Identification Of Circulating Fibrocytes Culture In Vitro And The Effect Of Serum With Chinese Medicine

We have researched the mechanism for interference on organs fibrosis by traditional Chinese medicine and analysis of traditional Chinese medicine theory—"Treating the different diseases with same therapy". So we can conclude that the pathologic basis of multiorgan fibrosis is the formation of fibrous focuses in the involved organs, accompanying with production of a large amount of collagen and extracellular matrix out of balance. The latest studies have showed that the key cells in the fibrous focuses in the involved organs are from a same origin, namely the marrow-originated circulating fibrocytes, theoretically based on which, we have put forward our"marrow-blood-target organ axis"hypothesis of fibrosis formation. The circulating fibrocytes are derived from the bone marrow , which circulating in the blood and migrated to the injured tissue. Circulating fibrocytes participate in various fibrosing disorder and produce matrix protein such as vimentin, collagenⅠ,collagenⅢ. It can further differentiate, and may represent a systemic source of the contractile myofibroblasts that appears in many fibrotic lesions.[Objectives] To establish an effective method of isolating, culturing and identification of circulating fibrocytes from human peripheral blood, and compare the morphological characters and collagen synthesis between circulating fibrocytes and fibroblasts, then study the effect of serum with Chinese medicine on circulating fibrocytes.[Methods] Total peripheral blood leukocytes were isolated from human peripheral blood by centrifugation over Ficoll-Paque and cultured in DMEM supplemented with 20% fetal calf serum. Adhered cells were detected by immunocytochemistry, FCM and electron microscopy. Then the collagen secreted into the medium was detected by measurement of the hydroxyproline concentration with enzymolysis, and the collagen accumulated on the cell layer was determined by the optical density at 540nm of picro-sirius red staining. The contents of collagen were assessed by Western blotting. Adding drug serum with different concentration to the DMEM, we observed the effects on proliferation and synthesis of collagen.[Results]1 Character of circulating fibrocytes cultured in vitroThe cells of leukocyte-rich buffy coat separated from peripheral blood adhered to the culture surface 1st day later, which was round and naive. After aspiration off the non-adherent cells, the remaining adherent cells appear as clusters of stellate on 3rd day, and then cells become elongated or spindle-shaped on 7th day like fibroblast. Finally the circulating fibroblast matured at 15th day and differentiated to be flat, thin and transparent as same as classic fibroblast. After 9 days culture in vitro, 83.5% of these cells were collagenⅠstaining positive detected by FCM.2 Effects of conditions in vitro on circulating cultureThe density of leukocyte seeded is very important, though circulating fibrocytes can be accelerated by high density, but it is also difficult to aspirate the non-adhere cells efficient, which is going to against the differentiation of circulating fibrocytes since 7th day culture in vitro. So we seed cells at 0.75×106PBMCs/cm2 which is good to mediate interaction between cells and decrease the pH of medium. Circulating fibrocytes can be cultured in 10% or 20% FBS medium, but cells in 20% FBS grew better.3 Electron microscope observation of circulating fibrocytesScanning electron microscopy showed these cells to be morphologically distinct from circulating blood leukocytes, to display spindle and prominent cell surface projections, and to be thick and hunch at the area of nuclear. Transmission electron microscopy show the cells are surrounded by abundant rough endoplasmic reticulum and free ribosome in the cytoplasm and abounded with active golgiosomes and mitochondrions.4 Immunocytochemical staining of circulating fibrocytesCirculating fibrcocytes expression CD34, a marker of stem/progenitor, and also have a leukocytes general marker CD45.The expression of CD34 and CD45 levels continued to decrease throughout the duration of culture in vitro, by contrast, COLⅠand COLⅢexpression remained elevated. As the cells matured in culture, circulating fibrocytes were founded to stain positive forα-SMA but negative for vWF on 30th day.5 The collagen synthesis of circulating fibrocytesThe COLⅠand COLⅢwere synthesized and secreted into medium of circulating fibrocytes as same as fibroblast. The collagen in the medium of circulating fibrocytes is less than fibroblast, but the collagen accumulated on the cell layer of circulating fibrocytes is more than fibrolast.6 Effects of serum with Chinese medicine on circulating fibrocytesWe used cultured circulating fibrocytes as a cell model for studying the effects of serum with Chinese medicine on cells .As a result, the serum with Radis Astragali can reducing the content of HYP in the medium and increasing the collagen in the cell layer, which suggest the serum with Radis Astragali can inhibit the collagen secreting. However, the serum with Rhizoma Zedoariae effected cells by another way, it can inhibit the cell proliferation. Serum with Radis Astragali and Rhizoma Zedoariae has no significant effects on these two factors.[Conclusions] We have establish a stability and reproducible system method to isolate and culture the circulating fibrocytes. On the basis of comparison with fibroblasts, endothelial cells, bone mesenchymal cells by immunocytochemical staining, we confirm further that there are a subunite progenitor cells from peripheral blood, circulating fibrocytes, can adhere the culture surface and differentiated into fibroblast. We study the morphological characters and collagen expression between circulating fibrocytes and fibroblasts, and use circulating fibrocytes as a cell model for pharmacological research. Then we found that the serum with Radis Astragali can inhibit the collagen secreting and the serum with Rhizoma Zedoariae can inhibit the cell proliferation, serum with Radis Astragali and Rhizoma Zedoariae has no significant effects on these two factors.