¹8F-FDG uptake by spleen helps rapidly predict the dose level after total body irradiation in a Tibetan minipig model.

OBJECTIVES: To investigate whether (18)F- FDG uptake can be applied in dosimetry to facilitate the rapid and accurate evaluation of individual radiation doses after a nuclear accident. METHODS: Forty-eight Tibetan minipigs were randomised into a control group (n = 3) and treatment groups (n = 45). (18)F-FDG combined positron-emission tomography and computed tomography (PET/CT) were carried out before total body irradiation (TBI) and at 6, 24 and 72 h after receiving TBI doses ranging from 1 to 11 Gy. Spleen tissues and blood samples were also collected for histological examination, apoptosis and blood analysis. RESULTS: Mean standardised uptake values (SUVs) of the spleen showed significant differences between the experimental and the control groups. Spleen SUV at 6 h post-irradiation showed significant correlation with radiation dose; Spearman's correlation coefficient was 0.97 (P < 0.01). Histological observations showed that damage to the splenic lymphocyte became more severe with an increase in the radiation dose. Moreover, apoptosis was one of the major routes of splenic lymphocyte death, which was also confirmed by flow cytometry analysis. CONCLUSIONS: In the Tibetan minipig model, radiation doses have a close relationship with the (18)F-FDG uptake of the spleen. This finding suggests that (18)F-FDG PET/CT may be useful for the rapid detection of individual radiation doses.

Terpenes isolated from Polyalthia simiarum and their cytotoxic activities.

Two new C31 triterpenes, polysimiaric acid A (1) and B (2) as well as one new clerodane diterpenoid, 16,16-dimethoxy-cleroda-3,13Z-dien-15-oic acid (3), together with six known compounds were isolated from Polyalthia simiarum. Their structures were determined by analysis of 1D and 2D NMR data. Three new compounds were tested for their cytotoxicity against five human tumour cell lines. Compound 3 showed cytotoxic activities against SMMC-7721 with the IC50 value of 22.43 µM.

[The effects of mesenchymal stem cells on the aging kidneys in rats].

OBJECTIVE: To study the effect of mesenchymal stem cells on the aging rat kidney and to explore the underlying mechanism. METHODS: Rat models of senile kidney were built with hypodermic injection of D-galactose daily. Injections of MSCs of 3 x 10(6) were given to each rat through vena caudalis and CFSE was used as a tracing label to detect the distribution of MSCs in vivo. After 24 h, rats were dissected and their kidneys were frozen for section. MSCs were observed with Fluophot and quantitative analysis of the various parameters of kidney was performed under a light microscope with BI2000 image analysis system. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and kidney were measured wtih hydroxylamine and chromatometry. The expression of VEGF and P16 mRNA in kidney tissue was detected with real-time PCR and Western blotting. RESULTS: MSCs was found homing in the rat kidney, and the glomerular size, sclerosis-rate and the average cell count of glomerulus in the treated group were different from those of the model group (P < 0.05). In the treated group, the activity of SOD was significantly higher and the content of MDA was lower in serum and kidney than that in the model control group (P < 0.05). The expression of VEGF mRNA and protein in the kidneys of MSCs group increased significantly as compared with the model group (P < 0.05). The expression of P16 mRNA and protein in the kidney of MSCs group decreased significantly compared with the model group (P < 0.05). CONCLUSION: MSCs can increase the expression of VEGF while decrease the expression of P16, so as to play a key role in the anti-aging on rat kidney.

MicroRNA-150 enhances radiosensitivity by inhibiting the AKT pathway in NK/T cell lymphoma.

BACKGROUND: Radioresistance is a major challenge during the treatment of NK/T cell lymphoma. This study aimed to investigate the potential role of MicroRNA-150 (miR-150) in increase the sensitivities of NK/T cell lymphoma to ionizing radiation. RESULTS: In this study, we found that miR-150 was significantly decreased in NK/T cell lymphoma tissues and cell lines. Low expression of miR-150 was positively associated with therapeutic resistance in 36 NK/T cell lymphoma cases. Our further in vitro and in vivo studies illustrated that overexpression of miR-150 substantially enhanced the sensitivity of NK/T cell lymphoma cells to ionizing radiation treatment. Furthermore, luciferase reporter assays in NK/T cell lymphoma cells transfected with the AKT2 or AKT3 three prime untranslated region reporter constructs established AKT2 and AKT3 as direct targets of miR-150. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit Akt to verify miR-150 increase NK/T cell lymphoma cell radiorsensitivity through suppress the PI3K/AKT/mTOR pathway. CONCLUSIONS: Taken together, this study demonstrates that miR-150 might serve as a potential therapeutic sensitizer through inhibition of the AKT pathway in NK/T cell lymphoma treatment.

[Blocking Two Signals of Immuno-Activated T Cells from Antigen Presenting Cells Can Prevent Chronic GVHD of Murine].

OBJECTIVE: To explore the effects of blocking TCR-CD3 and B7-CD28 signals on immune function of mice with chronic GVHD by using TJU103 and CTLA4-Ig. METHODS: On the basis of foregoing murine model of chronic GVHD, according to interference modes after infusion 6×107 spleen cells of donor mice, the recipients were divided into 5 groups: blank control, cGVHD, TJU103 interference, CTLA4-Ig interference and TJU103+CTLA4-Ig interference groups. The score of clinical manifestation and tissue histopathology were used to evaluate the effects of all the interferences on chronic GVHD. RESULTS: TJU103 and CTLA4-Ig could not influence the formation of the mouse chimera. The analysis of Kaplan survival curve of mice with chronic GVHD showed that the CTLA4-Ig and TJU103+CTLA4-Ig reduced the incidence of chronic GVHD, the TJU103 could delay the occurrence of chronic GVHD, but all the interference factors could not change the severity of chronic GVHD. CONCLUSION: TJU103 can delay the onset time of chronic GVHD, and the CTLA4-Ig can reduce the incidences of cGVHD, the combining use of TJU103 and CTLA4-Ig can significantly reduce the incidence of chronic GVHD, but can not change the severity of chronic GVHD.

[Synergistic Killing Effects of Arsenic Trioxide Combined with Itraconazole on KG1a Cells].

OBJECTIVE: To explore the effect of arsenic trioxide combined with itraconazole on proliferation and apoptosis of KG1a cells and its potential mechanism. METHODS: The cell morphology was observed with Wrighe-Giemsa staining; cell survival rate was examined by CCK-8; and colony formation capacity was measured by methylcellulose colony formation test; the flow cytometry was used to analyse the cell apoptosis rate and cell cycle; the protein expressions of BCL-2,caspase-3,BAX,SMO,Gli1 and Gli2 were detected by Western-blot. RESULTS: The arsenic trioxide and itraconazole alone both could inhibit the KG1a cell proliferation in dose-and time-dependent manner. In comparison between single and combined drug-treatment group, both the cell survival rate and the colony number of the single drug-treatment group were significantly lower(P<0.05), and the apoptosis rate was higher in the combined drug-treatment group. In the combined-treatment group, the protein expression of Caspase-3 and BAX was upregulated, while the protein expression of BCL-2,SMO,Gli1 and Gli2 was downregulated. CONCLUSION: Arsenic trioxide combined with itraconazole can inhibit the KG1a cell proliferation and induce apoptosis, which may be related with the inhibition of Hh signaling pathway and upregulation of both Caspase-3 and BAX protein expression, and provided experimental data of arsenic trioxide combined with itraconazole for the treatment of refractory AML.

Sunitinib Induces NK-κB-dependent NKG2D Ligand Expression in Nasopharyngeal Carcinoma and Hepatoma Cells.

Multitargeted tyrosine kinase inhibitors (MTKIs) have been shown to combine with natural killer (NK) cell adoptive transfer for the treatment in various cancers. MTKIs sensitize cancer cells to NK cell therapy through upregulation of nature killer group 2 member D ligands (NKG2DLs) on tumor cells. However, the molecular mechanism of MTKIs-mediated upregulation of NKG2DLs is still unknown. In this study, we confirmed sunitinib induced downregulation of its targets, such as vascular endothelial growth factor, platelet-derived growth factor, and c-kit in multiple-drug-resistant nasopharyngeal carcinoma cell line CNE2/DDP and hepatoma cell line HepG2. Then, we further showed sunitinib induced cell proliferation inhibition, apoptosis, and DNA damage in CNE2/DDP and HepG2 cells. Coculture experiments showed that sunitinib-treated CNE2/DDP and HepG2 cells were able to increase the activation and cytotoxicity of NK cells. Quantitative polymerase chain reaction results showed that sunitinib upregulated NKG2DLs, apoptotic genes, DNA damage repair genes, and nuclear factor (NF)-κß family genes. Silencing of NF-κß1, NF-κß2, or RelB (NF-κß pathway) inhibited sunitinib-induced upregulation of NKG2DLs. Taken together, we concluded that sunitinib upregulated NKG2DLs through NF-κß signaling noncanonical pathway which might mediate higher cytotoxic sensitivity of CNE2/DDP and HepG2 cells to NK cells.

[The outcomes of the thirty patients with refractory leukemia treated with related HLA haploidentical stem cells transplantation].

OBJECTIVE: To assess the outcomes of the therapy for patients with refractory leukemia with HLA haploidentical stem cells transplantation. METHODS: To analyze the outcomes of 30 patients with refractory leukemia who underwent HLA haploidentical peripheral blood stem cells transplantation from August 1998 to August 2004. RESULTS: Thirty refractory leukemia patients including 13 cases of acute non-lymphocytic leukemia, 10 cases of acute lymphocytic leukemia (ALL), 6 cases of chronic myeloid leukemia and 1 case of phase IV non-Hodgkin's lymphoma underwent HLA haploidentical peripheral blood stem cells transplantation. The median age was 25 years old (3-52 years old). Twelve patients received stem cells from parent donors, four from daughter or son donors and the remaining from sibling donors. Three HLA loci mismatched in twelve cases, two HLA loci mismatched in thirteen cases and one HLA locus mismatched in five cases. The conditioning regime consisted of fludara (25 mg/m(2) x 5 d), busulfan (4 mg/kg x 4 d) and cyclophosphamide (60 mg/kg x 2 d). Rabbit anti-human lymphocyte globulin (5 mg/kg x 5 d) was added in some patients in the conditioning regime. A mean of 5.0 (2.9-8.0) x 10(8)/kg mononucleated cells was grafted. The number of mean CD(34)(+) cells was 5.5 (3.0-6.5) x 10(6)/kg. Twenty-seven patients were successfully grafted, one failed to graft, one died from severe fungal infection at day 2 and one died from severe veno-occlusive disease at day 28. The mean time of white cell count more than 1.0 x 10(9)/L was 14 (11-18) days and platelet count more than 20 x 10(9)/L was 15 (11-18) days. ALL the 27 successfully grafted patients got complete remission. Severe acute graft versus host disease occurred in six patients and four of them died. Seven patients suffered from chronic graft versus host disease. Seven patients relapsed and died. The median relapse time was 10 (3-24) months. Fourteen patients are still surviving, and ten have disease free survival. CONCLUSION: It is concluded from our observation that HLA haploidentical peripheral blood stem cells transplantation may be an effective therapy for refractory and relapse leukemia. Some patients with refractory and relapse leukemia treated with HLA haploidentical stem cells transplantation may have disease free survival. Graft versus leukemia effect may be strong in patients receiving HLA haploidentical blood stem cells transplantation and leukemia will probably be relapsed when the patient without complete remission was treated with this therapy.

[HLA class I and II polymorphism and haplotypes in Guangdong Han population].

OBJECTIVE: To analyze the polymorphism and haplotypes of HLA class I and II in Guangdong Han population and detect the HLA-A, B, Cw and DRB1 allele frequencies. METHODS: An auto semi-quantitative PCR-sequence speacific oligonucleotide probe(PCR-SSOP) method was adopted in exploring the HLA-A, B, Cw and DRB1 genotypes of the samples from 160 bone marrow donors. RESULTS: Twelve HLA-A, 23 B, 11 Cw and 13 DRB1 alleles were obtained. A total of 9 HLA-A-B, 20 Cw-B, 7 A-Cw, and 8 A-DRB1, 9 B-DRB1, 10 Cw-DRB1 haplotypes were found. CONCLUSION: HLA class I and II alleles in Guangdong Han population have plenty of polymorphisms. The haplotype distribution possesses territory characteristic.

Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella.

To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice. After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells. Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection. Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes. The inhibition of the tumor and the survival time of the mice were also investigated. Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue. And the GFP expression was much stronger in spleen and tumor than in other tissues. Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo. The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05). Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy.

[Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model].

OBJECTIVE: To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro. METHODS: Human BM-MSCs were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum and 10 ng/ml epidermal growth factor. Flow cytometry (FCM) was used to examine the phenotypes of the cells. The eukaryotic expression vector pcDNA3.1(+)/NT3 was constructed and transferred into human BM-MSCs in vitro via liposomes. The genetically engineered BM-MSCs were selected several times with G418 and the clones were obtained and then amplified, followed by extraction of the RNA for detection of NT3 gene expression by reverse transcriptional (RT) PCR. The biological activity of the genetically engineered cells was examined by the collecting the supernatant of the culture medium for incubation of guinea pig cochlea hair cells. RESULTS: The cultured cells expressed CD13, CD29 and CD59, but no7 CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 or HLA-DR. The BM-MSCs genetically modified with pcDNA3.1(+)/NT3 not only expressed and produced NT3, but also promoted the survival of the guinea pig cochlea hair cells in vitro. CONCLUSION: It is possible to construct the genetically engineered BM-MSCs that excrete NT3 in vitro.

A controlled clinical trial of itraconazole for prevention of fungal infections secondary to chemotherapy.

To study the preventive effect of itraconazole against fungal infections following chemotherapy in tumor patients, we conducted a double-blind randomized clinical trial in 114 tumor patients receiving chemotherapy, who were divided into itraconazole treatment group (n=52) and control group (n=62) and the incidences of fungal infections after chemotherapy were recorded. In comparison with the control group, itraconazole treatment group exhibited a significant decrease in fungal infection episodes (12.8% vs 3.8%), illustrating the efficacy of itraconazole treatment as a preventive measure against fungal infections secondary to chemotherapy.

[Construction of MHC I gene retroviral expression vector and its expression in hematopoietic cells].

OBJECTIVE: To construct the retroviral expression vector of BALB/c mouse H-2Dd gene and study its expression in C57BL/6 mouse hematopoietic cells (MHC). METHODS: A retroviral vector pMSCV encoding H-2Dd gene was transduced into the packaging cell line PT67 by lipofectin, and the hematopoietic cells of C57BL/6(H-2Dd negative) were infected by the above viral supernatant. Reverse transcriptase-polymerase chain reaction and flow cytometry were employed to examine the expression of H-2Dd on the infected cells. RESULTS: The cDNA encoding H-2Dd was correctly inserted into the vector pMSCV, as confirmed by restriction endonuclease digestion. The H-2Dd gene was integrated into the C57BL/6 mouse hematopoietic cell genome and expressed on the cell surface. CONCLUSION: Recombinant H-2Dd of BALB/c mouse retroviral expression vector has been successfully constructed and its expression obtained in C57BL/6 mouse hematopoietic cells, which may facilitate further study of the function of MHC in transplantation immunology.

[HLA-Cw alleles polymorphism and haplotypes in Guangdong Han population].

OBJECTIVE: To analyze the polymorphism and haplotypes of HLA-Cw and detect HLA-A, B, Cw and DRB1 allele frequencies in Guangdong Han population. METHOD: An auto semi-quantitative PCR with reverse sequence-specific oligonucleo- tide was adopted to explore the HLA-A, B, Cw and DRB1 genotypes of 185 bone marrow donors. RESULT: Eleven HLA-Cw alleles were obtained in which Cw*03 (0.2580), 07 (0.1887), 01 (0.1732), and 08 (0.1071) had much higher allele frequencies. A total of 7 HLA-Cw-A, 20 HLA-Cw-B and 10 HLA-Cw-DRB1 haplotypes were found. CONCLUSION: HLA-Cw alleles have richer polymorphisms and their linkage disequilibrium with HLA-A, B, DRB1 exhibits geographic genetic characteristics.

[Prophylaxis of hepatic veno-occlusive disease by low-molecular-weight heparin and lipo- prostaglandin E1 after allogeneic peripheral blood stem cell transplantation].

To study the effects of low-molecular-weight heparin and lipid microspheres containing prostaglandin E(1) (lipo PGE) in preventing hepatic veno-occlusive disease after allogeneic peripheral blood stem cell transplantation, we observed 21 cases of allogeneic stem cell transplantation, in which the combined treatments were administered from day -7 to day +30. As a result, only 2 of these cases developed hepatic veno-occlusive disease, without obvious treatment-related adverse effects, suggesting the safety and effectiveness of the combined treatment using low-molecular-weight heparin and lipo PGE1 in the prevention of hepatic veno-occlusive disease after allogenetic stem cell transplantation.

In vitro culture of human bone marrow mesenchymal stem cell clonies and induced differentiation into neuron-like cells.

OBJECTIVE: To study the long-term in vitro culture of human bone marrow mesenchymal stem cells (hMSC) and their phenotypical and functional properties. METHODS: Adherent hMSC colonies were digested by 0.25% trypsin-EDTA with a clone cycle for in vitro subculture. Flow cytometry was employed to examine the phenotypes of the cells. Their committed differentiation potential to neurons, clone-forming ability and growth curves were all investigated. RESULTS: hMSCs could be subcultured under this culture condition for 20 passages, expressing CD13, CD29 and CD59 but not CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 and HLA-DR. The cells could be induced to differentiate into neurons when subcultured for 17 passages. CONCLUSION: hMSCs can be efficiently expanded under this culture condition, and the colony-derived hMSCs can maintain the differentiation potentials and retain their biological characteristics.

[Killer immunoglobulin-like receptor gene distribution in Guangdong Han population].

OBJECTIVE: To investigate the distribution of killer immunoglobulin-like receptor (KIR) gene in Guangdong Han population. METHODS: KIR phenotype was examined by PCR with sequence-specific primers in 96 subjects of Han nationality in Guangdong Province of China, and KIR frequency was calculated and compared with those in Caucasian, north Indian and Japanese populations. RESULT: The gene expression frequency of KIR in Guangdong Han people was 2DL1(0.85), 2DL2(0.12), 2DL3(0.58), 2DL4(1), 2DL5(0.24), 3DL1(0.96), 3DL2(1), 3DL3(1), 2DP1(0.97), 2DP2(0.98), 2DS1(0.10), 2DS2(0.30), 2DS3(0.02), 2DS4(0.28), 1D(0.65), 2DS5(0.19), and 3DS1(0.23) respectively. Comparison of the KIR recognizing the same HLA ligand suggested significantly higher expression frequency of inhibitory KIR than that of activating KIR. Compared with Caucasian and north Indian populations, Guangdong Han population had significantly lower expression frequency of activating KIR gene with the exception of KIR2DS4. CONCLUSION: Different KIR genes have different expression frequencies in Guangdong Han population, and KIR gene distribution varies between populations of different races.

[Effect of honokiol on proliferation and apoptosis in HL-60 cells and its potential mechanism].

This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P < 0.05). The expression of cyclin D1, cyclin A, cyclin E and CDK2/4/6 were significantly down-regulated (P < 0.05), the expression of P53 and P21 was significantly upregulated after treating for 24 h with HNK (P < 0.05). After 24 h treatment with HNK, HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P < 0.05). It is concluded that HNK arrestes the cells at G0/G1 phase and induces HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.

[Synergistic killing effect of arsenic trioxide combined with curcumin on KG1a cells].

This study was aimed to explore the effect of arsenic trioxide combined with curcumin on proliferation and apoptosis of KG1a cells and its potential mechanism. The cell survival rate was mesured by MTT; colony formation capacity was examined by methylcellulose colony formation test; flow cytometry was used to analyse the cell surface molecules, cell apoptosis rate and cell cycle; the cell morphology was observed with Wright-Giemsa staining and the protein expression of BCL-2, BAX, PARP was detected by Western blot. The results showed that the phenotype of KG1a cells was CD34(+)CD38(-), while the phenotype of HL-60 cell was CD34(+)CD38(+). The former possessed a stronger colony ability than the latter. Effect of curcumin and arsenic trioxide alone on cell proliferation and inhibition was in dose-dependent manner. Compared with single drug-treatment group, the cell survival rate and colony number were lower, and the apoptosis rate was higher in combined drug-treatment group. Protein expression of BCL-2 and PARP was upregulated, while the protein expression of PARP was downregulated in the combined treatment group. It is concluded that compared with HL-60 cells, KG1a cells are the earlier leukemia stem/progenitor cells. Arsenic trioxide combined with curcumin can effectively inhibit the KG1a cell proliferation and induce apoptosis, which may be associated with the downregulation of BCL-2 and PARP protein expression and the upregulation of BAX protein expression.

[Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis].

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.