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Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 08/2011; 32(8):548-50.
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ABSTRACT: To explore the effect of α- galactosyleramide( α-GalCer ) on immune reconstitution under acute graft-versus-host disease (aGVHD).
BALB/c mice were transplanted wit hallogeneic C57BL/6 bone marrow cells and splenocytes (both 1×10(7))after receiving lethal total-body irradiation. α-GalCer (100 ug/kg) or vehicle (dimethylsulfoxide) was administered intraperitoneally immediately after transplantation. The effects of α-GalCer on immune reconstitution,proliferation of T cells and B cells, hematopoiesis,and thymic microenvironment were assessed.
The α-GalCer group exhibited higher percentages of CD3(+),CD4(+), CD8(+), B220(+), CD40(+), and CD86(+)cells compared with the vehicle group . The number of colony forming unit per 1000 CD34(+) cells in the α-GalCer group was higher than in the vehicle group ( P=0.0012).In vitro proliferation assays showed that the α-GalCer group had higher percentages of CD3(+), CD4(+), CD8(+),and B220(+) cells compared with the vehicle group. As for the results of in vivo proliferation assays, the numbers of CD3(+), CD4(+), CD8(+), and B220(+)cells were higher in the α-GalCer group than in the normal group ,especially the number of B220(+) cells ( P=0.007).Significant difference was not found in thymocyte count between the α-GalCer group and the vehicle group, nor in the percentages of CD3(+), CD4(+), and CD8(+) cells.
Administration of α-GalCer after allogeneic bone marrow transplantation may promote immune reconstitution in the presence of aGVHD.
Chinese Medical Sciences Journal 06/2011; 26(2):91-7.
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ABSTRACT: It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.
The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.
The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05).
Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.
Chinese medical journal 05/2011; 124(9):1395-400. · 0.86 Impact Factor
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ABSTRACT: To investigate the effect of peroxisome proliferator-activated receptor (PPAR)α agonist bezafibrate and oxidized low density lipoprotein (ox-LDL) on fibroblast growth factor 21 (FGF21) expression and apoptosis in cardiac endothelial cells.
The mRNA level of FGF21 was determined by real time-PCR and the protein concentration of FGF21 in culture media was detected by enzyme-linked immunosorbent assay in cultured cardiac microvascular endothelial cells (CMECs) incubated with 10, 50, 100 µg/ml ox-LDL, 50, 100 or 200 µmol/L bezafibrate alone or in combination with 100 µg/ml ox-LDL. CMECs apoptosis in various treatment groups was also determined.
FGF21 mRNA and protein expressions were significantly upregulated in proportion to increased ox-LDL, and 200 µmol/L bezafibrate alone also significantly upregulated FGF21 expression and CMECs apoptosis was significantly reduced in 200 µmol/L bezafibrate + 100 µg/ml ox-LDL group compared to 100 µg/ml ox-LDL group (P < 0.05).
Our data suggest that bezafibrate and ox-LDL induced upregulation of FGF21 might mediate the protective effect against apoptosis. Endogenous FGF21 could thus play important roles in improving the endothelial function at the early stage of atherosclerosis and slowing the development of coronary heart disease.
Zhonghua xin xue guan bing za zhi [Chinese journal of cardiovascular diseases] 12/2010; 38(12):1113-7.
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ABSTRACT: Fibroblast growth factor 21 (FGF21) is a new member of FGF super family that is an important endogenous regulator for systemic glucose and lipid metabolism. This study aimed to explore whether FGF21 reduces atherosclerotic injury and prevents endothelial dysfunction as an independent protection factor.
The present study was designed to investigate the changes of FGF21 levels induced by oxidized-low density lipoprotein (ox-LDL), and the changes of apoptosis affected by regulating FGF21 expression. The FGF21 mRNA levels of cultured cardiac microvascular endothelial cells (CMECs) were determined by real time-PCR and the protein concentration in culture media was detected by enzyme-linked immunosorbent assay. We analyzed the different expression levels of untreated controls and CMECs incubated with ox-LDL, and the changes of CMECs apoptosis initiated by the enhancement or suppression of FGF21 levels.
The secretion levels of FGF21 mRNA and protein were significantly upregulated in CMECs incubated with ox-LDL. Furthermore, FGF21 levels increased by 200 µmol/L bezafibrate could reduce CMECs apoptosis, and inhibit FGF21 expression by shRNA induced apoptosis (P < 0.05).
FGF21 may be a signal of injured target tissue, and may play physiological roles in improving the endothelial function at an early stage of atherosclerosis and in stopping the development of coronary heart disease.
Chinese medical journal 12/2010; 123(23):3417-21. · 0.86 Impact Factor
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Guo-zhong Wang,
Shu-zheng Lv, Jing-hua Liu,
Yun-dai Chen,
Yong Huo,
Wei Gao,
Wei-min Wang,
Fang Chen,
Yu-jie Zhou,
Zhi-zhong Li,
Yuan-nan Ke,
Xin-chun Yang,
Shu-yang Zhang,
Hong-bing Yan,
Hong-wei Li,
Da-zhuo Shi,
Bu-xing Chen
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ABSTRACT: To determine whether the combination of traditional risk factors and quantitative coronary angiography (QCA) assessment could provide accurate prognostic information on a population-based study including 1137 adults with subclinical artherosclerosis and with coronary risk factors.
Participants underwent coronary angiography examination before the minimal stenotic diameters, segment diameters, percent stenosis, plaque areas. Other parameters were analyzed by the computer-assisted Coronary Angiography Analysis System. The Framingham Risk Score for each participant was assessed. During the 1 year follow-up period, all kinds of endpoint cardiovascular events were screened. Endpoint events were defined as death from coronary heart disease, nonfatal myocardial infarction (MI) or unstable angina pectoris.
During the 1 year of follow-up period, a total of 124 participants developed an endpoint event, which was significantly associated with the Framingham Risk Score, calcium of plaques and the plaque areas (all Ps<0.05). The QCA score incorporated with the QCA parameters was related to the endpoint events. The Framingham Risk Score was combined with QCA score through logistic regression for prediction of end-point events. Data from the ROC analysis showed the accuracy of this prediction algorithm was superior to the accuracy when variables themselves were used. The event-free survival rate was inferior to the control group in participates under high risk, when being screened with this prediction algorithm (P<0.05).
The risk of cardiovascular attack in subclinical artherosclerosis individual seemed to be associated with the Framingham Risk Score, calcium of plaques and the plaque areas. When the traditional risk factors (the Framingham Risk Score) were combined with QCA, the new method could provide more prognostic information on those adults with subclinical artherosclerosis.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 12/2010; 31(12):1383-8.
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ABSTRACT: Immune reconstitution is crucially relevant for patients receiving hematopoietic stem cell transplantation (HSCT). This study was purposed to investigate the ability of α-GalCer (α-galactosylceramide), a well-known activator of natural killer T cells (NK-T), to enhance immune and hematological reconstitution. Lethally irradiated BALB/c mice were transplanted with allogeneic C57BL/6 bone marrow cells and splenocytes. α-GalCer was administered immediately after HSCT. After transplantation, the weight, activity, hairs, diarrhea and survival time of mice were observed daily; the blood routine test was performed once weekly; the donor chimeras, amount of mononuclear cells in spleen (MNC) and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD11c(+), CD40(+), CD86(+) and CD80(+) cells were detected by FACS on day 2, 7, 14, 27, 70 after transplantation. The results indicated that the MNC counts and relative levels of CD3(+) and CD4(+) in group treated with α-GalCer on day 2 after transplantation were higher than those in control group; at the same time, the detected donor chimeras were complete recipient type chimeras, then gradually transformed into donor type, on day 7 - 14 donor chimeras in α-GalCer group were enhanced significantly as compared with control group, on day 27 the chimeras in two groups were complete donor type chimeras thereafter to day 70, the MNC count and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD40(+), CD86(+) cells in α-GalCer group were obviously higher than those in control group, at the same time, the hematopoietic reconstitution in α-GalCer group was accelerated as compared with control group. It is concluded that the α-GalCer administration after allogeneic bone marrow transplantations accelerates immune and hematological reconstitution.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2010; 18(6):1542-7.
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ABSTRACT: The study was purposed to understand immunological reconstitution of peripheral immune organs after transplantation, through establishing allogeneic murine bone marrow transplantation model and detecting the kinetic change of splenocytes after transplantation. C57BL/6 mice were donors, BALB/c mice were recipients. Recipient mice were divided into irradiation group (R), irradiation plus inoculating bone marrow mononuclear cells (MNC) group (B), and irradiation plus inoculating bone marrow mononuclear cells and spleno-MNC group (S). After transplantation, the mice were examined daily for the symptoms such as weight, hunched posture, activity, ruffled fur, diarrhea, and survival. Blood routine test was done once a week, splenocyte was counted and CD3, CD4, CD8, B220, CD11c positive cell relative count was detected by FACS on day 2, 7, 14, 27, 60 after transplantation, Liver, skin and intestine were biopsied for histopathological examination before dying. The results indicated that 89% mice in S group died of acute graft-versus-host disease (aGVHD) during day 6 to 78. The spleno-mononuclear cell count quickly decreased and reached to lowest level on day 2, then gradually recovered to level of pretransplantation on day 14; CD8 and B220 positive cells decreased to lowest level on day 12, in which CD8(+) cells quickly recovered and reached to level of pretransplantation, but the B220(+) recovered most slowly and sustained to be with low level, then gradually recovered to level of pretransplantation on day 60; CD3 and CD4 positive cells decreased relatively slowly, and reached to lowest level on day 14, then both gradually recovered to level of pretransplantation on day 60; CD11c positive cell count changed unstrikingly except day 14. It is concluded that when C57BL/6 mice are donors, and BALB/c mice are recipients treated with irradiation of 7.5 Gy and inoculated with 1 x 10⁷ bone marrow MNC and 1 x 10⁷ spleno-MNC, allogeneic murine bone marrow transplantation model can be thus set up. The spleno-mononuclear cells show obviously kinetic changes after transplantation, that CD8 positive cells recover ahead of all, then followed by CD3 and CD4 positive cells, and the reconstitution of B220 positive cells is the slowest.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 08/2010; 18(4):1013-6.
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ABSTRACT: To investigate whether the gene transfer of phospholamban antisense RNA could inhibit remodeling and preserve cardiac function after myocardial infarction.
Wistar rats received a ligation of left coronary with a direct intramyocardial injection of phospholamban antisense RNA eukaryote vector PcDNA4-asPLB. The cardiac function, hemodynamics and ventricular geometry of three groups (shame, saline injection and PcDNA4-asPLB injection) were studied by echocardiography and left ventricle hemodynamic recording. The levels of phospholamban (PLB) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) were analyzed by Western blot and the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) examined by RT-PCR. The histological study was performed to evaluate the collage content and cardiomyocyte fiber size.
The PcDNA4-asPLB injection group had significantly better systolic cardiac function and diastolic function [LVEF (39.4 +/- 7.8)% vs (30.9 +/- 7.4)%, P < 0.05; dp/dt Max (1545 +/- 127) mm Hg x s(-1) vs (1172 +/- 91) mm Hg x s(-1), P < 0.05)]. Compared with saline injection, the PLB expression was inhibited by 50% in PcDNA4-asPLB injection group (PLB/beta-actin ratio, 0.28 +/- 0.07 vs 0.57 +/- 0.11, P < 0.05) and the function of SERCA2a was enhanced [(1.47 +/- 0.21) micromol x min(-1) x g(-1) protein vs (0.34 +/- 0.13) micromol x min(-1) x g(-1) protein, P < 0.05]. The expressions of ANP and BNP in the saline injection group were elevated as compared to those in the PcDNA4-asPLB injection group. Histological study also showed that the collage density and the cardiomyocyte fiber size in the saline injection group were worse than those in the PcDNA4-asPLB injection group.
Intramyocardial injection of phospholamban antisense RNA eukaryote vector PcDNA4-asPLB after myocardial infarction results in PLB expression inhibition, attenuates ventricular remodeling and improves systolic and diastolic cardiac functions.
Zhonghua yi xue za zhi 05/2010; 90(20):1389-94.
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ABSTRACT: To make clear the repertoire of Killer immunoglobulin-like receptor at the level of DNA and RNA in NK-92MI and K562 cells and compare KIRs gene genotype and expression patterns in both cells.
KIRs gene genotype, include KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, was analyzed by PCR. KIRs gene phenotype, include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR2DS1, KIR2DS3, KIR2DS2/4 gene, was determined by RT-PCR and sequencing.
KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DS2, 2DS4*003-006, 2DS5, 3DL1, 3DL2 and 3DL3 gene were present in K562 cells, but all corresponding receptors were not expressed by K562 cells. KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*001/002, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in NK-92MI cells, but only transcripts of KIR2DL1, KIR2DL3 and KIR2DS2/4 were detectable.
KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in both K562 and NK-92MI cells. All corresponding KIR receptors were not expressed by K562 cells and only partial KIRs by NK-92MI cells. The repertoire of KIRs were specific for K562 and NK-92MI cells.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 10/2009; 25(9):767-9.
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ABSTRACT: The study was aimed to identify a new leukemia related gene zo-1 from leukemia and to explore its mechanism in leukemia. Methylation specific PCR (MSP) was used for testing gene zo-1 methylation in leukemia cells. The gene zo-1 specific siRNA was designed according to its sequence, and transfected into THP-1 cell, and the cells were cultured for 48 hours before harvesting. The effect of zo-1 siRNA was monitored by RT-PCR. The cellular proliferation activity was assayed by CCK-8, the apoptosis was detected by Annexin-V-fluorescence in isothiocyanate (FITC) assay, and cell cycle was observed by propidium iodide (PI). The results indicated that the gene zo-1 in patients with acute leukemia was hypermethylated, while the gene zo-1 in healthy persons was unmethylated. The THP-1 cells with unmethylation of zo-1 gene promoter overexpressed the gene zo-1, while the Molt4 and HL-60 cells with hypermethylation of gene zo-1 promoter did not express the gene zo-1. The silenced zo-1 gene in Molt4 and HL-60 leukemia cell lines could be reactivated by demethylation treatment with 5-AZA-dC. The oligofectamine-transfected siRNA for zo-1 gene successfully inhibited the expression of gene zo-1 in THP-1 cells, but did not interfere with cell proliferation, cell cycle and apoptosis. It is concluded that gene zo-1 is a leukemia-related gene. Gene zo-1 in acute leukemia was hypermethylated, the methylation status of gene zo-1 regulates the expression of gene zo-1. Lack of gene zo-1 expression in THP-1 cells does not influence the cell proliferation, apoptosis and cell cycle, which suggests that the methylation of gene zo-1 may be involved in the genesis of acute leukemia, its mechanism is worthy to be studied.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2009; 17(5):1140-3.
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ABSTRACT: To explore ZO-1 gene expression and methylation in leukemia cells and the involvement of ZO-1 gene in leukemogenesis.
Restriction landmark genomic scanning (RLGS) was used to identify new leukemia related gene, and methylation specific PCR (MSP) for ZO-1 methylation status. ZO-1 specific siRNA was designed and prepared by in vitro transcription and transfected into K562 cells, the transfected cells were cultured for 48 hours before harvesting. The effect of ZO-1 siRNA was monitored by Northern blot. Cellular proliferation capacity was assayed by CCK-8, cell apoptosis by Annexin V-fluorescence in isothiocyanate (FITC) assay, and cell cycle by phosphatidylinositol (PI).
The intensified spots in RLGS gel were subjected to bioinformatics analysis and one of the candidate spots was proved to be ZO-1 gene. In fresh leukemia cells, Molt4 cells and HL-60 cells, ZO-1 was hypermethylated, causing it reduced or silenced. ZO-1 gene was highly expressed with no methylation in normal peripheral blood MNC and K562 cells. There was a good correlation between promoter methylation and the gene silence. The silenced gene can be re-activated by demethylation treatment with 5-AZA-dC in most leukemia cell lines. RNA interference for ZO-1 gene in K562 cells did not interfere with cell proliferation, cell cycle and apoptosis.
ZO-1 gene methylation might be involved in the tumorigenesis of acute leukemia.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 07/2009; 30(7):473-6.
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Li-Ping Dou,
Jun-Hua Liu,
Chang Wang, Jing-Hua Liu,
Hui-Yuan Kang,
Yu Jing,
Yu Zhao,
Jian Bo,
Quan-Shun Wang,
Fang-Ding Lou,
Li Yu
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ABSTRACT: This study was aimed to obtain higher efficiency in gene transfection into K562 cells and to study the role of green fluorescence protein (GFP) as a reporter system. Transfection efficiencies with different methods including electroporation and lipofectamine 2000 transfection, were observed and calculated under fluorescent microscopy by using GFP as a reporter system. The results showed that the transfection efficiency with electroporation (10%) was higher than that with lipofectamine 2000 (1%). In conclusion, the electroporation is a more ideal method for introduction of foreign gene into K562 cells. GFP can be used as a reporter system for optimizing transfection of K562 cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2008; 16(5):1174-6.
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ABSTRACT: To evaluate the feasibility of gene therapy using bcl-2 as target in multiple drug resistance of leukemia, the small interfering RNA eukaryotic expression vector specific to human bcl-2 gene was constructed by gene recombination, then transfected into HL-60/VCR cells. Stable transfectants were obtained by G418 screening. The growth curve and drug sensitivity were detected by using MTT. The expression of Bax and ZNRD1 was analyzed by Western blot. The results showed that mU6pro-bcl-2 siRNA was successfully constructed and transfected into HL-60/VCR cells. The IC(50) of transfected cells to vincristine and adriamycin was significantly reduced as compared with that of the control. The expression of ZNRD1 in transfected cells was decreased as compared with that of the control, while Bax not. It is concluded that the bcl-2 siRNA restores the sensitivity of HL-60/VCR cells to conventional chemotherapeutic agents to a certain degree.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 01/2006; 13(6):1010-3.
