Table of Contents
PMNVs are an acronym for plasma membrane derived vesicles .they are tiny intact vesicle secreted by the cell membrane and help in the intercellular communication. Their role is examined in this paper on the terminal differentiation of monocytes .The agents of differentiation of cell such as al-trans retinoic acid /PMA and histamine, the mediators of inflammation that controls the proliferation of provoke the release of calcium dependent PMVS by the t-cell helper 1(THP-1)monocytes (Simons, 2001).
The released PMVS cause the TPH-1 cells to enter into the cell cycle of G0-G1 and cause endpoint monocyte to macrophage proliferation. The presence of the PMVS is ascertained by the us e of TGF –B1 receptor antagonist and the anti TGF-B1.thelevel of TGF however are thought to be variant depending on the cell type for example, the differentiating macrophages and maturing dendritic cell have increased TGF- B1.periferal blood monocytes will be used to assess the effect of PMVS on the proliferation of cell Lines. Flow cytometry autoclave cell counting techniques will be used in the analysis of the cell. The cell will be sorted after they have been subdivided by the ultra-centrifuge to obtain pure cell in proportion that are workable avoiding the effects of plasma proteins (Hugel, 2005).
The study of the effects of the PMVS is aimed to excavate the real factor in the proliferation of different cell lines in this case the need to study PMVS and their origin becomes scientifically vital. Microvesiculation is a normal process in cells. Plasma membrane derived vesicles are released by the cell. These vesicles aid in the intercellular communication within the body. PMVS is tiny of about 0.1-1um.the cell secretes the PMVS by the process of exocytosis.
PMVS are released by the proliferating dendritic cell and maturing macrophages, and even the platelets. The release of PMVS involves a number of physiological changes in the cells. PMVS release is however induced by increased calcium in cell and reduced lipid asymmetry. Outside the body microvesiculation can be induced by the release of sub lytic calcium (Eken, 2008).
PMVS have micro- RNA which is characteristic of the parent cells. The micro-RNA helps in the transport of proteins that are deficient of signal peptides such as epimorphin and fibroblast growth factor
In the TPH-1 PMVS can be demonstrated by the presence of these proteins .the micro-RNA are involved in the monocyte differentiation .PMVS are thought to be involved in the reprogramming the hematopoietic cells in their differentiation. Cells secrete PMVS from the cell membrane as either as exosomes or as direct plasma membrane derived vesicles (Gasser, 2004).
The effect of PMVS on the proliferation of monocytes originates from the fact that PMVS have been used in the therapies for acute monocytic leukemia. The study presented in this paper aims at determining whether the PMVS themselves are able to induce cell cycle or arrest terminal differentiation in the moncytic leukemia, this however represents the hallmark of differentiation therapy based on the cell line THP-1.recsent studies have shown that the micro vesicles from neutrophils are thought to interfere with maturation of immature dendritic cells of the TPH-1 type. The early breakthrough of the therapies of ATRA in the treatment of acute promyelocytic leukemia, no follow ups have been so far made on this concept whether there is any signs of resistance which can help develop new therapies.
This research paper aims at demonstrating the stand points of PMVS on the differentiation and proliferation of THP-1 cell lines. PMV mediated differentiation is a process involving several proteins and micro-RNAs. Studies on the proteomic of cytokines indicate that the TGF-B1 which is found in the platelets PMV that inhibits the proliferation of various cell lines, the presence of the TGF-B1on the PMVs has been intensively studied and shown to have effects on the proliferation of cells and also inhibits autocrine of proliferation in APL cells. Basing on the vital role played by the tgf-B1on the regulation of proliferation the quest to know if this could be influenced by the PMVS given the proliferation seen on the promonocytic leukemia (Ephraim A. Ansa-Addo, 2011).
All-trans retinoic acid coupled with the inflammatory mediator such as histamine and bacterial components like lipopolysaccharides induce cellular microvesiculation.The factor in place is that the cell should have receptors .since PMV inducers have been described to have a possible therapeutic importance in the treatment of monocytic leukemia the paper seeks to find if these PMV scan be directly be linked in inducing proliferation or arresting terminal differentiation (Del Buono BJ, 2008).
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Method and Materials
In order to show that the THP-1 cell proliferation is inhibited by PMVS and since THP-1 and monocytes can release PMVS the effect of PMVS will be measured on the monocytes .primary and continuous cell lines will be prepared to get the capacity and ability to induce differentiation. The monocyte will be culturered and allowed to grow in suspension as cells comparison with the control cell will be employed distinction on the morphology is expected in a while say 24 hours after addition of PMVS .
In this study two requirements will facilitate the structuring of the findings on the effects of PMVs, they include, peripheral blood monocyte isolation to monitor proliferation and terminal differentiation, and cell culture where cell are grown under favorable conditions and observed.
Cells will be maintained in a cell culture containing antibiotics such as kanamycin, streptomycin maintained at 37oc with an atmospheric humidity maintained with a 5%carbon dioxide. This maintained for two weeks.THP-1 cells are as well cultured in this manner with additional 5%FBS.all along the culturing procedure cell morphology is monitored with the aid of fluorescence microscope. Cell count and viability will be monitored (Anon., 2008).
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From the cells, PMVs will be isolated and moderated by use of normal human plasma. The proteins extracts will be analyzed by SDS-PAGE for or by western blot ting.
Cell expression of tgf-B1 molecules will be analyzed by flow cytometry, where thp-1 cells will be washed in the phosphate buffered saline; they are then incubated in the dark after which the cells will be stained with the use of isotope staining matched by controls (Jason R. Lane, 1999).
The cells are stain with annex inn and analyzed in a flow cytometer to assess whether all the cells TPH-1cell which were releasing PMV were undergoing apoptosis.
All samples for experiments will be put in plate with cover slips and centrifuged with an ultra-centrifuge; fixation is done by parafomaldhydeat37oc for ten minutes. Images will be collected by use of fluorescent microscope.
PMV derived from the experimental thp-1 will be labeled by use of octodecyl Rhoda mine chloride in the dark. PMVS will further be treated in the ethanoic solution and incubated at room temperature. Staining the intracellular cytokines thp-1will be suspended in buffer at room temperature, cell will permiabillzed by washing three times in buffer and labeled with a secondary antibody. The samples will then be suspended in the buffer and analyzed by flow cytometry (Anon., 1993).
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Negative staining will be employed for PMVS isolated from THP-1 cells. The PMV isolated without earlier sonication these will be stained with 2%aqueus unary acatete.alcian blue dye is used in staining the PMVS isolated for ten minutes .the digital images PMVS in this state will be recorded by use of AMT digital camera.