BACKGROUND: The determination of monoamine neurotransmitters and their metabolites is important in the experimental.research of Parkinson disease (PD) and experimental diagnosis of phaochromocytoma.OBJECTIVE: To develop a high performance liquid chromatography (HPLC) method for simultaneous and rapid determination of monoamine neurotransmitters and their metabolites which include epinephrine(E), norepinephrine(NE), dopamine(DA), 5'-hydroxytryptamine(5'-HT), 5-hydroxy3-indoleacetic acid(5-HIAA), homovanillic acid(HVA) and 3,4-dihydroxyphenylacetic acid(DOPAC).DESIGN: Case control observation and a randomized controlled animal experiment.SETTING: Department of Clinical Laboratory, Affiliated Hospital of Medical College, Qingdao University; Haiyang Branch, Affiliated Hospital of Medical College, Qingdao University.PARTICIPANTS: This experiment was carried out at Staff Room of Physiology, edical College, Qingdao University and Department of Clinical Laboratory, Affiliated Hospital of Medical College of Qingdao University from October to December 2003. ①Two patients with phaeochromocytoma were all confirmed by CT, MRI or pathologic section as well as physical sign. Twelve health volunteers were all university students and were excluded to suffer hypertension, endocrine system diseases and other diseases. ② Total 12 healthy female Wister rats weighed between 150 g to 180 g were chosen and randomly divided into control group and PD group with 6 rats in each.METHODS: The standard chromatograms of the standard solution of mixtures of seven substances (E, NE, DA, 5'-HT, 5-HIAA, HVA and DOPAC)were prepared and the precisions and recovery rates of the same samples were determined respectively. Urine samples of 24 hours obtained from 12 healthy volunteers and 2 patients with phaeochromocytoma were analyzed respectively by HPLC. The unilateral forebrain bundles in PD group were destroyed with 6-hydroxydopamine (6-OHDA) for monitoring release characteristics of monoamine neurotransmitters and their metabolites in healthy and marred caudate putamen nucleus striatum of normal and PD rats striatum.MAIN OUTCOME MEASURES: ①Determination of the precisions and recovery rates of monoamines. ②The contents of catecholamines in 24 hours urine of 12 health volunteers and 2 patients with phaeochromocytoma .③The release characteristics of monoamines and their metabolites in rats striatum. RESULTS: The results and analysis of catecholamines substances in 24hours urine were all obtained from 12 health volunteers and 2 patients with pheochromocytoma. There were no animals died in the experinment, and all of them were involved in the result analysis. ①There was a favorable linear relationship in the range of 2-1 000 μg/L for DA, E, NE and DOPAC, 10-1 000 μg/L for 5-HIAA, HVA and 5-HT for 30-1 000 μg/L with HPLC. The minimal detection limit of DA, E, NE and DOPAC was 2 μg/L, 5-HIAA 10 μg/L, HVA and 5-HT 30 μg/L. The coefficient of variation (CV) in the same batch was 3.0%-8.6%, and the CV in the different batchs was 3.8%-9.7%. The average rates of recovery of DA, NE, E and other 4 kinds of substances were 107.3%, 89.4%, 80.1% and 89.2%-95.7%, respectively.②The contents of CA in 24 hours urine of the 2 patients with phaeochromocytomas were higher than those of the 12 healthy volunteers. One of patient samples showed that E was mainly higher than that of control group. Other samples showed that NE was mainly higher than that of control group. Otherwise , E and NE were ten times higher than that of control group. ③The contents of DA and its metabolites such as DOPAC and HVA in intact sides of hemiparkinsonian rats had no significant changes ,while the contents of DA, DOPAC and HVA in lesion sides were significantly decreased compared with the controls (P ＜ 0.01 ).There were no significant changes in the contents of 5-HT and its metabolites 5-HIAA. ④The turnover rates of DA (DOPAC/DA and HVA/DA) in intact and lesion sides of hemiparkinsonian rats were all higher than those of the controls(P ＜ 0.05).CONCLUSION: The method we developed is suitable for the experimental research of normal and hemiparkinsonian rat models and for the diagnostic testing of phaeochromocytoma.

The total protein of Toxoplasma gondii RH strain tachyzoites was separated by two-dimensional electrophoresis (2-DE), and Western blotting was performed to find out distinct antigens. 209 spots were detected through Coomassie brilliant blue-stained small gels (7?8 cm, pH 3-10). Western blotting showed 17 specific antigen spots with sera of the experimental group and two nonspecific spots with the control sera.

Objective To evaluate the efficacy of total glucosides of paeony(TGP) in the treatment of undifferentiated spondyloarthropathies(uSpA) and investigate its mechanism.Methods One hundred and sixteen cases of uSpA were randomly assigned to 2 groups,one group was treated with TGP and diclofenac sodium,the other group with SASP and diclofenac sodium.The efficacy and side effects of the two groups were compared after 12 weeks treatment.The level of IL-6,IL-10 and TNF-? was determined at the same time.Result After 12 weeks treatment,the major clinical manifest and the indexes of disease activity were no difference of statistical significance in the two groups.The incidence of parenterally side effects was 8.6% in TGP group,and 25.9% in SASP group.There was difference at statistical significance between the two groups(? 2 =6.042,P=0.014).Before treatment,the level of IL-6,IL-10 and TNF-? in the serum of two groups were no statistical difference,while there was obviously difference after 12 weeks treatment(t= 2.672,2.483 and 2.088,P=0.009,0.014 and 0.039).Conclusion The efficacy of TGP and SASP has no significant difference in the treatment of uSpA.But the incidence of parenterally side effects in the TGP group was less than SASP group.The mechanism of efficacy of TGP may be related to inhibiting the production of IL-6,IL-10 and TNF-?.

Objective To improve the understanding of osteoarticular pain presented as rheumatic syndromes of cancers and malignant skeleton metastasis associated. Methods To analyse the clinical data of 32 patients initially manifested as osteoarticular pain. Results Forteen patients of 32 cases was diagnosed as cancer associated rheumatic syndromes, characterized by gradually deteriorated chronic recurrent vague pain. The effect of non-steroid anti-inflammatory drugs (NSAIDs) and glucocorticoid was not satisfactory. Pain subsided after removal of carcinous mass. Eighteen patients had malignant skeleton metastasis, vague pain at onset, but gradually progressed to be, severe persistent pain. NSAIDs and glucocorticoid were ineffective. Narcotic analgesics could produce short time relief. The nature and distribution of tumour were comparatively extensive. The clinical misdiagnosis rate was high. Conclusion Clinical doctors often neglect the coexistence of tumor because they are not familiar with the relation between osteoarticular pain and tumor. So patients who is over sixty and is not sensitive to commonly used analgesics and glucocorticoid with osteoarticular muscular pain should be considered the possibility of malignant tumor after excluding diffuse connective tissue disease.

Objective To detect the frequency of peripheral blood CD4+ CD25high Tr and CD4+CD25lowT cells and expression of programmed death-1 ( PD-1 ) on their surface in the patients with systemic lupus erythematosus(SLE) and their clinical signifcance is analyzed.Methods The expression of PD-1 on the CD4+ CD25highTr and CD4+CD25lowT cells was examined in patients with 33 active SLE,18 inactive SLE and 38 healthy controls(HC) by flow cytometry.Clinical manifestations and laboratory findings of SLE were collected.Patients were divided into two groups according to their disease activity.SLE disease activity index(SLEDAI) score≥10 was defined as high disease activity and ＜10 as inactivity.The percentage of CD4+ CD25highTr and CD4+CD25lowT cells and proportions of expression of PD-1 on their surface were compared between not only inactive or active SLE patients and healthy controls (HC),but also between patients with lupus nephritis and without lupus nephritis.Correlation with clincal manifestations and laboratory findings was analyzed.Results (1)The proportions of CD4+CD25highTr and CD4+CD25low T cells were significantly decreased in active and inactive SLE patients as compared with HC.(2)The percentage of CD4+ CD25lowPD-1 +Tr and in active and inactive SLE patients were higher than HC.The proportions of CD4+CD25lowPD-1+ T cells were significantly increased in HC and inactive SLE patients as compared with inactive SLE patients.(3) The proportions of CD4+CD25highPD-1+Tr in SLE patients with nephritis were higher than those in patients without nephritis.But the percentage of CD4+ CD25 lowPD-1 + T cells was significantly decreased in SLE patients with nephritis as compared those without nephritis.(4)A positive correlation was observed for proportions of CD4+CD25highPD-1 + T cells with SLEDAI score.Proportions of CD4+ CD25high Tr,CD4+ CD25low PD-1 + T cells was inversely correlated with SLEDAI score.Conclusion The aberrations of proportions of CD4+CD25highpD-1+,CD4+CD25lowPD-1+,CD4+CD25high and CD4+CD25low T cells were observed in patients with SLE.The abnormality of PD-1 and PD-L1 pathway may play an important role in the function and number of Tr.

Objective To obtain genes encoding the novel molecules for diagnosis of schistosomiasis.Methods Juvenile S.japonicum cDNA library was immunoscreened to obtain positive clones.By DNA sequencing and sequence analysis,the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a.The recombinant plasmid was transformed into E.coli BL21 followed by expression of the protein induced by IPTG.The protein was identified by Western blotting.Results 34 positive clones were obtained,24 of which were chosen to be sequenced,13 of which were Sj22600 gene.The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting.Conclusion The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library.The E.coli BL21 transformed with the recombinant plasmid can express the fusion protein,which shows immunoactivity.

Postoperative adjuvant therapy for primary invasive breast cancer patients with synchronous ax-illary metastases is mainly based on the characteristics of biomarker of the primary tumor. Recently, some studies have showed the discordance and clinical significance of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status between primary breast cancer and synchronous axillary lymph node metastases. As local metastasis, the synchronous axillary metastases may represent the potentially metastatic breast cancer cells much better than the primary tumor. Hence, determination of biomarkers status should be performed in synchronous axillary metastasis, together with primary tumor, to guide therapy management and evaluate the prognosis of primary invasive breast cancer patients with synchronous axillary metastases.

Objective To evaluate the patient-controlled paravertebral block (PCPB) in optimizing the cellular immune function when used after radical resection of pulmonary carcinoma performed via video-assisted thoracoscope in patients.Methods Forty-one ASA physical status Ⅰ or Ⅱ patients of both sexes,aged 50-64 yr,with body mass index of 20-25 kg/m2,of TNM staging Ⅰ or Ⅱ,undergoing radical resection of pulmonary carcinoma performed via video-assisted thoracoscope,were randomly divided into 2 groups using a random number table:PCIA group (n =21) and PCPB group (n =20).PCIA solution contained sufentanil 2 μg/kg in 100 ml of normal saline.The PCIA pump was set up to deliver a 2 ml bolus dose with a 15-min lockout interval and background infusion at 2 ml/h.In PCPB group,the patients received paravertebral injection of 0.2％ ropivocaine 5 ml at T5 level on the affected side under ultrasound guidance at the end of operation,and then received PCPB.PCPB solution contained 0.75％ ropivacaine 67 ml in 250 ml of normal saline,and the pump was set up to deliver a 5 ml bolus dose,with a 15-min lockout interval and background infusion at 5 ml/h.VAS score was maintained ≤ 3,and analgesia lasted until 50 h after operation.Before induction of anesthesia (baseline),at end of operation,and at 1,3 and 5 days after operation,peripheral venous blood samples were collected to determine the levels of regulatory T cells,natural killer cells and natural killer T cells (by flow cytometry) and plasma concentrations of interleukin-10 and transforming growth factor-β (by ELISA).Results Compared with group PCIA,the level of regulatory T cells was significantly decreased,the levels of natural killer cells and natural killer T cells were increased,and the plasma concentrations of interleukin-10 and transforming growth factor-β were decreased at 1 and 3 days after operation,and no significant change was found in the rate of cellular immune function decline after operation in group PCPB.Conclusion PCPB provides no significant difference clinically in optimizing the cellular immune function when used after radical resection of pulmonary carcinoma performed via video-assisted thoracoscope in the patients.

Objective To investigate the clinical manifestations and laboratory findings of patients with the late onset systemic lupus erythematosus(LOSLE) and the elderly onset SLE. Methods Forty one patients with SLE onset beyond the age of 50 years(including 20 patients SLE onset beyond the age of 60 years) were identified and compared with 41 patients who had SLE onset before the age of 50 (General SLE). Patients in the group of general SLE were selected by 1 to 1 individual matching according to sex, course of diseases, date of admission. The presenting clinical features, laboratory findings, incidence rate of complication, efficacy of glucocorticoid and the prognosis of patients in two groups of LOSLE and general SLE were compared and analyzed. In addition, 20 cases of elderly onset SLE were also compared and analyzed. Results There were significant differences in symptoms between groups of LOSLE and General SLE. The LOSLE group had less occurrence of rash (39 0% vs 73 2%, P

AIM: To observe the effects of H_2O_2 on apoptosis of skeletal muscle satellite cells (SMSC)and mitochondrial membrane potential (MMP), and protective effect of erythropoietin(EPO). METHODS: SMSC in vitro were divided into three groups: H_2O_2 group, H_2O_2+EPO group and control. Apoptosis rate and the means were obverted by monofluorescence flow cytometry. The morphological change of apoptosis cells were observed under fluorescence microscopy after Hoechst 33258 staining. RESULTS: The cells in H_2O_2 group show the highest apoptosis rate (22.13±1.79)%. In H_2O_2+EPO group, apoptosis rate were (16.47±2.53)%, (4.97±0.55)% and (2.93±0.47)% according to the EPO treated levels (10, 20 or 40 kU/L), respectively. MMP level in H_2O_2 group was the lowest 9.70±0.09. MMP levels in H_2O_2+EPO group were 12.67±0.32, 27.90±0.66, 44.53±0.93, respectively according to the EPO treated levels (10, 20 or 40 kU/L). In control group, apoptosis rate was 1.93±0.57 and MMP was 51.37±0.64. In H_2O_2 group and H_2O_2+ low dosage EPO group, Hoechst 33258 staining showed obvious apoptosis. CONCLUSION: EPO inhibits the apoptosis induced by H_2O_2 and stabilizes the MMP, which is related to the dosage of EPO.