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  本文關(guān)鍵詞：曲古抑菌素A對(duì)大鼠氣管干細(xì)胞增殖分化的影響　出處：《中國醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： TSA 5-Fu HDAC1 大鼠氣管干細(xì)胞 增殖 分化

【摘要】： 目的 目前,在氣管干細(xì)胞研究中,我們建立了氟脲嘧啶(Fluorouracil,5-Fu)引發(fā)離體氣管損傷修復(fù)模型,論證了氣管干細(xì)胞位于具有5-FU抗性的G0期細(xì)胞中。為闡明氣管干細(xì)胞增殖分化的調(diào)控機(jī)制,我們已檢測(cè)了wnt信號(hào)中的wnt1,β-catenin,cyclinD1等成員在氣管干細(xì)胞增殖分化過程中的時(shí)空變化情況,證明了Wnt/β-catenin信號(hào)傳導(dǎo)途徑參與調(diào)控氣管干細(xì)胞增殖分化全過程。另外,我們還觀察到了HDAC1在氣管干細(xì)胞增殖分化過程中的動(dòng)態(tài)表達(dá)情況。本文結(jié)合前段我研究組工作基礎(chǔ),利用TSA抑制HDAC1的作用,采用形態(tài)學(xué)、免疫組化及western blotting方琺觀察其在5-Fu模型中對(duì)氣管干細(xì)胞增殖分化的影響。 方法 1、制備氣管損傷模型及剝離上皮 取約200克左右的Wistar大鼠,雌雄不限(中國醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供),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(fù)沖洗,洗凈血液和粘液,置于DMEM/F12培養(yǎng)液中(含10%胎牛血清),于培養(yǎng)液中加入終濃度為100mg/ml的5-Fu,37℃,5%CO2孵育12小時(shí),棄去上述培養(yǎng)液,換成新鮮DMEM/F12液(含10%胎牛血清),同時(shí)加入終濃度為200nM的TSA繼續(xù)培養(yǎng),于換液后3、6、12、24、48小時(shí)分別10%中性福爾馬林固定,石蠟包埋,制成4μm厚的組織切片。同時(shí)各時(shí)間點(diǎn)取氣管組織,放于Eppendorf管中,-70℃保存。 2、HE染色動(dòng)態(tài)觀察各時(shí)間點(diǎn)氣管粘膜上皮組織學(xué)形態(tài)改變。 3、利用免疫組織化學(xué)染色觀察沒有TSA作用時(shí)HDAC1在5-Fu模型中的表達(dá)。 4、Western blotting檢測(cè)TSA對(duì)各時(shí)間點(diǎn)HDAC1的表達(dá)量變化的影響。 結(jié)果 1、HE:在沒有TSA作用情況下,去除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(xì)胞(即G0期細(xì)胞);去除5—FU后恢復(fù)3h,裸核細(xì)胞消失,細(xì)胞呈扁平狀,細(xì)胞核變長,細(xì)胞漿伸展幾乎可以將基底膜覆蓋;6h,細(xì)胞核變圓,有處可見變?yōu)榱⒎缴掀?細(xì)胞數(shù)增多;12小時(shí),上皮完全變?yōu)榱⒎缴掀?胞核增大,胞漿增多,上皮細(xì)胞數(shù)目繼續(xù)增多;恢復(fù)至24小時(shí),細(xì)胞表面出現(xiàn)多量纖毛;恢復(fù)48h,氣管上皮接近恢復(fù)假復(fù)層結(jié)構(gòu)。在有TSA作用情況下,去除5-Fu作用后0-12小時(shí),基底膜上殘留裸核細(xì)胞基本沒有變化,但是數(shù)量稍有增加;24-48小時(shí),裸核細(xì)胞消失,細(xì)胞成扁平狀,細(xì)胞核變長,細(xì)胞漿伸展覆蓋基底膜。 2、免疫組化檢測(cè)結(jié)果:HDAC1呈胞核陽性。無TSA作用時(shí)經(jīng)5-Fu作用后0小時(shí),未見HDAC1陽性細(xì)胞;去除5-Fu后3—6小時(shí),仍未見明顯HDAC1陽性細(xì)胞;12小時(shí),HDAC1陽性細(xì)胞數(shù)明顯增多,并可見幾個(gè)HDAC1陽性細(xì)胞圍繞著底部一個(gè)HDAC1陰性細(xì)胞和多個(gè)HDAC1陽性細(xì)胞間夾著幾個(gè)HDAC1陰性細(xì)胞分布的現(xiàn)象;24小時(shí),HDAC1陽性細(xì)胞數(shù)最多,累及全層。48小時(shí),HDAC1陽性細(xì)胞數(shù)略減少,陽性細(xì)胞分布集中于假復(fù)層結(jié)構(gòu)頂部即靠近管腔側(cè)。TSA作用后,基本沒有HDAC1陽性細(xì)胞出現(xiàn)。 3、Western blotting檢測(cè)結(jié)果:無TSA作用時(shí),HDAC1正常無表達(dá),去除5—FU后0小時(shí)無表達(dá),3、6、24小時(shí)表達(dá)量依次遞增,24小時(shí)達(dá)高峰,48小時(shí)表達(dá)量略減少。TSA作用時(shí),HDAC1表達(dá)明顯受到抑制。 結(jié)論 1、TSA可以阻斷經(jīng)過5-Fu處理后的氣管上皮損傷修復(fù)過程,抑制氣管干細(xì)胞的分化,造成氣管干細(xì)胞的增殖堆積。 2、本研究有可能為解決干細(xì)胞供體不足,提供一種新的方法,可能將對(duì)干細(xì)胞的來源和實(shí)際應(yīng)用帶來巨大優(yōu)勢(shì)。
[Abstract]:objective
At present, the tracheal stem cell research, we established the fluorouracil (Fluorouracil, 5-Fu) induced tracheal regeneration model, demonstrated that tracheal stem cells located with 5-FU resistant G0 cells. To elucidate the regulatory mechanism of tracheal stem cell proliferation and differentiation, we have detected the Wnt signal in Wnt1. Beta -catenin, cyclinD1 and other members of the tracheal stem cell proliferation and differentiation process of temporal and spatial change in the situation that Wnt/ beta -catenin signaling pathway involved in the regulation of cell proliferation and differentiation process of tracheal stem. In addition, we also observed the expression of HDAC1 in tracheal stem cell proliferation and differentiation in the dynamic process. This paper combined with the preceding research group I the work of the foundation, the inhibition of HDAC1 by TSA, the morphological, immunohistochemical and Western blotting methods to observe the effect of stem cells on proliferation and differentiation of trachea in 5-Fu model.
Method
1, the preparation of the model of tracheal injury and the exfoliation epithelium
Take about 200 grams of Wistar rats, male and female (provided by the experimental animal center of China Medical University), intraperitoneal injection of 10% chloral hydrate 0.4ml/100g, trachea removed under sterile conditions, aseptic PBS repeated washing, clean the blood and mucus in DMEM/F12 medium (containing 10% FBS), in the culture medium with a final the concentration of 100mg/ml 5-Fu, 37 C, 5%CO2 incubated for 12 hours, discard the culture liquid, replaced with fresh DMEM/F12 solution (containing 10% FBS), while continuing to cultivate 200nM TSA with the final concentration, to change liquid after 3,6,12,24,48 hours respectively in 10% neutral formalin fixed, paraffin embedded, made of 4 m thick tissue sections. At the same time each time point for tracheal tissue, put in the Eppendorf tube, -70 C preservation.
2, HE staining was used to observe the histological changes of tracheal epithelium at every time point.
3, immunohistochemical staining was used to observe the expression of HDAC1 in the 5-Fu model without the effect of TSA.
4, Western blotting detected the effect of TSA on the changes in the expression of HDAC1 at each time point.
Result
1, HE: without the effect of TSA, the removal of 5-Fu after 0h, most of the tracheal epithelial cells, residual naked nucleus intervals (G0 cells); removal of 5 - FU recovery after 3h, bare cells disappeared, the cells were flat, the nucleus becomes long, cytoplasm stretching almost the basement membrane covering; 6H, the nucleus became round, are visible into cuboidal epithelium, the number of cells increased; 12 hours, completely turned into cuboidal epithelium, enlarged nuclei, cytoplasm, epithelial cell number continues to increase; recovery to 24 hours, many ciliated cell surface; 48h recovery, tracheal epithelium to restore the pseudostratified structure. In the case of TSA, 0-12 hours after the removal of 5-Fu, the basement membrane residual bare cells did not change, but the number increased slightly; 24-48 hours, bare cells disappeared, flattened cells, the nucleus becomes long, cytoplasm extends over basal membrane.
2, the results of immunohistochemistry: the HDAC1 was located in the nucleus. TSA positive effect after 0 hours after 5-Fu, no HDAC1 positive cells; the removal of 3 - 6 hours after 5-Fu, still no obvious HDAC1 positive cells; 12 hours, the number of HDAC1 positive cells increased obviously, and visible a few HDAC1 positive cells around the bottom a negative HDAC1 cells and a number of HDAC1 positive cells between several negative cells distribution HDAC1; 24 hours, the number of HDAC1 positive cells, full-thickness.48 hours, the number of HDAC1 positive cells reduced slightly, the positive cells were concentrated on pseudostratified structure that is close to the top of the lumen side after.TSA, basic no HDAC1 positive cells appeared.
3, Western blotting test results: when there was no TSA action, HDAC1 was normal without expression. After 5 to FU removal, it didn't express at 0 hours, 3,6,24 hours increased gradually, reached peak at 24 hours, and decreased at 48 hours, HDAC1 expression was significantly inhibited when.TSA was used.
conclusion
1, TSA can block the repair process of tracheal epithelium injury after 5-Fu treatment, inhibit the differentiation of tracheal stem cells and cause the proliferation and accumulation of tracheal stem cells.
2, this study may provide a new way to solve the deficiency of stem cell donor, which may bring a great advantage to the source and practical application of stem cells.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

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本文編號(hào)：1358397