PurposeTo investigate the physical characteristics of the lumbar bone marrow fat content by analyzing the correlation of the lumbar bone marrow fat content with age, gender, body mass index (BMI) and waist circumference. It may be helpful to deepen the understanding the occurrence regularity of osteoporosis. Materials and Methods A total of 144 subjects were recruited including both healthy volunteers and the patients with chronic low back pain. The height, weight and waist circumference were measured, and the body mass index was calculated. All the subjects took spectroscopy sequence at the third lumber vertebra with single-voxel point resolved spectroscopy (PRESS) method. Lipid (1.3 ppm) to water (4.67 ppm) ratio (LWR) and lipid fractions (FF%) were measured. LWR and FF% of L3 were compared among the patients with different gender, age, BMI and waist circumference. The correlation of LWR and FF% of L3 and age were analyzed.Results The LWR and FF% of L3 showed no signiifcant difference between the male and female (t=-0.267 and-0.993,P>0.05). There was statistical difference of LWR and FF% among the different age groups (F=3.723 and 5.478,P<0.05). LWR and FF% of female in 61-70 year-old group showed signiifcant higher than that in 20-30, 31-40, 41-50 and 51-60 year-old group (P<0.05). FF% of L3 in 20-30 year-old female group showed signiifcant lower than that in >70 year-old group (P<0.05). LWR of L3 for both male and female in 60-70 year-old group also showed signiifcant higher than that in 20-30, 31-40, 41-50 and 51-60 year-old group (P<0.05). FF% for both male and female in 20-30 year-old group showed signiifcant lower than that in 31-40, 41-50, 51-60, 61-70 and>70 year-old group (P<0.05). FF% of 61-70 year-old group was signiifcant different from that in 31-40, 41-50, 51-60 year-old group (P<0.05). The LWR and FF% of L3 showed no signiifcant difference between the different BMI groups and waist circumference groups (P>0.05). Age was positive correlated with FF% of L3 (r=0.321,P<0.05).Conclusion The lumbar bone marrow fat content is correlated with age, but is not correlated with gender, BMI and waist circumference.

Objective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro and assess the influence of Naloxone on Morphine2s effect.Methods MT2 cells were randomly assigned into 4 groups: (1) Morphine treatment for MT2 group, (2) Morphine+Naloxone co-treatment for MT2 group, (3) Naloxone treatment for MT2 group and (4) MT2 Control;Macrophages were also randomly assigned into 4 groups: (5) Morphine treatment for Macrophage group, (6) Morphine+Naloxone co-treatment for Macrophage group, (7) Naloxone treatment for Macrophage group and (8) Macrophage Control. Group (2), (3), (6) and (7) were pre-treated with 10-8 mol/L Naloxone for 0.5 h, and then group (1) and (2) were treated with 10-12, 10-10 and 10-8 mol/L Morphine for 24 h;group (5) and (6) were disposed of 10-10 mol/L Morphine for 24 h.All 8 groups were added in HIV-1 viral strain with 50% tissue culture infective dose(TCID50).P24 antigen in MT2 cells culture supernatant at day 3, 4, 5 and 6, and in Macrophages culture supernatant at day 4, 6, 8, 10 and 12 after infection were determined with ELISA.Student2s t-test and ANOVA were used to compare the differential expression in different groups, and repeated measures ANOVA was used to compare the increasing or decreasing expression of p24 antigen in morphine treatment groups than that in the control group at different time points.Results On the 3rd day of infection with HIV-1 in MT2 cells, the expression of p24 antigen in 10-12, 10-10 and 10-8mol/L dose of group (1) were (4.44?.30), (5.59?.25) and (4.60?.24) ng/ml respectively, compared to control[(1.93?.05) ng/ml, t= 14.15, 24.74 and 19.14, all P<0.01].On the 4th day, 10-12, 10-10 and 10-8mol/L dose of group (1) resulted in a significant increase of p24 antigen expression [(24.30?.66), (31.73?.17) and (26.02?.37) ng/ml]in culture supernatants compared to control[(8.03?.09) ng/ml, t=10.59, 34.92 and 81.2, all P<0.01].On the 5th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (56.30?.26), (81.77?.49) and (63.66?.57) ng/ml respectively, compared to control [(15.30?.91) ng/ml, t= 45.83, 43.51 and 30.07, all P<0.01].On the 6th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (150.70?.97), (243.09?.93) and (173.72?.73) ng/ml respectively, compared to control [(41.01?.84) ng/ml, t= 21.09, 39.02 and 29.55, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of morphine treatment group compared to control increased with HIV-1 infected MT2 cells time, trend analysis of repeated measurements showed statistically significant time effect (F=842.18, P<0.01). On the 4th day of infection with HIV-1 in Macrophage cells, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (0.68?.15), (0.87?.41) and (0.75?.09) ng/ml respectively, compared to control [(0.60?.01) ng/ml, t= 7.27, 11.06 and 3.02, all P<0.05]. On the 6th day, 10-12, 10-10 and 10-8 mol/L dose of group (5) resulted in a significant increase of p24 antigen expression[(1.64?.57) , (2.07?.12 ) and (1.75?.17) ng/ml]in culture supernatants compared to control [(1.16?.07) ng/ml, t=8.93, 11.3 and 5.45, all P<0.01].On the 8th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (6.31?.17), (8.81?.34) and (7.19?.11) ng/ml respectively, compared to control [(3.84?.45) ng/ml, t=8.83, 15.11 and 12.42, all P<0.01]. On the 10th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of Morphine treated group were (32.30?7.55), (50.74?7.55) and (39.74?.56) ng/ml respectively, compared to control [(17.55?.86) ng/ml, t= 13.65, 17.84 and 36.69, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of group (5) compared to control increased with HIV-1 infected Macrophage cells time, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01).Conclusions Morphine has the ability to enhance HIV-1 replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression can be blocked by Naloxone.

OBJECTIVETo develop a HPLC-ESI-MS method for the determination of puerarin and its metabolite and study the metabolic kinetics in beagle dog liver microsomes.

METHODBeagle dog liver microsomes were prepared by using ultracentrifugation method. Chromatography was performed on a Shimadzu C18 column (2.0 mm x 150 mm, 5 microm). Amethanol-water gradient system was used. ESI interface was applied in the positive, and SIM m/z 417 was puerarin and m/z 531 was daidzein.

RESULTThe puerarin was metabolized by NADPH regenerating system in beagle dog microsomes. The Michaelis-Menten parameters Km and Vmax in beagle dog microsomes were initially estimated by analyzing Lineweave-Brurk plot. The Vmax Km of puerarin were (0.047 +/- 0.006) mg x min(-1) x g(-1), (1.22 +/- 0.53) mg x L(-1).

CONCLUSIONThe puerarin and daidzein can be rapidly determined by HPLC-MS in beagle dog microsomes and the puerarin was metabolized to daidzein by CY P450. The study can give help for Baige capsule.

Animals ; Chromatography, High Pressure Liquid ; Dogs ; Isoflavones ; pharmacokinetics ; Liver ; chemistry ; drug effects ; Microsomes, Liver ; chemistry ; drug effects ; Pharmacokinetics ; Spectrometry, Mass, Electrospray Ionization

Objective - To investigate the effect of lung flora dysbiosis on the process of pulmonary fibrosis and lung epithelial （ ） Methods - mesenchymal transition EMT in mice with silicosis. Male C57BL/6 mice of specific pathogen free grade were ， ， ， （ ） randomly divided into the blank control group silicosis model group solvent control group vancomycin VM + ampicillin （ ） ， （ ） （ ） ， AMP group metronidazole MNZ + neomycin NEO group and mixed treatment group 12 mice in each group. Except for ， ， the blank control group which was given 20.0 µL of 0.9% NaCl solution the other five groups of mice were dosed with 20.0 µL of silica dust suspension at a mass concentration of 250.0 g/L using a single tracheal drip to establish the silicosis mouse model. ： The intranasal drip method was used to treat silicosis mice in each group as following mice in the solvent control group were - ； ； given double distilled water mice in the VM+AMP group were given VM at a mass concentration of 0.5 g/L and AMP at 1.0 g/L ； mice in the MNZ+NEO group were given MNZ at a mass concentration of 1.0 g/L and NEO at 1.0 g/L mice in the mixed ， treatment group were given the same doses of the four antibiotics mentioned above all in a drip volume of 50.0 µL. Silicosis ， ， mice were treated seven days and half an hour before silica dusting and 7 14 and 21 days after silica dusting. Mouse lungtissue was collected aseptically 28 days after silica dusting. Hematoxylin eosin and Masson trichrome staining methods were - used to observe the pathological changes. Western blotting was used to detect the relative protein expression of α smooth muscle （ - ）， - （ - ） （ ） actin α SMA E cadherin E CAD and vimentin VIM . Immunohistochemistry was used to detect the relative expression of - - E CAD and VIM. Real time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of （Col1a2） Results collagen type Ⅰ alpha 2 mRNA in lung tissues. The histopathological results showed that the alveoli of the ， blank control group were thin and structurally intact with few surrounding infiltrating inflammatory cells and no abnormal ， distribution of collagen fibers. The alveoli of the silicosis model group were structurally disorganized with a large number of ， ， infiltrating inflammatory cells thickened alveolar walls and cellular fibrous nodules with abundant blue collagen deposit. In the ， ， VM+AMP group MNZ+NEO group and the mixed treatment group the inflammation and fibrosis were reduced with diferent degrees in the lung tissues compared to the silicosis model group and the solvent control group. The relative expression levels of - ， Col1a2 α SMA VIM protein and mRNA in lung tissues of mice in the silicosis model group were higher than those in the blank （ P ）， -CAD control group all <0.05 and the relative expression levels of E protein were lower than those in the blank control （P ） - ， Col1a2 group <0.05 . The relative expression levels of α SMA VIM protein and mRNA in lung tissues of mice in the MNZ+ （ P ）， -CAD NEO group and the mixed treatment group were lower all <0.05 and the relative expression levels of E protein were （P ）， Conclusion higher <0.05 when compared with the silicosis model group and the solvent control group. Pulmonary fibrosis ， - was reduced in silicosis mice with interventions in lung flora where anaerobic and gram negative bacteria affected pulmonary fibrosis and dysbiosis of the lung flora affected pulmonary EMT.

This study was aimed to investigate the influence of cryopreservation on biological properties and function of leukemic dendritic cells (L-DCs) derived from patients with acute or chronic leukemia. Some fresh leukemic cells were detected immediately; some were cultured immediately; some were cryopreserved in -80 degrees C with 5% DMSO-6% HES as cryopreservor. After being thawed, they were cultured. The combination of rhGM-CSF, rhIL-4, rhTNF-alpha and other cytokines were added into the culture system. 12 days later, L-DCs were assayed for morphology, immunophenotype, mixed lymphocytic reaction (MLR) and CTL cytotoxicity on autologous leukemic cells. The results showed that both fresh and cryopreserved leukemic cells obtained from patients with acute or chronic leukemia revealed typical DC morphologically by means of using combinations of cytokines in culture, but there was no significant difference between pre-or post cryopreservations. L-DCs also upregulated the expression of CD80, CD54, HLA-DR, CD1a, CD83 and CD86, and downregulated the expression of CD14, but there was also no difference as compared with L-DCs befor cryopreservation. L-DCs derived from leukemic cells were also capable of stimulating MLR and inducing CTL which could kill autologous leukemic cells obviously. It is concluded that leukemic cells, regardless of fresh or frozen, can induce L-DCs after culture with cytokine combination. The L-DCs can induce CTL targeting autologous leukemic cells, and may be used to treat MRD as immunotherapy. The induction and biological properties of L-DCs are not influenced by cryopreservation.
Bone Marrow Cells ; cytology ; CD8 Antigens ; metabolism ; Cryopreservation ; Dendritic Cells ; cytology ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Leukemia, Myeloid ; immunology ; pathology ; Lipopolysaccharide Receptors ; metabolism ; Lymphocyte Activation ; Recombinant Proteins ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo compare effects of integrated treatment traditional Chinese medicine and Western medicine (TCM-WM) and simple western medicine on TCM clincal symptoms in the patient of AIDS with pulmonary inflammation.

METHODA multicenter randomized controlled trials of 164 subjects evaluated the effects of clinical symptoms of AIDS with pulmonary inflammation of TWO regimens: the TCM-WM group (n = 111) and western medicine treatment group (n = 53), while incidence of TCM symptoms in different time points in two groups were analyzed.

RESULTTwenty eight days after treatment, the cured and markedly effective rate of TCM symptoms in the TCM-WM group significantly exceeding that in the western medicine treatment group (cured and markedly effective rate significant efficiency 44.55% vs 20.00%), while the incidence rate for the TCM symptoms of fever and headache in the TCM-WM group was significantly lower than that in western medicine group.

CONCLUSIONThe integrated treatment of traditional Chinese medicine and Western medicine helps to alleviate the TCM clinical symptoms of AIDS with pulmonary inflammation.

Acquired Immunodeficiency Syndrome ; complications ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; methods ; Multivariate Analysis ; Pneumonia ; complications ; drug therapy ; Treatment Outcome

AIMTo explore proper cryopreservative systems for hematopoietic stem cells.

METHODSPeripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.

RESULTSThe recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.

CONCLUSION5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Blood Preservation ; methods ; Cell Survival ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans

This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.
Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Fibroblasts ; Humans ; Stromal Cells ; cytology ; drug effects ; Thrombopoietin ; pharmacology

OBJECTIVEIn order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs).

METHODSImproved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process.

RESULTSTPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration.

CONCLUSIONThese findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.

Cells, Cultured ; Collagen Type III ; metabolism ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Fibrosis ; pathology ; Humans ; Laminin ; metabolism ; Megakaryocytes ; cytology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; pathology ; Thrombopoietin ; pharmacology

The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.
Bone Marrow Cells ; cytology ; Cell Survival ; Colony-Forming Units Assay ; Cryopreservation ; methods ; Cryoprotective Agents ; Humans ; Nitrogen ; Time Factors