本文關鍵詞： 氣管上皮損傷修復 干細胞 再編程 染色質(zhì)構像　出處：《中國醫(yī)科大學》2007年碩士論文　論文類型：學位論文

【摘要】： 前言 近年來，有學者研究提出胚胎干細胞相關基因組概念，它們僅在胚胎干細胞中表達，而在成熟體細胞中無表達，其中包括Oct3／4，Sox2，Nanog基因等。同源蛋白轉(zhuǎn)錄因子Nanog是最近在小鼠和人的胚胎干細胞(ESC)中發(fā)現(xiàn)的一類新的特異性分子標記，它是哺乳動物胚胎全能干細胞的標志物，在維持其未分化狀態(tài)中起重要作用，成體細胞中則不表達。那么，使用5-FU制造大鼠氣管損傷模型中的干細胞是否為機體應激狀態(tài)下形成，其中是否有Nanog的存在，在其增殖分化過程中Nanog的變化趨勢如何呢?國內(nèi)外尚無報道。本研究利用5-FU制造大鼠氣管損傷模型，觀察Nanog的動態(tài)變化，以探討氣管干細胞形成及保持未分化狀態(tài)的分子機制。另外本實驗采用掃描電鏡，透射電鏡及HE染色對照的方法觀察并探討了氣管上皮損傷修復過程中上皮細胞超微結(jié)構及細胞核中染色質(zhì)變化規(guī)律。 材料與方法 1、離體大鼠氣管損傷模型的制備。 取約250克左右Wistar大鼠，雌雄不限，，腹腔注射10％水合氯醛0.4ml／100g，無菌條件下取出氣管，無菌PBS反復沖洗，置于DMEM／F12培養(yǎng)液中(含10％胎牛血清)，剪成約2-3mm的氣管環(huán)，于倒置顯微鏡下觀察纖毛擺動良好，取一組織塊作正常對照；于培養(yǎng)液中加入終濃度為120mg／ml的5-FU，37℃，5％CO2孵育12小時，棄去上述培養(yǎng)液，換成新鮮DMEM／F12液(含10％胎牛血清)繼續(xù)培養(yǎng)，于換液后0、3、6、12、24、48、小時分別取出一組織塊，10％中性福爾馬林固定，石蠟包埋，制成2μm厚的組織切片。另取換液后各時間點的氣管組織及正常氣管組織，解剖顯微鏡下機械剝離上皮，放于1.5ml Eppendorf管中，-70℃保存，以備總蛋白提取。另外在換液后各時間點取氣管組織及正常氣管組織，2.5％戊二醛固定。 2、HE染色動態(tài)觀察各時間點氣管粘膜上皮組織學形態(tài)改變。 3、間接免疫熒光染色檢測氣管上皮損傷修復過程中Nanog表達的動態(tài)變化。 石蠟切片脫蠟至水，抗原修復，非免疫動物血清封閉，一抗用兔抗Nanog，二抗為FITC標記山羊抗兔IgG，用Hoechst復染細胞核。50％緩沖甘油封片，熒光顯微鏡Olympas-BX51下觀察并照相。陰性對照實驗：用等量的0.01mol／LPBS代替一抗，其余步驟同前。 4、氣管上皮Nanog蛋白的半定量檢測。 取5-FU作用前后的氣管上皮組織，提取總蛋白質(zhì)。用Western blot分析Nanog蛋白的表達情況。 5、掃描電鏡及透射電鏡觀察氣管上皮細胞超微結(jié)構，特別是染色質(zhì)的變化。 結(jié)果 1、組織學形態(tài)改變： 5-FU作用后0h，大部分氣管上皮脫落，殘留間隔分布的裸核樣細胞(即GO期細胞)；去除5—FU后恢復6h，裸核細胞消失，細胞數(shù)量增多，呈扁平狀，細胞核變長，細胞漿伸展幾乎可以將基底膜覆蓋；24小時，上皮細胞數(shù)目繼續(xù)增多，大部分變?yōu)榱⒎郊毎�，胞核增大，胞漿增多；恢復至48小時，細胞表面出現(xiàn)多量纖毛，局部上皮接近假復層結(jié)構。 2、免疫熒光檢測結(jié)果： 以胞核中出現(xiàn)明亮綠色熒光為Nanog陽性表達。正常氣管上皮中無Nanog表達。經(jīng)5-FU作用后0小時，G0期細胞中出現(xiàn)Nanog陽性細胞表達；去除5—FU后3-6小時，Nanog陽性細胞增多，表達增加，達到高峰；24小時，Nanog陽性細胞數(shù)明顯減少；48小時，Nanog表達量降至最低。 3、Western blot檢測結(jié)果： Nanog蛋白在氣管損傷修復過程中各時相的表達有明顯差異。去除5-FU后恢復0小時出現(xiàn)表達，恢復3小時表達量增高，至6小時達到高峰，24小時明顯減弱，至48小時只有微弱表達；正常氣管上皮中幾乎無表達。 4、掃描電鏡及投射電鏡結(jié)果： 掃描電鏡顯示G0期細胞為半球形，表面較光滑，無纖毛及微絨毛，恢復24小時細胞表面有微絨毛出現(xiàn)，48小時后長出纖毛。透射電鏡顯示殘存的G0期細胞核內(nèi)充滿致密、深染的異染色質(zhì)，細胞器不發(fā)達或缺乏，呈干細胞特點。隨著上皮細胞分化，細胞核內(nèi)異染色質(zhì)逐漸減少，常染色質(zhì)逐漸增多。 結(jié)論 Nanog在G0期干細胞中有表達，推測體細胞受到非常刺激后基因組再編程，具有干細胞的特點和功能，進而完成氣管上皮的損傷修復。Nanog在干細胞中高表達，隨著細胞分化，其表達下降，直至消失。說明Nanog對保持細胞未分化能力起重要作用。 在氣管干細胞分化過程中，染色質(zhì)構象出現(xiàn)有規(guī)律的變化。干細胞的分化伴隨著異染色質(zhì)的減少和常染色質(zhì)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.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R361

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