1.Expression of Hepatitis C Virus NS5A Gene In E.coli and Its Application in HCV Antibody Detection
Hua, RUAN ; Jin-rong, GAO ; Lin-Bai, YE ; Jing-ping, XU ; Xiao-ling, WANG ; Yue-e, ZHAO ; Zheng-hui, WU
Virologica Sinica 2001;16(2):190-192
Full-length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template. The amplified fragment (about 1.34 kb) was cloned into plasmid pQE32, and the recombinant plasmid pQENS5A was expressed in JM109 strain. The NS5A protein was purified by NiSO4 metal chelating resin, and characterized by Western-blot. Its antigenecity was determined by ELISA. The positive detection rate of anti-NS5A was 75% (69/92) in ninety-two clinic sera. The positive rate of anti-NS5A was 82.5% (33/40) in fourty positive standand sera, and the negative rate of anti-NS5A was 100% (40/40) in fourty negative standand sera. The results showed that the Full-length NS5A proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera, we suggested that NS5A protein was a useful antigen for blood screening.
2.Efficacy of intracutaneous methylene blue injection for moderate to severe acute thoracic herpes zoster pain and prevention of postherpetic neuralgia in elderly patients.
Ji-Zheng CUI ; Jin-Wei ZHANG ; Yun ZHANG ; Zheng-Liang MA
Journal of Southern Medical University 2016;36(10):1377-1381
OBJECTIVETo evaluate the clinical efficacy of intradermal injection of methylene blue for treatment of moderate to severe acute thoracic herpes zoster and prevention of postherpetica neuralgia in elderly patients.
METHODSSixty-four elderly patients with herpes zoster were randomized to receive a 10-day course of intradermal injection of methylene blue and lidocaine plus oral valaciclovir (group A, 32 cases) and intradermal injection of lidocaine plus oral valaciclovir (group B).Herpes evaluation index, pain rating index, incidence of postherpetic neuralgia, and comprehensive therapeutic effect were compared between the two groups at 11, 30 and 60 days after the treatment.
RESULTSThe baseline characteristics were comparable between the two groups (all P>0.05). Compared with that in group B, the time for no new blister formation, blister incrustation and decrustation, and pain relief was significantly shortened in group A (P<0.05) with also obviously lower pain intensity after the treatment. The incidence of postherpetic neuralgia was significantly lower in group A than in group B at 30 days (P<0.05), but not at 60 and 90 days after the treatment. The total clinical response rate was 93.8% in group A, much higher than that in group B (62.5%, P<0.05).
CONCLUSIONIntradermal injection of methylene blue can effectively shorten the disease course, reduce the pain intensity and prevent the development of postherpetic neuralgia in elderly patients with herpes zoster.
Acyclovir ; administration & dosage ; analogs & derivatives ; therapeutic use ; Aged ; Herpes Zoster ; complications ; Humans ; Incidence ; Injections, Intradermal ; Lidocaine ; administration & dosage ; therapeutic use ; Methylene Blue ; administration & dosage ; therapeutic use ; Neuralgia, Postherpetic ; therapy ; Pain Measurement ; Valine ; administration & dosage ; analogs & derivatives ; therapeutic use
3.Construction of a GFP/Puro double-labeled lentiviral vector containing CK8 interfering RNA and its effect on cell apoptosis in vitro.
Yanchao JIN ; Ronghua YIN ; Weiwei ZHENG ; Xiangzhen KONG ; Yiqun ZHAN ; Xiaoming YANG ; Changyan LI
Journal of Southern Medical University 2013;33(12):1761-1765
OBJECTIVETo construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the effects of CK8 silencing on cell apoptosis.
METHODSThe siRNA sequences of CK8 were inserted into the lentiviral expression vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24 and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis induced by cisplatin was detected with Annexin V/PI staining.
RESULTS AND CONCLUSIONWe successfully constructed CK8 interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced apoptosis.
Apoptosis ; Cell Line ; Down-Regulation ; Genetic Vectors ; Humans ; Keratin-8 ; genetics ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Small Interfering ; Transfection
4.Effects of acute myeloid leukemia cell supernatant on the proliferation and apoptosis of CD4+ and CD8+ T cell subsets.
Xing-Bing WANG ; Jun LIU ; Yan-Li HE ; Jun-Xia GU ; Jin-E ZHENG ; Jun-Xia YAO ; Jin YANG ; Xiao-Qing LI ; Shi-Ang HUANG
Journal of Experimental Hematology 2006;14(3):455-459
To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis
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physiology
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CD4-Positive T-Lymphocytes
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cytology
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immunology
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CD8-Positive T-Lymphocytes
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cytology
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immunology
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Cell Proliferation
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Cells, Cultured
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Culture Media
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HL-60 Cells
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Humans
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Leukemia, Myeloid, Acute
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immunology
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pathology
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T-Lymphocytes
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cytology
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Tumor Cells, Cultured
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U937 Cells
5.Effect of CCL23/myeloid progenitor inhibitory factor 1 (MPIF-1) on the proliferation, apoptosis and differentiation of U937 cells.
Qing GONG ; Jin-E ZHENG ; Wei LIU ; Li-Qiong LIU ; Yue-Ying LI ; Shi-Ang HUANG
Journal of Experimental Hematology 2007;15(3):496-500
CCL23 is a human CC chemokine with potential suppression effects on both human and murine myeloid progenitor cells both in vitro and in vivo, and only expressed and released by dendritic cells differentiated from monocytes in blood cells. However, recent study has shown that CCL23 was over-expressed in bone marrow and peripheral blood cells from pediatric patients with acute myeloid leukemia (AML). In order to investigate the effects of CCL23 on the development, therapy and prognosis of leukemia, the U937 cells, a leukemic cell strain, were adopted and cultured with rhCCL23 for 72 hours. The cell proliferation and apoptosis rate were detected by Cell Counting Kit-8 and FITC-AnnexinV/PI respectively; the morphologic changes and the expression of CCR1 (the only receptor of CCL23 known by now) were observed during the differentiation process. The results showed that no obvious effect on the proliferation, apoptosis and differentiation of U937 was found by using CCL23 alone (P > 0.05), but cultured in combination with CCL23 and PMA, the differentiation of U937 cells were promoted remarkably, during which the CCR1 expression increased (P < 0.05). It is concluded that CCL23 alone did not inhibit the proliferation and differentiation of U937, while its use in combination with PMA may possess synergistic effect on inducting differentiation of U937 through the increase of receptor CCR1 expression.
Apoptosis
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physiology
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Cell Proliferation
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drug effects
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Cell Transformation, Neoplastic
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drug effects
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Chemokines, CC
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pharmacology
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Humans
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U937 Cells
6.Cross-lineage expression in 505 patients with acute lymphoblastic leukemia by multiparametric flow cytometry analysis.
Xing-Bing WANG ; Wen DU ; Liang XIA ; Jin-E ZHENG ; Jun LIU ; Yan-Li HE ; Zi-Min SUN ; Shi-Ang HUANG
Journal of Experimental Hematology 2009;17(6):1419-1423
The expression of immunological markers of one hematopoietic lineage on the abnormal cells of another lineage (cross-lineage expression) is a known feature of leukemia. The present study was aimed to investigate the cross-lineage expression in ALL cells. The cross-lineage expression in ALL cells from 505 patients was detected by flow cytometry using 23 monoclonal antibodies (McAbs) in triple staining combinations. The results showed that in whole ALL, the expression of myeloid antigens occurred in 56.4% of the cases, and CD13 was the most frequently expressed myeloid marker (32.7%) followed by CD33 (29.5%), CD15 (19.2%) and CD11b (7.7%). CD13/CD33 expressions were more frequent in CD34(+) cases than in CD34(-) cases. In B-ALL, T-cell antigen CD4, CD5, CD7 and CD2 were found in 27 (6.3%), 12 (2.8%), 8 (1.9%), and 6 (1.4%) cases respectively, and CD7(+), CD2(+) and CD4(+) cases commonly expressed CD13/CD33. In T-ALL, B-cell antigen cCD79a, CD19 and CD22 were found in 6 (8.1%), 5 (6.8%), and 2 (2.8%) cases respectively, and all of CD19(+) and CD22(+) cases were all accompanied with CD13/CD33. It is concluded that cross-lineage expression in ALL mostly exists in the immature stages, ALL cells more frequently express phenotypes B(+)M(+), T(+)M(+) and occasionally B(+)T(+)M(+), but B(+)T(+)M(-) phenotype is extremely rare.
Adolescent
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Adult
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Aged
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Antigens, CD
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metabolism
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Child
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Child, Preschool
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Female
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Flow Cytometry
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methods
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Humans
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Immunophenotyping
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Infant
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Male
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Middle Aged
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Precursor Cell Lymphoblastic Leukemia-Lymphoma
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immunology
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metabolism
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Young Adult
7.Changes of sperm chromatin in oligo-astheno-teratozoospermia syndrome patients after treated by integrated Chinese and Western medicine.
Jiang-Ping DU ; Shu-Wen YANG ; Jin-E MEN ; Xia WANG ; Xiang-Yang ZHANG ; Hai-Ping ZHENG ; Yan LI
National Journal of Andrology 2008;14(4):334-337
OBJECTIVETo observe the changes of sperm chromatin in patients with oligo-astheno-teratozoospermia (OAT) syndrome after treated by integrated Chinese and Western medicine.
METHODSSixty patients with OAT syndrome were treated by integrated Chinese and Western medicine for 3 months. Their sperm samples were collected before and after the treatment, subjected to acridine orange staining and analyzed by fluorescent microscopy, flow cytometry and sperm routine detection.
RESULTSSignificant differences were shown in the master-group sperm signals (P < 0.01) and at and COMPalphat (P < 0.05) by flow cytometry, as well as in the green and the red groups (P < 0.05) by fluorescent microscopy before and after the treatment. Changes in sperm concentration, motility, vitality and deformity were noted after the treatment, with statistic difference between pre- and post-treatment (P < 0.05) except in forward sperm concentration.
CONCLUSIONTreatment by integrated Chinese and Western medicine can improve sperm chromatin in patients with OAT syndrome. Flow cytometry, along with fluorescent microscopy and sperm routine detection, plays an important role in the evaluation of male infertility therapy.
Adult ; Chromatin ; metabolism ; Combined Modality Therapy ; Flow Cytometry ; Humans ; Male ; Medicine, Chinese Traditional ; Microscopy, Fluorescence ; Oligospermia ; therapy ; Sperm Count ; Sperm Motility ; Spermatozoa ; metabolism ; pathology
8.Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation.
Zhao-Dong ZHONG ; Yi LUO ; Ping ZOU ; Jin-E ZHENG ; Jun-Xia YAO ; Shi-Ang HUANG ; Dong-Feng ZHOU ; Yong YOU
Chinese Medical Journal 2012;125(9):1669-1671
A female patient diagnosed with acute myelocytic leukemia M5a (AML-M5a) relapsed 986 days after her allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an unrelated male donor with matched human leukocyte antigen (HLA). Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer (CIK) cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion (DLI) and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.
Adult
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Cytokine-Induced Killer Cells
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transplantation
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Female
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Hematopoietic Stem Cell Transplantation
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Humans
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Leukemia
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therapy
9.Regulation of tissue factor expression in brain microvascular endothelial cells by PLA nanoparticles coating NF-kappaB decoy oligonucleotides.
Yu HU ; Hua-fang WANG ; Wang-qiang SUN ; Chang-sheng XIE ; Wen-ning WEI ; Jin-e ZHENG ; Jun-xia YAO
Chinese Journal of Hematology 2005;26(9):534-538
OBJECTIVETo investigate a new strategy of polylactic acid (PLA) nanoparticles delivery system coating nuclear factor-kappaB (NF-kappaB) decoy oligonucleotides (ODNs) for inhibiting TF expression in cultured brain microvascular endothelial cells(BMECs).
METHODSPLA nanoparticles coating FITC-labeled NF-kappaB decoy ODNs were formulated by nano-deposition method and the characteristics of nanoparticles were detected. BMECs were isolated and cultured in vitro. The cellular uptake and intracellular localization of nanoparticles in BMECs was detected by flow cytometry and confocal microscopy. Changes in the expressions of TF and nuclear protein P65 were examined by RT-PCR and Western blot in NF-kappaB decoy ODNs transfected BMECs by LPS stimulation.
RESULTSThe decoy-nanoparticles obtained were uniform spherical particles with an effective diameter of 162.1 nm and a polydispersity index of 0.118. NF-kappaB decoy ODNs encapsulated in nanoparticles could be released in a controlled manner in phosphate-buffered saline for up to 28 days. It was observed that the cellular uptake of nanoparticles were increased with the time of incubation and the concentration of nanoparticles in the medium. Nanoparticles localized mainly in the BMECs cytoplasm. LPS-induced upregulation of TF transcription was inhibited by NF-kappaB decoy ODNs transfection but not by missense ODNs transfection. Furthermore, changes in the transcription level of TF were paralleled by a reduction of capacity of P65 in nuclear extract of NF-kappaB decoy ODNs transfected cells.
CONCLUSIONSThese findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.
Animals ; Brain ; blood supply ; Capillaries ; cytology ; Cells, Cultured ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; Lactic Acid ; NF-kappa B ; genetics ; Nanoparticles ; Oligonucleotides ; genetics ; Polyesters ; Polymers ; Rats ; Thromboplastin ; genetics ; metabolism ; Transfection
10.Karyotyping and immunophenotyping analyses of the CD34+ CD38- cells isolated from human umbilical cord blood.
Hong TIAN ; Jin-e ZHENG ; Fei-li GONG ; Xing-bing WANG ; Shi-ang HUANG ; Zhong CHEN
Chinese Journal of Hematology 2005;26(5):257-260
OBJECTIVETo cultivate hematopoietic stem/progenitor cells (CD34(+)CD38(-)) isolated from umbilical cord blood (UCB) long for the observation of cell growth and expansion in vitro, surface marker expression, and chromosomal complements.
METHODSBy flow cytometry CD34-FITC and CD38-PE labeled CD34(+) and CD38(-) stem/progenitor cells were isolated from UCB. The cells were cultivated in vitro for 6 months in a stem cell culture system with addition of six kinds of cell growth factors (IL-3, IL-6, GM-CSF, Epo, SCF, IGF-1). One month after cultivation, cultured cells were investigated for surface marker expression by flow cytometry and karyotype by G banding method.
RESULTSAfter 7-12 days cultivation, the CD34(+)CD38(-) stem/progenitor cells began proliferation. The proliferation rate and the peak proliferation duration were greater in 1 cell/well cultivation conditions than in 10 cells/well. The cells remained CD34(+)CD38(-) and their karyotypic characteristics remained unchanged.
CONCLUSIONCD34(+)CD38(-) stem/progenitor cells from UCB may provide a larger than original amount of stem/progenitor cells for transplantation after long-term cultivation in vitro.
ADP-ribosyl Cyclase 1 ; immunology ; Adult ; Antigens, CD34 ; immunology ; Cell Proliferation ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunophenotyping ; Karyotyping