Taking Naegleria australiensis, a species of pathogenic free-living amoeba (isolated in Shanghai in 1986), as antigen, two cell lines which provided potentially permanent source of monoclonal antibodies were established by lymphocytic hybridoma technique. The results of identification showed that: (1) the two cell lines could secret two different kinds of McAbs; (2) both of the McAbs were IgG (by gel diffusion); (3) McAbs produced in BALB/c mice were at high concentrations. One of them had a titer of ≥1 : 8 192 and the other≥l : 1 024 (by enzyme linked immunosorbent assay). A decline of the titers after purification by salting-out method was shown. One of the purified McAbs had a titer of≥1: 2 024 and the other≥l : 128.We have adopted two ways of recovering cryopreserved cells : ordinary recovering and "direct" recovering. The latter way was more practical because it could reduce the cycle of antibody production, and avoid contamination and chromosome variation. Experiments with different doses of cells revealed that, if the latter way was used, the optimal dose was 3 to 5 ? 106 cells per mouse.

Objective To investigate the effects of ulinastatin with parecoxib sodium on early cellular immunology function and cognitive function in elderly patients undergoing total hip replacement.Methods One hundred patients (aged 65-80 years,ASA grade Ⅱ) undergoing elective total hip replacement were randomly assigned into 4 groups: group W(n=25),group P(n=25),group WP(n=25) and group C(n=25).The patients in group W received intravenous injection of ulinastatin with a dose of 5 000 U/kg before skin incision and at the moment of the end of operation.Patients in group P accepted intravenous injection of parecoxib sodium of 40 mg before anesthesia and at a 12 h intervals for six times.Patients in group WP accepted intravenous injection of ulinastatin with a dose of 5 000 U/kg before skin incision and at the moment of the end of operation,and accepted intravenous injection of parecoxib sodium with a dose of 40 mg before anesthesia and at a 12 h interval for six times.Patients in group C were given normal saline at the same time as placebo.The operation time,the volume of bleeding and autologous blood receiving and the intraoperative fentanyl consumption were recorded in four groups.Blood samples were obtained before induction (T0),at the end of operation (T1),on 6 h (T2),24 h (T3),72 h (T4) after operation for determination of plasma concentration of S100β protein,neuron-specific enolase(NSE),IL-6,TNF-α.The percentages of T lymphocyte subsets (CD3+,CD4+,CD8+) and CD4+/CD8+ ratio were detected with flow cytometry.The Mini-Mental State Examination (MMSE) was used to test the cognitive function of the patients at T0,T4 and 7 d after operation (T5).Results Compared with T0,the percentages of CD3+,CD4+ cells and CD4+/CD8+ in groups C,W and P were significantly decreased,the plasma concentrations of IL-6 and TNF-α significantly increased at T1-T4,concentration of NSE and S100 protein were significantly increased at T2 and T3 (P<0.05).the percentages of CD3+,CD4+T lymphocyte and CD4+/CD8+ ratio in groups W and P at T2,T3 and group WP at T1-T4 was significantly higher than that of group C,the concentration of IL-6,TNF-α,NSE and S100β protein content was significantly lower than in group C (P<0.05);the percentages of CD3+,CD4+T lymphocyte and CD4+/CD8+ ratio in group WP was significantly higher than those of groups W and P,the concentrations of TNF-α,IL-6,NSE and S100β protein content was significantly lower than those of group W and P at T2,T3 (P<0.05).Compared with T0,MMSE score in group C was significantly lower at T4,T5 (P<0.05).MMSE score of groups W,P and WP was significantly higher than that of group C at T4 (P<0.05).MMSE score of group WP was significantly higher than those of groups W and P at T4 (P<0.05).Conclusion Ulinastatin combined with parecoxib sodium ameliorates early cellular immunology function and cognitive function in elderly patients undergoing total hip replacement,and it is more effective than using ulinastatin or parecoxib sodium alone.

Objective To evaluate the effect of stellate ganglion block (SGB) on cellular immune function in diabetic rats.Methods Healthy male Sprague-Dawley rats,aged 3 months,weighing 240-280 g,were used in this study.Diabetes mellitus was induced by intraperitoneal 1％ streptozotocin 60 mg/kg and confirmed by blood glucose ≥ 16.7 mmol/L 3 days later.Forty-eight rats with diabetes mellitus were randomly divided into 2 groups (n=24 each) using a random number table:diabetes mellitus group (group DM) and group SGB.Another 24 healthy rats,aged 3 months,were selected and served as control group (group C).At 1 week after successful establishment of the model,unilateral transection of cervical sympathetic trunk (TCST) was performed in group SGB,while the right cervical sympathetic trunk was only exposed in C and DM groups.Before TCST (T0) and on 1,3,7 days after TCST (T1-3),6 rats were randomly selected from each group,and blood samples were collected from the inferior vena cava for determination of the blood glucose,plasma norepinephrine (NE) concentrations (by enzyme-linked immunosorbent assay),and levels of T lymphocyte subsets CD3+,CD4+ and CD8+ in whole blood (using FACSCalibur flow cytometer).C D4+/CD8+ratio was calculated.The rats were weighed before sacrifice,and the rats were sacrificed to obtain the thymus which was weighed.The thymus index (thymus weight/body weight) was calculated.Results Compared with group C,the blood glucose was significantly increased,and the levels of CD3+ and CD4+ in whole blood,CD4+/CD8+ ratio,and thymus index were significantly decreased at T0-3 (P＜0.05),and no significant change was found in CD8+ levels in DM and SGB groups (P＞0.05),the plasma NE concentrations were significantly decreased at T1-3 in group SGB (P＜0.05),and no significant change was found in plasma NE concentrations in group DM (P＞0.05).Compared with group DM,the blood glucose and plasma NE concentrations were significantly decreased,and the levels of CD3+ and CD4+ in whole blood,CD4+/CD8+ ratio,and thymus index were significantly increased at T1-3 (P＜0.05),and no significant change was found in CD8+ levels in group SGB (P＞0.05).Conclusion SGB can improve the cellular immune function in diabetic rats.

Objective To investigate the role of Rho/Rock signaling pathways in ventilator-in-duced lung injury in rats.Methods Ninety-six male SD rats,weighing 300-350 g,were equally and randomly divided into four groups using a random number table (n =24 each):control group (group C),fasudil group (group F),high tidal volume group (group H)and high tidal volume + fasudil group (group HF).Rats in group C and group F received no mechanical ventilation,rats in group H and group HF were intubated and mechanically ventilated (VT = 40 ml/kg,RR = 40 beats per minutes,FiO 2 =40%)for 4 h.The animals in group F and group HF were given intraperitoneal in-jection of fasudil 10 mg/kg at the time 1 h before mechanically ventilated.Six rats were chosen in each group at the time before ventilation (T0 )and at 4,8,24 h after ventilation (T1-T3 ),and blood sam-ples were taken for determination of the levels of serum tumor necrosis factor-α (TNF-α),IL-6 and IL-10,lungs were removed,bronchoalveolar lavage fluid (BALF)was collected to examine protein content,wet/drying (W/D)ratio was determined,which were then stained with haematoxylin and e-osin and examined under microscope,the pathological changes of lungs were scored.Myeloperoxidase (MPO)activity in lung tissue was determined by spectrophptometry.Protein and gene expression of RhoA and Rock2 in the lung tissue were detected by Western blot and reverse transcription PCR (RT-PCR).Results Compared with group C,the serum TNF-α,IL-6 and IL-10,BALF protein content, W/D ratio,the pathological scores,MPO activity,the expression of RhoA,Rock2 protein and mR-NA were significantly increased in group H and HF at T1-T3 (P <0.05 ).Compared with group H, the serum TNF-αand IL-6,BALF protein content,W/D ratio,the pathological scores,MPO activi-ty,the expression of RhoA,Rock2 protein and mRNA were significantly decreased,and the serum IL-10 was significantly increased in group HF at T1-T3 (P <0.05).Conclusion Fasudil can attenuate ventilator-induced lung injury in rats,and the mechanism may be associated with inhibition of the Rho/Rock signaling pathways reducing the inflammatory response.

Objective To investigate the impact of enhanced recovery after surgery (ERAS)on immune function in patients undergoing laparoscopic hepatectomy.Methods Sixty patients undergoing laparoscopic hepatectomy (34 males,26 females,aged 38-57 years,ASA Ⅰ or Ⅱ),were randomly divided into two groups:enhanced recovery after surgery group (group E)and non-enhanced recovery after surgery group (group C).The patients in group E received enhanced recovery after surgery,while the patients in group C received routine perioperative management and anesthesia methods.The operation method and time,the volume of bleeding,the intraoperative fentanyl con-sumption,the volume of fluid input,the preoperative and postoperative CVP and temperature were recorded in the two groups.Blood samples were obtained before induction (T0 ),at the end of opera-tion (T1 ),on day 1 (T2 ),day 3 (T3 ),day 7 (T4 )after operation for determination of plasma con-centration of IgA,IgM,IgG and the percentages of T lymphocyte subsets (CD3+ ,CD4+ ,CD8+ ) and CD4+/CD8+ ratio were detected with flow cytometry.Furthermore,the visual analogue scale (VAS)score and Ramsay score were evaluated 4 hours,8 hours,24 hours and 48 hours after opera-tion in two groups.Results Compared with group C,the intraoperative fentanyl consumption,the volume of fluid input and the postoperative CVP in group E were significantly decreased,while the postoperative temperature was significantly increased (P < 0.05 ).Compared with T0 , the percentages of CD3+ ,CD4+ ,the CD4+/CD8+ ratio and the plasma concentration of IgA,IgM,IgG in group E on T1-T3 were significantly decreased,the percentages of CD3+ ,CD4+ ,the CD4+/CD8+ratio and the plasma concentration of IgA,IgM,IgG in group C on T1-T4 were significantly decreased (P <0.05).Compared with group C,the percentages of CD3+ ,CD4+ ,the CD4+/CD8+ ratio and the plasma concentration of IgA,IgM,IgG in group E on T1-T4 were significantly increased (P <0.05),the visual analogue scale (VAS)score 4 hours,8 hours,24 hours after operation were signifi-cantly decreased (P <0.05).The comparision of Ramsay scores at all the time points between two groups were similar.Conclusion ERAS applied to patients undergoing laparoscopic hepatectomy can reduce the intraoperative fentanyl consumption,prevent the occurrence of hypothermia and provide satisfactory postoperative analgesia,which can significantly improve the immune function in patients.

Objective To investigate the effects of enhanced recovery after surgery (ERAS) pathway on early cognitive function of post-hepatectomy patients after hepatectomy.Methods Ninety-six patients undergoing elective hepatectomy for liver cancer were randomly assigned into two groups:group ERAS,n =48 and group C,n =48.patients in group ERAS accepted perioperative and anaes-thetic optimization of enhanced recovery after surgery pathway,and patients in group C received peri-operative and anaesthetic management of normal surgery concept.The mini-mental state examination (MMSE)was used to test the cognitive function of the patients one day before operation (T0 ),one day after operation (T4 ),three days after operation (T5 )and seven days after operation (T6 ),and the blood levels of S100βprotein,neuron-specific enolase(NSE),IL-1β,IL-6,and TNF-α were de-tected at T0 ,30 minutes after operation (T1 ),6 hours after operation (T2 ),12 hours after operation (T3 )and T4 .Results Compared with group C,MMSE scores in group ERAS were increased at T4 ,T5 , the blood levels of S100βprotein,IL-1βand IL-6 in group ERAS were decreased at T1 ,T2 ,the blood levels of NSE in group ERAS were decreased at T2 ,T3 ,the blood levels of TNF-αin group ERAS were decreased at T1-T3 ;Compared with the baseline value at T0 ,MMSE scores were decreased at T4 ,T5 in group C and decreased at T4 in group ERAS,the blood levels of S100 βprotein in two groups were increased at T1-T3 , the blood levels of NSE in two groups were increased at T2-T4 ,the blood levels of IL-1β,IL-6,and TNF-αin two groups were increased at T1-T4 ;and the postoperative length of stay in group ERAS were decreased. Conclusion Enhanced recovery after surgery pathway is helpful in improving early postoperative cognitive function of patients after hepatectomy,and the mechanism may be related to decreased the blood levels of S100βprotein,NSE,IL-1β,IL-6 and TNF-α.

Objective To evaluate the effects of age factors on hypothermia-induced reduction of ischemia-reperfusion(I/R)injury in isolated rat hearts.Methods Pathogen-free healthy aged male Sprague-Dawley rats,aged 18-20 months,weighing 400-600 g,and young rats,aged 4-6 months,weighing 280-350 g,were used in the study.After the animals were anesthetized with intraperitoneal chloral hydrate and heparinized,their hearts were excised and perfused with K-H solution in a Langendorff apparatus.Twenty-four isolated hearts of aged rats were assigned into 2 groups(n=12 each)using a random number table:I/R group(group AI/R)and hypothermia group(group AH).Twenty-four isolated hearts of young rats were assigned into 2 groups(n=12 each)using a random number table:I/R group(group YI/R)and hypothermia group(group YH).Perfusion was suspended for 30 min followed by 120 min reperfusion to establish the model of I/R.The temperature was maintained at 37 ℃ during the whole process in AI/R and YI/R groups.The hearts were perfused with 34 ℃ K-H solution until 120 min of reperfusion starting from onset of reperfusion in AH and YH groups.At 30 min of equilibration(T0)and 15,30,60 and 120 min of reperfusion(T1-4),heart rate(HR),left ventricular developed pressure(LVDP),the maximum rate of increase in left ventricular pressure(+dp/dtmax),and the minimum rate of increase in left ventricular pressure(+dp/dtmin)were recorded.Six hearts from each group were randomly selected at T4,and myocardial specimens were obtained for determination of ATP,superoxide dismutase(SOD)and malondialdehyde(MDA)levels and myocardial infarct size(IS).Results Compared with group YI/R,HR was significantly decreased at T1-4,ATP and SOD levels were increased,and the MDA content and myocardial IS were decreased in group YH,and the HR,LVDP,+dp/dtmax and +dp/dtmin at T0 and ATP and SOD levels at T4 were significantly decreased,and the MDA content and myocardial IS were increased in group AI/R(P<0.05).Compared with group YH,HR,LVDP,+dp/dtmax and +dp/dtmin at T0 and ATP and SOD levels at T4 were significantly decreased,and the MDA content and myocardial IS were increased in group AH(P<0.05).Compared with group AH,the levels of ATP and SOD were significantly decreased,and the MDA content and myocardial IS were increased in group AI/R(P<0.05).Conclusion Age factors affect the efficacy of hypothermia in reducing I/R injury in isolated rat hearts,and hypothermia provides better cardioprotection for young rats than for aged rats.