外源性人β防御素-2调节子宫内膜异位症细胞增殖/凋亡及炎性因子的表达

Exogenous Human β Defensin 2 Protein Regulate Proliferation/apoptosis and Expression of Inflammatory Factor in Endometriosis Cells

【目的】探讨体外条件下加入外源性人β防御素-2蛋白对子宫内膜异位症(EMs)细胞增殖/凋亡和炎性因子表达的影响。【方法】组织块消化法原代培养EMs细胞,Western Blot法检测EMs细胞hβD-2蛋白、TNF-α及IL-1β的蛋白表达;Western Blot法检测加入重组人hβD-2蛋白(40μg/mL)前后EMs细胞内TNF-α,IL-1β蛋白表达变化;按不同梯度浓度(80、40、20、10μg/mL)加入人β防御素-2蛋白(hβD-2)后,ELISA法检测EMs细胞培养液上清中TNF-α,IL-1β细胞因子的浓度;加入高剂量hβD-2蛋白(120μg/mL)并培养EMs细胞24H设为实验组,对照组为完全培养基培养的EMs细胞,MTT法检测实验组及对照组EMs细胞增殖情况;PI法检测实验组及对照组EMs细胞凋亡情况。【结果】原代培养的EMs细胞可表达hβD-2蛋白、TNF-α、IL-1β蛋白;加入外源性重组人hβD-2蛋白后,EMs细胞表达TNF-α、IL-1β蛋白下调;EMs细胞培养液上清中可检测出TNF-α,IL-1β的表达;在加入hβD-2后,EMs细胞培养液上清内TNF-α、IL-1β表达下降,且随着加入hβD-2浓度增高其下降趋势增大,差异有统计学意义(80>40>20>10μg/mL,P<0.05);在EMs细胞培养基中加入高浓度外源性人β防御素2蛋白孵育后(120μg/mL),与对照组相比,EMs细胞的增殖受到明显抑制,且凋亡明显增加(P<0.05)。【结论】hβD-2可以影响子宫内膜异位症细胞内炎症因子TNF-α,IL-1β的表达,并可抑制EMs细胞的增殖,促进其凋亡。为治疗EMs提供新的理论依据。

[Objective]This study was designed to discuss exogenous human 13 defensin 2 protein how to regulate the proliferation/apoptosis and the expression of inflammatory factor in endometriosis cells in vitro. [ Methods ] The endometriosis cells were cultured in vitro, the expression of TNF-α and IL-1β protein in endometriosis cells with/without 40μg/mL hl3D-2 protein was detected by Western blot analysis; when the endometriosis ceils were cultured with setting concentration gradient （80 μg/mL, 40 μg/ mL, 20 μg/mL, and 10 μg/mL） h13D-2 protein, on the concentration of TNF-α and IL-1β in the endometriosis cells conditioned medium was detected by ELISA; the endometriosis cells were cultured with adding high-dose h13D-2 （120 ~g/mL） protein 24H. as experimental group, the control group was endometriosis cells cultured with complete medium, the proliferation of two groups were detected by MTF. Cell apoptosis in the two groups were dyed by PI and detected by flow cytometry. [Results]The endometriosis cells expressed h13D-2, TNF-α, and IL-1β. The expression of TNF-α and IL-1β protein in the endometriosis ceils can be down-regulated by exogenous h13D-2 protein; the expression of TNF-α and IL-1β protein in endometriosis cells conditioned medium were downregulated also by exogenous h^D-2 protein, and with dose dependence, the difference have statistical significance （80 μg/mL〉40μg/mL〉20μg/mL〉10 μg/mL,P 〈 0.05）. Compared with control group, high concentration of h13D-2 protein （120 μg/mL） significantly inhibited cell proliferation and promoted apoptosis （P〈0.05）. [Conclusion] hD-2 can influence on expression of TNF-α and IL-1β inflammatory factors in the endometriosis cells, and which can inhibit the proliferation and promote the apoptosis of the endometriosis cells, all of that can provide new theoretical basis for treating endometriosis in the future.