Objective Human immunoglobulin combinatorial library was generated by using phage surface display expression system, and phage antibodies (Fabs) to platelet were screened.Methods:PBMC were separated from a patient who Were transfusion refractory to platelet. mRNA was isolated and cDNA was synthesized by reverse transcriptase.The immunoglobulin heavy chain Fd and the light chain ? genes were amplified by half nested PCR and the PCR products were cloned into the expression vector pCome3 respectively.The immunoglobulin combinatorial library was constructed and screened by 3 rounds of affinity selection.Results Human Immunoglobulin Combinatorial Library was successfully constructed.The specific phage antibodies were highly enriched after 3 rounds of biopanning selection against platelet membrane proteins.Conclusion The antibody library and human monoclonal antibodies to platelet may be useful as molecular tools to study the anti platelet drugs, platelet related diseases, epitope of human platelet antigens.

Objective To find out a way for culturing the myocardial cells of neonatal rats in vitro, and to study their biological characters. Methods Cardiomyocytes from the heart of 1-3 days old Sprague-Dawley rats were prepared by a modified protocol. Hearts were harvested and cut into pieces of about 1mm3 in size, and placed into cell culture bottles with nylon without pre-treatment of digestive enzyme. DMEM supplemented with 10% (v/v) FCS was used for culture, with thymidine (6mg/ml) added to inhibit fibroblast growth. Cells in monolayer were formed on the gauze and the bottom of the culture flask, and then cells were isolated by 0.25% trypsin digestion for 30 seconds. Cell suspension was transferred to 100cm2 cell culture flasks at a density of 104 cells /cm2 for identification, viability test and morphologic observation. The cells obtained were stained with monoclone anti-a-sarcomeric actinin and FITC to evaluate the purity of the myocardial cell preparation by flow cytometry, AO-PI fluorescein stain was used to evaluate the viability and Giemsa staining for examining the morphology of the myocardial cells. Results 24-48 hours after culture, the myocardial cells became adherent to bottle wall to form a sheet, and began to pulsate. The purity of myocardial cells in culture cell population was 94.13%, the ratio of viable myocardial cells was 95.3%, and 95%CI was 91.6%-99%. The output of myocytes from each neonatal heart was 3.3?106 and 95%CI is 2.1? 106- 4.5?106. Conclusion Neonatal myocytes culture in this way can generate large amount of viable single myocardial cells suitable for myocardial research in a short period.

We examined eutopic,ectopic endometria and peritoneal fluid macrophages from 22 patients with endometriosis(EMS) and 14 women without EMS.To obtain evidence for the induction of programmed cell death,apoptotic cells were identified using a modified terminal deixynucleotidyltransferasebiotin nick end labeling method(TUNEL).To evaluate cell death repressor activity,bcl 2,bax and fas genes expression was examined using immunohistochemical staining. The results showed that bcl 2 expression in eutopic,ectopic endometrium and peritoneal fluid macrophages with EMS was significantly increased compared with no EMS( P

We examined eutopic,ectopic endometria and peritoneal fluid macrophages from 22 patients with endometriosis(EMS) and 14 women without EMS.To obtain evidence for the induction of programmed cell death,apoptotic cells were identified using a modified terminal deixynucleotidyltransferasebiotin nick end-labeling method(TUNEL).To evaluate cell death repressor activity,bcl-2,bax and fas genes expression was examined using immunohistochemical staining. The results showed that bcl-2 expression in eutopic,ectopic endometrium and peritoneal fluid macrophages with EMS was significantly increased compared with no EMS(P＜0.01).Bax expression in eutopic,ectopic and peritoneal fluid macrophages with EMS was significantly decreased compared with no EMS(P＜0.05). Fas expression in eutopic,ectopic endometrium was decreased compared with no EMS(P＜0.05). The expression of apoptotic genes were different in eutopic,ectopic endometrium and peritoneal fluid macrophages from EMS and no EMS.Apoptotic rate in EMS was lower than no EMS,and its acceptance was decreased,which could bear implications for the growth and survival of ectopic endometrial tissue.

AIM:To study the electrophysiological effects of resveratrol from grape and polygonum cuspidate on autorhythmicity organization in left ventricular outflow tract of guinea pig and its intracellular mechanism.METHODS:In choosing guinea pig weighing 250~300 g,the action potential,including its MDP(maximal diastolic potential),APA(amplitude of AP),Vmax(maximal rate of zero phase depolarization),VDD[velocity of diastolic(phase 4)depolarization],RPF(rate of pacemaker firing)and APD_ 50,APD_ 90(the duration of 50% and 90% repolarization of action potential),were observed and recorded by means of the intracellular microelectrode technique.RESULTS:(1)Resveratrol could reduce the electric activity of left ventricular outflow tract.Resveratrol(30、60、120 ?mol/L)not only significantly decreased APA and Vmax,but also decreased VDD and RPF respectivily(P

Objective To screen the risk factors for postoperative residual neuromuscular blockade (RNMB) in the patients undergoing thoracic surgery.Methods A total of 733 patients undergoing elective thoracic surgery with general anesthesia,without neuromuscular disease,skin temperature ≥32 ℃,were transferred to the postanesthesia care unit (PACU) after surgery and given synchronized intermittent mandatory ventilation.Neuromuscular blockade was monitored immediately after admission to the PACU,and the occurrence of postoperative RNMB was defined as a train of four (TOF) ratio ＜90％ at the time of extubation.The patients were divided into RNMB group and nonRNMB group according to whether or not postoperative RNMB occurred.Each parameter of baseline patient characteristics,complications,sites and methods of surgery,anesthesia time,requirement for muscle relaxants during surgery,TOF ratio on arrival to the PACU,requirement for muscle relaxant antagonists in the PACU,and extubation time were recorded.The risk factors of which P values were less than 0.05 would enter the multivariable logistic regression analysis to stratify the risk factors for postoperative RNMB.Results A total of 385 patients developed postoperative RNMB,and the incidence was 52.5％.The results of multivariate logistic regression analysis showed that complications such as diabetes,intraoperative application of two kinds of muscle relaxants,average intraoperative consumption of cisatracurium ≥ 0.14 mg · kg-1 · h-1,TOF ratio on arrival to the PACU ≤ 0.5,and extubation time ≤ 30 min were independent risk factors for postoperative RNMB (P＜0.05).Conclusion Complications such as diabetes,intraoperative application of two kinds of muscle relaxants,average intraoperative consumption of cisatracurium 0.14 mg · kg-1 · h-1,TOF ratio on arrival to the PACU ≤ 0.5,and extubation time ≤ 30 min are independent risk factors for postoperative RNMB in the patients undergoing thoracic surgery.

Objective To establish a rat model of bone cancer pain by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. Methods Sixty female Wistar rots weighing 180-200 g were randomly divided into 4 groups (a=15each):groupⅠ normal control; group Ⅱsham operation; group Ⅲtumor cell inoculation + normal saline (NS) and group Ⅳtumor cell inoculation + flurbiprofen. NS 0.2 nd and flurbiprofen 10 mg/kg in 0.2 ml were injected IV at 2 h before determination of pain threshold on 14 and 17 d after inoculation oftumor cells in groupⅢand Ⅳ respectively. On day 0, 4, 7, 10, 14, 17 and 21 after inoculation pain threshold was measured after determination of body weight. X-ray examination of the tibia was performed on day 14 after inoculation. The animals were killed on day 21 after inoculation for microscopic examination of the inoculated tibia. Results The animals started losing weight and the threshold to yon Frey hair stimulation was decreased from dhy 10 after inoculation in group Ⅲand Ⅳ. X-ray examination showed destruction of bone and microscopic examination showed tumor growing in tibia. Flurbiprofen significantly decreased mechanical hyperalgesia in group Ⅳ. There was no significant difference in paw withdrawal latoney to radiant heat among the 4 groups. Conclusion A model of bone cancer pain can be made by inoculation of Walker 256 mammary gland carcinoma cells into tibia characterized by mechanical hyperalgesia.

ObjectiveTo investigate the protection effect of bifidobacterial adhesin for intestine ischemia/reperfusion (I/R) injury on gut barrier function in rat.MethodsSeventy-two male SD rats were randomly divided into sham operation group (n =24), I/R model group (n =24) and pretreatment group of bifidobacterial adhesin (pretreatment group, n = 24).Six rats were anatomized at 6 h, 1 d, 4 d and 7d after inducing I/R model in each group, respectively.The pathological changes of the terminal ilea and the blood levels of TNFα, IL-6, IL-10, diamine oxidase (DAO), and the activity and content of D-lactic acid were observed.ResultsThe blood levels of TNFα, IL-6, DAO and D-lactic acid in I/R model group were significantly higher than sham operation group at all time points (P ＜0.05) , while the blood level of IL-10 was no significantly change.The activity of IL-6 and DAO in pretreatment group was significantly lower than I/R model group at all time points (P ＜ 0.05), the blood level of TNFαt in pretreatment group was significantly lower than I/R model group at 1 d, the blood level of D-lactic was significantly lower than I/R model group at 4 d and 7 d (P ＜ 0.05). Intestinal pathological damages were obviously milder in pretreatment group than I/R model group at all time points (Chiu's pathological scores: 6 h, 3.22 ±0.22 vs 3.57 ±0.20;1 d,3.77 ±0.13 vs 3.90 ±0.12;4 d,2.93 ±0.23 vs 3.07 ±0.21;7 d,2.10 ±0.30 vs 2.22 ±0.17,all P ＜ 0.05).ConclusionThe pretreatment of bifidobacterial adhesin could protect the intestinal mucosa from I/R injury, and alleviate intestinal ischemic reperfusion injury.

AIM: To observe the immunoregulation of all ogeneic cornea on the human peripheral blood T lymphocytes in vitro. METHODS: After Co-culture of human peripheral blood lymphocytes and allogeneic cornea in vitro, T lymphocytes were labeled by monoclonal ant ibody, and analyzed by fluorescent activated cell sorter (FACS). RESULTS: CD25 expression on T lymphocytes in control was 25.2%, after stimulated by the allogeneic cornea or PDB, CD25 expression on T lymphocy tes was 56.8% and 80.9%, respectively. After stimulated by the allogeneic corn ea, CD25 expression on CD 4+ or CD 8+ T lymphocytes were 67.3% and 52.3% , respectively. CONCLUSION: Allageneic cornea stimulates CD25 expression on huma n peripheral blood T lymphocytes, and the CD25 expression on CD 4+ T lymphocy tes is more prominent than CD 8+ T lymphocytes.

[Objective] To observe the effect of Shuhou Kangyan Decoction (SKD), a liver-kidney nourishing, Qi-strengthening and blood-activating, wind-dispelling and heat-clearing prescription mainly composed of Radix Astragali, Radix Angelicae Sinensis, Fructus Lycii, Radix Rehmanniae, Radix Saposhnikoviae, Rhizoma Atractylodis Macrocephalae, etc. , on interleukin-2 (IL-2) and soluble IL-2 receptor (SIL-2R) in serum and tears of patients with cataract after operation. [Methods] Forty-seven cases of cataract after ultrasonic emulsification plus artificial lens implantation were randomized into two groups. Group A (n = 26) was treated with routine operation and eye drops and group B ( n = 21) with SKD additionally and other 15 healthy aged people ( n = 15) receiving physical examination in the out-patient department served as normal controls. [Results] Post-operative serum SIL-2R level was higher in groups A and B than that before operation ( P 0.05). Post-operative IL-2 and SIL-2R levels in the serum and tears were higher in group A than that in groups B and C (P