| Preoperative long-term antiglaucoma topical therapy would lead to sub-conjunctival inflammatory cell infiltration and accumulation of extracellular matrix, which would promote the fibrosis and correlate to the clinically reduced success of surgical procedures.Antihypertensive prostaglandin analogs are popularly used and become the first-line therapy of open angled glaucoma. Of them, latanoprost is used most frequently. The effect of long-term topical therapy of latanoprost on fibrosis of filtration tract attracts the ophthalmologists' attention .It was reported that prostaglandin analogs up-regulated CTGF (connective tissue growth factor) mRNA expression in ciliary smooth muscle cells. But the effect of prostaglandin analogs on CTGF by Tenon fibroblast has not been reported till now. CTGF is mitogenic for fibroblast-like cells and induces expression of extracellular matrix. The effect of prostaglandin analogs on CTGF by Tenon fibroblast was worth of being researched.Latanoprost stimulates secretion of matrix metalloproteinases in Tenon fibroblast both in vitro and in vivo, which may degrade collagen and other extracellular matrix. The amount of collagen is decided by degradation and synthesis. The effect of latanoprost on collagen synthesis by Tenon fibroblast has not been reported.To investigate the effect of prostaglandin analogs on CTGF expression and collagen synthesis, latanoprost free acid (LP-A) was administrated on cultured human Tenon fibroblast (HTF) in the present study.Part One: Culture and Identification of human Tenon FibroblastThe Tenon capsular tissue was obtained during cataract surgery and cultured using method of explanting. Low-glucosed DMEM contained 10% fetal bovine serum was used for subculture in 5%CO2, 37℃. Antibodies to Vimentin, fibroblast and cytokeratin were used in immunocytochemical staining or immunocyto-fluorescense staining for identification of fibroblasts.Result:The cultured cells were active growing, stable in manifestation, and confirmed to be fibroblast by positive staining with vimentin and fibroblast antibody, but negativestaining with cytokeratin antibody.Part Two: the Effect of Latanoprost Free Acid on CTGF mRNA and Protein Expression in Human Tenon Fibroblast1. HTFs were plated in 6-well plates and grew to confluence. Before treating, HTFs were starved in serum-free conditions for 24h.CD To observe the effect on CTGF mRNA expression of different concentration of LP-A: 6 groups were established (n=4), group 1 to group 5 treated by LP-A for 2 days in concentration of 10'5109M respectively, group 6 was control. ? To observe the effect on CTGF mRNA expression of different time of LP-A : 6 groups were established (n=4), group 1, 3, 5 treated by 1(T7M LP-A for Id, 2d, 3d, and the other groups were controls. The expression of CTGF mRNA was observed by RT-PCR.2. HTFs were plated in 24-well plates with cover slips laid, grew to confluence and starved. Six groups were established: group 1 to group 5 treated by LP-A for 2 days in concentration of 10"5, 10'7, 10"9, 10"and 10"13 M respectively, group 6 was control. The protein expression of CTGF was observed by immunocytofluorescense staining.Eight flask of confluenced HTF were starved and divided into 2 groups (n=4): group 1 treated by 1(T5M LP-A for 2 days, group 2 as control. The protein expression of CTGF was observed by immuno-Western blot.3. To investigate the role of FP receptor in LP-A induced CTGF expression, AL-8810, a FP inhibitor, was used to block FP receptor during LP-A treating.Results:1. After 10'9M<sup> 10"5M LP-A treating for 2 days, CTGF mRNA expression in group 1 5 was up-regulated 187%265% compared to control (p<0.05).After 10"7M LP-A treating for 1 day, CTGF mRNA was up-regulated 180±29% (P <0.01), for 2 days 166±25% (P<0.01), and for 3 days 59±27% (P<0.01).2. After LP-A treating for 2 days, the protein expression of CTGF was up-regulated 207±56% (P<0.01), 107±38% (P<0.01), 53±32% (P<0.05) and 87±53% (P< 0.01), respectively in group of 10"5M, 10'7M, 10"9M, 10"llM evaluated by immunocytofluorescense staining.Treated by 10"5M LP-A for 2 days, the protein expression of CTGF was up-regulated 59 8± 129% (P<0.01), detected by immuno-Western blot.3. AL-8810 did not have effect on the basal mRNA or protein expression of CTGF, neither could it block LP-A induced CTGF expression.Part Three: the Effect of Latanoprost Free Acid on Collagen Synthesis in Human Tenon FibroblastHTFs were plated in 6-well plates, grew to confluence and starved. CD To observe the effect on collagen I mRNA expression of different concentration of LP-A: 6 groups were established (n=4), group 1 to group 5 treated by LP-A for 2 days in concentration of 105 10"9M respectively, group 6 was control.(2) To observe the effect on collagen I mRNA expression of different time of LP-A: 6 groups were established (n=4), group 1, 3, 5 treated by 1(T7M LP-A for Id, 2d, 3d, and the other groups were controls.The expression of mRNA of a2 subunit of procollagen I was investigated by RT-PCR. Result:After 109M105M LP-A treating for 2 days, collagen I mRNA expression was down-regulated, 40.2±8.2% (P<0.01) in 10"5M group, 38.4±6.4% (P<0.01) in 10"6M group, 29.1±10.5% (P<0.01) in 10"7M group, and 23.3±10.6% (P<0.01) in 10"8M group respectively.After 10"7M LP-A treating for 1 day, collagen I mRNA was down-regulated 24.4±9.9%, 2 days 49.0±8.3% , and 3 days 94.8±7.1 %, in a time-dependent mannerPart Four: the Effect of Latanoprost Free Acid on the Proliferation of Human Tenon fibroblastHTFs were plated in 96-well plates (lx103cells/well). In serum-free conditions, 6 groups were established (n=8), group 1 to group 5 treated by LP-A for 2 days in con- centration of 10"510"9M respectively, group6 was control. The effect of LP-A on the proliferation of HTF was evaluated by MTT test.Same experiment was carried out in conditions containing 10% fetal bovine serum.Result:In serum-free conditions, the relative amount of cells increased byl7.7±27.8% (P=0.102) in 10"5M group and 17.3±17.9% (P = 0.095) inlO"6M group.In conditions with fetal bovine serum, the relative amount of cells increased by 13.6±5.5% (P<0.01) in 10'5M group.Conclutions:...
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