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.

[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.

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.

[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.

[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.

[Cytotoxicity of Naja Naja Actra Venom Component combined with activated immune cells on leukemia cell line KG1a].

This study was aimed to investigate the cytotoxic effect of the Naja Naja Actra Venom Component (NNAVC) combined with activated immune cells on human acute myeloblastic leukemia line KG1a cells. The cytotoxic effects of NNAVC at different concentrations on KG1a cells were measured by CCK-8 method. LDH releasing assay was used to detect the cytotoxic effects of activated immune cells, NNAVC combined with activated immune cells on KG1a cells and the sensitivity of KG1a treated with NNAVC to activated immune cells. The results showed that the inhibitory rate of NNAVC on KG1a cells increased with the concentration enhancement, the cytotoxicity of activated immune cells at the different effector to target (E:T) ratios(6.25:1, 12.5:1, 25:1) on KG1a cells were 12.30%, 24.85% and 52.26%. The cytotoxicity of NNAVC combined with activated immune cells at the different E:T cell ratios (10:1, 20: 1) on KG1a cells were 56.21% and 85.59%, which were higher than that of NNAVC or activated immune cells alone. The cytotoxicity of activated immune cells at the E: T cell ratio of 10:1 on KG1a cells treated with NNAVC at different concentrations were 25.65%, 31.33%, 28.63% and 16.78%, respectively, and that at the E:T cell ratio of 20: 1 were 40.62%, 44.70%, 44.62% and 40.72%. It is concluded that:both of NNAVC and activated immune cells have lethal effect on KG1a cells, and the combination of NNAVC and activated immune cells can strengthen their effect on KG1a.

[NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells].

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

Irradiation induced injury reduces energy metabolism in small intestine of Tibet minipigs.

BACKGROUND: The radiation-induced energy metabolism dysfunction related to injury and radiation doses is largely elusive. The purpose of this study is to investigate the early response of energy metabolism in small intestinal tissue and its correlation with pathologic lesion after total body X-ray irradiation (TBI) in Tibet minipigs. METHODS AND RESULTS: 30 Tibet minipigs were assigned into 6 groups including 5 experimental groups and one control group with 6 animals each group. The minipigs in these experimental groups were subjected to a TBI of 2, 5, 8, 11, and 14 Gy, respectively. Small intestine tissues were collected at 24 h following X-ray exposure and analyzed by histology and high performance liquid chromatography (HPLC). DNA contents in this tissue were also examined. Irradiation causes pathologic lesions and mitochondrial abnormalities. The Deoxyribonucleic acid (DNA) content-corrected and uncorrected adenosine-triphosphate (ATP) and total adenine nucleotides (TAN) were significantly reduced in a dose-dependent manner by 2-8 Gy exposure, and no further reduction was observed over 8 Gy. CONCLUSION: TBI induced injury is highly dependent on the irradiation dosage in small intestine and inversely correlates with the energy metabolism, with its reduction potentially indicating the severity of injury.

[A case report of myelodysplastic/myeloproliferative disease unclassifiable with karyotype aberration of trisomy 8 and JAK2 mutation].

This study aimed to investigate the relationship between clinical features of myelodysplastic/myeloproliferative disease, unclassifiable (MDS/MPD-U), karyotype of chromosome and JAK2 mutation in 1 case. The clinical features, karyotype and JAK2 mutation of the patient with MDS/MPD-U were studied by means of bone marrow biopsy, karyotype analysis and ARMS-PCR technique. The results indicated that the typical micromegakaryocytes and thrombocytosis, karyotype aberration of trisomy 8 as well as JAK2 V617F mutation were found in this patient. It is concluded that the patient was diagnosed as MDS/MPD-U with trisomy 8 and JAK2 V617F mutation. The data of this patient will provide evidence for studying correlation of chromosome karyotype aberration with JAK2 V617F mutation and for evaluating prognosis of MDS/MPD-U.

The diagnostic value of 18F-FDG-PET/CT in hematopoietic radiation toxicity: a Tibet minipig model.

This study was undertaken to assess the diagnostic value of 2-[(18)F]-fluoro-2-deoxy-D-glucose positron emission tomography with computed tomography ([(18)F]-FDG-PET/CT) in the detection of radiation toxicity in normal bone marrow using Tibet minipigs as a model. Eighteen Tibet minipigs were caged in aseptic rooms and randomly divided into six groups. Five groups (n = 3/group) were irradiated with single doses of 2, 5, 8, 11 and 14 Gy of total body irradiation (TBI) using an 8-MV X-ray linear accelerator. These pigs were evaluated with [(18)F]-FDG-PET/CT, and their marrow nucleated cells were counted. The data were initially collected at 6, 24 and 72 h after treatment and were then collected on Days 5-60 post-TBI at 5-day intervals. At 24 and 72 h post-TBI, marrow standardized uptake value (SUV) data showed a dose-dependent decrease in the radiation dose range from 2-8 Gy. Upon long-term observation, SUV and marrow nucleated cell number in the 11-Gy and 14-Gy groups showed a continuous and marked reduction throughout the entire time course, while Kaplan-Meier curves of survival showed low survival. In contrast, the SUVs in the 2-, 5- and 8-Gy groups showed early transient increases followed by a decline from approximately 72 h through Days 5-15 and then normalized or maintained low levels through the endpoint; marrow nucleated cell number and survival curves showed approximately the same trend and higher survival, respectively. Our findings suggest that [(18)F]-FDG-PET/CT may be helpful in quickly assessing the absorbed doses and predicting the prognosis in patients.

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.

Effect of total body X-ray irradiation on lymph node in Tibet minipig.

The purpose of this study was to determine the time-dose-effect of total body X-ray irradiation on lymphocytes in lymph nodes and peripheral blood in Tibet minipigs. Forty-eight Tibet minipigs were assigned into 6 groups including 5 experimental groups with 9 and the control group with 3. The minipigs in experimental groups were subjected to a total body X-ray irradiation of 2, 5, 8, 11, and 14 Gy respectively. Lymph nodes and peripheral blood samples were collected at 6, 24, and 72 hours after X-ray exposure and received histological microscopy examination and apoptosis analysis. Histology observation showed that the number of lymphocytes decreased within the lymph nodes with the increase of radiation doses and exposure time. The observation of transmission electron microscopy (TEM) showed typical apoptotic cells below 11 Gy while at 14 Gy necrotic cells were dominant. The apoptotic rate of lymphocytes in the lymph nodes was positively correlated with radiation dose in the range of 2-11 Gy, and reached the maximal level (39.4 ± 2.8) at 24 hours after 11 Gy irradiation, followed by a decrease in the apoptotic rate, but still higher than that of the control group. The number of lymphocytes in the peripheral blood samples was decreased significantly by increasing of the radiation dose and exposure time. We conclude that early damage of lymphocytes by total body X-ray irradiation is dose and time dependent below 11 Gy and before 24 hours post irradiation, and that the dosage of irradiation less than 11 Gy induced apoptosis, whereas the dose at 14 Gy resulted in necrosis in lymphocytes of the lymph nodes.

High incidence of severe influenza among individuals over 50 years of age.

Age-specific epidemiological data on asymptomatic, symptomatic, and severe infections are essential for public health policies on combating influenza. In this study, we incorporated data on microbiologically confirmed infections and seroprevalence to comprehensively describe the epidemiology of pandemic H1N1 2009 influenza. Seroprevalence was determined from 1,795 random serum samples collected in our hospital in January 2007 (before the first wave of the pandemic) and March 2010 (after the second wave). Data on microbiologically confirmed infection and severe cases were obtained from the Centre for Health Protection in Hong Kong. Severe cases were most common in the 51- to 60-year-old age group. The microbiologically confirmed incidence rate was highest for children aged ≤10 years and dropped sharply for the adult population (ρ = -1.0; P < 0.01), but the incidence rate for severe disease was highest for the 51- to 60-year-old age group. For the 51- to 60-year-old age group, the seroprevalence was similar to that for the younger age groups, but the proportion of severe cases relative to seroprevalence was significantly higher than that for 11- to 50-year-old age groups. As judged from the percentage of specimens positive for other respiratory viruses compared with that for pandemic H1N1 virus, the impact of symptomatic disease due to pandemic H1N1 virus was higher than that for other respiratory viruses in people aged ≤50 years. In conclusion, the 51- to 60-year-old age group, which had the highest overall incidence and the highest rate of severe disease but is currently not considered by the World Health Organization to be an at-risk group, should be prioritized for influenza vaccination in areas where universal influenza vaccination is not practiced.

[Effect of bone-marrow mesenchymal stem cells on the immune function of aging rats].

OBJECTIVE: To investigate the effect of transplantation of bone-marrow mesenchymal stem cells (MSCs) on the immune functions of aging rats. METHODS: Healthy SD rats were randomized into normal control, aging model group and MSCs group. The aging model was established by daily subcutaneous injection of D-galactose for 4 consecutive months. MSCs were isolated from the bone marrow of adult SD rats and injected (3×10(6) MSCs) in rats in the MSCs group via the tail vein once a week for 4 weeks. The spleen index, activity of T lymphocytes and the levels of IL-2 and IL-10 in spleen were measured, and the pathological changes of the spleen were observed after the treatments. RESULTS: MSCs transplantation enhanced the cellular immune function of aging rats manifested by obviously increased spleen index, activity of T lymphocyte and the level of IL-2, and lowered level of IL-10 in the spleen. The rats in the aging model group showed serious spleen injury, which was obviously lessened by MSCs injection. CONCLUSION: MSCs transplantation can improve the cellular immune function of aging rats and ameliorate spleen injury induced by D-galactose.

[Establishment of an erythroleukemia model in CB6F1 mice by transplant with haploidentical mouse leukemic cell line FBL-3].

The purpose of study was to investigate the feasibility for establishing erythroleukemia model in CB6F1 mice by transplant with haploidentical mouse leukemic cell line FBL-3 and to explore the biological characteristics of FBL-3 cells in CB6F1 mice, CB6F1 and C57BL/6 mice were inoculated intravenously at doses of 1×10(3)-1×10(7) FBL-3 cells respectively. The survival time, the count of peripheral white blood cells, the percentage of erythroblasts and chromosome of these mice were observed. The liver, spleen, lung and kidney were obtained from the dying CB6F1 mice for pathological examination. The ultrastructure of erythroblasts in bone marrow and spleen was observed by transmission electron microscopy as soon as these mice died. Expression of MHC molecules and karyotype of spleen and bone marrow cell were measured. The results showed that 100% and 92.5% incidences of erythroleukemia were observed when 1×10(3)-1×10(7) FBL-3 cells had been administrated intravenously to CB6F1 and C57BL/6 mouse, respectively. There was a linear relationship between the survival time and the number of inoculated leukemic cells. The survival time of CB6F1 was longer than C57BL/6 mice inoculated the same number cell. The main targets for FBL-3 cell infiltration were liver, spleen, marrow, lung and kidney. The reaction of FBL-3 cells to glycogen staining was positive, while the to reaction peroxidase, alkaline phosphatase and butyric acid staining were negative, reaction to chloroacetic acid staining partially was positive. Virus-like particles were found in the spleen and bone marrow cells under electron microscope. Chromosomes of spleen and bone marrow cells in the majority were non-diploid, and the expression of H-2b increased, H-2d expression decreased. It is concluded that the erythroleukemia mouse model can be established in CB6F1 mice transplanted with leukemic FBL-3 cells, that provides a convenience experimental erythroleukemia model for study.

[An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration].

OBJECTIVE: To establish an enzyme-linked immunosorbent assay (ELISA) for determining anti-themocyte globulin (ATG) levels in serum samples. METHODS: The microplate was coated with mouse anti-rabbit IgG monoclonal antibody, and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation. RESULTS: The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 microg/ml, 1:40 and 1:2500, respectively. The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection. A linear relationship was established within the concentration range from 40 to 1000 ng/ml, with the coefficients of variation of 7.91 within assay and 5.22 between assays, respectively. Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG, and after 90 days ATG could still be detected. CONCLUSION: The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting. In patients undergoing stem cell transplantation with ATG pretreatment, the ATG concentration gradually decreases but remains detectable 90 days after the administration.

[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.