【作者】 郭芳；

【导师】 张桂梅；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2022， 博士

【摘要】 目的:探讨中性粒细胞在早期子宫内膜异位症(Endometriosis,EMs)中的功能,揭示粒细胞集落刺激因子(Granulocyte colony-stimulation factor,G-CSF)和白细胞介素-6(Interleukin-6,IL-6)调节中性粒细胞功能改变的可能机制。方法:(1)建立EMs小鼠模型,募集或清除腹腔中性粒细胞,病理学检测病灶形成及中性粒细胞浸润的过程;检测血清及腹腔液中G-CSF与IL-6的含量。(2)构建G-CSF和/或IL-6、G-CSF受体/IL-6受体/糖蛋白130(G-CSFR/IL-6R/gp130)的真核表达载体,并转染小鼠。(3)建立中性粒细胞和子宫碎片共注射小鼠模型。(4)分析G-CSF/IL-6调节的中性粒细胞对病灶形成、血管变化的作用及阻断该因子对中性粒细胞功能的影响。(5)检测共注射小鼠腹腔中性粒细胞及病灶中与血管生成相关基因的表达。(6)在非异位症状态下小鼠转染p G-CSF和/或p IL-6质粒,通过体内、外实验探讨G-CSF和/或IL-6对中性粒细胞的直接作用。(7)探讨信号传导与转录激活因子3(Signal transducer and activator of transcription,STAT3)、磷脂酰肌醇-3激酶(Phosphatidylinositol 3-kinase,PI3K)信号分子在调节中性粒细胞中基质金属蛋白酶9(Matrix metalloproteinase 9,Mmp9)、Bv8(也称为促动素2,Bombina variegata 8k Da protein,Bv8)和肿瘤坏死因子相关的凋亡诱导配体(Tumor necrosis factor related apoptosis inducing ligand,Trail)表达的作用。结果:(1)建模后第3天粘附碎片形成异位病灶;未粘附碎片第5-6天消失。子宫碎片注入腹腔后12 h,中性粒细胞数量增多,碎片中中性粒细胞也增加。清除中性粒细胞,碎片中该细胞减少。EMs小鼠血浆及腹腔液中G-CSF、IL-6水平升高。(2)共注射小鼠EMs组和G-CSF/IL-6组中性粒细胞促进病灶形成;G-CSFR/IL-6R/gp130组则抑制病灶的形成;(3)共注射小鼠EMs组或G-CSF/IL-6组病灶内血管密度增大,其周围血管增粗,分支增多;去除中性粒细胞,血管密度减小;(4)共注射小鼠G-CSF/IL-6组与EMs组中性粒细胞Mmp9、Bv8 m RNA表达较对照组高,而Trail则低;并且(5)病灶中三基因m RNA和蛋白表达结果相同。(6)对中性粒细胞的直接作用中,单独表达G-CSF或IL-6均不影响Trail m RNA的表达,两者共同表达则降低Trail水平,并更增加Mmp9和Bv8 m RNA水平。此结果与EMs组相似,并得到体外实验证实。(7)当抑制STAT3,中性粒细胞Mmp9和Bv8表达降低,Trail增加。当抑制PI3K,Mmp9和Bv8表达与对照组无差异,但Trail的表达显著降低。而G-CSF在PI3K抑制剂存在下对Trail基因表达的抑制作用可被联合使用STAT3抑制剂后消除,Trail的表达升高。(8)G-CSF/IL-6组中性粒细胞中STAT3和磷酸化STAT3的表达明显高于G-CSF组或IL-6组,与EMs小鼠相比无统计学差异。结论:G-CSF/IL-6通过STAT3途径调节中性粒细胞,上调Mmp9、Bv8和下调Trail基因的表达,转变中性粒细胞的功能,促进小鼠早期异位病灶的形成。更多还原

【Abstract】 Objective To explore the function of neutrophils in early endometriosis(EMs),and to reveal the role of granulocyte colony-stimulation factor(G-CSF)and interleukin-6(IL-6)on modulating neutrophil.Methods(1)To establish EMs mouse model,recruit or remove peritoneal neutrophils,record and observe the process of lesion formation and neutrophil infiltration and detect the level of G-CSF and IL-6 in serum and peritoneal fluid.(2)To establish the eukaryotic expression vector of G-CSF and/or IL-6,G-CSF receptor/IL-6 receptor/glycoprotein 130(G-CSFR/IL-6R/gp130),and transfect mouse.(3)To establish a mouse model by co-injection a mixture of neutrophils and uterine fragments.(4)To analyze the effect of G-CSF/IL-6-regulated neutrophils on lesion formation and vascular changes,and the effect of blocking this factor on neutrophil function.(5)To detect the expression of angiogenesis-related genes in neutrophils and lesions in the peritoneal cavity of co-injected mice.(6)Mice were transfected with p G-CSF and/or p IL-6 plasmids before uterine fragments injection,and the direct effects of G-CSF and/or IL-6 on neutrophils were investigated through in vivo and in vitro experiments.(7)To investigate the role of signal transducer and activator of transcription 3(STAT3)and phosphatidylinositol3-kinase(PI3K)signaling molecules in the regulation expression of matrix metalloproteinase 9(Mmp9),bombina variegata 8k Da protein(Bv8,also known as kinesin2)and Tumor necrosis factor related apoptosis inducing ligand(Trail)in neutrophils.Results(1)Adhesive fragments formed ectopic lesions on the 3rd day after uterine fragments injection;non-adherent fragments disappeared on the 5th to 6th day.Twelve hours after the uterine fragments were injected into the abdominal cavity,the number of neutrophils increased,and the number of neutrophils in the fragments also increased.Once neutrophils were depleted,the cells were reduced in the fragments.The levels of G-CSF and IL-6 in plasma and peritoneal fluid of EMs mice increased.(2)Neutrophils originated from co-injected mouse EMs group and G-CSF/IL-6 group promoted the formation of lesions.On the contrary,neutrophils from G-CSFR/IL-6R/gp130 group inhibited the formation of lesions.(3)The blood vessels in the lesions and the surrounding were dense,thickened and branches increased in the co-injected group of G-CSF/IL-6 mouse or EMs mouse.While neutrophils were removed,and the blood vessel density decreased.The m RNA expression of Mmp9 and Bv8 in neutrophils in the G-CSF/IL-6 group and the EMs group was higher than that in the control group,while the Trail was lower,and(5)the m RNA and protein expressions of the three genes in the lesions were the same.(6)In the direct effect on neutrophils,the expression of G-CSF or IL-6 alone did not affect the expression of Trail m RNA,while the co-expression of the two decreased the level of Trail and increased the levels of Mmp9 and Bv8 m RNA.This result was similar to the EMs group and was confirmed by in vitro experiments.(7)When STAT3 was inhibited,the expression of Mmp9 and Bv8 in neutrophils decreased,and Trail increased.When PI3 K was inhibited,the expression of Mmp9 and Bv8 was no different compared with the control group,but the expression of Trail was significantly reduced.The inhibitory effect of G-CSF on the expression of Trail gene in the presence of PI3 K inhibitor could be eliminated by the combined use of STAT3 inhibitor,and the expression of Trail was increased.(8)The expression of STAT3 and phosphorylated STAT3 in neutrophils of G-CSF/IL-6 group was significantly higher than single one of G-CSF group or IL-6 group,and there was no statistical difference compared with peritoneal neutrophils of EMs mice difference.Conclusions G-CSF/IL-6 regulates neutrophils through the STAT3 pathway,up-regulates the expression of Mmp9,Bv8 and down-regulates Trail genes,transforms the function of neutrophils,and promotes the formation of early ectopic lesions in mice.更多还原