本文選題：薄型子宮內(nèi)膜 + PI3K�。� 參考：《湖北中醫(yī)藥大學》2017年碩士論文

【摘要】：第一部分薄型子宮內(nèi)膜PI3K、AKT m RNA的表達目的觀察薄型子宮內(nèi)膜組織中磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt/PKB)m RNA含量的表達情況,探討PI3K/AKT信號通路在薄型子宮內(nèi)膜中的作用。方法通過收集正常子宮內(nèi)膜30例及薄型子宮內(nèi)膜30例,正常子宮內(nèi)膜及薄型子宮內(nèi)膜的西醫(yī)診斷標準參照文獻,宮腔鏡手術(shù)術(shù)中收集子宮內(nèi)膜組織-70℃保存?zhèn)溆�。結(jié)果PI3K、AKT m RNA在薄型子宮內(nèi)膜組的表達明顯低于正常子宮內(nèi)膜組,兩組差異有統(tǒng)計學意義(P0.05)。結(jié)論PI3K/AKT信號通路存在于正常和薄型子宮內(nèi)膜組織中,且在薄型子宮內(nèi)膜增殖期PI3K、AKT表達下降,推測PI3K/AKT信號通路可能參與子宮內(nèi)膜組織的增殖、分化。第二部分左歸丸聯(lián)合臍帶間充質(zhì)干細胞修復(fù)受損子宮內(nèi)膜的實驗研究目的左歸丸是明代醫(yī)家張景岳的滋陰補腎代表方,該方有滋陰補腎、填精益髓等功效,能調(diào)節(jié)下丘腦-垂體-靶腺軸功能紊亂,臨床上廣泛用于婦科疾病、骨骼疾病、神經(jīng)系統(tǒng)疾病等的治療。臍帶間充質(zhì)干細胞(umbilical cord mesenchymal stem cells,UC-MSCs),是成體干細胞的新生代表,具有強大的自我增殖能力和多向分化潛能,且來源廣泛、不涉及倫理問題,可成為治療難治性子宮內(nèi)膜損傷疾病的種子細胞。研究證明PI3K/AKT信號通路在細胞的增殖、分化、凋亡、遷移等多種生物學功能的調(diào)節(jié)過程中,起著關(guān)鍵的信號分子作用,PI3K/AKT信號通路在子宮內(nèi)膜癌、子宮內(nèi)膜異位癥中表達活躍,可能參與介導(dǎo)了子宮內(nèi)膜的增長、修復(fù)過程。本實驗通過構(gòu)建大鼠的子宮內(nèi)膜損傷模型,探討中藥左歸丸及人臍帶間充質(zhì)干細胞對受損子宮內(nèi)膜的修復(fù)作用。研究左歸丸及人臍帶間充質(zhì)干細胞修復(fù)受損子宮內(nèi)膜的可能作用機制,為臨床上治療子宮內(nèi)膜受損疾病提供理論依據(jù)。方法目前尚無子宮內(nèi)膜受損模型的統(tǒng)一建模標準,故參照已有文獻,采用100℃熱水燙傷子宮內(nèi)膜方法建立SD大鼠子宮內(nèi)膜受損模型,根據(jù)摸索實驗結(jié)果,確定子宮內(nèi)膜受損模型最佳熱水燙傷時間。所有造模組均燙傷SD大鼠Y型子宮左側(cè)部分,共分5組,每組6只大鼠。A組:假手術(shù)組,即暴露Y型子宮后,用頭皮針導(dǎo)管穿過左側(cè)子宮,后拔掉導(dǎo)管,不用流動的熱水經(jīng)過宮腔;B組:模型組,在大鼠造模完成后常規(guī)飼養(yǎng),不做任何處理;C組:藥物組,造模完成后第3天開始左歸丸溶液灌胃,按1.62g/Kg的劑量每日灌胃一次,連續(xù)灌胃28天;D組:宮腔注射組,造模完成后第三天再開腹,經(jīng)宮腔原位注射5×107個人臍帶間充質(zhì)干細胞;E組:聯(lián)合組,造模完成后第三天經(jīng)宮腔原位注射5×107個人臍帶間充質(zhì)干細胞,并于同一天開始左歸丸水溶液灌胃,按1.62g/Kg劑量每日一次,連續(xù)灌胃28天;治療28天后處死各組大鼠,取出每只SD大鼠Y型子宮左側(cè)造模部分,棄去靠近卵巢及陰道部分,剪成2段,一段子宮內(nèi)膜組織作HE染色、天狼猩紅染色及免疫熒光檢測,另一部分子宮內(nèi)膜組織作Western Blot檢測PI3K、AKT、p-AKT蛋白表達情況。結(jié)果1.HE染色結(jié)果:與模型組比較,中藥組、宮腔注射組、聯(lián)合組子宮內(nèi)膜厚度均有不同程度增厚、腺體個數(shù)不同程度增加,差異有統(tǒng)計學意義,其中宮腔注射組與聯(lián)合組的內(nèi)膜厚度及腺體個數(shù)增加最顯著(P0.05),但與假手術(shù)組比較,宮腔形態(tài)仍未完全恢復(fù)(P0.05)。天狼猩紅染色結(jié)果:與模型組比較,中藥組、宮腔注射組、聯(lián)合組子宮內(nèi)膜纖維化程度均不同程度減輕,差異有統(tǒng)計學意義(P0.05),其中宮腔注射組及聯(lián)合組纖維化的程度減輕最明顯;與假手術(shù)組比較,纖維化程度仍沒有完全接近,差異有統(tǒng)計學意義(P0.05)。2.免疫熒光結(jié)果顯示:標記臍帶間充質(zhì)干細胞的CD29紅色熒光主要集中在SD大鼠子宮內(nèi)膜的間質(zhì)部,少部分出現(xiàn)在子宮內(nèi)膜靠近腺上皮及腔上皮處。標記CD29紅色熒光主要出現(xiàn)在宮腔注射組及聯(lián)合組,但是在宮腔注射組及聯(lián)合組CD29熒光強度比較,差異不顯著(P0.05);假手術(shù)組、模型組、中藥組子宮內(nèi)膜組織中可見少量標記CD29的紅色熒光,三組之間相互比較,無統(tǒng)計學差異(P0.05)。標記AKT的綠色熒光主要集中在子宮內(nèi)膜腔上皮處及腺上皮處,少量分散在子宮內(nèi)膜間質(zhì)部。標記AKT的熒光強度在中藥組、宮腔注射組、聯(lián)合組較假手術(shù)組及模型組均增強,且在宮腔注射組及聯(lián)合組比中藥組增強明顯,但宮腔注射組及聯(lián)合組比較,差異無統(tǒng)計學意義。3.Western Blot結(jié)果顯示PI3K、AKT、p-AKT蛋白在中藥組、宮腔注射組、聯(lián)合組較假手術(shù)組及模型組均不同程度增強,且宮腔注射組及聯(lián)合組的表達較中藥組均增強,但宮腔注射組與聯(lián)合組比較,差異無統(tǒng)計學意義。結(jié)論中藥左歸丸、臍帶間充質(zhì)干細胞對受損子宮內(nèi)膜均有一定程度的修復(fù)作用,可以增加子宮內(nèi)膜厚度、腺體個數(shù)及抑制子宮內(nèi)膜纖維化的趨勢。經(jīng)宮腔原位注射的臍帶間充質(zhì)干細胞在經(jīng)過治療28天后仍有部分存在于子宮內(nèi)膜中。臍帶間充質(zhì)干細胞及左歸丸修復(fù)受損子宮內(nèi)膜可能跟激活信號通路PI3K/AKT有關(guān),且AKT蛋白的活化可能是PI3K/AKT信號通路下游一系列反應(yīng)的關(guān)鍵。左歸丸聯(lián)合臍帶間充質(zhì)干細胞可有效修復(fù)受損子宮內(nèi)膜,可為臨床上難治性薄型子宮內(nèi)膜、宮腔粘連等婦科疾病提供一種新思路。
[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.
【學位授予單位】：湖北中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R711.74

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