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.

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.

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.

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.

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 results and effects of freezing phrenic nerves for the patients of pulmonary lobectomy.To optimize the best freezing time by studying the effects of different time at-65 ℃.Methods 50 patients of pulmonary lobectomy were randomly entered into 5 groups,including control group,30 seconds group,60 seconds group,90 seconds group,120 seconds group.After operation,the patients' cardiotach,blood pressure,SaO2,breath rate,time period of pulling out drainage tube and VAS about referred pain of scapular region were noted.After pulling out the intrathoracic drain tube,the routine chest normal X ray film and chest ultrasonic inspection were processed and the post-operation remnant cavity were observed.The chest normal fluoroscopy was inspected in 90 days after operation in order to observe the motion information of trouble side diaphragmatic muscle.Results The chest fluid [first day (329±178) ml,(345±150)ml,(268±51) ml,(227±36) ml,second day (251±131) ml,(269±112) ml,(208±61) ml,(158±110) ml,time of pulling out intrathoracic drain tube (5.8±1.75) days,(4.6±1.77) days,(3.9±0.74) days,(3.6±1.07) days] and VAS [(3.6±2.9) scores,(2.2±2.4) scores,(1.0±1.3) scores,(0.7±1.2) scores] about referred pain of scapular region of freezing groups were obviously lower than those in the control group [(375±136) ml,(309±132) ml,(5.7±2.36) days,(4.0±3.3) scores].90 s and 120 s freezing groups were lower than that of 30 s and 60 s groups,andthe 90 s freezing group did not significantly different from the 120 s group.The heart rate,blood pressure,saturation of blood oxygen and breathing rate of each group also had no difference.The remnant cavity sizes were larger in the control group (＞200 ml),they also had more fluidity (＞200 ml),and one case had been taken punctuation.The remnant cavity sizes of freezing groups were small and didn' t need special treatment.The diaphragmatic muscle' s movement of each group were fine after 3 months of operation.Conclusion The freezing phrenic nerves can effectively reduce the chest fluid,the post-operation remnant cavity and the time of pulling out intrathoracic drain tube.The freezing phrenic nerves can reduce the pain of referred pain of scapular region.The best freezing time should be 90 s.The freezing phrenic nerves do not influence the respiration function,and should be advantages of for the clinic researches and applications.

Objective: To establish a method for the content determination of naringin and neohesperidin in Weitong pills.Methods: An HPLC method was adopted.The determination was performed on an Agilent TC-C18 column with the mobile phase consisting of acetonitrile-water(20∶80)at the flow rate of 1.0 ml·min-1.The detection wavelength was set at 283nm.The column temperature was 30 ℃.Results: Naringin and neohesperidin respectively within the concentration range of 0.027-4.552 μg (r=0.999 9) and 0.029-4.016 μg(r=0.999 8) had good linear relationship with the peak area, the average recovery was 102.19% and 103.60%,and the RSD was 2.88% and 1.79%(n=9), respectively. Conclusion: The method is simple,accurate and reproducible, and suitable for the content determination of naringin and neohesperidin in Weitong pills.

Objective To investigate the effect of Janus kinase2/signal transducer and activator of transcription 1 (JAK2/STAT1) signaling pathway on acute kidney injury induced by liver cold ischemia reperfusion (IR) in rats.Methods Thirty healthy male Sprague-Dawley rats,weighing 220-250 g,were assigned randomly to 3 groups (n =10/group):sham operation group (Sham group);liver cold ischemia reperfusion model group (I/R group);JAK2 kinase inhibitor AG490 group (AG490 group) (AG490 at dose of 10 mg/kg was intraperitoneally injected 30 min before establishment of the model).Other groups were given the equal volume of normal saline at the same time points.Then the rats were sacrificed at 6 h after reperfusion (at 6 h after the end of operation in Sham group).The renal function and oxidative stress level were observed.The pathological changes of the renal tissues and nephritic cell apoptosis were analyzed,and the expression of p-JAK2,pSTAT1,Bcl-2 and Bax was detected by Western blotting.Results As compared with Sham operation group,renal histological lesion and renal dysfunction were aggravated,level of oxidative stress and apoptosis rate were increased in I/R group,the Bcl-2/Bax ratio was decreased and the expression of pJAK2 and p-STAT1 was up-regulated.As compared with I/R group,AG490 dramatically attenuated histological lesions and oxidative stress,restored the renal function,and reduced the number of apoptotic tubular epithelial cells.AG490 significantly increased the Bcl-2/Bax ratio,and inhibited the expression of p-JAK2 and p-STAT1.Conclusion Blockage of JAK2/STAT1 signaling pathway can alleviate acute kidney injury after liver cold ischemia reperfusion probable through inhibiting the oxidative stress and apoptosis.

Objective To evaluate the effect of dexmedetomidine on myocardial injury in pediatric patients undergoing living-related liver transplantation. Methods Fifty-eight pediatric patients of both sexes,aged 5-20 months,weighing 4.5-15.0kg,of American Society of Anesthesiologists physical status Ⅲ or Ⅳ,scheduled for elective living-related liver transplantation,were divided into 2 groups(n=29 each)using a random number table:control group(group C)and dexmedetomidine group(group D).In group D,dexmedetomidine was intravenously infused at a dose of 0.5 μg/kg over 10min starting from the time point immediately before skin incision,followed by an infusion of 0.8 μg·kg-1·h-1 until the end of surgery. The equal volume of normal saline was given instead in group C. Immediately before skin incision(baseline,T0),at 10min of anhepatic phase(T1),at 30min of neohepatic phase(T2)and at the end of surgery(T3),blood samples were obtained from the central vein for determination of serum cardiac troponin I(cTnI),lactate dehydrogenase(LDH),alpha-hydroxybutyrate dehydrogenase(α-HBDH),interleukin-6(IL-6)and IL-10 concentrations. The changing rate of serum cTnI concentrations were calculated at T2. The occurrence of myocardial ischemia and ventricular premature beat and requirement for dopamine were recorded during surgery. Results Compared with the baseline at T0,the serum concentrations of cTnI,LDH and α-HBDH were significantly increased at T2,3,and the serum concentrations of IL-6 and IL-10 were increased at T1-3 in both groups(P<0.05).Compared with group C,the serum concentrations of cTnI,LDH,α-HBDH and IL-6 were significantly decreased at T2,3,the serum concentration of IL-10 was increased at T1-3,the changing rate of serum cTnI concentrations was decreased(P<0.05),and no significant change was found in the incidence of myocardial ischemia and ventricular premature beat and requirement for dopamine in group D(P>0.05).Conclusion Dexmedetomidine can attenuate the myocardial injury to some extent in pediatric patients undergoing living-related liver transplantation.