Influence on the immune function of the human peripheral blood mononuclear cells transfected by retrovirus-mediated HSV-tk gene.
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Cytokines↗
Biomedical subjects
Zhen Cai
Publications and source records attributed to Zhen Cai.
Homoharringtonine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of telomerase.
Homoharringtonine (HHT), first isolated from the Chinese evergreen Cephalotaxus Harringtonia, has been shown inhibiting activity in leukemia in initial studies in China and in later studies in the US, but the detailed mechanism of action is still unclear. The goal of the experiments shown here is to explore the effect of HHT on the telomerase activity and apoptosis of human leukemia HL-60 cells. The telomerase activity of HL-60 cells was examined by the telomeric repeat amplification protocol (TRAP)--an enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed by morphological observation, DNA agarose gel electrophoresis, flow cytometry (FCM), and TdT-mediated dUTP-biotin nick end labeling (TUNEL). After treatment with HHT at 5-500 microg/l for 48 hours, the level of telomerase activity in HL-60 cells decreased in a dose-and time-dependent manner. Simultaneously, HL-60 cells underwent apoptosis. In conclusion, our data suggest that HHT can inhibit the telomerase content of HL-60 cells effectively and induce apoptosis.
Apoptosis↗
Heat-inducible autolytic vector for high-throughput screening.
In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage lambda was placed downstream of heat-inducible promoters, lambda cI857/pR promoter and its mutant, c1857/pR(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter; cI857/pR, the SRRz lysis cassette was expressed by temperature up-shift from 28 degrees to 38 degrees C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter beta3-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/pR(M) was utilized to allow bacteria growth at 35 degrees C and lysis at 42 degrees C. However; this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further inserted into the multiple cloning sites of the autolytic vector containing cI857/pR, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demonstrating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.
Bacillus subtilis↗
[Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine].
OBJECTIVE: To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation. METHODS: The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay. RESULTS: A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control. CONCLUSION: It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.
Antibodies, Monoclonal↗
[Study on unrelated donor allogeneic bone marrow transplantation with Bu-CY2 conditioning regimen for myelodysplastic syndrome].
OBJECTIVE: To evaluate the efficacy and safety of Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation (BMT) with unrelated donor for myelodysplastic syndrome. METHODS: Six patients received chemotherapy regimen of busulfan (Bu) and cyclophosphamide (CY) before allogeneic BMT (Bu 4 mg . kg(-1) . d(-1), -7 d - -4 d, CY 60 mg . kg(-1) . d(-1), -3 d - -2 d). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E(1)was used in prophylactic regimen for hepatic veno-occlusive disease. RESULT: Neutrophil count began to be higher than 0.5 x 10(9)/Lat the 18th day after BMT. Platelet count began to be higher than 20 x 10(9)/Lat the 21st day after BMT. Disease-free survival in the six patients was 27 months. CONCLUSION: Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation with unrelated donor is an effective therapy for patients with myelodysplastic syndrome.
Adolescent↗
[Inhibition effect of fludarabine on the growth of myelodysplastic syndrome cell line MUTZ-1 and its mechanism].
OBJECTIVE: To investigate the inhibition effect of fludarabine on the growth of human MDS-RAEB cell line MUTZ-1 and to explore the possible cellular and molecular mechanism. METHODS: The apoptosis of MUTZ-1 cells induced by fludarabine was studied by transmission electron microscope, MTT assay, DNA ladder test, flow cytometry and RT-PCR method. RESULT: Treatment with fludarabine remarkably inhibited the growth of MUTZ-1 cells, the 24 h IC(50), 48 h IC(50) and 72 h IC(50) of fludarabine for MUTZ-1 cells were 137.65 mg/L, 6.27 mg/L and 0.51 mg/L, respectively. Fludarabine inhibited the growth of MUTZ-1 cells in a dose-dependent and time-dependent manner. After treated by fludarabine (1 mg/L-16 mg/L)for 24 h, MUTZ-1 cells showed the typical features of apoptosis. After fludarabine treatment the mRNA expression of Bcl-2, Bax, survivin, XIAP, cIAP-1 and cIAP-2 was not changed, but the mitochondrial membrane potential (MMP) was decreased. CONCLUSION: With a certain range of dose fludarabine (1 mg/L-16 mg/L)could inhibit MUTZ-1 cell growth by inducing cells apoptosis. MMP may play a certain role in apoptosis of MUTZ-1 cells induced by fludarabine.
Antineoplastic Agents↗
[Expression of survivin mRNA in HHT-induced cell apoptosis of hematological malignancy cell lines].
OBJECTIVE: To investigate the expression of survivin mRNA in hematological malignancy cells and its correlation with HHT-induced cell apoptosis. METHODS: Hematological malignancy cell lines MUTZ-1, K562, Jurkat, RPMI and HL60 were treated with HHT in vitro. Cell apoptosis was observed by flow cytometry (FCM) and the expression of surviving and XIAP mRNA was evaluated with semi-quantitative RT-PCR. RESULT: The expression of survivin mRNA on cell lines was negatively correlated to HHT-induced cell apoptotic rate (r=-0.980, P=0.003). There were no significant differences in XIAP expression among these 5 cell lines. CONCLUSION: Survivin could be used as a new marker for drug-sensitivity and a new target for treatment of hematological malignancies.
Apoptosis↗
[Relationship between the IFN-gamma producing cells specific for recipient and acute graft versus host disease].
OBJECTIVE: To study the relationship between the IFN-gamma producing cell specific for recipient (IFN-gamma-PCSR) in allogeneic stem cell transplantation (allo-HSCT) and acute graft versus host disease (aGVHD). METHODS: In 37 consecutive HLA-identical sibling allo-HSCT pairs, peripheral blood mononuclear cells (PBMNC) from donors before allo-HSCT and recipients after allo-HSCT were taken as responder cells (RC), and PBMNC from recipients before allo-HSCT as allogeneic stimulator cells (allo-SC) in mixed lymphocyte reaction (MLR). IFN-gamma-PCSR in PBMNC were assayed using enzyme linked immunospot assay (ELISPOT). RESULTS: Pretransplantation frequencies of IFN-gamma-PCSR in donor PBMNC were significantly higher in aGVHD group than in non-aGVHD group (P < 0.01) and IFN-gamma PCSRs (>or= 20/2 x 10(5)RC) were significantly associated with the occurrence of grade II-IV GVHD (P < 0.05). Compared with that before allo-HSCT, IFN-gamma PCSR in PBMNC of aGVHD patients was significantly increased (P < 0.05). When PBMNC from aGVHD patients reacted with donor PBMNC, the IFN-gamma PC was significantly lower than that with recipient PBMNC before allo-HSCT. Longitudinal analysis of IFN-gamma PCSR following allo-HSCT showed that compared with that in patients without aGVHD, the IFN-gamma PCSR were significantly higher in patients with that in aGVHD on day +14 (P < 0.01) and day +28 (P < 0.01), respectively. After immunosuppressive therapy for 7 days, IFN-gamma PC declined significantly (P < 0.05). CONCLUSION: The recipient-specific IFN-gamma PC is closely correlated with the allo-response during allo-HSCT and may be helpful for the prediction, diagnosis and monitoring of aGVHD.
Adolescent↗
Correlation between survivin mRNA expression and homoharringtonine induced apoptosis of malignant hematopoietic cells.
BACKGROUND: The inhibitor of apoptosis (IAP) gene family is involved in the suppression of apoptotic cell death as well as an increasing number of seemingly unrelated cellular functions. It is not known, however, whether IAP expression in malignant hematopoietic cells is affected by chemotherapeutic agents such as homoharringtonine (HHT). In this study, we investigated mRNA expression levels of IAPs, especially survivin, in various hematopoietic cell lines in relation with apoptosis induced by HHT. METHODS: Semiquantitative reverse transcriptase polymerase chain reaction was used to determine survivin mRNA levels. Cell apoptosis was examined by flow cytometry. Cell viability and proliferation assay was evaluated by MTT. The experiments were performed on the malignant hematopoietic cell lines MUTZ-1, K562, Jurkat, RMPI and HL60, with or without survivin antisense-oligodeoxynucleotides (AS-ODN) and HHT. RESULTS: The expression levels of survivin mRNA were variable in the cell lines and negatively correlated to HHT induced cell apoptosis. Survivin AS-ODN significantly decreased mRNA level of survivin, but not those of bax and bcl-2. Survivin also inhibited MUTZ-1 cell growth and induced apoptosis in a dose dependent manner. AS-ODN and HHT showed synergistic effect on MUTZ-1 cell growth. CONCLUSION: The apoptotic effect of HHT on the hematopoietic cell lines is associated with decreased level of survivin expression. Survivin could be a new marker for drug sensitivity and a new target for cancer treatment.
Anemia, Refractory, with Excess of Blasts↗
[Tandem autotransplants of peripheral blood stem cells following sequential high-dose CHOEP chemotherapy for aggressive lymphoma].
The purpose of this study was to evaluate the effecacy and safety of CHOEP mobilization regimen, and the effect and tolerance of sequential chemotherapy combined with tandem autotransplants of peripheral blood stem cells for aggressive lymphoma. The clinical data of 5 patients with recurrent, aggressive lymphoma treated with of sequential chemotherapy combined with tandem autotransplants were analyzed retrospectively. The patients included 1 HD and 4 NHL. Mobilization regimen was CHOEP combined with G-CSF 5 microg/(kg x d). The conditioning regimen for the tandem transplantation was high-dose CHOEP. The interval of the tandem autotransplantation was 9 (5 - 31) weeks. In tandem autotransplant, the cell number of MNC transfused was 3.05 (1.91 - 4.14) x 10(8)/kg and 3.55 (2.23 - 6.0) x 10(8)/kg; CD34(+) cells were 4.11 (2.59 - 4.94) x 10(6)/kg and 5.70 (2.77 - 10.6) x 10(6)/kg; CFU-GM was 2.96 (2.01 - 4.54) x 10(5)/kg and 2.44 (1.78 - 2.9) x 10(5)/kg respectively (P > 0.05). The results showed that all patients gained prompt and sustained hemotopoietic reconstitution. The interval of ANC >or= 0.5 x 10(9)/L was 10 (8 - 12) days and 10.5 (9 - 12) days; Pt >or= 2.0 x 10(9)/L was 11 (10 - 14) days and 12.5 (10 - 15) days respectively (P > 0.05). Four patients survived, three patients among them were alive in disease-free for median of 46 (9 - 88) months. The overall survival was 80%, and the disease-free survival was 60%. In conclusion, the method of sequential high-dose CHOEP chemotherapy combined with autotransplants of peripheral blood stem cells in tandem for aggressive lymphoma is probably safe and effective.
Adolescent↗
[Purification of human endothelial overexpressed lipopolysaccharide-associated factor 1 protein].
OBJECTIVE: To investigate the feasibility of obtaining of a highly pure protein of human endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) with metal chelation chromatography. METHODS: Inclusion bodies of the E. coli transformed with EOLA1 gene were extracted and washed with BugBuster Protein Extraction Reagent. The primary purified products were purified by His. Bind Resin Chromatography under denaturing condition and dialyzed for renaturation, and then were analyzed with SDS-PAGE, Western blotting and peptide mass fingerprinting (PMF). RESULTS: EOLA1 was mainly expressed in E. coli as insoluble inclusion bodies. The protein content in the primary extracted inclusion bodies accounted for over 75%, and it accounted for more than 90% after chromatography and renaturation. It was indicated by PMF that the targeted protein peptide overlaid many of designed protein peptide. CONCLUSION: The method of EOLA1 protein purification and renaturation was convenient and efficient, and by this method sufficient amount of highly pure EOLA1 protein could be obtained for the preparation of EOLA1 monoclonal antibody and for the study of its gene function.
Antibodies, Monoclonal↗
Human cytomegalovirus induces alteration of beta-actin mRNA and microfilaments in human embryo fibroblast cells.
OBJECTIVE: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on beta-actin mRNA and microfilaments. METHODS: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, beta-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. RESULTS: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of beta-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. CONCLUSION: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of beta-actin mRNA and the microfilaments.
Actin Cytoskeleton↗
Application of proteomics in the study of tumor metastasis.
Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular and cellular mechanisms underlying tumor metastasis are still elusive. The identification of protein molecules with their expressions correlated to the metastatic process would help to understand the metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions and clinical management of cancer. Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. Proteomic technology has been widely used in biomarker discovery and pathogenetic studies including tumor metastasis. This article provides a brief review of the application of proteomics in identifying molecular factors in tumor metastasis process. The combination of proteomics with other experimental approaches in biochemistry, cell biology, molecular genetics and chemistry, together with the development of new technologies and improvements in existing methodologies will continue to extend its application in studying cancer metastasis.
Gene Expression Profiling↗
[Inhibition effect of arsenic trioxide on the growth of human MDS cell line MUTZ-1 cells].
OBJECTIVE: To investigate the inhibition effect of arsenic trioxide (AS(2)O(3)) on the growth of human MDS-RAEB cell line MUTZ-1 cells and to explore the possible cellular and molecular mechanisms. METHODS: The apoptosis and differentiation of MUTZ-1 cells induced by AS(2)O(3) solution of different concentrations were studied with cell morphology, MTT, DNA fragmentation assay, RT-PCR, Nitroblue tetrazolium (NBT) reduction method and flow cytometry. RESULT: (1) Low concentration ofAS(2)O(3) (0.05 - 0.25 micromol/L) had no marked growth inhibition effect on MUTZ-1 cells; after 14 d treatment, it down-regulated the expression of positive cell differentiation antigens CD38, CD7, CD10, HLA-DR (P<0.05), but did not up-regulate the expression of CD11b (P>0.05). (2) After treatment with 1.0 - 20.0 micromol/L of AS(2)O(3), MUTZ-1 cells presented typical features of apoptosis with a dose dependent manner (r=-0.999, P<0.05). The expression of bcl-2 mRNA and the ration of bcl-2/bax were decreased after AS(2)O(3) treatment (P<0.05). CONCLUSION: Low concentration of (2)O(3) may have partial differentiation inducement on MUTZ-1 cells. With a certain range of dose (1.0 - 20.0 micromol/L), (2)O(3) can induce apoptosis of MUTZ-1 cells. (2)O(3) can significantly down-regulate bcl-2 and it might be one of the mechanisms of (2)O(3) treatment.
Antineoplastic Agents↗
[A comparison of clinical outcomes between HLA allele matched and 1 - 2 alleles mismatched unrelated allogeneic bone marrow transplantations].
OBJECTIVE: To compare the clinical outcomes between HLA allele matched (HLA-M) and 1 approximately 2 alleles disparity mismatched (HLA-mis) unrelated allogeneic bone marrow transplantation (URD-BMT). METHODS: Thirty-nine patients received HLA-M and 21 received HLA-mis URD-BMT for the treatment of acute leukemia, chronic myeloid leukemia in chronic phase (CP) and myelodysplastic syndromes (MDS) in our hospital between November 1998 and December 2002. Conditioning regimen was Bu 16 mg/kg plus CTX 120 mg/kg, and mycophenolate mofetil (MMF), CsA and MTX were given to prevent aGVHD. RESULTS: Thirty-eight of the HLA-M group and 18 of the HLA-mis group were engrafted successfully. The median follow-up duration was 11 (2.5 - 52.0) months for HLA-M group and 9 (2 - 46) months for HLA-mis group. The 3-year probabilities of disease-free survival (DFS) for HLA-M and HLA-mis group were (79.2 +/- 7.1)% and (45.8 +/- 15.5)%, respectively (P < 0.05). Grade II - IV aGVHD occurred in 10 (26.3%) patients in HLA-M group and 6 (33.3%) in HLA-mis group, respectively (P > 0.05). CONCLUSION: URD-BMT is an effective modality for the treatment of leukemia and MDS. The outcome after URD-BMT can be optimized by matching the HLA-A, B and DR alleles between the donor and recipient.
Adolescent↗
[Expression and significance of apoptosis protein inhibitor survivin and XIAP, in patients with myelodysplastic syndromes and in the cell line MUTZ-1].
OBJECTIVE: To investigate the expression of apoptotic protein inhibitors, survivin and XIAP, in patients with myelodysplastic syndromes (MDS) and in the cell line MUTZ-1, as well as to explore the possible mechanisms of homoharringtonine (HHT) in the treatment of MDS. METHODS: Bone marrow samples from 47 patients with de novo MDS at diagnosis were examined and bone marrow samples from 15 normal donors were used as control. A MDS-RAEB cell line MUTZ-1 was used as in vitro model. Detection of apoptotic cells and cell cycle analysis were performed with flow cytometry (FACS). The expression of apoptotic protein inhibitor survivin and XIAP in the MDS cells were detected by RT-PCR technique. MUTZ-1 were treated with antisense oligodeoxynucleotide (AS-ODNs) of survivin and or HHT, the effects were evaluated by cell viability and cell apoptosis. RESULTS: Survivin mRNA positive rate in MDS were significantly higher than that in normal controls (38.3% and 0, respectively, P < 0.01), and the positive rate in high risk group (RAEB, RAEBT and CMML) was significantly higher than that in RA/RAS group (53.6% and 16.7%, respectively, P < 0.05). XIAP was expressed in all untreated MDS and healthy controls. XIAP mRNA expression in high risk group was significantly higher than that in RA/RAS subtypes and healthy controls (1.55 +/- 0.34, 0.74 +/- 0.24, and 1.01 +/- 0.28, respectively, P < 0.01). However, XIAP mRNA expression was significantly lower in RA/RAS subtypes than in healthy control (0.74 +/- 0.24 and 1.01 +/- 0.28, P < 0.054). Apoptosis peak detected by FACS analysis and positive Annexin V FITC staining on cell membrane indicated that HHT could induce MUTZ-1 cell undergoing apoptosis in dose- and time-dependent manners. Treatment of MUTZ-1 cells with HHT revealed that HHT could significantly down-regulate survivinexpression but had no significant effect on XIAP expression in the cells. AS-ODNs of survivin could inhibit MUTZ-1 cells growth, induce them to apoptosis and sensitize them to HHT. CONCLUSION: The expression levels of survivin; Institute of Hematology, Oncology and Tumor Immunology, Robert Roessle Clinic, Humboldt University, Berlin, Germany (Wolf Dieter Ludwig, Christian Wuchter) and XIAP vary in different subtypes of MDS patients, suggesting that the proteins may play an important role in the pathogenesis of the disease. Down-regulation of survivin in MUTZ-1 cells may be one of the mechanisms that HHT induces apoptosis of MDS cells.
Apoptosis↗
[Electrically stimulated olfactory evoked potential in olfactory-lesioned rabbit].
OBJECTIVE: To develop olfactory dysfunctioned animal model by injuring olfactory blub(s) and to investigate the characteristics of olfactory evoked potential. METHODS: The changes of OEP in rabbits were observed at 24 h, 48 h and 1 w after olfactory bulb(s) were electrolytically injured. The pathological and ultrastructural changes of olfactory mucosa and olfactory bulb were also observed. RESULTS: When unilateral olfactory bulb was damaged, N2 vanished with elongated latencies and lowered amplitudes of N1 and P1. When both olfactory bulbs were damaged, N1 and N2 were absent, only P1 could be recorded. The latency was extended obviously and the amplitude had a big change. Histopathology and ultrastructure showed that there were a lot of inflammatory cells and degenerated neurons scattered around OB 24 hours to 48 hours after injure. It was observed that the olfactory cilia had distraction perversion and colliquefaction changes. CONCLUSION: Different degree of olfactory bulb injury caused different effect on OEPs. These changes of potential were based on the histopathology and ultrastructure.
Animals↗
[The effects of mycophenolic acid on the maturation and immunologic function of murine bone marrow-derived dendritic cells].
Mycophenolate mofetil (MMF) is a newly developed immunosuppressor, widely used in allogeneic bone marrow transplant. The purpose of this study was to evaluate the effects of mycophenolic acid (MPA), the active metabolite of MMF in vivo, on the maturation and immunologic function of murine bone marrow-derived dendritic cells (DC), and to explore the underlying mechanisms of MMF in graft versus host disease. Cultured DC were treated with MPA at doses of 0.01 and 0.1 micro mol/L. The immunophenotype of DC in control and treated groups was analyzed by flow cytometry. The capability of antigen presentation and the stimulatory activity of the DC on allogeneic T cells were tested by incorporation of (3)H-TdR and mixed lymphocyte reaction respectively. IL-12 production in culture supernatant and the levels of Th1/Th2 cytokines such as IL-2, IFN-gamma, IL-4 and IL-10 in mixed lymphocyte reaction (MLR) supernatant were examined by ELISA assay. The results showed that DCs cultured in the presence of MPA expressed low levels of CD40, CD80 and CD86, and exhibited weak activity in stimulating the proliferation of allogeneic T cells and antigen presenting function with a concurrent reduction of IL-12 production. Allogeneic T cells stimulated by MPA-treated DC expressed higher levels of Th2 cytokines such as IL-4 and IL-10 but lower levels of Th1 cytokines such as IL-2 and IFN-gamma than those stimulated by DC without MPA treatment. It is concluded that MPA, and hence MMF, exerts a negative effect on the maturation and immunologic functions of DC in culture, and drives a shift of Th1 to Th2 cytokines in MLR.
Animals↗