胚胎干细胞修复受损子宫内膜的实验研究

发布时间：2018-09-13 08:20

【摘要】：背景：子宫内膜修复障碍与许多临床问题有关，这些疾病的临床上处理十分棘手。近年来有学者提出大胆假设，认为子宫内膜的生理性再生修复是由存在于子宫内膜基底层的干细胞始发的[1]。可以进一步假设：子宫内膜修复障碍可能由于局部干细胞枯竭或功能障碍导致。由此推测：加入外源胚胎干细胞可能有助于子宫内膜修复。目的：了解同种异体胚胎干细胞是否有助于急性子宫腔损伤模型小鼠的子宫损伤修复，移植后胚胎干细胞能否定植于子宫内膜及存在时间；胚胎干细胞能否在受损宫腔内膜修复过程中被诱导分化；以及胚胎干细胞的成瘤性。方法：复苏带有eGFP基因的小鼠胚胎干细胞（ESC），用小鼠胚胎成纤维细胞（MEF）做饲养层培养并肾被膜下移植鉴定其全能性。将ESC移植到内膜损伤模型鼠体内，试验分三组：第一组ESC移植入单侧内膜损伤模型鼠双侧宫腔；二组，尾静脉注射ESC到单侧内膜损伤模型鼠体内。第三组用小鼠子宫内膜细胞与ESC共培养，将混合细胞移植入双侧内膜损伤模型鼠单侧宫腔，另一侧宫腔移植等量PBS；各组分别在1W、2W、3W、4W处死小鼠，荧光显微镜直接观察和HE染色、免疫组化GFP鉴定，观察ESC在内膜损伤鼠宫腔定植迁徙情况及成瘤性和ESC在小鼠体内定植情况及损伤对ESC定植的影响。结果：ESC在MEF饲养层上生长状态良好，且可维持未分化状态，荧光显微镜下可见绿色荧光克隆；ESC肾被膜下注射后，可形成畸胎瘤，荧光显微镜下可见瘤体发绿色荧光，HE染色及免疫组化鉴定可见三胚层分化；子宫内膜细胞与ESC共培养后，胚胎干细胞呈扁平状克隆样生长，荧光显微镜下见绿色荧光；ESC内膜损伤鼠宫腔内移植后不同时间处死小鼠，体式荧光显微镜下观察可见损伤侧宫腔见绿色荧光，未损伤侧宫腔无荧光，子宫内膜比PBS对照侧较厚且腺体较丰富；且随时间的延长，ESC可在宫腔内形成畸胎瘤，荧光镜下见绿色荧光；ESC内膜损伤鼠尾静脉注射后处死小鼠，荧光显微镜下可见腹壁切口，损伤侧子宫见绿色荧光，未损伤侧宫腔未见荧光；ESC与子宫内膜共培养后移植，可见移植侧宫腔内见绿色荧光，在观察期内未见肿瘤生长；荧光组织取材后固定，GFP免疫组化鉴定，可见ESC损伤宫腔内移植后，形成的肿瘤组织可见向脂肪、软骨、腺体、神经等组织分化，GFP阳性；共培养ESC移植后宫腔修复良好，可见完整子宫内膜上皮及腺体，GFP染色阳性；尾静脉ESC注射后，子宫修复亦好，GFP染色细胞呈巢状分布于子宫内膜；共培养ESC移植后宫腔修复良好，可见完整子宫内膜上皮及腺体，GFP染色阳性。结论：ESC可以在内膜损伤鼠宫腔内定植，，并可成瘤；损伤对ESC在小鼠体内迁徙有重要作用。ESC与子宫内膜细胞共培养后再植入子宫内膜损伤鼠宫腔，可在宫腔内定植，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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】