Objective To study proliferation,secreted cytokines,immune phenotypes and cytotoxicity on Raji cells by cytokine induced killer (CIK) cells co-cultured with dendritic cells (DC).Methods The mononuclear cells from peripheral blood of healthy individuals were extracted,then cultured the cells under 5 ％ CO2 at 37 ℃ for 2 hours.DC were induced from suspended cells,and CIK cells were from adherent cells.After 9 days of nurturing,two types of cells were mixed.CIK cells were cultured alone as the control.The cytotoxic activity of CIK and DC-CIK cells were detected by MTT assay.The morphologies,proliferation,secreted cytokines,and immune phenotypes of the two cells in day 0,3,6,9,12,15 in culture were monitored.Results In day 12 in culture,comparing with CIK cells,DC-CIK cells significantly enhanced the proliferation rate [(42.44±2.68) fold vs (30.01±2.05) fold] (t =11.64,P ＜ 0.05) and had increased IL-2,IFN-γ,IL-12 and TNF-α secretion [(124.34±12.57) ng/L vs (56.32±6.58) ng/L,(496.60±95.32) ng/L vs (247.80± 69.45) ng/L,(84.92±6.07) ng/L vs (24.18±3.31) ng/L,(380.6±45.95) ng/L vs (196.61±24.19) ng/L] (t =15.16,P ＜ 0.05; t =6.67,P ＜ 0.05; t =27.78,P ＜ 0.05; t =11.20,P ＜ 0.05),and there were more CD3+ CD8+ cells and CD3+ CD56+ cells in the co-culture [(71.79±1.73) ％ vs (60.37±3.24) ％,(48.54±3.30) ％ vs (33.07±2.22) ％](t =9.83,P ＜ 0.05; t =12.30,P ＜ 0.05),and DC-CIK cells had a significandy increased cytotoxicity on Raji cells in vitro at the same ratio of effector cells to target cells.Conclusion CIK cells have higher proliferation rate and cytotoxicity against Raji cells when co-cultured with DC.

Objective To analyze effects of different anesthesia procedures on heart rate variability(HRV) as an index of autonomic nervous activity and hemodynamics during CO2 pneumoperitoneum pressure in laparoscopic cholecystectomy(LC).Methods 45 patients of ASA I or II phage scheduled for LC were divided into general anesthesia group(Group I,Control Group),general anesthesia combined with esmolol group(Group II) and general anesthesia combined with epidural block group(Group III) according to operation date.HRV,HR and MAP were measured before anesthesia(T1),before pneumoperitoneum(T2) and at 5 min(T3),10 min(T4),20 min(T5),30 min(T6) after pneumoperitoneum.Results As compared with pre-pneumoperitoneum(T2),low frequency(LF),low frequency/high frequency(LF/HF) in the Group I increased significantly(P0.05).Comparisons among the three groups,LF,LF/HF in the Group I at all time points after pneumoperitoneum were significantly higher than those in the II and III Group(P0.05).Conclusions Esmolol can relieve stress reaction induced by pneumoperitoneum,but it can't completely block increasing of sympathetic nerve activity;using general anesthesia combined with epidural block in LC can inhibit sympathetic nerve excitation,sustain automatic never stability.

Objective To evaluate the predictive performance of the target controlled infusion (TCI) system for propofol using Marsh pharmacokinetic parameters in the elderly patients. Methods Sixteen ASA grade I - Ⅱ patients of both sexes (9 male, 7 female) aged 65-75 yr weighing 49-76 kg scheduled for elective lower abdominal surgery were enrolled in this study. The patients were unpremedicated. Right internal jugular vein was cannulated for drug and fluid administration and left radial artery was cannulated for BP monitoring and blood sampling. Anesthesia was induced with TCI propofol (target plasma concentration was set at 3 ?g?ml-1 ) and TCI fentanyl (target plasma concentration set at 2 ng?ml-1 ) . Tracheal intubation was facilitated with succinyl choline 1.5mg?kg-1 . The patients were mechanically ventilated ( VT 8 ml?kg-1 , RR 10 bpm) . Anesthesia was maintained with TCI propofol and fenlanyl and intermittent i.v. boluses of vecuronium. The target plasma concentration of propofol and fentanyl were the same as that for induction. MAP, HR, SpO2 and BIS were continuously monitored during operation. Blood samples were taken at 5, 10, 15, 20, 30, 45, 60, 90, 120 min during TCI of propofol for determination of plasma propofol concentration with HPLC. The predictive performance was evaluated by PE% [measured concentration (Cm) - predicted concentration (Cp)] /Cp ? 100% , MDPE, MDAPE and wobble. Results BIS was maintained between 38-55. The MDPE was 6%, MDAPE 14% and wobble 18 % with this TCI system incorporating Marsh pharmacokinetic parameters for propofol. Conclusion The TCI system incorporating Marsh pharmacokinetic parameters for propofol is acceptable for elderly patients.

Objective Corneal endothelial decompensation is caused by many corneal diseases. It often results in severe clinical complications. Endothelial keratoplasty (EK) is a new therapy for corneal endothelial decompensation. This study aimed to investigate a new approach to establishing corneal endothelial decompensation animal model with Descemetorhexis technique in order to better understand the tissue response to EK. Methods Thirty New Zealand white rabbits were randomly divided into three groups according to different surgical procedures; corneal endothelial cells (CEC), Descemet's membrane and corneal endothelial cells (DM + CEC) as well as Descemet' s stripping with endothelial keratoplasty(DSEK) group and 10 eyes for each. The right eyes of rabbits were as surgery eyes. Other 10 rabbits were as DSEK donors. Corneal transparency, anterior chamber response and graft location were examined once per day for two weeks under the slit lamp. Comeal thickness was measured by ultrasound biomicroscope. Corneal endothelial cells were analyzed using vital staining with alizarin red and trypan blue in 2, 4 and 8 weeks after operation. Results The cornea in DM + CEC group remained opaque throughout the observation period. In CEC and DSEK group, corneal clarity was gradually restored and corneal thickness was significantly less than that in the DM + CEC group during the postoperative 8 weeks. There were significant differences in corneal thickness between the DM + CEC group and CEC group or DSEK group during the postoperative 8 weeks (P ＜0. 05). The vital staining showed that most Descemetorhexis area was not covered by endothelial cells even 2 months after surgery. Conclusion A new corneal endothelial decompensation model is successfully established for the study of corneal endothelial keratoplasty, which is helpful for understanding the wound-healing of rabbit corneal endothelium after Descemel' s membrane damage.

BACKGROUND:Due to the different transplantation time after myocardial infarction, the homing ability of bone marrow mesenchymal stem cels to damaged tissues as wel as the repairing role wil be very different. OBJECTIVE:To explore the optimal window time for the homing of bone marrow mesenchymal stem cels to the myocardial tissue after myocardial infarction. METHODS: Eighteen Chinese miniature pigs were modeled by the ligation of left anterior descending coronary artery. BrdU-labeled bone marrow mesenchymal stem cels were injectedvia the coronary artery at 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks after modeling, respectively. Then, the animals were kiled at 3 days after cel transplantation to detect the home of bone marrow mesenchymal stem cels in the infarct zone. RESULTS AND CONCLUSION:BrdU-labeled positive cels with brown nuclei were visible at 1 day, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after myocardial infarction, especialy at 1 week after myocardial infarction (P < 0.05). It indicates that the best homing window for bone marrow mesenchymal stem cels was at week after myocardial infarction, when the stem cel transplantation is given to be able to promote myocardial repair.

Objectives To observe the pre and post-operational changes of the expressions of survivin, caspase-3 and CD44V6 in patients with colorectal cancer after intra-abdominal implantation of sustained releasing fluorouracil. Meth-ods Sixty-four patients with colorectal cancer (Dukes’stage of B and C) were divided into treatment group and control group, 32 patients in each group. The standard radical surgery was performed in two groups of patients. The fluorouracil im-plants were implanted intra-abdominally in treatment group. The peripheral blood levels of surviving and caspase-3 were de-tected by RT-PCR. The level of CD44V6 was detected by flow cytometry in two groups of patients. Results There were no significant differences in levels of survivin, caspase-3 and CD44V6 before surgery between two groups (P>0.05). The level of survivin (0.362 ± 0.183) was significantly lower at 14 days after operation in treatment group than that of control group (0.585±0.207), but the level of caspase-3 (2.001±0.146) was significantly higher than that of control group (1.654±0.111). The levels of CD44V6 were significantly lower in treatment group (1.857±0.535) and control group (3.471±0.496) after opera-tion than those before operation (9.557±1.170 and 9.729±0.943, P＜0.05), and the level of CD44V6 was significantly lower in treatment group than that of control group (P＜0.05). Conclusion The implant for the sustained release of fluorouracil showed a positive impact on micrometastases and prognosis of colorectal cancer, while improved the long-term efficacy of postoperative colorectal cancer.

Objective To investigate the expressions of apoptosis stimulating protein of P53 (ASPP)family in colorectal carcinoma tissue and to explore their relationship with the clinicopathological characteristics of colorectal carcinoma,and to clarify the effect of ASPP family in the development of colorectal carcinoma.Methods 45 cases of colorectal carcinoma tissue and 20 cases of healthy controls were selected. Among 45 cases of colorectal carcinoma tissue, there were 1 1 cases of well differentiated colorectal carcinoma, 2 1 cases of moderately differentiated colorectal carcinoma,and 13 cases of poorly differentiated colorectal carcinoma;7 cases of T1 stage colorectal carcinoma,8 cases of T2 stage colorectal carcinoma,25 cases of T3 stage colorectal carcinoma,and 5 cases of T4 stage colorectal carcinoma;19 cases of N1 stage with lymph node metastasis,26 cases of N0 stage without lymph node metatasis.The expressions of ASPP1,ASPP2,and iASPP in 45 cases of colorectal carcinoma tissue and 20 cases of normal colon tissue were detected by immunohitochemistry SP method,and the correlations between the expressions of ASPP family and the pathologic typing,infiltrative depth,and lymph node metastasis of colorectal carcinoma were analyzed. Results ①The immunohitochemical staining results showed that the ASPP family members expressed in colorectal carcinoma tissue and normal colon tissue, and there were no significant differences in ASPP1 and ASPP2 positive rates between colorectal carcinoma tissue and normal colon tissue (P>0.05);the positive expression rate of iASPP in colon cancer tissue was higher than that in normal colon tissue (P<0.01).② The expression of ASPP1 in colon caner tissue had no correlation with the differentiation degree of tumor cells (rs=0.163,P>0.05);the expression of ASPP2 positive rate was decreased when the differentiation degree of tumor cells reduced,they had positive correlation (rs=0.454,P=0.002);the expression of iASPP in colon cancer tissue had no correlation with the differentiation degree of tumor cells (rs=-0.171,P>0.05).③ The expression of ASPP1 in colon caner tissue had no correlation with the infiltrative depth of tumor (rs=-0.268,P>0.05);the expression of ASPP2 positive rate was decreased when the tumor infiltrative depth increased,they had negative correlation (rs=-0.348,P<0.05);the expression of iASPP in colonic carcinoma caner tissue had no correlation with the infiltrative depth of tumor (rs=0.231,P>0.05).④The expressions of ASPP1,ASPP2, and iASPP in colon caner tissue had no correlation with lymph node metastasis (rs=0.089,rs=0.044,rs=0.210, P>0.05).Conclusion The expression levels of iASPP in colon cancer and normal colon tissues are different,it may be useful for the diagnosis, differential diagnosis and evaluation in benign and malignant colorectal diseases. The expression of iASPP is negatively correlated with the pathologic typing and neoplasm staging of colorectal carcinoma,it indicates that iASPP can be used as a indicator in judging the prognosis of colorectal carcinoma.

Objective To study the killing effects of the killer cell immunoglobulin-like receptors (KIR) KIR2DS1-positive natural killer (NK) cells against leukemia cells.Methods High-purified NK cells separated by RosetteSep NK cell enrichment kit from healthy donor peripheral blood were taken as effector cells,and the freshly isolated bone marrow mononuclear cells from newly diagnosed acute myelogeneous leukemia (AML) patients were taken as target cells.The cytotoxic activity of NK cells were detected by CCK8 kit assay.HLA-Cw,KIR gene of the healthy donors and patients were detected by polymerase chain reaction and sequence specific primer (PCR-SSP) genotyping techniques,respectively.NK cells were divided into KIR2DS1-positive group and KIR2DS1-negevitive group,and then anti-KIR2DS1 mononuclear antibody was used to block KIR2DS1 of NK cells.Meanwhile based on HLA-Cw,KIR2DS1-positive NK cells and target cells were divided into C1 homozygote group(expressing HLA-Cw 01,03,07,08,12,14,16 alleles),C2 homozygote group (expressing HLA-Cw 02,04,05,06,15,17,18 alleles) and the C1/C2 heterozygote group (co-expressing the alleles in C1 group and C2 group).Results The purity of NK cells was (91.2±5.94) ％ through flow cytometry analysis.At the same effector-target ratio,cytotoxicity of KIR2DS1-positive NK cells against target cells was higher than that of KIR2DS1-negetive NK cells.While the E:T =10:1,cytotoxicity of KIR2DS1-positive NK cells against target cells [(2.82±6.81) ％] was significant higher than that of KIR2DS1-negetive NK cells [(28.61±5.14) ％] against target cells.The killing effects of KIR2DS1-positive NK cells was significantly weakened after KIR2DS1 blockaded with specific antibody (t =-3.00,P =0.05).When focusing on the C1 group NK cells,KIR2DS1-positive NK cells against C2 group [(4.39±3.46) ％] target cells was significantly higher than than against C1 group [(41.22±3.68) ％] (t =8.33,P ＜ 0.05) and C1/C2 group [(41.32±5.09) ％] (t =6.37,P ＜ 0.05) target cells.Conclusions The killing effects of KIR2DS1-positive NK cells are significantly higher than that of KIR2DS1-negetive NK cells.The HLA-C1 group KIR2DS1-positive NK cells could recognize HLA-C2 loci and then kill the target cells.

Objective To investigate the effect of tongue tie corrective surgery on the development of language in children with short lingual frenulum.Methods 94 children with short lingual frenulum were selected,and under the permission of the patients'family they were randomly divided into two groups.The treatment group received ankylotomy and language training.The control group was only given language training.The clinical efficacy,improvement time of pronunciation and adverse reactions were compared between the two groups.Results The total effective rate of the treatment group was 91.49％,which was significantly higher than 72.34％ of the control group (x2 =5.816,P ＜0.05).The improvement time of pronunciation in the treatment group was significantly shorter than the control group(t =21.250,P ＜ 0.01).There were no serious adverse reactions observed in two groups.Conclusion Tongue tie correction surgery can significantly affect the language development in children with short lingual frenulum,the improvement time of pronunciation is short,which is worthy of clinical application.

Objective To study the killing effects of the killer cell immunoglobulin-like receptors (KIR)2DS1-mediated natural killer (NK) cells against dendritic cells (DCs) and analysis the expression of chemokine receptor CCR7 after KIR2DS1-positive NK cells interact with the C2 epitope DCs in vitro,and explore the mechanism of alloreactive NK cell to limit graft-versus-host disease (GVHD).Methods NK cells were separated by Rosettesep NK sorting kit from healthy donor peripheral blood and taken as effector cells.The mononuclear cells from peripheral blood of newly diagnosed acute myeloid leukemia (AML) patients,and DCs were induced as target cells.Polymerase chain reaction and sequence specific primers (PCR-SSP) genotyping techniques and direct sequencing SBT were used to detect KIR gene,HLA-Cw of the healthy donors and patients.The cytotoxic activity of KIR2DS1-positive NK cells or KIR2DS1-negative NK cells against DCs were detected by CCK-8 assay.The expression of CCR7 after NK cells interacted with DCs was detected by flow cytometry.Results The purity of NK cells analyzed by flow cytometry was (94.20±1.23) ％.Under same target ratio (10∶1),the killing effect of KIR2DS1+ NK cell against DCs was higher than that in KIR2DS1-NK cells (t =3.70,P ＜ 0.05).Under same target ratio (10∶1),the cell lysis in C1/C1 group,KIR2DS1+ NK cells against C2/C2 group DCs (15.06±1.81) ％ was significantly higher than that against C1/C1 group (10.07±1.03) ％ and C1/C2 group (8.65±0.93) ％ DCs (F =56.368,P =0.001).KIR2DS1 + NK cells efficiently acquired CCR7 upon coculture with C2 epitope DCs.Conclusions NK cell expressing the KIR2DS1-activating receptor efficiently kill DCs,prevent GVHD,while enable NK cell acquire CCR7,obtaining migratory properties in vitro.