Objective To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice.Methods PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout(Rictor-KO)mice and wild-type mice(n=65),whose expression levels of Rictor,protein kinase B(AKT)and p-AKT were detected using Western blotting.Platelet counts of the mice were determined using routine blood tests,and hemostatic function was assessed by tail vein hemorrhage test.Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis.Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice,and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets.Plasma PF4 levels were determined with ELISA.Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes,respectively.Platelet elongation on collagen surface was observed with scanning electron microscopy.Results Compared with the wild-type mice,Rictor-KO mice showed significantly decreased AKT phosphorylation,decreased platelet production,reduced thrombosis,and decreased platelet activation in response to ADP and thrombin stimulation.The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension,reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow.The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice.Conclusion Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice,suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Objective To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice.Methods PF4-Cre and Rictorfl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout(Rictor-KO)mice and wild-type mice(n=65),whose expression levels of Rictor,protein kinase B(AKT)and p-AKT were detected using Western blotting.Platelet counts of the mice were determined using routine blood tests,and hemostatic function was assessed by tail vein hemorrhage test.Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis.Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice,and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets.Plasma PF4 levels were determined with ELISA.Megakaryocytes from Rictor-KO and wild-type mice were incubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes,respectively.Platelet elongation on collagen surface was observed with scanning electron microscopy.Results Compared with the wild-type mice,Rictor-KO mice showed significantly decreased AKT phosphorylation,decreased platelet production,reduced thrombosis,and decreased platelet activation in response to ADP and thrombin stimulation.The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension,reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow.The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice.Conclusion Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice,suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.