ABSTRACT:Objective To analyze the effects of antisurvivin oligonucleotide (Survivin ASODN)on IL-6/STAT3 signaling pathway and down-stream cancer genes of ovarian cancer SKOV3 cell line and to detect the changes of invasive ability of cell line in order to explore the role of Survivin ASODN in reducing metastasis and recurrence of ovarian cancer and its potential clinical value.Methods ASODN was transfected into SKOV3 cells by lipofectamineTM2000 in ASODN group,and lipofectamineTM 2000 was introduced in control group.The invasive ability in ASODN and control groups was detected by Transwell chamber.The expressions of interieukin-6 (IL-6 ), signal transducer and activator of transcription 3 (STAT3 ), Survivin, and vascular endothelial growth factor (VEGF)in ovarian cancer cell line at mRNA level were detected by Real-time PCR.The expressions of IL-6 , STAT3 ,signal transducer and activator of transcription 3 phosphorylation (p-STAT3 ),Survivin,and VEGF-A in ovarian cancer cell line at protein level were detected by Western blot.Results The expressions of IL-6,STAT3, Survivin,and VEGF in ovarian cancer cell line at mRNA level were significantly down-regulated in ASODN group (P<0 .0 5 ).IL-6 ,STAT 3 ,p-STAT 3 ,Survivin and VEGF-A protein levels in ovarian cancer cell line were significantly down-regulated in ASODN group (P<0.05).The invasive ability was significantly reduced by ASODN (P<0.01).Conclusion ASODN targeting Survivin could reduce the invasive ability of ovarian cancer cell line. The mechanism may be blocking IL-6/STAT3 signaling pathway and its down-stream cancer genes of ovarian cancer cell line.ASODN targeting Survivin may have clinical value in preventing and treating the metastasis and recurrence of ovarian cancer.

Objective To develop a HPLC-MS/MS methodfor determination of cyclosporin A(CSA)and AM1 in bone marrow transplant patient,and explore the relationship of CSA and its main metabolite AM1 within individual and between individuals,and provide reliable basis for clinical rational use of monitoring in CSA blood drug concentration.Methods CSD was used as internal standard,and whole blood samples were treated with internal standard methanol precipitated protein.The column was Ultimate XB-C18 with a column temperature of 65 ℃ and the mobile phase was eluted with 0.1％ of formic acid and 2 mmol/L ammonium acetate in water and methanol containing 0.1％ formic acid.The mass spectrometry was detected by electrospray ion trap positive ion mode,MRM scanning,monitoring CSA 1219.9 to 1203.1 m/z,AM1 1236.1 to 1219.1 m/z,CSD 1234.0 to 1217.0 m/z.Results The concentration of CSA was linear in the range of 16 to 1600 ng/mL,Y=0.0143X+0.0213(r=0.9976).The concentration of AM1 was linear in the range of 10～1000 ng/mL,Y=0.00363X-0.00528(r=0.9973).The ratio of CSA to AM1 (AM1/CSA) between individual ranged from 32％ to 356％.The ratio of CSA to AM1 within indiviolual(AM1/CSA) ranged from 27 ％ to 147 ％.Conclusion HPLC-MS/MS method for the simultaneous detection of CSA and AM1 was established.The variation of CSA exists in the bone marrow transplant patient between individuals and within individual;the HPLC-MS/MS method can be used for monitoring of whole blood concentration of CSA in bone marrow transplant patients.

Endothelium-derived hyperpolarizing factor (EDHF) is the third factor released by en-dothelial cell other than NO and PGI2. It relaxes smooth muscle accompanied by a hyperpolarization in the membrane potential. EDHF may be epoxye-icosatrienoic acids (EETs) formed from arachidon-ic acid by the action of cytochrome P450. It is synthesized and/or released by endothelial cell as a result of an cytosolic Ca2+ increase, which is stimulated by the action of acetylcholine or bradykinin on endothelial cell. EDHF is shown to activate Ca2+-activated K+ channels and induce a hyperpolarization in the membrane potential in vascular smooth muscle. The hyperpolarization of the membrane inhibits the opening of voltage-dependentcalcium channels, allows calcium sequestration and removal mechanisms to lower intracellular calcium, and leads smooth muscle to relaxation. In large conducing arteries, EDHF may provide a secondary system to NO, which assumes primary importance in endothelium-dependent relaxation and inhibits the release of EDHF. However, in small resistance arteries, EDHF appears to be a major determinant of vascular calibre, and may be of primary importance in the regulation of vascular resistance.

Objective To develop a HPLC-MS/MS method for determination of Tacrolimus(FK506) in bone marrow trans plant patient,and explore the relationship of HPLC-MS/MS with CMIA and Elecsys in the measurement of FK506 blood drug concentration,and provide reliable basis for clinical rational use of monitoring method in FK506 blood drug concentra tion.Methods The separation was performed on a Ultimate xB-C18 column with a mobile phase of water (containing 2 mmol/L ammonium acetate and 0.1 ml/dl formic acid) and methanol (containing 0.1 ml/dl formic acid).The way of eluting was gradient.Mass spectrum detection method was ESI positive ion mode and the MRM transitions of FK506 m/z 821.5～ 768.5 and Ascomycin m/z 809.4～756.4.Selected part of the whole blood samples of FK506 in bone marrow transplant patient and respectively were tested by HPLC/MS/MS method,CMIA method and Elecsys method.Then,three methods for the detection of FK506 density were compared.Results The standard curve of FK506 was linear over the range of 0.5～50 ng/ml,Y=0.228X-0.003 08 (r=0.999 0).FK506 blood concentrations were not significantly different in HPLC/MS/MS method,CMIA method and Elecsys method.Conclusion The HPLC-MS/MS method in the detection of FK506 was established and can be used for monitoring of whole blood concentration of FK506 in bone marrow transplant patients.

Objective To explore the effects of osteoblasts on the recovery of hematopoiesis and angiogenesis in acute irradiation injury mice.Methods The femurs of 18 male BALB/c mice were used to prepare the bone marrow osteoblasts, and the rest mice were divided into 3 groups as normal group, saline group and osteoblast group.The mice in normal group received no treatment, and the other two groups were received 6.0 Gy 60Co γ-ray irradiation.After irradiation each mouse of osteoblast group was administered with 2 × 106 osteoblasts through tail vein injection, and equal volume saline was given to each mouse of saline group by the same way.The following factors were measured at 7, 14, 21 d after irradiation, they were the counts of peripheral blood cells and bone marrow mononuclear cells ( BMMNC ) , the percentage of CD34 + cells in BMMNC, the histology changes and micro vascular density (MVD) of bone marrow tissue.Results The counts of peripheral blood cells, BMMNC and hematopoietic tissue area in osteoblast group were higher than those in saline group.The percentage of CD34 + cells in BMMNC and the MVD of bone marrow in osteoblast group were also higher than those in saline group at 7, 14, 21 d after irradiation ( t = 2.46 - 64.51, P ＜ 0.05 ).Conclusions Osteoblasts could significantly promote the recovery of hematopoiesis and angiogenesis in mice after acute irradiation injury.

Objective To explore the effects of cotransplantation with osteoblasts on hematopoietic reconstitution in mice after bone marrow transplantation (BMT). Methods The typical model of syngeneic BMT was established. 18 Balb/c mice were used to prepare the bone marrow nuclear cells and osteoblasts for BMT. The 42 Balb/c mice were randomly divided into 3 group:normal group (6 mice, without any treatment), the single BMT group ( 18 mice, given 2 × 106 bone marrow nuclear cells/each mouse) and the cotransplantation group of HSC with osteoblaats (18 mice,given 2 × 106 bone marrow nuclear cells and osteoblasts/each mouse). The following factors were measured on day 7, 14, 21 after BMT: peripheral blood cells, bone marrow mononuclear cells (BMMNC), the percentage of CD34+ cells in BMMNC (assayed by flow cytometry), the hematopoietic tissue changes (detected by HPIAS-1000 image analysis system) and micro vascular density (MVD) of bone marrow tissue (with immunohistochemistry). Results The levels of periphral WBC, RBC, PLT, BMMNC in the contransplantation group were higher than those in the single BMT group (P＜0. 01 or P＜0. 05). In the contransplantation group, the percentage of CD34+ cells in BMMNC, the hematopoietic tissue area and the MVD of bone marrow were also higher than the single BMT group on the 7th, 14th, 21st day after BMT(P＜0.01 or P＜0.05). Conclusion Cotransplantation with osteoblasts could significantly promote hematopoietic reconstruction in mice after BMT. Cotransplantation may represent a promising means of achieving higher engraftment rate after BMT.

To study the expression of the bFGF and its receptor in the mouse bone marrow by treatment with acute radioactive injury and Ligustrazine, 56 mice were divided into 3 groups: normal group, radiation-injured group and Ligustrazine group. After irradiation by 6.0 Gy 60Co gamma-ray, each mouse was orally given 0.1 ml Ligustrazine twice a day for 13 days in Ligustrazine group, and each mouse in radiation injured group was orally given equal amount of saline. On the 3rd, 7th, 14th day after irradiation, bone marrow mono-nuclear cells (BMMNC) were counted, and the expression levels of bPGF and bFGFR in bone marrow were evaluated by immunohistochemistry and flow cytometry analysis respectively. On the 3rd, 7th, 14th day after irradiation, expression of bFGF in bone marrow were significantly lower than in normal group (P<0.05 or P<0.01). Expressions of bFGF and bFGFR were much higher in Ligustrazine treated group than that in the control group (P<0.05 or P<0.01). Ligustrazine potentiate the expression of bFGF and bFGFR in bone marrow MNC to recover the bone marrow hematopoiesis inductive microenvironment, which is one of the mechanisms by which Ligustrazine rebuild the bone marrow hematopoiesis after acute radioactive injury.
Bone Marrow Cells/metabolism ; Fibroblast Growth Factor 2/*biosynthesis ; Hematopoiesis/drug effects ; Pyrazines/*pharmacology ; Radiation Injuries, Experimental/*metabolism ; Radiation-Protective Agents/pharmacology ; Receptors, Fibroblast Growth Factor/*biosynthesis

To investigate p120 catenin mRNA expression in Non-Hodgkin's lymphoma (NHL) cell lines (U937, Raji, Jurkat and Molt4) and normal lymphocytes and explore the relationship between p120 catenin and Non-Hodgkins lymphoma, total RNA sample was extracted by using TRIzol and reversely transcripted into cDNA. Polymerase chain reaction was performed to detect mRNA expression of p120 catenin in NHL cell lines U937, Raji, Jurkat and Molt4. Normal lymphocytes were used as control. It was found expressions of p120 catenin 1A and 3A mRNA were high in above-mentioned NHL cell lines, but neither p120 catenin 1A nor 3A was found in normal lymphocytes as shown by RT-PCR. It is concluded that both P120ctn1A and P120ctn3A mRNA transcripts were found in all NHL cell lines U937, Raji, Jurkat and Molt4 but they don't exist in normal lymphocytes, suggesting p120ctn possibly is of importance in diagnosis and therapy of lymphoma.
Catenins/genetics ; Cell Adhesion Molecules/*genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Jurkat Cells ; Lymphoma, Non-Hodgkin/genetics ; Lymphoma, Non-Hodgkin/pathology ; Phosphoproteins/*genetics ; RNA, Messenger/genetics ; RNA, Messenger/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Markers, Biological/genetics ; U937 Cells

Objective To investigate the correlation between MRI characteristics and tumor pathologic manifestitions of ovarian tumors.Methods 38 cases of benign ovarian tumor with clinic and MRI data were enrolled in this study.The correlation between the results of postoperative pathology and MRI characteristics was analyzed.Results 38 cases showed as ovarian tumor by MRI was diagnosed as ovarian tumor by pathology.So the accuracy of qualitative diagnosis was 100%;48 lesions were detected by surgery and pathology, of which one case had 3 lesions.The detection rate was 87.5%(42/48).Conclusion MRI is a relatively reliable method for the localization and qualitative diagnosis of ovarian tumor,which provides the guidance for surgery.

To modify and improve the quality standard of Fufang Shilintong capsules. Methods: Lonicerae Japonicae Caulis and Pyrrosoae Folium in Fufang Shilintong capsules were identified by TLC, and the content of chlorogenic in the capsules was determined by HPLC. Results: Specific spots of Lonicerae Japonicae Caulis and Pyrrosoae Folium were shown in TLC. Chlorogenic showed a good linear relationship within the range of 0. 020-1. 530 μg(r=1. 000 0), and the average recovery was 99. 2%(RSD=0. 8%, n=6). Conclusion:The method is accurate and reliable, which can be more effectively used in the quality control of Fufang Shilintong capsules.