Simultaneous determination of two constituents in five parts of Belladonnae Herba by RP-HPLC / 中成药

AIM To establish an RP-HPLC method for the simultaneous content determination of two constituents in five parts (whole grass,roots,stems,leaves and fruits) of Belladonnae Herba.METHODS The analysis of Belladonnae Herba methanol extract was carried out on a 35 ℃ thermostatic Agilent TC-C18 column (4.6 mm × 150 mm,5 μm),with the mobile phase comprising of acetonitrile-phosphate buffer flowing at 1.0 ml/min in an isocratic elution manner,and the detection wavelength was set at 216 nm.RESULTS Atropine sulfate and scopolamine hydrobromide showed good linear relationships within the ranges of 51.60-1 290 mg/L (R2 =0.999 9)and 2.90-72.00 mg/L (R2 =0.999 5),whose average recoveries were 101.3％ and 101.5％ with the RSDs of 2.5％ and 1.3％,respectively.The contents of two constituents were the highest in the roots and leaves,respectively,and both of them demostrated the lowest contents in the stems.CONCLUSION The contents of atropine sulfate and scopolamine in different parts of Belladonnae Herba show obvious differences.

Effect of Intensive Core Muscles Training on General Function in Patients after Hip Arthroplasty / 中国康复理论与实践

Objective To observe the effect of the intensive training of core stabilization on the general function of patients with femoral neck fractures in old patients after hip arthroplasty. Methods From January, 2011 to December, 2012, 60 old patients accepted hip arthroplas-ty for femoral neck fractures were randomly divided into control group (n=30) and observation group (n=30). The control group received routine rehabilitation training 50 minutes each time, and the observation group received intensive training of core muscles 20 minutes each time based on the routine rehabilitation training 30 minutes each time, twice a day, five days a week for two weeks. Both groups were evalu-ated with Harris Hip Score (HHS), Charnley Hip Score (CHS) and modified Barthel Index (MBI) before training and one week, two weeks and three months after training. Results There was no significant difference in the scores of HHS, CHS and MBI before and one week after training (P>0.05). All the scores increased with time in both groups (F>248.165, P<0.001). The scores of HHS, CHS and MBI were signifi-cantly higher in the observation group than in the control group two weeks and three months after training (t>3.211, P<0.001). Conclusion Early intensive training of core stabilization may facilitate to recover hip function and activity of daily living in old patients after hip arthro-plasty.

Effect of Intensive Core Muscles Training on General Function in Patients after Hip Arthroplasty / 中国康复理论与实践

Objective To observe the effect of the intensive training of core stabilization on the general function of patients with femoral neck fractures in old patients after hip arthroplasty. Methods From January, 2011 to December, 2012, 60 old patients accepted hip arthroplas-ty for femoral neck fractures were randomly divided into control group (n=30) and observation group (n=30). The control group received routine rehabilitation training 50 minutes each time, and the observation group received intensive training of core muscles 20 minutes each time based on the routine rehabilitation training 30 minutes each time, twice a day, five days a week for two weeks. Both groups were evalu-ated with Harris Hip Score (HHS), Charnley Hip Score (CHS) and modified Barthel Index (MBI) before training and one week, two weeks and three months after training. Results There was no significant difference in the scores of HHS, CHS and MBI before and one week after training (P>0.05). All the scores increased with time in both groups (F>248.165, P<0.001). The scores of HHS, CHS and MBI were signifi-cantly higher in the observation group than in the control group two weeks and three months after training (t>3.211, P<0.001). Conclusion Early intensive training of core stabilization may facilitate to recover hip function and activity of daily living in old patients after hip arthro-plasty.

Effects of Ilizarov technique on the histopathological changes of articular cartilage / 中国骨伤

<p><b>OBJECTIVE</b>To study the effects of pressure changes on the experimental articular cartilage by utilizing Ilizarov distraction technique.</p><p><b>METHODS</b>The Ilizarov external fixators were fixed at the left ankle joints of 18 experimental dogs, and the fixators were stretched with a speed of 1 mm/d to create a varus deformity. After three weeks, the ankle varus was 15 degree; when the degree was confirmed by X-ray film, the same traction was retained for 3 weeks. Then the traction decreased at the same speed of 1 mm/d to correct the deformity for 3 weeks. At the 3rd, 7th and 9th weeks, articular cartilages were separated from medial foot of the experimental and normal side to do morphology observation after HE stain, which were group A, group B, group C and group D respectively separately. The gray scale values were obtained using Image-Pro Plus 6.0 software and compared among the 4 groups using SPSS 13.0. The microstructure of cartilage was observed through transmission electron microscopy.</p><p><b>RESULTS</b>(1) The Mankin scores: there was significant difference between group A and group B (P=0.043<0.05); and significant difference between group B and group C (P=0.038<0.05). (2) The values of gray scale: there was significant difference between group A and group B (P=0.047<0.05); significant difference between group B and group C (P=0.045<0.05); significant difference between group A and group D (P=0.039<0.05); no significant difference between group C and group D (P=1.23>0.05).</p><p><b>CONCLUSION</b>Using Ilizarov technique, when the pressure between the cartilage surface of the ankle joint increases, the necrosis of articular cartilage occurred; and when the stress between the articular cartilage surface decreases, the damaged articular cartilage can be restored.</p>

Contribution of SDF-1/CXCR4 axis on proliferation of megakaryocyte co-cultured with human umbilical cord blood-derived stromal cells / 中国实验血液学杂志

In order to investigate the effect of stromal cell derived factor-1 (SDF-1)/CXCR4 on the proliferation of megakaryocytic line-HEL cells co-cultured with human umbilical cord blood-derived stromal cells (hUCBSCs) and to further elucidate the mechanism of SDF-1/CXCR4-mediated functions, the HEL cells were co-cultured with hUCBSCs or human bone marrow stromal cells (hBMSCs), the suspended HEL was used as control. The concentrations of SDF-1 in supernatant of hUCBSCs and hBMSCs were detected by ELISA assay. The expression of CXCR4 membrane-bound protein of HEL cells was detected by laser confocal scanning microscopy and flow cytometry, and the expression of CXCR4 mRNA was detected by RT-PCR. The result showed that the concentrations of SDF-1 in different groups were the same at the early stage of culturing. But at 6 days after seeding, the concentrations of SDF-1 increased significantly in the hUCBSCs group, even though the passage was done. By means of laser confocal microscopy, the expression of CXCR4 protein and also red dots of fluorescence could be detected in cytoplasm of HEL cells co-cultured with hUCBSCs. However, there was no significant differences of the CXCR4 mRNA level between different groups (p > 0.05). It is concluded that hUCBSCs may play important roles in secreting high level of SDF-1 and regulating megakaryocyte expression of CXCR4.

Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP / 中国实验血液学杂志

This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

Influence of different gelatin concentration and lymphocyte isolation liquid on primary culture of umbilical cord blood derived adhesive cells / 中国实验血液学杂志

In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.

Effect of the NHE-1-specific inhibitor DMA on pHi, proliferation and apoptosis of HL-60/ADM cells in vitro / 中国实验血液学杂志

The aim of this study was to evaluate the effect of dimethyl amiloride (DMA), a specific inhibitor of Na(+)/H(+) exchanger-1 (NHE-1), on intracellular pH value (pHi), proliferation and apoptosis of HL-60/ADM cells in vitro. After treatment with DMA at different doses, pHi of HL-60 and HL-60/ADM cell lines were determined by using pH-sensitive fluorescence dye BECEF-AM; the rate of growth inhibition of cells was detected with MTT assay; cell cycle was detected by flow cytometric DNA analysis; cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results showed that pHi in HL-60/ADM cells was higher than that in HL-60 cells. After treatment with DMA at different doses, pHi decreased, the rate of growth inhibition and the rate of apoptotic cells in HL-60/ADM cells were all higher than those in HL-60 cells. Meanwhile, after treatment with DMA during 100 micromol/L to 150 micromol/L, the increase amplitude of G(0)/G(1) phase cells and the decrease amplitude of S + G(2)/M cells in HL-60/ADM cells were higher than those in HL-60 cells. It is concluded that by causing intracellular acidification, the NHE-1-specific inhibitor DMA inhibits proliferation of HL-60/ADM cells and induces apoptosis of HL-60/ADM cells, and the degree of this growth inhibition of HL-60/ADM cells is higher than that of HL-60 cells.

In vitro effect of all-trans retinoic acid on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells in patients received peripheral blood stem cell transplantation / 中国实验血液学杂志

The aim of this study was to evaluate the effect of all-trans retinoic acid (ATRA) on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells (BMSC) in patients after conditioning treatment for peripheral blood stem cell transplantation (PBSCT). BMSC of 27 patients before and after conditioning treatment for PBSCT were cultured in vitro. After treated with ATRA at 0.01, 0.1, or 1 micromol/L, expression of intercellular adhesion molecule-1 (ICAM-1) protein and vascular adhesion molecule-1 (VCAM-1) protein were detected by flow cytometry, and soluble ICAM-1 (sICAM-1) protein was determined by using radioimmunoassay. Then BMSC was co-cultured with CD34+ cells, and adhesion rate of BMSC to CD34+ cells was measured. The results showed that after pretreatment with conditioning regimen for PBSCT, the expressions of ICAM-1 and VCAM-1 proteins in BMSC and the expression level of sICAM-1 protein in supernatant of BMSC culture were down-regulated, and the adhesion rate of BMSC to CD34+ cells was decreased, after administration of ATRA, the expression of ICAM-1 protein in BMSC, sICAM-1 protein in culture medium and adhesion rate of BMSC to CD34+ cells all increased significantly, but expression of VCAM-1 protein changed no significantly. It is concluded that the ATRA can partly restore adhesion function of BMSC injured by pretreatment for PBSCT and contribute to hematopoietic reconstitution.

Observation on the biological behavior of human umbilical cord blood adherent cells / 中国实验血液学杂志

To study the possibility of separation and culture of human umbilical cord blood adherent cell (HUCBAC), the umbilical cord blood CD34(+) cells were cultured in Dexter system in order to evaluate and observe the biological behavior of adherent cells in vitro. The results showed that all cells were cultured with Dexter system. By day 9-14 (at a median of 11.2 days), adherent cell colonies formed and reached their maximum at 15-22 days (mean 19.6 days), by day 28, all adherent cells spread over the bottom of Petri dish. By means of light microscopy, these cells were found to differentiate into three kinds of cells in culture of 28 days: fibroblast-liked cell, macrophage liked cell and small-round cells. The ratio of these three kinds of cells was 56.8%, 38%, 5.5% respectively. Cytochemistry assay revealed that the positive rate reached 100% in NSE stain and PAS stain; the adherent cell by ALP stain were shown 35% positive, but in POX stain the result was negative. Immunohistochemistry stain revealed that the positive rate of cord adherent cells for CD106, CD29, CD44, CD45, CD50, Fn, Ln, collagen IV etc reached 96%, 93%, 98%, 68%, 72%, 92%, 74%, 83% respectively. It is concluded there are hematopoietic adherent precursors in cord blood CD34(+) cells and the HUCBAC shows some biological behavior of hematopoietic stromal cells.

Influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside to HL-60 cell / 中国实验血液学杂志

This study was aimed to explore the influence of anti-CXCR4 monoclonal antibody 12G5 on killing effect of cytosine arabinoside (Ara C) to HL-60 cell, and to assess its therapeutic value in marrow residual disease. HL-60 cells were cultured and co-cultured with leukemic stromal cells, and SDF-1 activity was inhibited with 10 microg/ml 12G5, then, killing effects of Ara C on HL-60 cells were investigated by MTT and morphology assay. Curves by MTT assay revealed that in the test group of 20 microg/ml Ara C, A(540) values decreased slowly but straightly, however, in control group A(540) values decreased markedly for the first two days, and increased from day 3 or 4. In the test group of 40 microg/ml Ara C, although increasing at constricted range of 7 - 9 days, A(540) values decreased in whole observing period of 12 days, while in control group A(540) values decreased markedly at day 0-3, and increased from day 4. Furthermore, two curves go across each other at day 5, and continue the increasing tendency. Morphology results showed that in both treated groups, the number of HL-60 cell decreased markedly and increased gradually in control group, but just contrary to test group. It is concluded that 12G5 may weaken the killing effect of Ara C on HL60 cell in earlier period, but reinforce the total killing effect and delay the occurrence of drug resistance simultaneously. Thus 12G5 has the therapeutic potential on marrow residual disease.

Expression of stromal cell derived factor-1(SDF-1) and its receptor CXCR4 in hematologic malignancies / 中国实验血液学杂志

The aim was to analyze the expression level of stromal cell derived factor-1 (SDF-1) and its functional chemokine receptor CXCR4 in the patients with hematologic malignant tumor and their clinic significance. 28 patients with hematologic malignant tumor and 12 normal controls were chosen to be experimental objects. CXCR4 expressed on the cell membrane in bone marrow was enumerated by flow cytometry and serum level of SDF-1 was determined by ELISA assay. The result showed that the expression of SDF-1 and CXCR4 in hematologic malignant tumor were higher than that in normal controls, and the expression levels of two molecules were correlated. What is more, the different hematologic malignant tumor had different CXCR4 expression. In conclusion, the high expression of SDF-1 and CXCR4 in serum and bone marrow cells can be used as detective factors to hematologic malignant tumor. A correlation exists between the high expression of CXCR4 and the infiltration of hematologic malignant cells.

Effects of RNA interference inhibiting SDF-1 expression in bone marrow stromal cells on the proliferation and apoptosis of co-cultured Jurkat cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells.</p><p><b>METHOD</b>Inhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transferring SDF-1 specific short hairpin RNA (shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles, apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The un-transfected acute leukemic (group B) and normal (group C) bone marrow stromal cells were taken as controls.</p><p><b>RESULTS</b>The content of SDF-1 protein in supernatant of group A\[(384 +/- 41) pg/ml] was significantly lower than that in group B[(2474 +/- 271) pg/ml] or group C[(1324 +/- 154) pg/ml]. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B: 29 h, group C: 33 h), and G(0)/G(1) stage cells increased [group A: (28.47 +/- 2.39)%, vs group B: (19.43 +/- 2.80)%, group C: (27.15 +/- 2.07)%], S stage cells decreased [group A: (25.57 +/- 1.90)%, vs group B: (74.48 +/- 3.23)%, group C: (60.99 +/- 2.33)%], G(2)/M stage cells increased [group A: (45.96 +/- 3.24)%, vs group B: (6.09 +/- 1.96)%, group C: (11.86 +/- 1.98)%], the apoptosis rate increased [group A: (15.2 +/- 0.8)%, vs group B: (5.4 +/- 0.7)%, group C: (9.5 +/- 0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased.</p><p><b>CONCLUSION</b>The inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.</p>

Effects of inhibiting SDF-1 expression by RNA interference on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effects of inhibiting stromal cell derived factor-1 (SDF-1) expression by RNA interference (RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells.</p><p><b>METHODS</b>SDF-1 specific short hairpin RNA (shRNA) expressing plasmid was transferred into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A) , SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The un-transfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control.</p><p><b>RESULTS</b>The level of secreted SDF-1 protein (pg/10(5) cells/week) in the supernatants of Group A, B and C were 1920 +/- 205, 12,370 +/- 1355 and 6620 +/- 770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8 +/- 2.6)%, (57.4 +/- 3.8)% and (45.2 +/- 4.0)%, respectively, and the IC50 values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively.</p><p><b>CONCLUSION</b>Down-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.</p>

Effects of anti- CXCR4 monoclonal antibody on adhesiveness of human acute myelocytic leukemia cell line HL-60 and expression of Bcl-2, Fas proteins / 中国实验血液学杂志

To study the importance of chemokine SDF-1 in surviving of acute myelocytic leukemia cells HL-60, the adhesion ability of HL-60 and expression of Bcl-2, Fas protein when SDF-1 activity was blocked by anti-CXCR4 monoclonal antibody (12G5) were compared with those detected before MAb incubation, in this experiment, HL-60 cell were cultured and co-cultured with normal marrow stromal cell. The adhesion rate was detected while the expression of Bcl-2 and Fas proteins were assayed by immunohistochemical technique when SDF-1 activity was inhibited. The results showed that cell adhesion rate of HL-60 decreased while the expression Bcl-2 decreased and Fas increased. It is concluded that inhibition of SDF-1 activity increases cell apoptosis and thus reduces life-span of leukemia cell at certain level.

Inhibiting effects of stroma cell drived factor 1 (SDF-1) on proliferation of human acute myelocytic leukemia cell HL-60 / 中国实验血液学杂志

This study was aimed to investigate the importance of chemokine SDF-1 in maintaining proliferation ability of acute myelocytic leukemia cell line HL-60 when the effects of SDF-1 on HL-60 cell proliferation were inhibited. Marrow stromal cells were cultured and co-cultured with HL-60 cells, and SDF-1 activity was blocked with anti-CXCR4 McAb. HL-60 cell activity was detected by MTT while cell cycle and the expression of CXCR4 on HL-60 cell membrane were observed by flow cytometry meanwhile. The internal calcium ionic concentration in HL-60 cell was detected as well before and after treated with 12G5. The results showed that 12G5 down-regulated the expression of CXCR4 on HL-60 cell membrane; HL-60 cells at G(0)/G(1) phase increased, but decreased at S phase; survive rate of leukemia cells reduced; the intercellular calcium ionic concentration of HL-60 cell decreased after treated with 12G5. It was concluded that brockage of the SDF-1 activity may inhibit proliferation of leukemia cell at certain level.

Effects of leukemia bone marrow stromal cells on resistance of co-cultured HL-60 to idarubicin / 中国实验血液学杂志

To study the role of hematopoietic microenvironment abnormality in development of minimal residual disease and its mechanism, the viability of HL-60 cells was investigated by means of bone marrow stromal cell culture system or co-culture system of bone marrow stromal cell with HL-60 cells and idarubicin (IDA), flow cytometry and ELISA. The results showed that viability of HL-60 cells gradually decreased along with the increase of IDA dose and prolongation of culture time. Amount of HL-60 cells co-cultured with leukemia bone marrow stramal cells was significantly increased as compared with that of the control (P < 0.05). Bone marrow stromal cells or stromal cell conditioned medium reduced the effect of IDA on HL-60 cells in culture. In conclusion, leukemia bone marrow stromal cells contribute to increasing resistance of HL-60 cells to chemotherapeutic agents, and play some role in developing minimal residual disease.