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目的:探讨常通口服液(CTOL)对体外培养的粘连腹膜成纤维细胞(AF)增殖的影响。方法:(1)药物血清的制备:雄性SD大鼠10只,随机分为2组:正常血清组、CTOL组。大鼠灌服相应药物7 d后,腹主动脉采血,分离含药血清。(2)成纤维细胞的体外培养:采用组织块贴壁培养法进行成纤维细胞培养,本实验所用细胞为第3~8代。(3)成纤维细胞的增殖:取生长状态良好的AF,按5×105/孔接种于24孔培养板中,分别加入:5%含药血清;5%正常血清;纤溶酶原活化剂(PAI-1),浓度为1 ng/mL;纤溶酶原活化物抑制因子(tPA),浓度为1 ng/mL。同时设置10%胎牛血清作空白对照孔。培养24 h后,加入B rdU,37℃、5%CO2孵箱中继续孵育4 h,细胞用4%多聚甲醛固定,B rdU免疫荧光检测细胞增殖情况。结果:与胎牛血清组比较,CTOL、PAI-1、tPA组的阳性率均有统计学意义(P<0.001);而正常血清组无统计学意义。结论:CTOL与tPA可以抑制体外培养的AF增殖,而PAI-1则促进AF增殖。
Objective: To investigate the effect of Changtong oral liquid (CTOL) on the proliferation of adherent peritoneal fibroblasts (AF) cultured in vitro. Methods: (1) Preparation of drug serum: Ten male SD rats were randomly divided into two groups: normal serum group and CTOL group. Seven days after the rats were fed with the corresponding drugs, blood was collected from the abdominal aorta and the drug-containing serum was separated. (2) In vitro culture of fibroblasts: fibroblasts were cultured using adherent tissue culture method. The cells used in this experiment were from the 3rd to the 8th generation. (3) Proliferation of fibroblasts: Take good growth of AF, inoculate in 24 well culture plates at 5×105/well, add: 5% drug-containing serum; 5% normal serum; plasminogen activator (PAI-1) at a concentration of 1 ng/mL; plasminogen activator inhibitor (tPA) at a concentration of 1 ng/mL. At the same time set 10% fetal bovine serum as a blank control well. After cultured for 24 h, B rdU was added and incubated at 37° C. in a 5% CO 2 incubator for 4 h. The cells were fixed in 4% paraformaldehyde, and the proliferation of cells was detected by B rdU immunofluorescence. Results: Compared with the fetal bovine serum group, the positive rates of CTOL, PAI-1, and tPA groups were statistically significant (P<0.001). However, the normal serum group was not statistically significant. Conclusion: CTOL and tPA can inhibit the proliferation of AF cultured in vitro, while PAI-1 promotes the proliferation of AF.