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RESULTS
The inhibition rate of HPF growth changed after the intervention of Tet, which was seen in Table 1. Cells in control group were shaped like pavingstones, neatly arranged and growing adherent to the wall, while cells in Tet group became round and distributed loosely. Moreover, the number of cells in high dose group decreased markedly. When the concentration of Tet exceeded 20μmol/L, Tet could significantly inhibit the proliferation of HPF (P<0.05). In the range of 20μmol/L to 160μmol/L, the inhibitory intensity increased with the rising of concentration (P<0.05). During 24 to 72 hours of drug acting, the inhibitory intensity increased with the prolonging of time (P<0.05). 48 hours later, the inhibition rate did not increase, which showed the feature of saturated pharmacodynamics. At 48 hours with the concentration of 160μmol/L, the inhibition of Tet on HPF proliferation was significant (P<0.05) and the inhibitive rate reached 91.0%. At 72 hours, the inhibitive rates of HPF proliferation were respectively 89.9% and 90.6% with the concentrations of 80μmol/L and 160μmol/L. With the increase of Tet concentration and the prolongation of time, the inhibition rate of fibroblast growth increased (P<0.05).
Within the range of 2060μmol/L acting for 24 to 72 hours, Tet could be in a dose and timedependent manner. Based on the inhibition rates of HPF proliferation under various concentrations, the timedose effect curve was produced (Figure 4).
The expression of PCNA of HPF cells was seen in Figure 5 and Table 2. Cell positive expression showed brown, yellow or pale yellow uniform fine particles, distributing in the whole nucleus. No nuclear staining and very pale yellow color of cytoplasm were characteristics of negative control group. The nuclei of HPF cells appeared brown, and the expression of PCNA was strongly positive; after the intervention of Tet, the color of nuclear staining became light, and in a dosedependent manner (P<0.05).
DISCUSSION
Pterygium was a common disease of ophthalmology. Pathological study indicated that its main ingredient was abnormal proliferating fibroblasts and neovascularization[2], with the nature of pretumorigenesis. The scope and extent of fiber proliferation and neovascularization of pterygium were reliable morphological indicators for prediction of recurrence after pterygium resection surgery[3]. In the present study cell culture was directly from human pterygium tissue, so its biological characteristics were in accordance with human eye diseases.
Furthermore, the immunohistochemical method was used to identify the cells. Its expression of vimentin was positive, and its expression of keratin was negative. In view of organization sources and cell growth characteristics, they could be identified as fibroblasts. There are a lot of surgical methods to treat pterygium currently, but they are unable to effectively control its relapse rate. In clinic the immunosuppressive agents, antiproliferative and antimetabolic drugs were used as adjuvant therapies, such as dexamethasone, mitomycin C and 5fluorouracil alone or in combination to reduce the recurrence rate after pterygium surgery. Clinical observation showed that they had certain effects on delaying the development of pterygium, but these drugs had great local and systemic sideeffects. Therefore it is a research topic facing many scholars to find safe and effective antiproliferation drugs.
Tet is a kind of alkaloid separated from traditional Chinese medicine such as Menispermaceae, Tetrandrine or Stephania, derivative of dualbenzylisoquinolin. It had antiinflammatory, analgesic, antipyretic, antiallergic, antifree radicals, anticancer and other pharmacological effects. It could also inhibit fibroblast proliferation and collagen synthesis[4]. Researches found that Tet in the concentration of above 5μg/mL could significantly inhibit the proliferation and collagen synthesis of human scar fibroblasts cultured in vitro[5]. The antiscar function of Tet may be accomplished by directly inhibiting the DNA synthesis of fibroblast, regulating and controlling collagen metabolism, indirect acting on cytokines and other aspects[6].
In this experiment we used Tet of different concentrations to intervene human pterygium fibroblasts and found that Tet could inhibit the growth of human pterygium fibroblast. With the increase of concentration and prolongation of time, the inhibition rate increased. In addition, the expression of PCNA protein was inhibited and the degree of its decrease was negatively correlated with Tet concentration. PCNA is a nuclear protein, coenzyme of D polymerase in the DNA replication. It is directly involved in the nuclear DNA synthesis and mainly expressed in the S phase of cell proliferation cycle, which was closely related to cell proliferation activities. Therefore it could be used as an accurate and simple indicator of cell proliferation status[7]. Our study found that when the concentration of Tet≥20μmol/L, it could dosedependently inhibit the expression of cell PCNA. It was consistent with the results of Tet inhibiting cell proliferation activity detected by MTT in this experiment. The present experimental study showed that Tet could inhibit the proliferation of HPF and induce the apoptosis in timeand dosedependent manner, suggesting that the treatment of pterygium by Tet has potential application prospect. In short, this experiment has provided a new way of thinking for adjuvant therapies of pterygium, but the current study is limited to in vitroexperiments and mechanisms of Tet are complex, so further studies are still expected.
【参考文献】
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Materia Medica 2004;20(4):1011
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