact co-culture group increased and showed the following trends on the 7th day:the above-mentioned indexes in group MSC + HSC, group shh + HSC were higher than those in group HSC, while those in MSC + shh + HSC Group was higher than those in MSC + HSC and shh + HSC group.</p><p><b>CONCLUSION</b>Angiogenic factors help MSC to proliferate HSC and amplify the CD34 hematopoietic stem/progenitor cells by shh and MSC synergism in vitro coculture system which may be related with angiogenic factors.</p>

16.
Article | WPRIM | ID: wpr-777870

ABSTRACT

@#Amebiasis is one of the major causes of diarrhea in the developing countries and it can present with a wide range of gastrointestinal symptoms depending on the phase of infection. We described a case of 50 year-old male patient who presented with abdominal pain, diarrhea and vomiting. After right hemicolectomy for appendicular abscess with tumour over the ileum, histopathological examinations revealed numerous trophozoites of Entamoeba histolytica in a background of inflammations (Figure 1). Following resection of the ameboma, he received intravenous metronidazole treatment for total of two weeks duration.

17.
Article in Chinese | WPRIM | ID: wpr-690975

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the influencing factors of nosocomial infection in adult patients with acute myeloid leukemia (AML) and its control strategies.</p><p><b>METHODS</b>The clinical data of 109 patients with AML treated in our hospital from June 2014 to June 2017 were retrospectively analyzed. The clinical factors were analyzed retrospectively, and the influencing factors of infection after chemotherapy were explored.</p><p><b>RESULTS</b>A total of 109 patients received chemotherapy for 267 case-times, the infection occurred in 168 case-times, the nosocomial infection rate was 62.92%, the main affected sites included upper respiratory tract and lung. 155 samples from 168 case-times infection patients were collected and cultured, 32 pathogens were obtained with a positive rate 20.6% (32/155), including 14 Gram-negative bacteria, 9 Gram-positive bacteria and 4 strains of fungi and 5 strains of other pathogens. Statistics showed that the patient's age over 40 years old, hospitalization in spring and summer, glucocorticoid therapy, high intensity chemotherapy, neutrophil count, white blood cell count and hemoglobin content were the independent risk factors for infection after chemotherapy in AML patients (P<0.05).</p><p><b>CONCLUSION</b>The age of more than 40 years old, hospitalization in spring and summer, glucocorticoid therapy, high-intensity chemotherapy, white blood cell count, neutrophil count and hemoglobin content are the independent risk factors for infection after chemotherapy in the AML patients with the above-mentioned characteristics, they should be closely monitored, and chemotherapy intensity should be controled, so as to control the occurrence of infection; and in the event of infection, a timely powerful anti-infective treatment would be indispensable.</p>


Subject(s)
Adult , Cross Infection , Hospitalization , Humans , Leukemia, Myeloid, Acute , Retrospective Studies , Risk Factors
18.
Article in Chinese | WPRIM | ID: wpr-663013

ABSTRACT

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

19.
Article in Chinese | WPRIM | ID: wpr-661223

ABSTRACT

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

20.
Military Medical Sciences ; (12): 796-799, 2017.
Article in Chinese | WPRIM | ID: wpr-694258

ABSTRACT

Objective To explore the role of microRNA-155 ( miR-155 )/suppressor of cytokine signaling 1 ( SOCS1 ) pathway combined with IL-17 in modulating the early progression of Schistosoma japonicum.Methods BALB/c mice were infected subcutaneously with cercariae of S.japonicum before being sacrificed at 8 and 14 weeks after infection to collect liver samples for pathological observation by H&E staining .The spleens of mice were collected to calculate the spleen index.The level of IL-17 in serum was determined using ELISA.Furthermore, qRT-PCR was used to detect the expression of miR-155 and SOCS1 mRNA in the spleens of mice .Results Compared with normal group , the spleen index in the model group was increased significantly along with the loss of relative body mass .H&E staining revealed that the deposition of parasite eggs began to appear in the liver tissue at week 8 post-infection.Meanwhile, there were a large number of inflammatory cells infiltrating to form the eosinophilic granuloma.Over time, the formation of egg granulomas and the infiltration of inflammatory cells were alleviated on week 14 post-infection.The level of IL-17 in serum and the expression of miR-155 in the spleen were peaked on week 8 post-infection, and decreased sharply on week 14, but were still evidently higher than those in normal group .The expression of SOCS1 mRNA was progressively increased both on week 8 and 14. Conclusion The data indicates that miR-155 might cooperate with IL-17 to modulate SOCS1 in the development of S.japonicum infection.

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