雌激素對去勢小鼠子宮內膜NOTCH1基因表達的影響
發(fā)布時間：2018-09-04 05:42
【摘要】：目的：建立一個去雙側卵巢完整保留子宮的小鼠模擬絕經期雌激素缺少模型，探討外源性高劑量β-E2持續(xù)刺激下，小鼠子宮內膜組織中NOTCH1基因變化的情況，從病因學上推測E2和NOTCH1信號通路的關系。 方法：（1）未孕成熟雌鼠(25-30g)隨機分為四個實驗組，每組10只，一組（正常對照組）：不做任何處理；二組（假手術組）：只切除卵巢組織旁邊的脂肪，而完整保留子宮和卵巢，術后腹腔注射0.9%NaCl；三組（去卵巢組）：1.5%戊巴比妥鈉麻醉下手術去除雙側卵巢完整保留子宮，，術后腹腔注射0.9%NaCl溶液；四組（去卵巢加β-E2組）：切除卵巢完整保留子宮，術后腹腔注射1mg/kg β-E2；每組隔天注射一次，持續(xù)10天；（2）對手術切下的組織標本進10%福爾馬林固定，常規(guī)脫水，透明，浸蠟，包埋制成蠟塊，石蠟連續(xù)切片，進行HE染色。在光學顯微鏡下進行觀察拍照，以確定所切組織標本是否為小鼠的卵巢；（3）術前、術后分別抽取尾靜脈血液0.3ml，分離出血清凍存于-20℃冰箱備用。藥物干預10天后，無菌條件下取出子宮，稱取子宮濕重計算子宮系數(shù)，并分離子宮內膜，樣本保存液浸沒于EP管中，-20℃冰箱保存?zhèn)溆�。ELISA檢測各組小鼠前后體內血清E2的變化水平及各組小鼠藥物干預后體內E2的水平。（4）采用TRIZOL一步法提取組織總RNA，使用特異性引物對目的基因NOTCH1和內參基因GAPDH進行RT-PCR擴增，然后產物進行1.5%瓊脂糖凝膠電泳，運用FR-200A全自動紫外與可見分析裝置觀察電泳結果并拍照，采用QuantityOne-4.6.2凝膠分析軟件對NOTCH1基因與GAPDH基因的目標帶進行半定量分析，測定Normal組、Sham+NS組、OVX+NS組和OVX+β-E2組NOTCH1基因與GAPDH基因目標帶的光強度(Adj.Vol.)值。 結果：（1）HE染色石蠟切片結果顯示去卵巢手術造模所切下的組織標本確定為卵巢組織。（2）四個實驗組小鼠前后血清E2水平分別為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組和Sham+NS組前后差異無統(tǒng)計學意義（P0.05），OVX+NS組和OVX+β-E2組造模前后差異有統(tǒng)計學意義（P0.05）。（3）高劑量的β-E2的刺激下，四組間的統(tǒng)計量F=48.21，OVX+β-E2組的雌激素水平顯著高于Control組，Sham+NS組和OVX+NS組（P=0.00，P=0.00，P=0.00），差異有統(tǒng)計學意義（P0.05）。OVX+NS組雌激素水平顯著低于Control組和Sham+NS組（P=0.01，P=0.01），差異有統(tǒng)計學意義（P0.05），Control組與Sham+NS組比較，P=0.87，差異無統(tǒng)計學意義（P0.05）。（4）在高劑量β-E2的刺激下，OVX+β-E2組的子宮系數(shù)（%）顯高于其它各組（P0.05）。各組間的統(tǒng)計量F=9.23，OVX+β-E2組和Control組比較，P=0.00；和Sham+NS組比較，P=0.00；和OVX+NS組比較，P=0.00；（5）所有標本均擴增出目的基因NOTCH1和內參基因GAPDH的目標條帶，片段大小分別為247bp和398bp。8例Control組的OD值為0.45±0.22，7例Sham+NS組的OD值為0.24±0.12，9例OVX+NS組的OD值為0.27±0.08，8例OVX+β-E2組的OD值為0.16±0.06。各組間的檢驗統(tǒng)計量F=7.26，OVX+β-E2組NOTCH1的表達水平顯著低于Control組、OVX組，P=0.03，P=0.01，差異具有統(tǒng)計學意義（P0.05）。 結論：(1)ELISA可用于檢測去卵巢前后小鼠體內E2水平的變化，去卵巢后的小鼠血清E2的變化水平顯著低于去卵巢前，結合所切組織標本為卵巢組織確定去卵巢造模成功。（2）外源性β-E2刺激可促使子宮肌肉細胞肥大，子宮增重。（3）OVX+β-E2組NOTCH1基因的表達水平顯著低于Control組、OVX組，這可能暗示著在子宮內膜細胞中高劑量的β-E2抑制NOTCH1基因的表達活性， NOTCH1基因的活性和雌激素之間的相互作用可能在子宮內膜疾病的發(fā)展機制中發(fā)揮了重要作用。
[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.
【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
[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.
【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965
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