The Effect Of Doxycycline On The Bovine Corneal Myofibroblasts In Vitro

Corneal transparency is essential for vision. The cornea’s unique structural anatomy acts as the eye’s first refactive media, accounting for up to90%of the total refractive state in human beings. Therefore, any ocular disease that affects the cornea’s transparency affects vision. Corneal disorders are one of the most significant causes of vision loss in human. Corneal wound healing resulting from inflammation, infection, ocular trauma, autoimmune disease or neoplasia, affects corneal transparency, and therefore visionSome careful literatures review done by Berryhill, B.L.、Wilson, S.E.、 Netto, M.V.support that the hypothesis of the process of wound contracture and corneal healing relies on the stromal cell activation, protein synthesis, cytokine release, loss of cell contact, and culminates in the transformation of local keratocytes and fibroblasts to myofibroblasts. Stop the transformation or inhibit the proliferation of myofibroblasts is the key of preventing the formation of corneal scar.Doxycycline is a broad spectrum antibiotic that chelates metal ions and is frequently used as part of the treatment of ocular surface diseases. Its therapeutic value has been ascribed to an ability to inhibit matrix metalloproteinase (MMP) activity and both MMP and IL-1synthesis Recently doxycycline has been shown to posses the effects on inhibition of corneal epithelial cell proliferation and the process of neovascularization. The aim of this study was to investigate the effect of doxycycline on the inhibition of cell proliferation and the Nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms.PART Ⅰ ISOLATION AND CULTIVATION OF BOVINE CORNEAL STROMAL FIBROBLASTSOBJECTIVE:Developed a new protocol to isolate and culture bovine corneal stromal fibroblasts, prepare for the next study. METHODS:The bovine corneal stromal layer was digested twice by type Ⅰ collagenase, then the cell suspension was cultured and passaged in vitro. Cell vitality was measured by Trypan blue staining. The morphology of the bovine corneal stromal fibroblast cell cultures was characterized by inverted phase-contrast microscope. Immunohistochemical staining with Vimentin was used to confirm the presence of bovine corneal fibroblasts. The cell proliferation was tested by methyl thiazolyl tetrazolium (MTT) method. Bovine corneal stromal fibroblasts in logarithmic growth phase were obtained to draw growth curve and calculate doubling time.RESULTS:Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal stromal fibroblasts. Microscopic examination and immunohistochemical staining comfirmed that the cells cultured were bovine corneal stromal fibroblasts. Trypan blue exclusion demonstrated that the immediately survival rate of bovine corneal stromal fibroblasts was93.5%. Cell growth curve approximated the "S" shape, and cell population doubling time was38.70hours.CONCLUSION:This technique provides a simple and economic means for the reproducible initiation of primary cultures of bovine corneal stromal fibroblasts for us in a variety of experiments. PART II EFFECT OF DOXYCYCLINE ON THE PROLIFERATION OF BOVINE CORNEAL MYOFIBROBLASTS IN VITROOBJECTIVE:The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms.METHODS:Bovine corneal fibroblasts were treated with different concentrations of doxycycline (10mg/L,20mg/L,40mg/L,60mg/L,80mg/L),a bland control group and the DXM group (120mg/L) were set up. Cell proliferation was evaluated by MTT and the cell cycle was performed on BD FACScan flow cytometer assay when the cells were treated for24hours and48hours.RESULTS:For the MTT investigation shows that the inhibition rate of bovine corneal myofibroblasts progressively increased as the concentrations of doxycycline was increased (P<0.05).And60mg/L of doxycycline had an obviously stronger inhibitory action than120mg/L DXM in the same treated hours(P<0.05). As the concentrations investigated, doxycycline can inhibit cell growth of bovine corneal myofibroblasts, and that inhibition of cell growth was due to cell cycle arrest at the G0/G1phase progression. What’s more,60mg/L of doxycycline had an obviously effection as120mg/L DXM.(P>0.05).CONCLUSION:As the concentrations of doxycycline was increased from1Omg/L to80mg/L,the proliferation of cultured bovine corneal myofibroblasts can be significantly inhibited in vitro. Doxycycline significantly suppressed the proliferation of bovine corneal myofibroblasts in a dose-and time-dependent positive trend, and60mg/L of doxycycline had an obviously inhibitory action as120mg/L DXM. PART Ⅲ INHIBITORY EFFECTS OF DOXYCYCLINE ON ARGYROPHILIC NUCLEOLAR ORGANIZING REGIONS AND A-SMOOTH MUSCLE ACTIN EXPRESSION IN PROLIFERATIVE BOVINE CORNEAL MYOFIBROBLASTS IN VITROOBJECTIVE:The purpose of the present study was to investigate the effect of doxycycline on the Nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms.METHODS:Bovine corneal fibroblasts were treated with different concentrations of doxycycline (10mg/L,20mg/L,40mg/L,60mg/L,80mg/L),a bland control group and the DXM group (120mg/L) were set up. The AgNORs staining and the immunohistochemistry for a-SMA were performed when the cells were treated for24hours and48hours.RESULTS:The AgNORs count and AgNORs area of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increased (P<0.05).And60mg/L of doxycycline had an obviously inhibitory action as120mg/L DXM in the same treated hours(P<0.05). As the concentrations investigated, doxycycline can inhibit the expression of a-SMA in the bovine corneal stromal fibroblasts,while DXM can not.CONCLUSION:As the concentrations of doxycycline was increased from10mg/L to80mg/L, the AgNORs count and AgNORs area of bovine corneal myofibroblasts can be significantly reduced in vitro. Doxycycline significantly suppressed the expression of a-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.