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放射治疗(简称放疗)是恶性肿瘤重要的治疗手段,卵巢对放射线高度敏感,较低的剂量即可能导致卵巢早衰[1-2]。如何消除或减轻放疗对卵巢功能损伤的研究具有重要临床意义。相关研究及我们前期研究结果已证明,在放化疗前给予促性腺激素释放激素激动剂(gonadotropin releasing hormone agonist, GnRH-a)能够减少其对卵巢功能的损害[3-4]。但由于GnRH-a存在“点火效应”,因此在GnRH-a应用后约2周才可以进行放化疗,否则会使卵巢受到更严重的损害,这一缺点易导致治疗时机的延误,造成不良结果[5-6]。促性腺激素释放激素拮抗剂(gonadotropin releasing hormone antagonist,GnRH-ant)是促性腺激素释放激素(gonadotropin releasing hormone,GnRH)类似物家族中的拮抗剂,相比激动剂,它可快速抑制性腺轴和卵巢,且无明显“点火效应”[7]。但对于其能否对放疗中的卵巢起保护作用,目前国内外相关研究甚少。本研究拟采用大鼠盆腔照射模型,通过在放疗前应用GnRH-ant抑制卵巢功能,观察其对放疗所致卵巢功能损伤的保护作用,并对其防护作用机制展开研究。
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由图 1可见,GnRH-a组在给药后LH水平先表现为一过性升高,6 d左右达最高峰[(37.70±7.50)IU/L],在第11天左右达到最大抑制状态[(0.37±1.12)IU/L];GnRH-ant组在给药后血清LH水平迅速下降,4 d左右即可达到最大抑制状态[(0.46±1.37)IU/L],停药后5 d(1个动情周期)左右,LH即可恢复治疗前水平[(15.20±4.26)IU/L],上述两组的血清LH水平变化有明显不同。
图 1 GnRH-a组与GnRH-ant组给药后大鼠血清LH水平的变化
Figure 1. Dynamic change of serum luteinizing hormone(LH) of the rats after administration of gonadotropin releasing hormone agonist(GnRH-a) and gonadotropin releasing hormone antagonist (GnRH-ant) group
血清E2水平的变化滞后于LH的变化。由图 2可见,GnRH-ant组血清E2水平在5 d左右达最大抑制状态[(0.40±0.52)pg/mL],停药后5 d(1个动情周期)E2水平恢复到治疗前水平[(46.80±6.71)pg/mL];GnRH-a组在给药后E2的水平表现为一过性升高,7 d左右达最大值[(146.00±23.70)pg/mL],12 d左右达到最大抑制状态[(0.60±0.73)pg/mL]。GnRH-a组和GnRH-ant组的E2水平变化与LH的变化趋势相同。
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经数据分析结果显示,注射醋酸西曲瑞克后大鼠卵巢功能迅速受到抑制,4 d前后即可达到最大抑制水平。因此,将大鼠卵巢照射时间确定在GnRH-ant组给药后第4天,并在照射后继续连续给药6 d。
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解剖各组大鼠,肉眼可见放疗组大鼠卵巢明显萎缩,表面呈暗红色且布满微小出血点,质地较硬;GnRH-ant+放疗组卵巢表面呈淡粉红色且无明显皱缩,质地柔软。称取各组大鼠双侧卵巢湿重,对照组[(64.3±13.5)mg]、GnRH-ant组[(59.7±11.2)mg]和GnRH-ant+放疗组[(58.3±9.1)mg]比较,差异均无统计学意义(t=-0.31~-1.16,均P>0.05);GnRH-ant+放疗组与放疗组[(37.8±7.1)mg]比较,卵巢湿重较高且差异有统计学意义(t=5.61,P=0.000);GnRH-ant组与对照组比较,在卵巢外观、形态、质地等方面无明显差异,GnRH-ant组卵巢湿重略小于对照组,但差异无统计学意义(t= 0.829,P=0.418)。
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在GnRH-ant组停药后第15天,测量各组大鼠血清FSH、E2和AMH水平,结果见表 1。放疗组在照射后卵巢功能受损,与对照组比较,血清E2的水平降低、FSH水平呈反馈性升高;GnRH-ant+放疗组的激素水平较对照组也表现为卵巢功能受损,即E2的水平降低和FSH的升高,但与放疗组相比,其E2水平较高、FSH水平较低,且差异均有统计学意义(t=8.64、7.75,均P=0.000)。除了E2及FSH这两个易受月经周期影响的指标外,在目前认为反映卵巢储备功能较稳定的指标AMH的测定中,GnRH-ant+放疗组与放疗组比较,AMH水平显著较高且差异有统计学意义(t=6.81,P=0.000)。
组别 FSH/(mIU/mL) E2/(pg/mL) AMH/(mIU/mL) 对照组 8.12±1.23 73.55±15.78 7.72±4.B3 GnRH-ant组 9.24±1.37 69.63±4.81 8.34±3.43 放疗组 27.74±7.75 16.64±6.54 2.23±B.72 GnRH-ant+放疗组 B8.35±1.43a 57.49±13.45a 5.47±1.32a 注:表中,a:与放疗组比较,差异均有统计学意义(t=7.75、8.64、6.81,均P<0.01)。FSH:卵泡刺激素;E2:雌二醇;AMH:抗缪靳管激素;GnRH-ant:促性腺激素释放激素拮抗剂。 表 1 醋酸西曲瑞克治疗后4组大鼠血清FSH、E2和AMH的水平(x ± s)
Table 1. Levels of serum estradiol(E2), follice-stimulating hormone(FSH) and auti-mullerian hormone(AMH) in 4 groups after cetrorelix acetate treatment(x ± s)
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通过统计分析,我们发现GnRH-ant+放疗组与放疗组相比,生长卵泡和成熟卵泡的数量差异均无统计学意义(t=0.67~0.88,P>0.05);原始、初级卵泡的数量较高,且差异有统计学意义(t=4.42,P=0.000)。具体数据见表 2。
分组 原始、初级卵泡 生长卵泡 成熟卵泡 对照组 73.7±7.6 41.5±5.2 12.6±43.2 GnRH-ant组 67.4±11.4 24.3±4.1 9.7±2.6 放疗组 27.6±5.1 7.5±1.9 3.9±1.3 GnRH-ant+放疗组 46.2±12.3a 6.8±2.7 4.5±1.7 注:表中,a:与放疗组比较,差异有统计学意义(t=4.42,P=0.000)。GnRH-ant:促性腺激素释放激素拮抗剂。 表 2 4组大鼠各型卵泡的计数比较(x ± s)
Table 2. Comparison of various types of follicular counts in rats of 4 groups (x ± s)
促性腺激素释放激素拮抗剂对盆腔放疗所致卵巢功能损伤的防护作用
Protective effect of gonadotropin releasing hormone antagonist on ovarian function injury induced by pelvic radiotherapy
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摘要:
目的 研究促性腺激素释放激素拮抗剂(GnRH-ant)对盆腔放疗所致大鼠卵巢功能损伤的防护作用及潜在保护机制。 方法 1.雌性Wistar大鼠10只,采用随机数字表法分为促性腺激素释放激素激动剂(GnRH-a)组与GnRH-ant组,分别皮下注射醋酸戈舍瑞林0.25 mg(1次)、醋酸西曲瑞克5 μg(每天,连续10 d),动态观察两组大鼠血清黄体生成素(LH)和雌二醇(E2)水平的变化。2.雌性Wistar大鼠40只,采用随机数字表法分为对照组、GnRH-ant组、放疗组、GnRH-ant+放疗组,分别给药及盆腔60Co照射200 cGy后,检测卵巢湿重与血清卵泡刺激素(FSH)、E2和抗缪勒管激素(AMH)的水平;分类计数卵巢内各级卵泡总数,评价卵巢功能的保护情况。两组间比较采用独立样本t检验,多组间比较采用最小显著性差异法。 结果 1.GnRH-a组的LH、E2水平短暂升高后逐渐下降,10 d左右达最低值;GnRH-ant组的LH、E2水平迅速下降,4 d左右达最低值,且无“点火效应”。2.与放疗组比较,GnRH-ant+放疗组的卵巢湿重更高[(37.8±7.1)mg vs.(58.3±9.1)mg,t=5.61,P=0.000]、E2水平较高[(16.64±6.54)pg/mL vs.(57.49±13.45)pg/mL,t=8.64,P=0.000]、FSH水平较低[(27.74±7.75)mIU/mL vs.(8.35±1.43)mIU/mL,t=7.75,P=0.000]、AMH水平较高[(2.23±0.72)mIU/mL vs.(5.47±1.32)mIU/mL,t=6.81,P=0.000]、原始及初级卵泡数明显更高(27.6±5.1 vs.46.2±12.3,t=4.42,P=0.000),且差异均有统计学意义。 结论 GnRH-ant能迅速有效地抑制大鼠卵巢功能的损伤,且无给药后“点火效应”。放疗前给予GnRH-ant,会将大鼠卵巢内的卵泡发育停滞于原始及初级卵泡阶段,减轻放疗辐射对卵巢功能的损伤。 -
关键词:
- 辐射耐受性 /
- 放射疗法 /
- 卵巢 /
- 辐射防护 /
- 促性腺激素释放激素拮抗剂
Abstract:Objectives To investigate the protective effect and mechanism of a gonadotropin-releasing hormone antagonist (GnRH-ant) against an ovarian function injury induced by pelvic radiotherapy in a rat model. Method 1. Ten female Wistar rats were randomly assigned to gonadotropin-releasing hormone agonist (GnRH-a) and GnRH-ant groups using the random number table method. The rats were subcutaneously injected with goserelin (0.25 mg once) or cetrorelix (5 μg/day for 10 days). Changes in luteinizing hormone (LH) and estradiol (E2) in each group were dynamically observed. 2. Forty female Wistar rats were randomly divided into four groups (control, GnRH-ant, R, and GnRH-ant+R) and then given corresponding treatments. Ovarian wet weight, levels of serum LH, E2, and AMH, and the number of follicles at every stage were compared between groups through analysis of variance and independent sample t-test. Results1. In the GnRH-a group, LH and E2 levels increased initially and then gradually decreased, reaching a low value in approximately 10 d. In the GnRH-ant group, LH and E2 levels decreased rapidly, reaching the minimum value in 4 d without flare-up effect. 2. After pelvic radiotherapy, the ovarian wet weight in the GnRH-ant+R group was significantly higher[(58.3±9.1) mg vs. (37.8±7.1) mg, t=5.61, P=0.000] than that in group R. In the GnRH-ant+R group, the levels of E2[(57.49±13.45) pg/mL vs. (16.64±6.54) pg/mL, t=8.64, P=0.000] and AMH[(5.47±1.32) mIU/mL vs. (2.23±0.72) mIU/mL, t=6.81, P=0.000] were significantly higher than those in group R. The FSH level in GnRH-ant+R group was significantly lower[(27.74±7.75) mIU/mL vs. (8.35±1.43) mIU/mL, t=7.75, P=0.000] than that in group R. The number of primordial and primary follicles in group GnRH-ant+R was significantly higher (46.2±12.3 vs. 27.6±5.1, t=4.42, P=0.000) than that in group R. Conclusions GnRH-ant can rapidly induce ovarian inhibition without flare-up effect. Subcutaneous injection of GnRH-ant before pelvic radiotherapy can inhibit the ovary and stop the follicles in the primary and primordial follicle stages, thus reducing the damage induced by radiotherapy. -
表 1 醋酸西曲瑞克治疗后4组大鼠血清FSH、E2和AMH的水平(x ± s)
Table 1. Levels of serum estradiol(E2), follice-stimulating hormone(FSH) and auti-mullerian hormone(AMH) in 4 groups after cetrorelix acetate treatment(x ± s)
组别 FSH/(mIU/mL) E2/(pg/mL) AMH/(mIU/mL) 对照组 8.12±1.23 73.55±15.78 7.72±4.B3 GnRH-ant组 9.24±1.37 69.63±4.81 8.34±3.43 放疗组 27.74±7.75 16.64±6.54 2.23±B.72 GnRH-ant+放疗组 B8.35±1.43a 57.49±13.45a 5.47±1.32a 注:表中,a:与放疗组比较,差异均有统计学意义(t=7.75、8.64、6.81,均P<0.01)。FSH:卵泡刺激素;E2:雌二醇;AMH:抗缪靳管激素;GnRH-ant:促性腺激素释放激素拮抗剂。 表 2 4组大鼠各型卵泡的计数比较(x ± s)
Table 2. Comparison of various types of follicular counts in rats of 4 groups (x ± s)
分组 原始、初级卵泡 生长卵泡 成熟卵泡 对照组 73.7±7.6 41.5±5.2 12.6±43.2 GnRH-ant组 67.4±11.4 24.3±4.1 9.7±2.6 放疗组 27.6±5.1 7.5±1.9 3.9±1.3 GnRH-ant+放疗组 46.2±12.3a 6.8±2.7 4.5±1.7 注:表中,a:与放疗组比较,差异有统计学意义(t=4.42,P=0.000)。GnRH-ant:促性腺激素释放激素拮抗剂。 -
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