| Objective:To observe and investigate the effects of pseudolaric acid B (PAB) on the proliferation and apoptosis of cultured human subconjunctival Tenon's capsule fibroblasts (HTCF) in vitro, between different concentrations the drug's strength, in order to search for a auxiliary treatment and a new way to prevent recurrence after glaucoma filtering surgery.Methods:The Tenon's capsule tissues, which were obtained from Glaucoma patients drawn-subconjunctival, were cultured by means of sticking tissue-pieces on the culture bottle wall.And the cells from generations 3 to 6 were used. We employ the following methods:1. Normal Tenon's capsule fibroblasts identification:Preparation the climbing cells film, the give the HE staining and using anti-a-SMA antibody, anti-vimentin antibodies for cell identification by the streptavidin-perosidase (SP) immunohistochemical staining techniques.2. Investigating the effects of pseudolaric acid B on the proliferation and apoptosis of cultured HTCF in vitro by MTT colorimetric assay:HTCF were incubated with 0-160umol/L PAB for 12-96h. The MTT colorimetric assay was used to assay the biologic activities of the HTCF which PAB incubated in different doses and time.3. By the SP immunohistochemical staining techniques, expression of the B-cell lymphoma gene 2 (bcl-2) in the HTCF was detected:incubated with 0-160umol/L PAB after 48h, using SP immunohistochemical staining techniques detected the expression of Bcl-2 protein situation.Results:1. Normal Tenon's capsule fibroblasts were cultured in vitro. Generally the cells were fusiform or triangular. And there is oval-shaped nucleus located in the rich central cell cytoplasm. The cells grow capacity strong and its radial as weaving. waterfall-like or spiral arrangement.2. By the SP immunohistochemical staining techniques, it shows the positive expression of vimentin, and cytokeratin staining was negative. The positive expression of vimentin in the cell cytoplasm, showing the long axis of the same cell bundle or brown mesh node.3. After 4-60umol/L PAB effect within 12-72h, MTT assay that the surg can significantly inhibite the proliferation of HTCF in a dose and time dependent manner (P<0.05). When the reaction time was 12h, the inhibition ratio of PAB were 2.33%, 4.33%,8.33%,18.00%,56.00%,61.00%,64.67%,67.67%,68.33%,71.33%and 77.00%correspond to 0.5μmol/L, lμmol/L,2μmol/L,4μmol/L,8μmol/L,16μmol/L, 25μmol/L,40μmol/L,60μmol/L, 80μmol/L and 160μmol/L. When the time was 48h, the inhibition ratio of PAB were 1.48%,6.14%,9.11%,51.69%,75.21%,77.33%, 81.57%,83.35%,84.75%,86.65and 88.56%. When the time was 72h, the inhibition ratio of PAB were 1.41%,4.08%,8.63%,65.78%,83.05%.84.14%, 86.81%,87.28%,88.38%,90.42%and 91.21%。When concentration of PAB≥4 umol/L, we see the proliferation of fibroblasts was inhibited (P<0.05). When concentration of PAB range in the 4-60 umol/L, inhibition increased with the enhance of drug concentration (P<0.05). When the concentration of PAB>60 umol/L, the role of inhibition increased with enhance of drug concentration, but as longer than 60 umol/L, the inhibitory effect is not so obvious increase (P>0.05).When the reaction time range in the 12-72h, the role of inhibition increased with strengthen of time, but as longer than 72h, the inhibitory effect is not so obvious increase, showing saturation kinetics. Among them, when the drug concentration is 160umol/L and the reaction time is 72h, inhibition is the most significant (P<0.05), its inhibition rate was 91.21%.4. When the concentration of PAB≥4umol/L during 48h, the HTCF can be concentration-dependent inhibition of the expression of bcl-2 (P<0.05). In the negative control group, the positive rate of Bcl-284±1.23%, while the drug group, the positive rates were:81±1.02%(0.5umol/L group),76±0.78%(1umol/L group), 70±1.10%(2umol/L group),47±0.09%(4umol/L group),21±0.64%(8umol/L group),15±0.89%(16umol/L group),12±0.25%(25umol/L group),9±1.03% (40umol/L group).5±0.98%(60umol/L group),4±0.07%(80umol/L group) and 2±0.11%(160umol/L group). After the effect of PAB, the expression of Bcl-2 in fibroblast was significantly decreased, adopting the statistical analysis, the inhibition was dose dependent (P<0.05).Conclusion:Pseudolaric acid B can inhibit the proliferation of cultured HTCF, and the effect was dose and time dependent. It is expected to be assisted treatment and a new drugs on prevention of recurrence after glaucoma filtration surgery. |
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