雌激素对去势小鼠子宫内膜NOTCH1基因表达的影响

发布时间：2018-09-04 05:42

【摘要】：目的：建立一个去双侧卵巢完整保留子宫的小鼠模拟绝经期雌激素缺少模型，探讨外源性高剂量β-E2持续刺激下，小鼠子宫内膜组织中NOTCH1基因变化的情况，从病因学上推测E2和NOTCH1信号通路的关系。方法：（1）未孕成熟雌鼠(25-30g)随机分为四个实验组，每组10只，一组（正常对照组）：不做任何处理；二组（假手术组）：只切除卵巢组织旁边的脂肪，而完整保留子宫和卵巢，术后腹腔注射0.9%NaCl；三组（去卵巢组）：1.5%戊巴比妥钠麻醉下手术去除双侧卵巢完整保留子宫，，术后腹腔注射0.9%NaCl溶液；四组（去卵巢加β-E2组）：切除卵巢完整保留子宫，术后腹腔注射1mg/kg β-E2；每组隔天注射一次，持续10天；（2）对手术切下的组织标本进10%福尔马林固定，常规脱水，透明，浸蜡，包埋制成蜡块，石蜡连续切片，进行HE染色。在光学显微镜下进行观察拍照，以确定所切组织标本是否为小鼠的卵巢；（3）术前、术后分别抽取尾静脉血液0.3ml，分离出血清冻存于-20℃冰箱备用。药物干预10天后，无菌条件下取出子宫，称取子宫湿重计算子宫系数，并分离子宫内膜，样本保存液浸没于EP管中，-20℃冰箱保存备用。ELISA检测各组小鼠前后体内血清E2的变化水平及各组小鼠药物干预后体内E2的水平。（4）采用TRIZOL一步法提取组织总RNA，使用特异性引物对目的基因NOTCH1和内参基因GAPDH进行RT-PCR扩增，然后产物进行1.5%琼脂糖凝胶电泳，运用FR-200A全自动紫外与可见分析装置观察电泳结果并拍照，采用QuantityOne-4.6.2凝胶分析软件对NOTCH1基因与GAPDH基因的目标带进行半定量分析，测定Normal组、Sham+NS组、OVX+NS组和OVX+β-E2组NOTCH1基因与GAPDH基因目标带的光强度(Adj.Vol.)值。结果：（1）HE染色石蜡切片结果显示去卵巢手术造模所切下的组织标本确定为卵巢组织。（2）四个实验组小鼠前后血清E2水平分别为59.57±31.70和70.35±22.38，t=0.39，P=0.70；56.62±24.36和67.90±32.04，t=1.96，P=0.10；60.51±39.39和28.73±5.58，t=2.41，P=0.04；61.21±27.55和36.62±7.73，t=2.90，P=0.02；Control组和Sham+NS组前后差异无统计学意义（P0.05），OVX+NS组和OVX+β-E2组造模前后差异有统计学意义（P0.05）。（3）高剂量的β-E2的刺激下，四组间的统计量F=48.21，OVX+β-E2组的雌激素水平显著高于Control组，Sham+NS组和OVX+NS组（P=0.00，P=0.00，P=0.00），差异有统计学意义（P0.05）。OVX+NS组雌激素水平显著低于Control组和Sham+NS组（P=0.01，P=0.01），差异有统计学意义（P0.05），Control组与Sham+NS组比较，P=0.87，差异无统计学意义（P0.05）。（4）在高剂量β-E2的刺激下，OVX+β-E2组的子宫系数（%）显高于其它各组（P0.05）。各组间的统计量F=9.23，OVX+β-E2组和Control组比较，P=0.00；和Sham+NS组比较，P=0.00；和OVX+NS组比较，P=0.00；（5）所有标本均扩增出目的基因NOTCH1和内参基因GAPDH的目标条带，片段大小分别为247bp和398bp。8例Control组的OD值为0.45±0.22，7例Sham+NS组的OD值为0.24±0.12，9例OVX+NS组的OD值为0.27±0.08，8例OVX+β-E2组的OD值为0.16±0.06。各组间的检验统计量F=7.26，OVX+β-E2组NOTCH1的表达水平显著低于Control组、OVX组，P=0.03，P=0.01，差异具有统计学意义（P0.05）。结论：(1)ELISA可用于检测去卵巢前后小鼠体内E2水平的变化，去卵巢后的小鼠血清E2的变化水平显著低于去卵巢前，结合所切组织标本为卵巢组织确定去卵巢造模成功。（2）外源性β-E2刺激可促使子宫肌肉细胞肥大，子宫增重。（3）OVX+β-E2组NOTCH1基因的表达水平显著低于Control组、OVX组，这可能暗示着在子宫内膜细胞中高剂量的β-E2抑制NOTCH1基因的表达活性， NOTCH1基因的活性和雌激素之间的相互作用可能在子宫内膜疾病的发展机制中发挥了重要作用。
[Abstract]:AIM: To establish a model of estrogen deficiency in postmenopausal mice with bilateral ovariectomy and intact uterus preservation. To investigate the changes of NOTCH1 gene in endometrium of mice stimulated by exogenous high dose of beta-E2, and to speculate the relationship between E2 and NOTCH1 signaling pathway.
Methods: (1) Immature female rats (25-30g) were randomly divided into four experimental groups, 10 rats in each group and one group (normal control group): without any treatment; two groups (sham operation group): only the fat beside the ovarian tissue was removed, while the uterus and ovary were preserved intact, intraperitoneal injection of 0.9% NaCl after operation; three groups (ovariectomized group): 1.5% pentobarbital sodium anesthesia. Removal of bilateral ovaries and preservation of uterus, intraperitoneal injection of 0.9% NaCl solution after surgery; four groups (ovariectomy plus beta-E2 group): ovariectomy complete preservation of uterus, intraperitoneal injection of 1 mg/kg beta-E2 after surgery; every other day injection, lasting 10 days; (2) tissue samples under the operation into 10% formalin fixed, routine dehydration, transparent, wax immersion, package. Embedded into wax block, paraffin continuous section, HE staining. Observed and photographed under the optical microscope to determine whether the tissue samples were ovaries of mice; (3) Preoperative, postoperative blood from caudal vein were extracted 0.3 ml, bleeding was separated and frozen in - 20 C refrigerator for reserve. The uterine coefficients were calculated by wet weight, and the samples were immersed in EP tube. The samples were stored in refrigerator at - 20 C for reserve. The internal reference gene GAPDH was amplified by RT-PCR, and then the product was electrophoretized by 1.5% agarose gel. The electrophoresis results were observed and photographed by using FR-200A automatic ultraviolet and visible analyzer. The target bands of NOTCH1 gene and GAPDH gene were semi-quantitatively analyzed by Quantity One-4.6.2 gel analysis software. Normal group, Sham+NS group, OVX+NS group were determined. The light intensity (Adj.Vol.) value of NOTCH1 gene and GAPDH gene target band in OVX+ and -E2 group.
Results: (1) HE staining and paraffin section showed that the tissue samples were ovarian tissue. (2) The serum E2 levels of mice in the four experimental groups were 59.57 (+ 31.70) and 70.35 (+ 22.38), t = 0.39, P = 0.70, 56.62 (+ 24.36) and 67.90 (+ 32.04), t = 1.96, P = 0.10, 60.51 (+ 39.39) and 28.73 (+ 5.58), t = 2.41, P = 0.04, respectively. There was no significant difference between the control group and the Sham+NS group (P 0.05). There was significant difference between the OVX+NS group and the OVX+beta-E2 group (P 0.05). (3) Under the stimulation of high dose of beta-E2, the statistical value F=48.21 between the four groups, the estrogen level of the OVX+beta-E2 group was significantly higher than that of the control group, the Sham+NS group and the OVX+beta-E2 group. The estrogen level of OVX + NS group was significantly lower than that of control group and Sham + NS group (P = 0.01, P = 0.01), and the difference was statistically significant (P = 0.05). Compared with Sham + NS group, the uterine coefficient of OVX + beta - E2 group was significantly lower (P = 0.87, P = 0.05). The statistical value of F = 9.23, P = 0.00 in OVX + beta-E2 group and Control group, P = 0.00 in Sham + NS group, P = 0.00 in OVX + NS group, P = 0.00 in comparison with OVX + NS group, and 0.00 in OVX + NS group. (5) Target bands of NOTCH1 and GAPDH were amplified from all specimens, with OD values of 247 BP and 398 bp, respectively. The OD value of 7 cases of Sham+NS group was 0.24+0.12,9 cases of OVX+NS group was 0.27+0.08,8 cases of OVX+beta-E2 group was 0.16+0.06.
Conclusion: (1) ELISA can be used to detect the level of E2 in mice before and after ovariectomy. The level of E2 in serum of ovariectomized mice was significantly lower than that before ovariectomy. Combined with the ovariectomized tissues, the ovariectomized mice were successfully established. (2) Exogenous beta-E2 stimulation can promote the hypertrophy of uterine muscle cells and the weight of uterus. (3) OVX + beta-E2 group N The expression level of OTCH1 gene was significantly lower than that of control and OVX groups, which may indicate that high dose of beta-E2 inhibits the expression of NOTCH1 gene in endometrial cells. The interaction between NOTCH1 gene activity and estrogen may play an important role in the development of endometrial diseases.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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