同种异体子宫内膜异位症大鼠模型用于GnRH激动剂类药物的药效评价研究

Application of Allograft Endometriosis Rat Model in Pharmacodynamic Evaluation of GnRH Agonists

目的用同种异体移植的方法构建大鼠子宫内膜异位症模型,并评价促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)激动剂GenSci006对实验大鼠子宫内膜异位症模型的影响。方法取供体SPF级雌性SD大鼠的子宫内膜移植于受体雌性大鼠的腹腔壁上,构建同种异体的子宫内膜异位症模型。3周后,测量异位内膜的长、宽、高,计算给药前异位内膜的体积V1。将进行假手术操作的大鼠设为假手术组,将造模大鼠随机分为模型组、曲普瑞林组(0.25mg/kg)、GenSci006-1组(0.125mg/kg)和GenSci006-2组(0.25mg/kg)共4组,每组大鼠16只。各组大鼠单次给予相应的药物,假手术组和模型组给予同等体积的溶剂。3周后,再次测量异位内膜,计算给药后异位内膜的体积V2和抑制率;通过比较脏器系数及病理切片的变化,判断GenSci006对大鼠子宫和卵巢组织的影响;ELISA法测定血清中雌二醇(estradiol,E_(2))、孕酮(progesterone,P_(4))、卵泡刺激素(follicle stimulating hormone,FSH)和黄体生成素(luteinizinghormone,LH)水平的变化;实时荧光定量PCR法测定下丘脑和垂体中GnRH受体(GnRHreceptor,GnRHR)mRNA的表达水平。蛋白质印迹法检测异位内膜组织中雌激素受体(estrogen receptor,ER)α、ERβ和孕激素受体(progesterone receptor,PR)蛋白的表达。结果给药3周后发现,与模型组相比,曲普瑞林组和GenSci006-2组大鼠的体重显著增加(P<0.05),而异位内膜的体积显著减小(P<0.05)。与假手术组相比,模型组子宫、卵巢脏器系数及子宫内膜厚度无明显变化(P>0.05);与模型组相比,曲普瑞林组和GenSci006-2组子宫脏器系数及子宫内膜厚度显著降低(P<0.05)。与假手术组相比,模型组血清中E_(2)、P_(4)、FSH、LH水平无明显变化(P>0.05);与模型组相比,曲普瑞林组、GenSci006-1组和GenSci006-2组大鼠的卵巢脏器系数和血清P4水平均显著降低(P<0.05),而GenSci006-1组大鼠的血清LH水平明显升高(P<0.05),但各组血清E_(2)和FSH水平并无显著变化(P>0.05)。与模型组相比,曲普瑞林组和GenSci006-2组大鼠垂体GnRHRmRNA表达水平显著下调(P<0.05);各组大鼠下丘脑组织中GnRHRmRNA表达及异位内膜组织中ERα、ERβ、PR蛋白表达水平无明显变化(P>0.05)。结论大鼠同种异体子宫内膜异位症模型适宜作为筛选和评价子宫内膜异位症治疗药物的动物模型,而且异位内膜体积、抑制率、子宫及卵巢脏器系数、血清E_(2)水平均可作为检测药物疗效的指标。

Objective To establish an allogeneic rat model of endometriosis and to evaluate the effects of gonadotropin-releasing hormone(GnRH)agonist GenSci006 on experimental rat endometriosis.Methods Endometrium from SPF grade donor female SD rats were transplanted onto the abdominal wall of recipient female rats to construct an allogeneic endometriosis model.The rats undergoing sham surgery were divided into the sham group.Three weeks later,the length,width and height of the ectopic endometrium were measured,and the volume of the endometrium(V_(1))was calculated before drug administration.The modeling rats were randomly divided into four groups:model group,triptorelin group(0.25 mg/kg),GenSci006-1 group(0.125 mg/kg)and GenSci006-2 group(0.25 mg/kg).Each group had 16 rats and received a single dose of the corresponding drug.The sham group and model group were administered an equal volume of solvent.Three weeks after administration,ectopic endometrium was measured to calculate the volume V_(2) and inhibition rate.The effect of GenSci006 on rat uterus and ovarian tissues was assessed by comparing organ coefficients and changes in pathological sections.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of serum estradiol(E_(2)),progesterone(P_(4)),follicle stimulating hormone(FSH),and luteinizing hormone(LH).Real-time fluorescent quantitative PCR was used to detect the expression of GnRH receptor(GnRHR)mRNA in the hypothalamus and pituitary.Western blot was used to detect the expression of estradiol receptor alpha(ERα),beta(ERβ)and progesterone receptor(PR)in ectopic endometrium.Results Three weeks after administration,compared with the model group,the body weight of rats in the triptorelin and GenSci006-2 groups significantly increased(P<0.05),while the volume of ectopic endometrium significantly decreased(P<0.05).Compared with the sham group,the model group showed no significant changes in uterine and ovarian organ coefficients or endometrial thickness(P>0.05).Compared with the model group,the uterine organ coefficients and endometrial thickness were significantly reduced in the triptorelin and GenSci006-2 groups(P<0.05).Compared with the sham group,the serum levels of E_(2),P_(4),FSH and LH in the model group showed no significant changes(P>0.05).Compared with the model group,the ovarian organ coefficient and serum P_(4) levels of rats in the Triptorelin,GenSci006-1,and GenSci006-2 groups were significantly reduced(P<0.05),while the serum LH levels of rats in the GenSci006-1 group were significantly increased(P<0.05).However,there were no significant changes in serum E_(2) and FSH levels in each group(P>0.05).Compared with the model group,the expression levels of GnRHR mRNA in the pituitary tissue of rats in the triptorelin and GenSci006-2 groups were significantly downregulated(P<0.05),with no significantly changes in the hypothalamus(P>0.05).There were no significant changes in the expression level of GnRHR mRNA in the hypothalamus or the protein levels of ERα,ERβand PR in the ectopic endometrial tissue in any group(P>0.05).Conclusion The allogeneic endometriosis rat model is a suitable animal model for screening and evaluating drugs for treating endometriosis.The volume of ectopic endometrium,inhibition rate,uterine and ovarian organ coefficients,and serum E_(2) levels may serve as indicators for detecting drug efficacy.