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目的观察缓激肽(BK)对体外培养的人角质形成细胞(HKC)增殖、凋亡和分化的影响并探讨其机制。方法以1×10-4~1×10-9mol/LBK作用于体外培养的HKC,采用噻唑蓝法、台盼蓝染色法观察到1×10-4mol/LBK抑制作用最强,以此浓度BK进行余下实验。当HKC达对数生长期后,一部分加入1×10-4mol/LBK作为实验组,另一部分不加BK作为对照组,分别培养24、48h后采用流式细胞仪检测细胞早期凋亡情况及细胞周期,用链霉亲和素-生物素复合物(SABC)免疫细胞化学法检测HKC分化标志物角蛋白10(K10)及内披蛋白的表达情况。用含1×10-4mol/LBK的无血清培养基KC-SFM培养HKC作为实验组,对照组细胞仅加KC-SFM,分别用激光扫描共聚焦显微镜结合钙荧光探针Fluo-3/AM技术检测细胞内游离Ca2+浓度即[Ca2+]i的变化。结果与对照组比较,实验组G0/G1期细胞比例相对上升34.57%,S期相对下降58.91%,其细胞早期凋亡率为15.34%,明显高于对照组(5.60%,P<0.05)。实验组HKCK10阳性细胞百分比为2.20%,明显低于对照组(6.89%,P<0.05)。BK作用3min后实验组HKC[Ca2+]i较对照组上升163.0%,之后开始下降,5min后接近对照组。结论高浓度BK可抑制HKC周期进程、明显促进其凋亡及诱导[Ca2+]i升高,这可能是其使HKC体外生长受抑的部分机制。BK还可抑制表皮再生和HKC分化。
Objective To observe the effect of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocytes (HKC) cultured in vitro and to explore its mechanism. Methods 1 × 10-4 ~ 1 × 10-9mol / LBK was used to culture HKC in vitro. The inhibitory effect of 1 × 10-4mol / LBK was observed by trypan blue staining with thiazolyl blue staining. The concentration of BK Carry on the remaining experiments. When HKC up to logarithmic growth phase, part of the added 1 × 10-4mol / LBK as the experimental group, the other part without BK as a control group were cultured for 24,48 h after the use of flow cytometry cells early apoptosis and cell Cycle, and the expression of keratin 10 (K10) and the putative protein of HKC were detected by immunocytochemistry with streptavidin-biotin complex (SABC). HKC was cultured in KC-SFM containing 1 × 10-4mol / LBK serum-free medium and KC-SFM was added to the control group. Laser scanning confocal microscopy combined with Fluorescence probe Fluo-3 / AM Detection of intracellular free Ca2 + concentration [Ca2 +] i changes. Results Compared with the control group, the proportion of cells in G0 / G1 phase increased by 34.57% and that in S phase decreased by 58.91% and 15.34%, respectively, which was significantly higher than that in control group (5.60%, P <0.05). The percentage of HKCK10 positive cells in experimental group was 2.20%, which was significantly lower than that in control group (6.89%, P <0.05). After 3 minutes of BK treatment, the HKC [Ca2 +] i in experimental group increased by 163.0% compared with that in control group, then began to decline, and then approached to control group after 5 minutes. Conclusions High concentration of BK can inhibit the cycle of HKC, promote its apoptosis obviously and induce the increase of [Ca2 +] i, which may be part of the mechanism of inhibiting the growth of HKC in vitro. BK also inhibits epidermal regeneration and HKC differentiation.