胚胎干細(xì)胞修復(fù)受損子宮內(nèi)膜的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-09-13 08:20
【摘要】：背景：子宮內(nèi)膜修復(fù)障礙與許多臨床問題有關(guān)，這些疾病的臨床上處理十分棘手。近年來有學(xué)者提出大膽假設(shè)，認(rèn)為子宮內(nèi)膜的生理性再生修復(fù)是由存在于子宮內(nèi)膜基底層的干細(xì)胞始發(fā)的[1]。可以進(jìn)一步假設(shè)：子宮內(nèi)膜修復(fù)障礙可能由于局部干細(xì)胞枯竭或功能障礙導(dǎo)致。由此推測：加入外源胚胎干細(xì)胞可能有助于子宮內(nèi)膜修復(fù)。 目的：了解同種異體胚胎干細(xì)胞是否有助于急性子宮腔損傷模型小鼠的子宮損傷修復(fù)，移植后胚胎干細(xì)胞能否定植于子宮內(nèi)膜及存在時(shí)間；胚胎干細(xì)胞能否在受損宮腔內(nèi)膜修復(fù)過程中被誘導(dǎo)分化；以及胚胎干細(xì)胞的成瘤性。方法：復(fù)蘇帶有eGFP基因的小鼠胚胎干細(xì)胞（ESC），用小鼠胚胎成纖維細(xì)胞（MEF）做飼養(yǎng)層培養(yǎng)并腎被膜下移植鑒定其全能性。將ESC移植到內(nèi)膜損傷模型鼠體內(nèi)，試驗(yàn)分三組：第一組ESC移植入單側(cè)內(nèi)膜損傷模型鼠雙側(cè)宮腔；二組，尾靜脈注射ESC到單側(cè)內(nèi)膜損傷模型鼠體內(nèi)。第三組用小鼠子宮內(nèi)膜細(xì)胞與ESC共培養(yǎng)，將混合細(xì)胞移植入雙側(cè)內(nèi)膜損傷模型鼠單側(cè)宮腔，另一側(cè)宮腔移植等量PBS；各組分別在1W、2W、3W、4W處死小鼠，熒光顯微鏡直接觀察和HE染色、免疫組化GFP鑒定，觀察ESC在內(nèi)膜損傷鼠宮腔定植遷徙情況及成瘤性和ESC在小鼠體內(nèi)定植情況及損傷對ESC定植的影響。 結(jié)果：ESC在MEF飼養(yǎng)層上生長狀態(tài)良好，且可維持未分化狀態(tài)，熒光顯微鏡下可見綠色熒光克�。籈SC腎被膜下注射后，可形成畸胎瘤，熒光顯微鏡下可見瘤體發(fā)綠色熒光，HE染色及免疫組化鑒定可見三胚層分化；子宮內(nèi)膜細(xì)胞與ESC共培養(yǎng)后，胚胎干細(xì)胞呈扁平狀克隆樣生長，熒光顯微鏡下見綠色熒光；ESC內(nèi)膜損傷鼠宮腔內(nèi)移植后不同時(shí)間處死小鼠，體式熒光顯微鏡下觀察可見損傷側(cè)宮腔見綠色熒光，未損傷側(cè)宮腔無熒光，子宮內(nèi)膜比PBS對照側(cè)較厚且腺體較豐富；且隨時(shí)間的延長，ESC可在宮腔內(nèi)形成畸胎瘤，熒光鏡下見綠色熒光；ESC內(nèi)膜損傷鼠尾靜脈注射后處死小鼠，熒光顯微鏡下可見腹壁切口，損傷側(cè)子宮見綠色熒光，未損傷側(cè)宮腔未見熒光；ESC與子宮內(nèi)膜共培養(yǎng)后移植，可見移植側(cè)宮腔內(nèi)見綠色熒光，在觀察期內(nèi)未見腫瘤生長；熒光組織取材后固定，GFP免疫組化鑒定，可見ESC損傷宮腔內(nèi)移植后，形成的腫瘤組織可見向脂肪、軟骨、腺體、神經(jīng)等組織分化，GFP陽性；共培養(yǎng)ESC移植后宮腔修復(fù)良好，可見完整子宮內(nèi)膜上皮及腺體，GFP染色陽性；尾靜脈ESC注射后，子宮修復(fù)亦好，GFP染色細(xì)胞呈巢狀分布于子宮內(nèi)膜；共培養(yǎng)ESC移植后宮腔修復(fù)良好，可見完整子宮內(nèi)膜上皮及腺體，GFP染色陽性。 結(jié)論：ESC可以在內(nèi)膜損傷鼠宮腔內(nèi)定植，，并可成瘤；損傷對ESC在小鼠體內(nèi)遷徙有重要作用。ESC與子宮內(nèi)膜細(xì)胞共培養(yǎng)后再植入子宮內(nèi)膜損傷鼠宮腔，可在宮腔內(nèi)定植，并且成瘤性降低。
[Abstract]:Background: endometrial repair disorders are associated with many clinical problems, and the clinical management of these diseases is very difficult. In recent years, some scholars have proposed a bold hypothesis that the physiological regeneration of endometrium is originated from stem cells existing in the basal layer of endometrium [1]. It can be further assumed that endometrial repair disorders may result from the depletion of local stem cells or dysfunction. It is inferred that the addition of exogenous embryonic stem cells may contribute to endometrial repair. Objective: to investigate whether allogeneic embryonic stem cells can contribute to the repair of uterine injury in mice with acute uterine cavity injury, and whether embryonic stem cells can be transplanted into endometrium and exist for a long time after transplantation. Whether embryonic stem cells can be induced to differentiate during the repair of damaged endometrium, and the tumorigenesis of embryonic stem cells. Methods: mouse embryonic stem cells (ESC),) with eGFP gene were resuscitated by using (MEF) as feeder layer and renal submembrane transplantation to determine its totipotency. ESC was transplanted into the model mice of endometrial injury. The experiment was divided into three groups: the first group was transplanted into the bilateral uterine cavity of unilateral endometrial injury model mice, and the second group was injected with ESC through tail vein into unilateral endometrial injury model mice. The third group was co-cultured with ESC and mixed cells were transplanted into the unilateral uterine cavity of the model rats with bilateral endometrial injury. The mice were killed at 1W ~ 2W ~ 3W ~ 3W ~ 4W by transplantation of the same amount of PBS; on the other side of the uterine cavity. The mice were observed directly by fluorescence microscope and HE staining. The migration and tumorigenesis of ESC in uterine cavity of injured mice and the effect of ESC on ESC colonization in mice were observed by immunohistochemical GFP identification. Results the growth of MEF was good and it could remain undifferentiated. Green fluorescent clone ESC could form teratoma after being injected under the renal capsule under fluorescence microscope. He staining and immunohistochemical analysis showed that the embryonic stem cells grew flat like clone after co-culture of endometrial cells and ESC, and green fluorescence was observed under fluorescence microscope. The mice were killed at different times after intrauterine transplantation of ESC endometrium. Green fluorescence was observed in the injured uterine cavity, no fluorescence was found in the uninjured uterine cavity, and the endometrium was thicker and richer in glandular body than that in the control side of PBS. ESC could form teratoma in uterine cavity with the extension of time. The mice were killed after injection of green fluorescent ESC into caudal vein under fluorescence microscope. The abdominal wall incision was observed under fluorescence microscope, and green fluorescence was observed in the injured uterus. No fluorescent ESC was found in the uninjured uterine cavity after co-culture with endometrium. Green fluorescence was observed in the transplanted uterine cavity, and no tumor growth was observed in the observed period. After intrauterine transplantation of ESC injury, the tumor tissue was positive for differentiation to fat, cartilage, gland, nerve and so on, the uterine cavity was repaired well after co-cultured ESC transplantation, and the intact endometrial epithelium and glandular tissue were positive for GFP staining. After ESC injection into caudal vein, ESC staining cells were nestled in endometrium, and the intact endometrial epithelium and glandular tissue were positive after co-cultured ESC transplantation. ConclusionESC can be colonized and tumorigenic in the uterine cavity of rats with endometrial injury, and the injury may play an important role in migration of ESC in mice. After co-culture with endometrial cells, it can be implanted into the uterine cavity of rats with endometrial injury, and can be implanted in uterine cavity. And the tumorigenesis decreased.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
本文編號：2240592
[Abstract]:Background: endometrial repair disorders are associated with many clinical problems, and the clinical management of these diseases is very difficult. In recent years, some scholars have proposed a bold hypothesis that the physiological regeneration of endometrium is originated from stem cells existing in the basal layer of endometrium [1]. It can be further assumed that endometrial repair disorders may result from the depletion of local stem cells or dysfunction. It is inferred that the addition of exogenous embryonic stem cells may contribute to endometrial repair. Objective: to investigate whether allogeneic embryonic stem cells can contribute to the repair of uterine injury in mice with acute uterine cavity injury, and whether embryonic stem cells can be transplanted into endometrium and exist for a long time after transplantation. Whether embryonic stem cells can be induced to differentiate during the repair of damaged endometrium, and the tumorigenesis of embryonic stem cells. Methods: mouse embryonic stem cells (ESC),) with eGFP gene were resuscitated by using (MEF) as feeder layer and renal submembrane transplantation to determine its totipotency. ESC was transplanted into the model mice of endometrial injury. The experiment was divided into three groups: the first group was transplanted into the bilateral uterine cavity of unilateral endometrial injury model mice, and the second group was injected with ESC through tail vein into unilateral endometrial injury model mice. The third group was co-cultured with ESC and mixed cells were transplanted into the unilateral uterine cavity of the model rats with bilateral endometrial injury. The mice were killed at 1W ~ 2W ~ 3W ~ 3W ~ 4W by transplantation of the same amount of PBS; on the other side of the uterine cavity. The mice were observed directly by fluorescence microscope and HE staining. The migration and tumorigenesis of ESC in uterine cavity of injured mice and the effect of ESC on ESC colonization in mice were observed by immunohistochemical GFP identification. Results the growth of MEF was good and it could remain undifferentiated. Green fluorescent clone ESC could form teratoma after being injected under the renal capsule under fluorescence microscope. He staining and immunohistochemical analysis showed that the embryonic stem cells grew flat like clone after co-culture of endometrial cells and ESC, and green fluorescence was observed under fluorescence microscope. The mice were killed at different times after intrauterine transplantation of ESC endometrium. Green fluorescence was observed in the injured uterine cavity, no fluorescence was found in the uninjured uterine cavity, and the endometrium was thicker and richer in glandular body than that in the control side of PBS. ESC could form teratoma in uterine cavity with the extension of time. The mice were killed after injection of green fluorescent ESC into caudal vein under fluorescence microscope. The abdominal wall incision was observed under fluorescence microscope, and green fluorescence was observed in the injured uterus. No fluorescent ESC was found in the uninjured uterine cavity after co-culture with endometrium. Green fluorescence was observed in the transplanted uterine cavity, and no tumor growth was observed in the observed period. After intrauterine transplantation of ESC injury, the tumor tissue was positive for differentiation to fat, cartilage, gland, nerve and so on, the uterine cavity was repaired well after co-cultured ESC transplantation, and the intact endometrial epithelium and glandular tissue were positive for GFP staining. After ESC injection into caudal vein, ESC staining cells were nestled in endometrium, and the intact endometrial epithelium and glandular tissue were positive after co-cultured ESC transplantation. ConclusionESC can be colonized and tumorigenic in the uterine cavity of rats with endometrial injury, and the injury may play an important role in migration of ESC in mice. After co-culture with endometrial cells, it can be implanted into the uterine cavity of rats with endometrial injury, and can be implanted in uterine cavity. And the tumorigenesis decreased.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 曲軍英;呂一帆;林茵;江一平;;建立小鼠子宮內(nèi)膜損傷模型的研究[J];福建醫(yī)科大學(xué)學(xué)報(bào);2011年01期
2 胡菲菲;廖聯(lián)明;劉嘉茵;;產(chǎn)后小鼠子宮內(nèi)膜邊緣群干/祖細(xì)胞的分離純化[J];生殖醫(yī)學(xué)雜志;2008年04期
3 謝模英;魯文果;陳紅;楊慧民;張?zhí)K明;;Duchenne肌營養(yǎng)不良模型鼠肌肉內(nèi)移植小鼠胚胎干細(xì)胞后分化潛能的初步研究[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年01期
本文編號：2240592
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