5-FU诱发大鼠离体气管上皮损伤修复过程中Nanog表达及超微结构观察

发布时间：2018-02-27 18:36

本文关键词： 气管上皮损伤修复干细胞再编程染色质构像　出处：《中国医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 前言近年来，有学者研究提出胚胎干细胞相关基因组概念，它们仅在胚胎干细胞中表达，而在成熟体细胞中无表达，其中包括Oct3／4，Sox2，Nanog基因等。同源蛋白转录因子Nanog是最近在小鼠和人的胚胎干细胞(ESC)中发现的一类新的特异性分子标记，它是哺乳动物胚胎全能干细胞的标志物，在维持其未分化状态中起重要作用，成体细胞中则不表达。那么，使用5-FU制造大鼠气管损伤模型中的干细胞是否为机体应激状态下形成，其中是否有Nanog的存在，在其增殖分化过程中Nanog的变化趋势如何呢?国内外尚无报道。本研究利用5-FU制造大鼠气管损伤模型，观察Nanog的动态变化，以探讨气管干细胞形成及保持未分化状态的分子机制。另外本实验采用扫描电镜，透射电镜及HE染色对照的方法观察并探讨了气管上皮损伤修复过程中上皮细胞超微结构及细胞核中染色质变化规律。材料与方法 1、离体大鼠气管损伤模型的制备。取约250克左右Wistar大鼠，雌雄不限，，腹腔注射10％水合氯醛0.4ml／100g，无菌条件下取出气管，无菌PBS反复冲洗，置于DMEM／F12培养液中(含10％胎牛血清)，剪成约2-3mm的气管环，于倒置显微镜下观察纤毛摆动良好，取一组织块作正常对照；于培养液中加入终浓度为120mg／ml的5-FU，37℃，5％CO2孵育12小时，弃去上述培养液，换成新鲜DMEM／F12液(含10％胎牛血清)继续培养，于换液后0、3、6、12、24、48、小时分别取出一组织块，10％中性福尔马林固定，石蜡包埋，制成2μm厚的组织切片。另取换液后各时间点的气管组织及正常气管组织，解剖显微镜下机械剥离上皮，放于1.5ml Eppendorf管中，-70℃保存，以备总蛋白提取。另外在换液后各时间点取气管组织及正常气管组织，2.5％戊二醛固定。 2、HE染色动态观察各时间点气管粘膜上皮组织学形态改变。 3、间接免疫荧光染色检测气管上皮损伤修复过程中Nanog表达的动态变化。石蜡切片脱蜡至水，抗原修复，非免疫动物血清封闭，一抗用兔抗Nanog，二抗为FITC标记山羊抗兔IgG，用Hoechst复染细胞核。50％缓冲甘油封片，荧光显微镜Olympas-BX51下观察并照相。阴性对照实验：用等量的0.01mol／LPBS代替一抗，其余步骤同前。 4、气管上皮Nanog蛋白的半定量检测。取5-FU作用前后的气管上皮组织，提取总蛋白质。用Western blot分析Nanog蛋白的表达情况。 5、扫描电镜及透射电镜观察气管上皮细胞超微结构，特别是染色质的变化。结果 1、组织学形态改变： 5-FU作用后0h，大部分气管上皮脱落，残留间隔分布的裸核样细胞(即GO期细胞)；去除5—FU后恢复6h，裸核细胞消失，细胞数量增多，呈扁平状，细胞核变长，细胞浆伸展几乎可以将基底膜覆盖；24小时，上皮细胞数目继续增多，大部分变为立方细胞，胞核增大，胞浆增多；恢复至48小时，细胞表面出现多量纤毛，局部上皮接近假复层结构。 2、免疫荧光检测结果：以胞核中出现明亮绿色荧光为Nanog阳性表达。正常气管上皮中无Nanog表达。经5-FU作用后0小时，G0期细胞中出现Nanog阳性细胞表达；去除5—FU后3-6小时，Nanog阳性细胞增多，表达增加，达到高峰；24小时，Nanog阳性细胞数明显减少；48小时，Nanog表达量降至最低。 3、Western blot检测结果： Nanog蛋白在气管损伤修复过程中各时相的表达有明显差异。去除5-FU后恢复0小时出现表达，恢复3小时表达量增高，至6小时达到高峰，24小时明显减弱，至48小时只有微弱表达；正常气管上皮中几乎无表达。 4、扫描电镜及投射电镜结果：扫描电镜显示G0期细胞为半球形，表面较光滑，无纤毛及微绒毛，恢复24小时细胞表面有微绒毛出现，48小时后长出纤毛。透射电镜显示残存的G0期细胞核内充满致密、深染的异染色质，细胞器不发达或缺乏，呈干细胞特点。随着上皮细胞分化，细胞核内异染色质逐渐减少，常染色质逐渐增多。结论 Nanog在G0期干细胞中有表达，推测体细胞受到非常刺激后基因组再编程，具有干细胞的特点和功能，进而完成气管上皮的损伤修复。Nanog在干细胞中高表达，随着细胞分化，其表达下降，直至消失。说明Nanog对保持细胞未分化能力起重要作用。在气管干细胞分化过程中，染色质构象出现有规律的变化。干细胞的分化伴随着异染色质的减少和常染色质的增多。
[Abstract]:Preface
In recent years, some scholars put forward the concept of embryonic stem cell related genome, they only expressed in embryonic stem cells, but no expression in mature somatic cells, including Oct3 / 4, Sox2, Nanog genes. The homologous protein transcription factor Nanog is recently stem cells in mouse and human embryos (ESC) of a class new specific molecular markers found in mammalian embryos, it is totipotent stem cell markers, to maintain their undifferentiated state play an important role in somatic cells was not expressed. Then, using 5-FU manufacturing rat tracheal injury model whether stem cells into the body under stress, which is with the presence of Nanog, how to change the trend of Nanog on the proliferation and differentiation in the process? There is no report at home and abroad. This study uses 5-FU manufacturing tracheal injury model of rats, to observe the dynamic changes of Nanog, to explore the formation and preservation of tracheal stem cells The molecular mechanism of undifferentiated state was also observed. In addition, scanning electron microscopy, transmission electron microscopy and HE staining were used to observe and explore the ultrastructure of epithelial cells and chromatin in nucleus during the repair process of tracheal epithelium.
Materials and methods
1, the preparation of the isolated rat model of tracheal injury.
Take about 250 grams of Wistar rats, male or female, intraperitoneal injection of 10% chloral hydrate 0.4ml / 100g, the trachea removed under sterile conditions, aseptic PBS repeated washing, in DMEM / F12 medium (containing 10% FBS), trachea cut into 2-3mm, was observed by inverted microscope cilia beat good take a piece of tissue, normal control group; in the culture medium with a final concentration of 120mg / ml 5-FU, 37 C, 5%CO2 incubated for 12 hours, discard the culture liquid, replaced with fresh DMEM / F12 solution (containing 10% fetal bovine serum to culture, to change the liquid) after 0,3,6,12,24,48 hours respectively removed a piece of tissue, 10% neutral formalin fixed, paraffin embedded, made of 2 m thick slices. Another tracheal tissue fluid change after each time point and normal tracheal tissue under a dissecting microscope, mechanical stripping epithelium, put in the 1.5ml Eppendorf tube, -70 temperature preservation, extraction for total protein. In addition, the trachea tissue and normal trachea tissue were taken at each time point after the change of liquid, and 2.5% glutaraldehyde was fixed.
2, HE staining was used to observe the histological changes of tracheal epithelium at every time point.
3, the dynamic changes of Nanog expression during the repair of tracheal epithelia were detected by indirect immunofluorescence staining.
Paraffin sections were dewaxed to water, antigen repair, non immune animal serum closed with Rabbit anti Nanog antibody, anti FITC Two Goat anti rabbit IgG, using Hoechst staining of nuclear.50% buffered glycerol under fluorescence microscope and photographed under Olympas-BX51. The negative control experiment: with the amount of 0.01mol / LPBS instead of a resistance the remaining steps, as before.
4, the semi quantitative detection of Nanog protein in the tracheal epithelium.
The total protein was extracted from the tracheal epithelium before and after the action of 5-FU, and the expression of Nanog protein was analyzed by Western blot.
5, scanning electron microscopy and transmission electron microscopy were used to observe the ultrastructure of tracheal epithelial cells, especially the changes of chromatin.
Result
1, morphological changes in histology:
After 5-FU 0h, most of the tracheal epithelial cells, residual naked nucleus intervals (GO cells); removal of 5 - FU recovery after 6h, bare cells disappeared, the number of cells increased, the nucleus is flat, variable length, cytoplasm stretch to almost 24 hours, covering the basement membrane; epithelium the number of cells continue to increase, most into cubic cells, the nucleus increased, cytoplasm; recovered to 48 hours, many cilia appeared on cell surface, close to the local epithelial pseudostratified structure.
2, the results of immunofluorescence detection:
In the nucleus of bright green fluorescence positive for Nanog expression. No Nanog expression in normal tracheal epithelial cells. After 0 hours after 5-FU, the expression of Nanog positive cells in G0 phase cells; removal of 5 - 3-6 hours after FU, Nanog positive cells increased, increased expression reached the peak; 24, Nanog positive cells the number was significantly reduced; 48 hours, the expression of Nanog is reduced to the minimum.
3, Western blot detection results:
Nanog protein in the healing process of each phase was significantly different in the trachea. The removal of 5-FU after 0 hours was 3 hours, the expression increased, reached a peak after 6 hours, 24 hours and 48 hours decreased significantly, only weak expression in normal tracheal epithelium; almost no expression.
4, scanning electron microscope and projective electron microscope results:
Scanning electron microscopy showed that the cells in G0 phase was hemispherical, smooth surface, no cilia and microvilli, restore the microvilli on cell surface in 24 hours, 48 hours after the long cilia. Transmission electron microscopy showed that the remnants of the G0 nucleus with dense, dark stained heterochromatin, organelles are underdeveloped or lack, showed the characteristics of stem cells with epithelial cells, nuclear heterochromatin decreased, euchromatin increased gradually.
conclusion
Nanog in the G0 period of stem cells are expressed that body cells are very exciting after genome reprogramming, has the characteristics and functions of stem cells, and then complete the repair of damaged.Nanog in tracheal epithelial stem cells with high expression, cell differentiation, the expression decreased until disappeared. Nanog to maintain cell undifferentiated ability an important role.
During the differentiation of the stem cells of the trachea, the chromatin conformation changes regularly. The differentiation of stem cells is accompanied by the decrease of heterochromatin and the increase of the normal chromatin.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R361

【参考文献】