左归丸联合脐带间充质干细胞修复受损子宫内膜的研究

发布时间：2018-07-04 20:33

本文选题：薄型子宫内膜 + PI3K　；参考：《湖北中医药大学》2017年硕士论文

【摘要】：第一部分薄型子宫内膜PI3K、AKT m RNA的表达目的观察薄型子宫内膜组织中磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt/PKB)m RNA含量的表达情况,探讨PI3K/AKT信号通路在薄型子宫内膜中的作用。方法通过收集正常子宫内膜30例及薄型子宫内膜30例,正常子宫内膜及薄型子宫内膜的西医诊断标准参照文献,宫腔镜手术术中收集子宫内膜组织-70℃保存备用。结果PI3K、AKT m RNA在薄型子宫内膜组的表达明显低于正常子宫内膜组,两组差异有统计学意义(P0.05)。结论PI3K/AKT信号通路存在于正常和薄型子宫内膜组织中,且在薄型子宫内膜增殖期PI3K、AKT表达下降,推测PI3K/AKT信号通路可能参与子宫内膜组织的增殖、分化。第二部分左归丸联合脐带间充质干细胞修复受损子宫内膜的实验研究目的左归丸是明代医家张景岳的滋阴补肾代表方,该方有滋阴补肾、填精益髓等功效,能调节下丘脑-垂体-靶腺轴功能紊乱,临床上广泛用于妇科疾病、骨骼疾病、神经系统疾病等的治疗。脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs),是成体干细胞的新生代表,具有强大的自我增殖能力和多向分化潜能,且来源广泛、不涉及伦理问题,可成为治疗难治性子宫内膜损伤疾病的种子细胞。研究证明PI3K/AKT信号通路在细胞的增殖、分化、凋亡、迁移等多种生物学功能的调节过程中,起着关键的信号分子作用,PI3K/AKT信号通路在子宫内膜癌、子宫内膜异位症中表达活跃,可能参与介导了子宫内膜的增长、修复过程。本实验通过构建大鼠的子宫内膜损伤模型,探讨中药左归丸及人脐带间充质干细胞对受损子宫内膜的修复作用。研究左归丸及人脐带间充质干细胞修复受损子宫内膜的可能作用机制,为临床上治疗子宫内膜受损疾病提供理论依据。方法目前尚无子宫内膜受损模型的统一建模标准,故参照已有文献,采用100℃热水烫伤子宫内膜方法建立SD大鼠子宫内膜受损模型,根据摸索实验结果,确定子宫内膜受损模型最佳热水烫伤时间。所有造模组均烫伤SD大鼠Y型子宫左侧部分,共分5组,每组6只大鼠。A组:假手术组,即暴露Y型子宫后,用头皮针导管穿过左侧子宫,后拔掉导管,不用流动的热水经过宫腔;B组:模型组,在大鼠造模完成后常规饲养,不做任何处理;C组:药物组,造模完成后第3天开始左归丸溶液灌胃,按1.62g/Kg的剂量每日灌胃一次,连续灌胃28天;D组:宫腔注射组,造模完成后第三天再开腹,经宫腔原位注射5×107个人脐带间充质干细胞;E组:联合组,造模完成后第三天经宫腔原位注射5×107个人脐带间充质干细胞,并于同一天开始左归丸水溶液灌胃,按1.62g/Kg剂量每日一次,连续灌胃28天;治疗28天后处死各组大鼠,取出每只SD大鼠Y型子宫左侧造模部分,弃去靠近卵巢及阴道部分,剪成2段,一段子宫内膜组织作HE染色、天狼猩红染色及免疫荧光检测,另一部分子宫内膜组织作Western Blot检测PI3K、AKT、p-AKT蛋白表达情况。结果1.HE染色结果:与模型组比较,中药组、宫腔注射组、联合组子宫内膜厚度均有不同程度增厚、腺体个数不同程度增加,差异有统计学意义,其中宫腔注射组与联合组的内膜厚度及腺体个数增加最显著(P0.05),但与假手术组比较,宫腔形态仍未完全恢复(P0.05)。天狼猩红染色结果:与模型组比较,中药组、宫腔注射组、联合组子宫内膜纤维化程度均不同程度减轻,差异有统计学意义(P0.05),其中宫腔注射组及联合组纤维化的程度减轻最明显;与假手术组比较,纤维化程度仍没有完全接近,差异有统计学意义(P0.05)。2.免疫荧光结果显示:标记脐带间充质干细胞的CD29红色荧光主要集中在SD大鼠子宫内膜的间质部,少部分出现在子宫内膜靠近腺上皮及腔上皮处。标记CD29红色荧光主要出现在宫腔注射组及联合组,但是在宫腔注射组及联合组CD29荧光强度比较,差异不显著(P0.05);假手术组、模型组、中药组子宫内膜组织中可见少量标记CD29的红色荧光,三组之间相互比较,无统计学差异(P0.05)。标记AKT的绿色荧光主要集中在子宫内膜腔上皮处及腺上皮处,少量分散在子宫内膜间质部。标记AKT的荧光强度在中药组、宫腔注射组、联合组较假手术组及模型组均增强,且在宫腔注射组及联合组比中药组增强明显,但宫腔注射组及联合组比较,差异无统计学意义。3.Western Blot结果显示PI3K、AKT、p-AKT蛋白在中药组、宫腔注射组、联合组较假手术组及模型组均不同程度增强,且宫腔注射组及联合组的表达较中药组均增强,但宫腔注射组与联合组比较,差异无统计学意义。结论中药左归丸、脐带间充质干细胞对受损子宫内膜均有一定程度的修复作用,可以增加子宫内膜厚度、腺体个数及抑制子宫内膜纤维化的趋势。经宫腔原位注射的脐带间充质干细胞在经过治疗28天后仍有部分存在于子宫内膜中。脐带间充质干细胞及左归丸修复受损子宫内膜可能跟激活信号通路PI3K/AKT有关,且AKT蛋白的活化可能是PI3K/AKT信号通路下游一系列反应的关键。左归丸联合脐带间充质干细胞可有效修复受损子宫内膜,可为临床上难治性薄型子宫内膜、宫腔粘连等妇科疾病提供一种新思路。
[Abstract]:The expression of phosphatidylinositol -3- kinase (phosphatidylinositol 3-kinase, PI3K) in thin endometrium and the expression of protein kinase B (protein kinase B) in thin endometrium, in the first part of the endometrium of thin endometrium, were observed to explore the role of the protein kinase B (protein kinase B) in the thin endometrium of the endometrium, and to explore the role of the PI3K pathway in the thin endometrium. 30 cases of normal endometrium and 30 cases of thin endometrium, normal endometrium and thin endometrium were referred to western medicine. The endometrial tissue was collected at -70 C during hysteroscopy. The results showed that the expression of PI3K, AKT m RNA in the thin endometrium group was lower than that of the normal endometrium group, and the difference between the two groups was statistically significant. Significance (P0.05). Conclusion PI3K/AKT signaling pathway exists in normal and thin endometrium tissues, and the expression of PI3K and AKT decreases in the proliferation of thin endometrium. It is presumed that PI3K/AKT signaling pathway may participate in the proliferation and differentiation of endometrium. The experiment of the second part Zuo GUI pill combined with umbilical cord mesenchymal stem cells to repair the damaged endometrium Objective Zuo GUI pill is the representative of the nourishing yin nourishing kidney of Zhang Jingyue, the Ming Dynasty physician, which has the efficacy of nourishing yin and tonifying the kidney, filling the lean pulp and so on. It can regulate the dysfunction of the hypothalamus pituitary - target gland axis, and is widely used in the treatment of gynecologic diseases, bone diseases, and nervous system diseases. Umbilical cord mesenchymal stem CE LLS, UC-MSCs), a new representative of adult stem cells, has a strong ability to proliferate and multidirectional differentiation potential, and has a wide range of sources, without ethical issues, and can be a seed cell for the treatment of refractory endometrium injury diseases. Research has proved that the PI3K/AKT signaling pathway has many biological functions, such as cell proliferation, differentiation, apoptosis, migration and so on. In the process of regulation, it plays a key role of signal molecules. PI3K/AKT signaling pathway is active in endometriosis and endometriosis. It may be involved in mediating the growth and repair of endometrium. In this experiment, the rats' endometrial injury model was constructed to explore the effect of the Chinese medicine Zuo GUI pill and human umbilical cord mesenchymal stem cells. The possible mechanism of the action of Zuo GUI pill and human umbilical cord mesenchymal stem cells to repair the damaged endometrium is studied. It provides a theoretical basis for the clinical treatment of endometrial damaged diseases. The endometrial damage model of SD rats was established by endometrial method. According to the experimental results, the best hot hot time of the endometrium damaged model was determined. All the modules were burned to the left part of the Y type uterus in SD rats. A total of 6 rats in each group were divided into 5 groups, 6 rats in each group: the sham operation group, that is, after the Y uterus was exposed, the scalp needle was used through the left uterus and pulled back. Group B: model group: model group, routine feeding after rat model completion, without any treatment; group C: drug group, third days after the completion of the model, Zuo GUI pill solution gavage, according to the dose of 1.62g/Kg daily stomach one time, continuous perfusion for 28 days; group D: uterine cavity injection group, third days after the completion of the model, the uterus, the uterus, the palace after third days of the abdominal, via uterus after the completion of the palace 5 x 107 human umbilical cord mesenchymal stem cells were injected into the cavity in situ; group E: combined group, 5 x 107 human umbilical cord mesenchymal stem cells were injected into the uterine cavity in situ third days after the completion of the model, and the gavage of the left GUI pill water solution began on the same day, once every day for 28 days according to the dose of 1.62g/Kg, and the rats were killed for 28 days, and each SD rat was taken out of Y. The left building part of the uterus was abandoned near the ovary and vagina part, cut into 2 segments, one segment of the endometrium was stained with HE, Sirius scarlet and immunofluorescence, and the other part of the endometrium was detected by Western Blot for the expression of PI3K, AKT, p-AKT protein. Results the result of 1.HE staining: compared with the model group, Chinese medicine group, intrauterine injection In the group, the thickness of endometrium in the combined group was thickened in varying degrees, and the number of glands increased in different degrees. The difference was statistically significant. The endometrial thickness and the number of glands in the combined group increased most significantly (P0.05), but compared with the sham operation group, the morphology of the uterine cavity was not completely recovered (P0.05). The result of Sirius scarlet staining: with the model group. Comparison, the degree of endometrial fibrosis in the Chinese medicine group, the intrauterine injection group and the combined group were all relieved in varying degrees, and the difference was statistically significant (P0.05). The degree of fibrosis in the intrauterine injection group and the combined group was most obvious. Compared with the sham operation group, the degree of fibrosis was not completely close, and the difference was statistically significant (P0.05).2. immunofluorescent junction. The results showed that the CD29 red fluorescence of the labeled umbilical cord mesenchymal stem cells mainly concentrated in the interstitial part of the endometrium of the SD rats, and the few appeared in the endometrium near the gland epithelium and the cavity epithelium. The red fluorescence of the labeled CD29 appeared mainly in the intrauterine injection group and the combined group, but the difference in the fluorescence intensity of the CD29 in the intrauterine injection group and the combined group was different. In the endometrium of the sham operation group, the model group and the Chinese medicine group, the endometrium of the sham operation group, the model group and the Chinese medicine group were marked with a small amount of red fluorescence labeled CD29. There was no statistical difference between the three groups (P0.05). The green fluorescence of the labeled AKT was mainly concentrated in the endometrium epithelium and the gland epithelium and scattered in the endometrium interstroma. The fluorescence intensity of AKT was marked. In the traditional Chinese medicine group, the intrauterine injection group and the combined group were enhanced compared with the sham operation group and the model group, and the intrauterine injection group and the combined group were significantly enhanced than the traditional Chinese medicine group. However, there was no significant difference between the intrauterine injection group and the combined group. The difference was not statistically significant.3.Western Blot results showed PI3K, AKT, and p-AKT protein in the Chinese medicine group, the intrauterine injection group and the combined group compared with the sham operation group and the sham operation group. The expression of the intrauterine injection group and the combined group were all enhanced, but there was no difference between the intrauterine injection group and the combined group, but the difference between the intrauterine injection group and the combined group was not statistically significant. Conclusion the traditional Chinese medicine Zuo GUI pill and umbilical cord mesenchyme stem cells have a certain degree of repair on the damaged endometrium, which can increase the thickness of the endometrium and the number of glands. The tendency of endometrial fibrosis to inhibit endometriosis. Intrauterine intrauterine mesenchymal stem cells still exist in the endometrium after 28 days of treatment. Umbilical cord mesenchymal stem cells and left return pills may be related to the activation of the damaged endometrium PI3K/AKT, and the activation of AKT protein may be PI3K/AKT signaling. The key to a series of reactions in the lower reaches of the road is that Zuo GUI pill combined with umbilical cord mesenchymal stem cells can effectively repair the damaged endometrium, which can provide a new way of thinking for the clinical refractory thin endometrium, uterine cavity adhesion and other gynecological diseases.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.74

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