补肾活血方对子宫内膜干细胞移植治疗薄型子宫内膜大鼠子宫内膜形态学的影响

Effect of Bushen Huoxue Decoction on endometrial morphology of thin endometrial rats treated with endometrial stem cell transplantation

目的探讨补肾活血方对子宫内膜干细胞(EDSCs)移植治疗薄型子宫内膜模型大鼠子宫内膜形态学的影响。方法从8周龄雌性SD大鼠子宫内膜中分离提取EDSCs,采用机械分离与两步酶消法,实验选用纯度较高的第3代细胞,分别采用尾静脉(IV)和宫腔注射(IU)的方法移植到实验大鼠体内。采用宫腔灌注95%无水乙醇及灌胃羟基脲加尾静脉注射高分子右旋糖酐方法建立肾虚血瘀薄型子宫内膜联合模型。将实验大鼠分为正常组、模型组、戊酸雌二醇组、EDSCs+IV组、EDSCs+IU组、EDSCs+IV+中药组、EDSCs+IU+中药组,每组8只。各组大鼠给予相应的方法干预后,经过3个动情周期处死后取材。采用HE染色观察各组子宫内膜厚度、腺体和血管分布情况,免疫组化、Western blot检测角蛋白、波形蛋白的表达情况。结果在改善子宫内膜厚度方面,EDSCs+IU组、EDSCs+IV+中药组及EDSCs+IU+中药组较模型组内膜厚度均增加,差异有统计学意义(P<0.01);在增加子宫内膜腺体数方面,EDSCs+IV组较模型组腺体数增加,差异有统计学意义(P<0.05),EDSCs+IU组、EDSCs+IV+中药组、EDSCs+IU+中药组较模型组腺体数增加,差异有统计学意义(P<0.01);在增加子宫内膜血管数方面,EDSCs+IV组、EDSCs+IU组、EDSCs+IV+中药组、EDSCs+IU+中药组较模型组血管数增加,差异有统计学意义(P<0.01);在改善角蛋白表达方面,EDSCs+IV组、EDSCs+IU组、EDSCs+IV+中药组、EDSCs+IU+中药组较模型组表达量升高,差异有统计学意义(P<0.01);在改善波形蛋白表达方面,EDSCs+IV组、EDSCs+IU组、EDSCs+IV+中药组、EDSCs+IU+中药组较模型组表达量升高,差异有统计学意义(P<0.01);在改善子宫内膜厚度、免疫组化检测波形蛋白表达方面宫腔注射EDSCs与尾静脉注射EDSCs比较,差异有统计学意义(P<0.01);EDSCs+IV+中药组与EDSCs+IV组比较、EDSCs+IU+中药组与EDSCs+IU组比较子宫内膜厚度显著增加,差异有统计学意义(P<0.01)。结论宫腔注射EDSCs在增加子宫内膜厚度、免疫组化检测波形蛋白表达方面更优于尾静脉注射EDSCs;加用补肾活血方后有助于增加子宫内膜厚度。

Objective To investigate the effect of Bushen Huoxue Decoction on endometrial morphology in rats with thin endometrium treated by transplantation of endometrial stem cells(EDSCs).Methods EDSCs were isolated from the endometrium of 8-week-old female SD rats by the methods of mechanical separation and two-step enzyme elimination.The third generation cells with high purity were selected and transplanted into the experimental rats by tail vein and intrauterine injection respectively.The combined model of thin endometrium with kidney deficiency and blood stasis was established by infusing 95%ethanol without water into the uterine cavity and injecting hydroxyurea and high molecular dextran into the tail vein of female SD rats.The experimental rats were divided into normal control group,model group,estradiol valerate group,EDSCs+IV group,EDSCs+IU group,EDSCs+IV+traditional Chinese medicine(TCM)group and EDSCs+IU+TCM group,with 8 rats in each group.After intervention with corresponding methods,the rats in each group were killed after 3 estrous cycles.HE staining was used to observe the thickness of uterus,the number of glands and blood vessels,and the expressions of keratin and vimentin by immunohistochemistry and Western blot.Results In terms of improving endometrial thickness,the endometrial thickness in the EDSCs+IU group,the EDSCs+IV+TCM group and the EDSCs+IU+TCM group was significantly higher than that in the model group(P<0.01);in terms of increasing the number of endometrial glands,the number of glands in the EDSCs+IV group was significantly higher than that in the model group(P<0.05),and that in the EDSCs+IU group,the EDSCs+IV+TCM group and the EDSCs+IU+TCM group was significantly higher than that in the model group(P<0.01);in terms of increasing the number of endometrial blood vessels,the number of blood vessels in the EDSCs+IV group,the EDSCs+IU group,the EDSCs+IV+TCM group and the EDSCs+IU+TCM group was significantly higher than that in the model group(P<0.01);in terms of improving the expression of keratin,the expression of keratin in the EDSCs+IV group,the EDSCs+IU group,the EDSCs+IV+TCM group and the EDSCs+IU+TCM group was significantly higher than that in the model group(P<0.01);in terms of improving the expression of vimentin,the expression of vimentin in the EDSCs+IV group,the EDSCs+IU group,the EDSCs+IV+TCM group and the EDSCs+IU+TCM group was significantly higher than that in the model group(P<0.01).There was significant difference between intrauterine injection of EDSCs and tail vein injection of EDSCs in improving the thickness of endometrium and the expression of vimentin detected by immunohistochemistry(P<0.01).The endometrial thickness in the EDSCs+IV+TCM group was significantly higher than that in the EDSCs+IV group,and that in the EDSCs+IU+TCM group was significantly higher than that in the EDSCs+IU group(P<0.01).Conclusion Intrauterine injection of EDSCs is better than tail vein injection in increasing endometrial thickness and vimentin expression detected by immunohistochemistryl,and adding Bushen Huoxue Decoction helps to increase the thickness of endometrium.