| Background and Objective:Thrombopenia is a common clinical syndrome in hemorrhagic disease,which usually can be caused by radiotherapy and chemotherapy of tumours,bone marrow depression after hematopoietic stem cell transplantation and the injury of MK cell by the chemical,and all of them may interfere the releasing of platelet.Moreover,some heritage and immunity disease can also result in disfunction of platelet,such as MYH9-related disorders, idiopathic thrombocytopenic purpura,and et al.At present,platelet transfusion is an effective and broadly used treatment of these kinds of thrombopenia.But the problem is that transfusion reaction and isoimmunity may induce anti-platelet antibody if repeating transfusion.How to promote platelet quantity as well as quality quickly without antibody production or with less adverse reactions has become an urgent problem to be resoleved.These years,the discovery of some cytokines,such as granulocyte colony-stimulating factor,granulocyte-macrophage colony-stimulating factor,erythropoietin,has provided a better method to solve agranulocytosis and anemia,at the same time,it shines a new light to thrombopenia treatment.In 1994, thrombopoietin,as one of the most important cytokines in clinical,was successfully purified,but as TPO has not uesd for long in clinic as well as using TPO alone can not promote platelet quickly or effectively,and what is the worse is the unpredictable side effect,such serious bleeding because of anti-TPO antiboby producing.All of above limit its clinical application.To develop a new drug which can stimulate thrombopoiesis and platelet releasing directly and effectively has become the badly need for us.Cytoskeleton plays an important role in the formation of proplatelet from megacaryocyte and the releasing of platelet at the late phase of megakaryocytopoiesis.Cytoskeleton is composed of microtuble,microfilament and intermediate fibre,and they provide motive power for proplatelet formation and platelet releasing from MK.Microtubule propels the extension and elongation of proplatelet,while microfilament provides the motor for proplatelet bending,branching and for the amplification of proplatelet ends,and microtuble inhibitor nocodazole and microfilament inhibitor cytochalasin B or D all can block the PPT formation.Serotonin,as an indolamine compounds,does not only have a critical physiologic function in both central nerveous system and peripheral organs as a neurotransmitter and vasoactive agent, but also has been proved to be a selective grow factor of megakaryocytopoiesis.On one hand, serotonin has effects to support megakaryocyte proliferation and differentiation via 5-HT2 receptors,on the other hand,serotonin takes part in the regulation of cytoskeleton reorganization in smooth muscle cells and nerve cells and then influence their movement and differentiation. Our latest study has observed that:human megakaryocytic leukemia cell line Meg-01 appears reorganization of F-actin as well as proliferation and differentiation by serotonin.For this reason,we presume that serotonin has the ability to promote proplatelet formation and platelet releasing in megakaryocytes through cytoskeleton reorganization via 5-HT2 receptors.Therefor,in this experiment we will detect PPT formation,cytoskeleton reorganization,and expression of cell surface marker of MK cells CD41 by serotonin,and we hope we can consummate the mechanism of thrombopoiesis as well as explain the effect of serotonin in this process.Furthermore,we hope it can profit to explore new agent to stimulate thrombopoiesis and promote platelet quickly to satisfy the clinical need.Methods:This experiment includes two parts:experiment for serotonin promoting pseudopodia formation and elongation to form PPT from MK and reorganising cytoskeleton,and then researching whether serotonin has effect on the expression of MK surface mark CD41.Four groups was divided in the experiment:control group,serotonin(200nmol/1) group, serotonin+kentaserin(serotonin 200nmol/l,ketanserin 10μmol/l) group,and TPO(50ng/ml) group. Firstly,inverted phase contrast microscope and MTT method are used to prove our protophase study that serotonin promotes MK cell proliferation.Next,inverted phase contrast microscope and transmission electron microscope are used to observe the effects of serotonin on the morphology change,PPT formation and reorganization of cytoskeleton respectively.In the end, immunocytochemical method is taken to approach whether serotonin can increase the expression of cell surface marker CD41 on MK. Results:Observing Meg-01 cell with inverted phase contrast microscope,the cell density of serotonin group and TPO group are both obviously higher than control group and serotonin+KE group,and at the same time MTT shows the same result as above:there are significant difference from serotonin group to control group and serotonin+KE group(both P≤0.01),while no difference versus TPO group(P＞0.05).Inverted phase contrast microscope also shows that the spread and elongated MK cells in serotonin group and TPO group are obviously more than those in the other two groups,what is more,some PPT stalks elongate several times longer than the original body.The ratios of spread but not elongated cells and spread and elongated cells in serotonin group are 22.7±5.98%and 15.4±5.97 respectively,which have significant differences versus control group(7.3±3.27%and 3.6±1.56%) and serotonin+KE group(8.8±4.5%and 5.3±3.31%)(both P≤0.05),however it is not statistically significant versus TPO group (22.4±7.30%and 7.5±3.59)(P＞0.05).Observation the morphologic details of the cytoskeleton reorganization in Meg-01 cells by transmission electron microscope shows that in serotonin group cytoplasm extended and formed axis while microtubules were longitudinally oriented in the cytoplasmic extension;dense bands that were apparently accumulations of actin microfilaments were present in the filopodia and in the periphery of the extended cytoplasm,and some have bifurcated.Some vacuolar inclusions andαgranules were found in the nodes,which are similar to the demarcation membrane systems usually found in proplatelets The granules were also found in a few particles which looked like platelets.MK cells in control group and serotonin+KE group can be found little cytoplasm extension or bifurcation,and there were little or no vacuolar inclusions orαgranules.The same change appeared in the TPO group. Immunocytochemical method was used to detect the cell surface mark CD41,and the the result shows that the ratios of CD41 positive cells in serotonin group and TPO group(18.94±3.94 and 20.92±7.03%respectively) are higher than the other group(control 14.28±3.79%and 5-HT+KE group 14.38±7.56%),however,there is no statistical significance among them(P＞0.05). Conclusions:Serotonin not only has the function to stimulate MK cells proliferatian,at the end phase of MK cell releasing platelet,it but also has distinguished effect to reorganise MK cytoskeleton and facilitate PPT formation,but the expression of cell surface marker CD41 on Meg-1 didn't have significant increase by serotonin.
|
PDF Full Text Request