Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.

Objective To investigate the influencing factors involved in the establishment of a C57BL/6 J model of metastatic melanoma in the lung,including the way of tumor inoculation,the number of inoculated cells and the time of tumor formation. Methods Mouse melanoma B16F10 cells were cultured in vitro. 1)Eighteen healthy male C57BL/6 J mice were randomly divided into three groups. Mice in each group received 100 μL cell suspension(including 3 ×106 melanoma cells)via intravenous,intraperitoneal and subcutaneous injection,respectively. After two weeks,the mice were killed and dissected,and the tumor growth and metastasis were observed. 2)Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 3 ×106cells,1 ×106cells, and 3 ×105cells through the tail vein,respectively. After two weeks,mice were killed and dissected,and the tumor growth and metastasis were observed. 3)Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 1×106cells though the tail vein. Mice were killed and dissected after one week, two weeks and three weeks, respectively. The growth and metastasis of tumor were observed. Results 1)The success rate of lung metastasis was 100% in the mice with intravenous injection,but not in mice receiving intraperitoneal injection and subcutaneous injection. 2)The size of metastatic melanoma nodules were moderate in mice inoculated by 1 ×106cells. The number of melanoma metastatic foci was too high in the mice inoculated with 3 ×106cells,but too low in the mice inoculated with 3 ×105cells. 3)Significant metastatic melanoma foci were observed in the mice killed and dissected after two weeks with no death. The number of melanoma foci in the lung was too high in the mice killed after three weeks,while was too low in the mice killed at one week after tumor cell inoculation. Conclusions Intravenous injection of 1×106mouse melanoma cells into C57BL/6 J mice and killed after two weeks is an optimal method for establishment of a mouse model of metastatic melanoma in the lung, and is worth of recommendation.

Since its inception, anti-platelet antibodies have been an important tool for studying the interaction of platelets with blood components and blood vessels. At the same time, anti-platelet antibodies also play an important role in the detection and diagnosis of hemorrhagic and thrombotic diseases, and become a kind of powerful anti-thrombotic drugs. With the further understanding of the role of platelets in physiological hemostasis and pathological thrombosis, anti-platelet and anti-thrombotic antibodies also seek a balance between better anti-thrombotic effects and less bleeding side effects.

OBJECTIVETo investigate the in vitro effects of immune inhibitor tacrolimus on platelet function.

METHODSFresh venous blood was collected from healthy volunteers at ages of 18-25 years old, who are not taking antiplatelet drugs within two weeks. The platelets were isolated from the blood and incubated with different concentrations of tacrolimus (0.06, 0.6, 6, 60, 120, 240 μmol/L) at 37 °C for 2 hours, and then the changes of mitochondrial membrane potential and P-selection of platelets were detected by flow cytometry, the expression of apoptosis related protein by Western Blot, and the change of the platelet aggregation function by platelet aggregation analyzer.

RESULTSTacrolimus at concentration of 0.06 μmol/L could promote collagen induced platelet aggregation, inhibit thrombin induced platelet aggregation, have no effect on ristocetin and vWF induced platelet aggregation function. Tacrolimus at concentration of 120 μmol/L and 240 μmol/L could reduce the platelet mitochondrial membrane potential and induce the expression of apoptosis protein caspase-3.

CONCLUSIONIn vitro experimental results showed that high concentration of tacrolimus could lead to platelet apoptosis. But the current therapeutic dose of tacrolimus at 0.06 μmol/L (which is equivalent to 50 ng/ml blood concentration) could have different effects on platelet aggregation function according to different stimulating agents.

Adolescent ; Adult ; Blood Coagulation Tests ; Blood Platelets ; drug effects ; Caspase 3 ; Humans ; In Vitro Techniques ; Platelet Aggregation ; Tacrolimus ; pharmacology ; Thrombin ; Young Adult

Objective: To establish primary immune thrombocytopenia (ITP) animal model induced by anti-platelet membrane glycoprotein GPⅠbα antibodies AN51 and R300. Methods: Twenty guinea pigs (6-8 week) were divided into 4 groups. Five guinea pigs in each group were intravenously injected with different doses of AN51 (0.05, 0.1, 0.2 μg/g) and 0.2 μg/g IgG as control. The whole blood was collected from inner angular venous plexus. Platelets number was determined by an automated cell counter and Swiss-Jim method. Then, the similar protocol was used to establish ITP nude mice model by intraperitoneal injection of different concentrations of anti-platelet GPⅠbα antibody R300, respectively. Results: ①Five minutes after intravenous injection of AN51 at 0.05, 0.1 and 0.2 μg/g, the platelet counts of guinea pigs reduced about 0-5%, 50%-60% and 70%-80% compared to the control group, respectively. The difference was statistically significant (P<0.01) . ②Six hours after intraperitoneal injection of R300 at 0.05, 0.1, 0.2 μg/g, the platelet counts of nude mice decreased about 20%-30%, 60%-70% and 80%-90% compared to the control group, respectively. The difference was statistically significant (P<0.01) . The nude mice, injected 0.2 μg/g R300 once a day for 2 weeks, showed typical ITP clinical manifestations including large number of petechiaes or ecchymoses on limbs, head and abdomen. Conclusion AN51 at 0.2 μg/g and R300 at 0.2 μg/g could establish stable ITP model in guinea pigs and nude mice respectively.

OBJECTIVETo study the effect of reactive oxygen species (ROS) on the regulation of platelet apoptosis.

METHODSWashed healthy human platelets were pre-incubated with N-caetyl-Lcysteine (NAC), and then stimulated with dibucaine or thrombin. The production of ROS and depolarization of mitochondrial membrane potential (∆ ψm) were detected by flow cytometry. The activation of caspase-3 and expression of Bcl-xL were analyzed by Western blot.

RESULTS(1)The average ROS fluorescence value of NAC+dibucaine group was lower than that of dibucaine group(0.66 ± 0.11 vs 1.06 ± 0.08, P<0.01), while that of NAC+thrombin group was also lower than that of thrombin group(0.45 ± 0.05 vs 0.71 ± 0.11, P=0.001). (2)The percentage of platelets with normal ∆ψm in NAC+Dibucaine group was higher than that of dibucaine group[(86.30 ± 9.37)% vs (13.52 ± 3.01)%, P=0.000], while that of NAC+thrombin group was also higher than that of thrombin group[(93.00 ± 3.03)% vs (76.58 ± 5.28)%, P=0.000]. (3)Fragmentation generated by caspase-3 activation in dibucaine group was much more than that in DMSO control group, while the fragmentation in NAC+dibucaine group was significantly decreased. (4)The expression of anti-apoptosis protein Bcl-xL of NAC+dibucaine group was significantly higher than that of the dibucaine group, while that of NAC+thrombin group was also higher than that of thrombin group.

CONCLUSIONThrough the regulation of ROS, NAC could inhibit the platelet apoptosis induced by dibucaine or thrombin.

Acetylcysteine ; pharmacology ; Apoptosis ; drug effects ; physiology ; Blood Platelets ; drug effects ; metabolism ; physiology ; Caspase 3 ; metabolism ; Dibucaine ; pharmacology ; Humans ; Membrane Potential, Mitochondrial ; physiology ; Reactive Oxygen Species ; metabolism ; Thrombin ; pharmacology ; bcl-X Protein ; metabolism