Objective To prepare and identify monoclonal antibodies (mAb) against histidine-tag (His-tag ) .Methods Balb/c mice were immunized with polypeptides containing 15 histindine(His)-coupled BSA and this fusion was prepared according to con-ventional methods .Indirect ELISA was used to screen the positive clones and limited dilution for further cloning .After purified with Protein G affinity chromatography ,these antibodies for His-tag were detected for antibody titer ,relative affinity as well as subtypes by using ELISA .The specificity of these mAb was identified by ELISA and Western blotting analysis .Double-antibody sandwich ELISA was composed of these mAb paired with the corresponding recombinant protein of specific antibody ,which was used to de-tect the recombinant protein quantitatively .Results 5 hybridoma cell lines stably secreting anti-His-tag IgG1(κ) were screened .A-mong these antibodies ,1-G2 was of the highest affinity ,reaching 1 .27 × 1010 ,which could combine with different recombinant pro-teins containing His-tag in the experiment of ELISA and Western blotting .1-G2 also could be used to detect recombinant PCT pro-tein containing His-tag quantitatively by using double-antibody sandwich ELISA with antibody of PCT ,the sensitivity of detection reached 4 .6 ng/mL .Conclusion The mAb against for His-tag with high specificity ,affinity and secreted stably are successfully prepared .This prepared antibody could be used in ELISA and Western blotting to detect a variety of His-tag .

Objective To investigate the effect of sevoflurane postconditioning on mitochondrial connexin 43 (Cx43) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts and the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in it.Methods Forty-five adult male SpragueDawley rats,weighing 200-250 g,were used in the study.Their hearts were excised and retrogradely perfused with K-H solution in a Langendorff apparatus.The 45 isolated hearts were assigned into 5 groups (n =9 each) using a random number table:control group (group C),I/R group,sevoflurane postconditioning group (group Sev),and sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD,a specific mitoKATp channel blocker) group (group Sev+5-HD) and I/R plus 5-HD group (group I/R+5-HD).The hearts were subjected to 20 main of global ischemia followed by 90 min of reperfusion to establish the model of myocardial I/R injury.From the beginning of reperfusion,the hearts were perfused with K-H solution saturated with 3％ sevoflurane for 15 min in group Sev,with K-H solution saturated with 3％ sevoflurane and containing 100 μ mol/L 5-HD in group Sev+5-HD,and with K-H solution containing 100 μ mol/L 5-HD in group 5-HD.The heart rate (HR),left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and the maximum rate of increase and decrease of ventricular pressure (±dp/dtmax) were recorded at the end of equilibration (T1) and 30 and 60 min of reperfusion (T2,3).At 90 min of reperfusion,the myocardial infarct size was measured by TTC staining,and the expression of total Cx43 (tCx43)and phosphorylated Cx43 (p-Cx43) in mitochondria was determined by Western blot analysis.The percentage of myocardial infarct size and p-Cx43/tCx43 ratio were calculated.Results Compared with group C,the HR,LVDP and ±dp/dtmax were significantly decreased,and the LVEDP was increased at T2,3,and the percentage of myocardial infarct size was increased in the other 4 groups,the expression of mitochondrial tCx43 and p-Cx43 was significantly down-regulated in I/R,Sev+5-HD and 5-HD groups (P＜0.05),and no significant change was found in the expression of mitochondrial tCx43 and p-Cx43 in group Sev (P＞0.05).Compared with group I/R,the HR,LVDP and ±dp/dtmax were significantly increased,and the LVEDP was decreased at T2,3,the percentage of myocardial infarct size was decreased,and the expression of mitochondrial tCx43 and p-Cx43 was up-regulated in group Sev (P＜0.05),and no significant change was found in the parameters mentioned above in Sev+5-HD and 5-HD groups (P＞0.05).Compared with group Sev,the HR,LVDP and ±dp/dt were significantly decreased,and the LVEDP was increased at T2,3,the percentage of myocardial infarct size was increased,and the expression of mitochondrial tCx43 and p-Cx43 was down-regulated in group Sev+5-HD (P＜0.05).There was no significant change in the pCx43/tCx43 ratio between the five groups (P＞0.05).Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R injury may be related to induction of mito-KATP channel opening and up-regulation of the expression of mitochondrial Cx43 in cardiomyocytes of rats.

Objective To evaluate the effect of sevoflurane postconditioning on the expression of Pim-1 kinase during myocardial ischemia-reperfusion (I/R) in rats.Methods Male Sprague-Dawley rats,weighing 250-300 g,were used in this study.After the animals were anesthetized,their hearts were immediately removed and retrogradely perfused with an oxygenated K-H solution at 37 ℃ in a Langendorff apparatus.Thirty-six isolated rat hearts were assigned into 3 groups (n=12 each) using a random number table:control group (group C),group I/R and sevoflurane postconditioning group (group SP).The hearts were subjected to ischemia for 30 min followed by 120 min of reperfusion to establish the model of myocardial I/R injury.In group SP,the hearts were perfused with K-H solution saturated with 3％ sevoflurane for 15 min starting from the beginning of reperfusion.Heart rate (HR),left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and the maximum rate of increase and decrease of left ventricular pressure (±dp/dtmax)were recorded at the end of equilibration and 30,60,90and 120 min of reperfusion.Myocardial tissues were obtained at T2 for determination of the expression of Pim-1 kinase,Bcl-2 and cytochrome c (Cyt c) in cytoplasm and mitochondria by Western blot.The hearts were selected at T4 for determination of myocardial infarct size (by TTC staining) and for examination of mitochondrial ultrastructure (with transmission electron microscope).Results Compared with group C,HR,LVDP and ±dp/dtmax were significantly decreased,and LVEDP was increased at T1-4,the myocardial infarct size was enlarged,the expression of Pim-1 and Bcl-2 in cytoplasm and mitochondria was down-regulated,the expression of Cyt c in cytoplasm was up-regulated,and the expression of Cyt c in mitochondria was down-regulated in group I/R (P＜0.05).Compared with group I/R,HR,LVDP and ±dp/ dtmax were significantly increased,and LVEDP was decreased at T1-4,the myocardial infarct size was decreased,the expression of Pim-1 kinase and Bcl-2 in cytoplasm and mitochondria was up-regulated,the expression of Cyt c in cytoplasm was down-regulated,and the expression of Cyt c in mitochondria was upregulated in group SP (P＜0.05).The damage to mitochondria was significantly attenuated in group SP as compared with group I/R,and was aggravated as compared with group C.Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R may be related to up-regulation of Pim-1 kinase expression in rats.

Objective To compare the myocardial protective effects of post-treatment with sevoflurane and isoflurane on myocardial ischemia-reperfusion injury(MIRI) in adult rats.Methods Twenty-four adult male SD rats were divided into four groups (n =6) by using the random number table,control group (C),isehemia-reperfusion group (R),sevoflurane post-treatment (S) and isoflurane post-treatment group(I).The Langendorff isolated heart perfusion model was established.The heart rate(HR),left ventricular end-diastolic pressure(LLVEDP),left ventricular developed pressure(LVDP),maximum rate of rise of left ventricular pressure(LV+-dp/dtmax),and maximum rate of decrease of left ventricular pressure(LV-dp/dtmax) were recorded at the end of equilibrium perfusion,and at 30,90 min of reperfusion,respectively.At the end of infusion,1 mm3.of apical myocardial tissue was removed for observing mitochondrial structure under electron microscopy and scoring.The myocardial infarct size(MIS) in the remaining heart tissue was measured by TTC staining.Results Compared with the R group,the S and I groups showed improved cardiac function indicators,decreased MIS,and reduced mitochondrial damage after reperfusion(P＜0.05).Compared with the S group,the I group showed worse heart function,increased MIS,and more severe mitochondrial damage after reperfusion(P＜0.05).Conclusion Post-treatment with sevoflurane and isoflurane has a protective effect on MIRI in adult rats.Post-treatment with sevoflurane has a better cardioprotective effect than that with isoflurane.

Objective: To study the efficacy and safety of the intrapleural injection of nedaplatin combined with recombinant hu-man endostatin (Endostar) in the treatment of malignant pleural effusion in non-small cell lung cancer (NSCLC) patients. Methods:Seventy patients diagnosed with NSCLC with malignant pleural effusion were treated by intrapleural injection. The subjects were divid-ed into two groups, as follows: 35 cases were injected with nedaplatin and Endostar (the treatment group) and 35 cases were injected with nedaplatin only (the control group). One month after the treatment, the clinical efficacy and toxicity were evaluated. The clinical ef-ficacy, quality of life, and toxic reaction were compared between the two groups. Results: The short-term clinical efficacy rate of the treatment group was 74.28%, whereas that of the control group was 48.57%, and the difference between the two groups was significant (P<0.05). Adverse effects such as nausea vomiting and leukopenia, occurred in both groups, but no significant differences were found between the two groups (P>0.05). Conclusion: The efficacy of the intrapleural injection of recombinant human endostatin combined with nedaplatin is superior to that of cisplatin alone in the treatment of malignant pleural effusion, without increasing chemotherapeutic toxicity.

Objective:To investigate the clinical efficacy of mechanical solitaire AB stents thrombectomy combined with intra-arterial thrombolysis in the treatment of patient with acute ischemic stroke.Methods:Fifteen patients with acute ischemic stroke admitted into our hospital from August 2014 to August 2016 were treated with mechanical thrombectomy with solitaire AB stents plus intra-arterial thrombolysis.The National Institutes of Health Stroke Scale score (NIHSS) of all patients were evaluated before and after treatment to compare the clinical efficacy.The prognosis ofpatients between two groups were compared via evaluating modified Rankin score (mRS)and gelasijia coma score (GCS).Results:After mechanical thrombectomy with solitaire AB stents plus intra-arterial thrombolysis treatment,14 patients achieved complete or part recanalization,and 1 patient was terminated treatment due to vital signs instability,and the rate ofrecanalization was 93.3％.The NIHSS score of patients before treatment was 12.93± 4.25,which was much higher than that after treatment (4.33± 1.45,P＜0.05).After follow-up by 3 months,the good mRS scores were obtained in all 18 patients,including 2 patients with mRS score of 2,5 patients with 1,and 8 patients with 0.Additionally,there was no patient with re-obstruction during follow-up period.Conclusion:Mechanical thrombectomy with solitaire AB stents combined with intra-arterial thrombolysis had a good capability and safety in the treatment of patients with acute ischemic stroke.

Objective To identify interleukin 17 (IL-17) and B cell activating factor (BAFF) that could influence B cell biology by detecting the expression of BAFF in the serum and labial salivary glands from primary Sj(o)gren's syndrome (pSS) patients and to test the apoptosis rates of B cells cultured with Th17 cells which were transfected with IL-17-siRNA,BAFF-siRNA.Methods A total of 40 patients with pSS who were referred to the Department of Rheumatology and Immunology at Anhui Provincial Hospital from June 2011 to June 2012 were enrolled into this study.The expression of BAFF on salivary gland and serum from pSS patients and healthy controls were detected by ELISA and immunohistochemical examination (22 patients with pSS).Flow cytometry was used to detect B cell's apoptosis,BAFF and IL-17 interfered with amplified Th17 cells,and co-cultured with B cells.Immunoblot was used to detect supernatant antibody in 5 patients with pSS.Independent samples t test was used for statistical analysis.Results In all pSS specimens,infiltrating inflammatory cells expressed BAFF,so did some ductual cells,but acinar cells did not express these markers.There was no expression of BAFF in the controls.BAFF-positive cell numbers in the labial salivary glands of pSS patients with focal infiltrating lymphocytes were more than that with non-focal infiltrating lymphocytes (888±372 vs 164±161,t=5.94,P＜0.05),and the percentage of BAFF-positive lymphocytes over the total infiltrating lymphocytes in the salivary glands of pSS patients with focal infiltrating lymphocytes [(0.18 ±0.08) ％] was higher than those with non-focal infiltrating lymphocytes [(0.09 ±0.07) ％] (t =3.03,P＜0.05).The level of soluble BAFF in patients with pSS [(6.0±2.8) ng/ml] was significantly higher than the controls [(3.8±1.7) ng/ml,t=3.26,P＜0.05].BAFF or IL-17 transfected group,B cell apoptosis rate [(24± 5)％,(23±5)％] were significantly higher than the non-transfected group [(7±4)％],t=4.6,4.4; P＜0.05].And there was no significant difference when compared with cultured B cells (P＞0.05).Compared with the controls,no antibody could be detected in the supernatants.Conclusion BAFF may be involved in the process of local inflammatory damage of the pSS,it may have a synergistic effect with IL-17 on abnormal B cell function.

Objective:To determine the relationship among tumor malignancy, invasion, and plasma D-dimer (DD) levels via a retrospective analysis of the correlation between preoperative plasma DD levels and postoperative pathology parameters in esophageal cancer patients. Methods:A total of 101 esophageal cancer patients without coagulation among preoperative plasma DD levels and postoperative pathology parameters in esophageal cancer patients who underwent radical operation between January 2009 to April 2010 were enrolled. The preoperative plasma DD levels, postoperative pathology T, N Grade, number of metastatic lymph nodes, tumor posi-tion, tumor length, and TNM stage were recorded. A statistic analysis of the above parameters was performed to reveal the correlation between plasma DD levels and tumor pathology features. Results:Preoperative plasma DD level has no correlation with Grade and tu-mor length, but shows positive correlation with pathology T, N status, and TNM stage, and has strong positive correlation with numbers of metastatic nodes. In addition, lower-third thoracic esophageal cancer patients have higher DD level than middle-third thoracic cases. Conclusion:Plasma DD level has a positive correlation with T stage, N stage, TNM stage, and number of positive lymph nodes. It also has important clinical significance.

Objective To explore the effect of heating temperature and heating time on the dissolution characteristics of phenolic acids in water extract of Danshen Decoction. Methods The contents of tanshinol, protocatechuic aldehyde and salvianolic acid B in water extract of Danshen Decoction were determined by reverse-phase high performance liquid chromatography (RP-HPLC) . The changes of the contents were monitored by classic constant temperature acceleration method. Results Of the three components, the content of salvianolic acid was decreased with the increase of heating temperature and the prolongation of heating time, and the degradation characteristics of the salvianolic acid B met the first-order kinetics equation. Conclusion The water-soluble components have less stability in the water extracts of Danshen Decoction. Lignum Santali Albi and Fructus Amomi accelerate the degradation speed of Radix Salviae Miltiorrhizae, suggesting that we should pay attention to the influence of heating temperature and heating time on the content of salvianolic acid B in the extraction and concentration process.

OBJECTIVETo investigate the feature of histomorphology and biology of uterine bizarre leiomyoma from the clinical, pathological features as well as the immunohistochemical expression.

METHODSTotally 25 cases of leiomyomas were studied. Among them, immuno-histochemical staining (SP and ABC methods) for smooth muscle actin (SMA), proliferative cell nucleus antigen (PCNA), estrogen receptors (ER), and progesterone receptors (PR) were performed in 20 cases. The clinical features and follow-up records were analyzed.

RESULTSThe main clinical findings were irregular vaginal bleeding, pain and pelvic tumor. One case was with immense amount of ascitis and the other two were with pregnancy. All the cases had no history of taking pregestine. Light microscopy showed that part of all the cell nuclei were bizarre, accompanying with double or multiple nuclei in which rather big and reddish staining inclusion bodies were obtained (D = 7 - 26 micro m), and the mitotic figures were 0 - 2/10 HPF. Among 20 cases with immunohistochemical staining, markers indicating muscle cells in origin were positive in the bizarre cells, 15 of which (75%) with negative or weak positive PCNA, and 18 of which (90%) with negative ER. There were significant difference between the studied and control groups (P = 0.027, P < 0.005 respectively) and in addition, PR was positive in both these two groups. A majority of the nucleus inclusion bodies was SMA positive. Follow-up records demonstrated so far no recurrence cases obtained.

CONCLUSIONSUterine atypical leiomyoma belongs to benign tumor, although its shape is bizarre. In this group, the morphology changes are correlated with pregnancy, but not with pregestine. The expression of immunohistochemistry shows certain features, and is important to identify uterine bizarre leiomyoma with leiomyosarcoma and STUMP in pathological diagnosis.

Adult ; Female ; Humans ; Immunohistochemistry ; Leiomyoma ; metabolism ; pathology ; Middle Aged ; Pregnancy ; Uterine Neoplasms ; metabolism ; pathology