Objective To apply fluorescence-activated cell sorting (FACS) and magnetic activated cell sorting (MACS) in sorting natural killer (NK) cells from human peripheral blood. The efficiency of these two methods were compared so that the basic conditions for the further study on the lower proportion of peripheral blood cells could be created. Methods Twenty cases of normal peripheral blood and 3 cases of post-hematopoietic stem cell transplantation (post-HSCT) were collected and NK cell were sorted by FACS and MACS. Results Before sorting the normal peripheral blood, the proportion of the NK cell in the leukocyte was (12.86±3.62) %, the purity of NK cell was up to (96.15±2.03) % and the recovery was (95.08±2.16) %after sorting by FACS, the purity was up to (93.35±3.61) % and the recovery was (94.11±3.01) % by MACS.And before sorting the post-HSCT peripheral blood, the proportion of the NK cell in the leukocyte was (11.01±2.08) %, the purity of NK cell was up to (96.22±2.16) % and the recovery was (95.27±1.18) % after sorting by FACS. The purity was up to (90.98±1.94) % and the recovery was (94.54±3.52) % by MACS. Thereis difference in purity of NK cell (t = 5.925, P ＜0.05), while no difference in rate of recovery between 2methods (t = 0.789, P ＞0.05). Conclusion FACS is more efficient, rapidly and easily than MACS in sorting NK cell, especially for post-HSCT cases.

Objective To observe the changes of CD+8CD+28,CD+8CD-28 and CD+4CDhigh25CDlow127 regulate T (Treg)cellsin peripheral blood of lung cancer patients,and to analyze the correlation between CD+8CD-28 and CD+4CDhigh25CDlow127 Treg cells to reveal the role and clinical significance of them in lung cancer patients.Methods Flow cytometry was applied to evaluate the level of CD+8CD+28,CD+8CD-28 and CD+4CDhigh25CDlow127 Treg cells in peripheral blood of 60 untreated lung cancer patients and 60 healthy controls group.The association of each term with clinical features was analyzed.Results The percentage of CD+8CD-28 and CD+4CDhigh25CDlow127 Treg cells in lung cancer group[(58.430:15.749) ％,(7.365±2.025) ％]was significantly higher than those in healthy group [(41.057±15.436)％,(6.648±1.669)％,(t=6.102,P＜0.05;t=2.115,P＜0.05)],while the percentage of CD+8CD+28cells is lower(41.570±15.739)％ than that in healthy group[(58.700±15.298)％,(t=-6.043,P＜0.05)].No close associations were found between three index and gender,age,and biological characteristics.With the increase of TNM stage,The percentage of CD+4CDhigh25 CDlow127 Treg cells increased gradually,which was remarkably higher in patients rith stage Ⅳ than that with stage ⅢA(t=-3.898,P＜0.05).The percentage of CD+4CDhigh25 CDlow127 Tregcells was uncorrelated with CD+8CD-28 cells(r=-0.169,P＞0.05).Conclusion The higher percentage of CD+8CD-28 and CD+4CDhigh25 CDlow127 Treg cells,the lower percentage of CD+8CD+28 cells may be the important reasons of immune suppression in lung cancer patients.Though there is no correlation between CD+8CD-28 and CD+4CDhigh25 CDlow127 Treg cells,it is may be helpful to understand immunologic function and it may look for more specific therapy and provide a new reference in the prognosis of lung cancer.

Objective To explore the clinical significance of T lymphocyte subsets and NK cells' detection in peripheral blood of patients with non-Hodgkin' s lymphoma (NHL).Methods 62 newly diagnosed NHL and 30 healthy adults were enrolled for analyzing.T lymphocyte subsets and NK cells were deteced by flow cytometry and compared between different groups according to age,sex and pathology.Multiple linear regression was used to analyze the correlation among the levels of T lymphocyte subsets and NK cells and several clinical factors,such as clinical stage,IPI and B symptoms.Results The proportions of CD3,CD4 and NK cells in peripheral blood of NHL group [(64.13±19.83) ％,(27.10±8.20) ％,(13.51± 10.59) ％] were significantly lower than those of normal control [(65.78±10.69) ％,(31.95±12.74) ％,(18.76± 7.36) ％] (P ＜ 0.05),while the proportions of CD8 and Treg cells in peripheral blood of NHL [(32.15±13.83) ％,(10.44±3.00) ％] were significantly higher than those of normal control [(29.25±12.35) ％,(7.51±4.36) ％] (P ＜ 0.05).In NHL,the proportions of CD3 and NKT cells [(67.06±19.24) ％,(4.91±3.69) ％] in ≤60 years old group were significantly higher than those in ＞ 60 years old group [(59.18±20.33) ％,(4.89±3.05) ％] (P ＜ 0.05).The proportions of Treg cells in T-NHL was significantly higher than that in B-NHL [(8.17±6.41) ％ vs (7.11±2.53) ％] (P ＜ 0.05).The proportions of CD3,CD4,NK and NKT cells in peripheral blood of NHL were significantly correlated with clinical stage (P ＜ 0.05),that of CD8 cells were significantly correlated with IPI (P =0.000),that of Treg cells were significantly correlated with IPI and B symptoms (P ＜ 0.05).Conclusion The cellular immune is suppressed in NHL patients.The proportions of T lymphocyte subsets and NK cells in peripheral blood of patients with NHL are different between different groups according to age,sex and pathology.The correlations among that and clinical factors need to be further explored.

Objective To detect the levels of CD4+CD25HiCD127Low regulatory T cells (Treg) in the peripheral blood and its clinical significance in patients with esophageal cancer. Methods The levels of Treg in the peripheral blood were detected by three-color flow cytometry (FCM) in 80 patients with esophageal cancer and 20 healthy controls. Among the 80 patients, 30 patients were also further studied for preoperative and postoperative comparison after operation. The clinical and pathological data of each patient were collected and analyzed for the correlation with the Treg levels. Results The level of Treg in the peripheral blood of the control group was lower than that of the esophageal cancer patients [(3.36±1.14) % and (5.70±1.96) %, respectively], with significant difference (P <0.01). The levels of Treg in the peripheral blood was higher in the patients with metastasis of lymph node (n=40) than that in the patients without metastasis of lymph node (5.96±1.36) % and (4.23±1.18) %, respectively] (n=30), with significant difference (P <0.01). The levels of Treg in the peripheral blood of the patients were negatively correlated with their TNM classification. As the TNM classification advanced, the level of the Treg in the peripheral blood increased. Conclusion The levels of Treg in the peripheral blood of the patients with esophageal cancer are significantly higher than that of the healthy subjects, which is correlated with the clinical and pathological conditions. The development of esophageal cancer may relate with suppression of immune function.

Effect and safety of repeated embryo aspiration for reduction of muhifetal cervical pregnancy Was retrospectively evaluated.Three women with muhifetal cervical pregnancy successfully received conservative treatment of repeated embryo aspiration under the guidance of transvaginal ultrasound,and the gestational sac contents were aspirated completely.Local(2.5～5.0 mg injectable methotrexate,MTX) or systemic medication was used.Our findings showed that repeated embryo aspiration for reduction of cervical multifetal pregnancy could improve the coupe of disease,decrease systemic medication of MTX,and discontinue a cervical pregnancy within 8 weeks effectively and safely.However,the mminimum therapeutic dose of MTX remained to be explored.

This study established an HPLC fingerprint of Tibetan medicine Shaji Gao from different habitats and lay a foundation for Shaji Gao varieties identification and preparation process. The chromatographic condition was as follow: Agilent zorbax SB-C18 (4.6 mm x 250 mm, 5 μm) eluted with the mobile phases of acetonitrile and 0.4% phosphoric acid water in gradient mode. The flow rate was 1.0 mL x min(-1), and the detection wavelength was set at 360 nm. The fingerprints of 15 batches Shaji Gao were carried out by similarity comparation, 7 chromatographic peaks were extracted as the common peaks of fingerprint, 3 peaks were identified, which were quercetin, kaempferol and isorhamnetin. The similarity degrees of 14 batches of samples were above 0.9 and 1 batch of samples was below 0.9. This is the first established fingerprint of Shaji Gao by using HPLC. This method has good precision, stability and repeatability that it could provide basis for quality control and evaluation of Shaji Gao.
Chromatography, High Pressure Liquid ; methods ; Medicine, Tibetan Traditional ; Quality Control

Objective To investigate the effect of idarubiein to imensify the MAC (IMAC) as preparative regimen for autologous peripheral blood stem cell transplantation(APBSCT)in acute myeioid leukemia.Methods Fourteen patients with acute myeloid leukemia who underwent APBSCT were analysed. IMAC was used as preparative regimens.Results All patients were engrafted successfully.The disease-free survival could be Ben in 8 cases(57.1%),the median disease-free survival duration were 26(8-72)months and no treatment-related mortality was present.Conclusion This study suggested that addition of Idarubicin to the MAC preparative regimens Was safe,effective and feasible for patients with acute myeloid leukemia,and may improve disease-free survival and overall survival.

Bioimprinting is a new developed technique to improve the characteristics of enzymes.Bioimprinting by lauric acid was conducted to improve the esterification activity of lipase PS in sol-gel immobilization process with methyltrimethoxysila(MTMS) and tetramethoxysila(TMOS) as the precursors.Results generated by checking the esterification activity and scanning electron microscope showed that bioimprinting can enhance the specific activity and thermal stability of lipase PS.The bioimprinting system was optimized by orthogonal experiment,and the optimal condition for lipase bioimprinting is water/silane molar ration(R) 12,polyethylene glycol(PEG) 120?l,and lauric acid 0.15 mmol.Compared with the free enzyme and the non-imprinted enzymes,the specific activity of imprinted enzymes has been improved 44.3 fold and 2.4 fold,respectively.Imprinted lipase show better thermal stability,and the relative activity is 58% after incubated in 80 ℃ for 0.5 h,while no activity was detected for the free enzyme.

Objective To evaluate the short-term curative effect and the safety of transcatheter arterial chemoembolization (TACE) therapy by using microspheres and lipiodol for hepatocellular carcinoma (HCC).Methods A total of 87 patients with pathologically proved HCC were randomly divided into the study group (n=44,using embospheres of 100-300 μm in diameter together with lipiodol) and the control group (n=43,using gelfoam particles of 350-560 μm in diameter together with lipiodol).Postopertaive biochemical (liver function and AFP) findings and imaging (CT and/or MRI) manifestations were recorded,and the clinical efficacy and adverse reactions were analyzed.Results TACE was performed in all 87 patients.After the treatment,both the disease benefit rate and the postoperative reduction in AFP level in the study group were remarkably better than those in the control group (P＜0.05),but postoperative liver function indexes were not significantly different from the preoperative ones (P＞0.05).The average number of interventional therapy within the follow-up period of 6 months in the study group was smaller than that in the control group (P＜0.05).No statistically significant differences in 6-,12-and 18-month survival rates existed between the two groups (P＞0.05).Conclusion In treating HCC,TACE by combination use of microspheres and lipiodol is safe,its short-term curative effect is more obvious than TACE by combination use of gelfoam particles and lipiodol,and it can reduce the times of interventional procedure.Before TACE,careful planning of the pre-treatment of hepatic artery-portal vein fistula and the superselective catheterization with micro catheter should be taken into consideration.

Objective To evaluate the recognition and uptake of transglutaminase 3 (TG3) by dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptors on the membrane surface of DC-SIGN-transfected human embryonic kidney (HEK) 293T cells and monocytederived dendritic cells (MDDCs).Methods The eukaryotic expression vector pGCMV-enhanced green fluorescent protein (EGFP) containing DC-SIGN gene fragments was transfected into HEK293T cells to prepare DC-SIGN-EGFP-HEK293T cells by using liposome transfection method.CD14+ monocytes were isolated from peripheral blood samples by magnetic bead-based negative selection,and then were induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to prepare MDDCs.Laser confocal microscopy and flow cytometry were performed to evaluate the recognition and uptake of TG3 protein by DC-SIGN receptors on the surface of HEK293T cells and MDDCs.MDDCs treated without Alexa Fluor 647 dye-tagged TG3 served as blank control group,and those treated with Alexa Fluor 647 dye alone served as negative control group.Results After co-culture with TG3 for 3 hours,laser confocal microscopy and flow cytometry both showed that TG3 could be recognized by and uptaken through DC-SIGN receptors into HEK293T cells and MDDCs.Flow cytometry also revealed that the binding of TG3 to MDDCs could be partially blocked by DC-SIGN blocking antibodies.Neither the negative control group nor the blank control group showed the recognition and binding of TG3 to HEK293T cells and MDDCs.Conclusion TG3 can serve as a kind of autoantigen to be recognized and bound by DC-SIGN receptors,followed by uptake by dendritic cells.