本文選題：氣管干細(xì)胞 + 5-FU�。� 參考：《中國醫(yī)科大學(xué)》2008年博士論文

【摘要】： 前言 成體干細(xì)胞數(shù)量稀少但對機(jī)體卻至關(guān)重要,它們是生后機(jī)體損傷修復(fù)、自我更新的重要來源。氣管上皮存在著氣管干細(xì)胞,它對氣管上皮的損傷修復(fù)和自我更新及多種呼吸系統(tǒng)疾病具有重要意義,但由于氣管上皮結(jié)構(gòu)的復(fù)雜性及所處微環(huán)境的特殊性,氣管干細(xì)胞的研究進(jìn)展極為緩慢。 利用經(jīng)典的5-FU打擊的方法,我們在體外建立了氣管上皮再生修復(fù)的模型,這一模型成功再現(xiàn)了氣管干細(xì)胞的增殖分化過程,使氣管干細(xì)胞增殖分化機(jī)制的初步研究成為可能。離體氣管在5-FU的作用下,增殖期細(xì)胞脫落,基底膜上僅殘余散在分布的GO期細(xì)胞;去除5-FU作用后,氣管上皮經(jīng)裸核樣細(xì)胞、扁平細(xì)胞、立方細(xì)胞,至48-72h時,基本恢復(fù)氣管上皮的假復(fù)層纖毛上皮正常結(jié)構(gòu)。可見,5-FU作用后的GO期細(xì)胞中含有氣管干細(xì)胞,氣管上皮的修復(fù)過程正是通過氣管干細(xì)胞的增殖分化完成的,先前的研究已經(jīng)證實(shí)了這一點(diǎn)。但我們對這一過程的調(diào)控機(jī)制還所知甚少。 Notch是脊椎動物和無脊椎動物發(fā)育過程中一類十分重要的信號受體蛋白家族,它通過與鄰近細(xì)胞間的相互作用來精確調(diào)控各譜系細(xì)胞的分化、增殖和凋亡,進(jìn)而決定細(xì)胞命運(yùn)和發(fā)育過程,其表達(dá)與功能具有組織特異性。近年來研究報道,Notch信號通路在干細(xì)胞增殖分化過程中起著重要的作用,Notch信號對氣管干細(xì)胞增殖分化的作用尚未闡明。 本研究利用5-FU介導(dǎo)的大鼠氣管上皮損傷修復(fù)模型,檢測了大鼠氣管干細(xì)胞增殖分化過程中Notch信號分子的表達(dá)及意義。 材料和方法 1、離體大鼠氣管損傷修復(fù)模型的制備及DAPT和Jag-1處理 取約200克左右的Wistar大鼠,雌雄不限,水合氯醛麻醉,無菌條件下取出氣管,PBS沖洗,置于DMEM/F12培養(yǎng)液中(含10%胎牛血清)。取正常氣管,剪取一氣管環(huán)固定,留做石蠟切片,灌入蛋白酶ⅩⅣ(0.5mg/ml),結(jié)扎另一端,4℃消化過夜,收集消化液,加FCS至終濃度為2.5%終止酶反應(yīng)收集正,常氣管上皮細(xì)胞于2mlEppendorf管中,-70℃凍存,留做mRNA。其余氣管組織分為DAPT預(yù)處理組、Jag-1預(yù)處理組、對照組、DAPT處理組和Jag-1處理組。DAPT及Jag-1預(yù)處理組先在培養(yǎng)液中加入5μM DAPT或40μM Jag-1,37℃,5%CO_2孵育12小時,棄去上述培養(yǎng)液,于DMEM/F12(含10%胎牛血清)培養(yǎng)液中加入終濃度為120mg/ml的5-FU,37℃,5%CO_2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);對照組單純5-FU作用;DAPT處理組和Jag-1處理組在5-FU作用后換為含有5μM DAPT或40μM Jag-1的DMEM/F12(含10%胎牛血清)繼續(xù)培養(yǎng),方法同上,分別于換液后0、3、6、12、24、48小時取出氣管組織分別固定,留做石蠟切片;消化,收集細(xì)胞于2mlEppendorf管中,—70℃保存,以備RNA和蛋白提取。 2、RT-PCR檢測 按Trizol~(TM)試劑使用說明提取氣管上皮細(xì)胞總RNA,檢測它的濃度,純度和完整性。按逆轉(zhuǎn)錄試劑盒使用說明操作,選用Oligo—dT做引物合成第一鏈cDNA,逆轉(zhuǎn)錄條件為:30℃10min,40℃40min,99℃5min,5℃5min;PCR反應(yīng)條件為:94℃變性2min后,94℃30s,退火,72℃1min,進(jìn)行30個循環(huán),最后于72℃延伸7min。擴(kuò)增產(chǎn)物使用2%瓊脂糖凝膠電泳,溴化乙啶染色分析表達(dá)結(jié)果,測灰度值,統(tǒng)計。相同條件下用β-actin作為內(nèi)對照。 3、蛋白印跡檢測 按蛋白抽提試劑盒說明抽提氣管上皮細(xì)胞總蛋白,SDS-PAGE電泳,50V恒壓濕轉(zhuǎn)120mins,BSA室溫封閉2hrs,一抗4℃孵育過夜,二抗37℃孵育2hrs,DAB顯色。轉(zhuǎn)膜后各步之間均用洗膜液洗膜3次,每次5mins。相同條件下用β-actin作為內(nèi)對照。 4、間接免疫熒光檢測氣管上皮ABCG2,CK19,Notch3和Jagged1的表達(dá) 連續(xù)切片,石蠟切片脫蠟至水,抗原修復(fù),非免疫動物血清封閉,一抗分別為Goat anti-ABCG2,Rabbit anti-CK19,Rabbit anti-Notch3和Goat anti-Jagged1;二抗分別TRITC標(biāo)記Rabbit anti Goat IgG和FITC標(biāo)記Goat anti Rabbit IgG。DAPI復(fù)染細(xì)胞核。50%緩沖甘油封片,熒光顯微鏡OlympasBX51下觀察并照相。陰性對照實(shí)驗(yàn):用等量的0.01mol/L PBS代替一抗,其余步驟同前。 5、統(tǒng)計分析 所有的RT-PCR實(shí)驗(yàn)和蛋白印跡實(shí)驗(yàn)均獨(dú)立重復(fù)三次,所得數(shù)據(jù)的值用均數(shù)(means)±標(biāo)準(zhǔn)偏差(SD)來表示。用SPSS 11.5軟件對所得數(shù)據(jù)進(jìn)行單因素方差分析,當(dāng)P＜0.05認(rèn)為有統(tǒng)計學(xué)意義。 結(jié)果 1、5-FU誘導(dǎo)損傷修復(fù)過程中ABCG2、CK19和PCNA的表達(dá)變化 正常氣管上皮幾乎沒有ABCG2表達(dá),去除5-FU作用后Oh,ABCG2少量表達(dá),隨后表達(dá)量逐漸增高,至6h達(dá)到峰值,隨后逐漸降低,至48h幾乎恢復(fù)正常水平;正常氣管上皮CK19高表達(dá),去除5-FU作用后Oh,CK19表達(dá)極微量,之后表達(dá)逐漸增加,至12h后CK19表達(dá)量迅速上升,至48h幾乎恢復(fù)正常水平;正常氣管上皮PCNA微量表達(dá),去除5-FU作用后Oh表達(dá)量仍然很低。3h后PCNA開始表達(dá)并逐漸增高,至6h達(dá)到峰值,隨后下降,至48h時,基本恢復(fù)正常水平。 2、Notch信號分子在大鼠氣管上皮的表達(dá) RT-PCR結(jié)果顯示,正常大鼠氣管上皮中Notch-1、Notch-3、Jagged-1、Hes1、Hey1和Hey2均有表達(dá);Notch-2、Jagged-2、Hes3和Hes5在正常氣管上皮及去除5-FU作用后氣管干細(xì)胞增殖分化過程中均沒有明顯表達(dá)。Notch-3、Jagged-1和Hey1在5-FU打擊修復(fù)過程中表達(dá)量有顯著差異。 3、5-FU誘導(dǎo)損傷修復(fù)過程中N3ICD,Jagged1及Hey1的表達(dá)變化 蛋白印跡檢測大鼠氣管上皮損傷修復(fù)過程中N3ICD,Jagged1及Hey1的蛋白水平發(fā)現(xiàn),正常氣管上皮N3ICD、Jagged-1和Heyl僅有微量表達(dá)。5-FU打擊后Oh,其表達(dá)增高,并隨著氣管上皮的修復(fù)逐漸增加,分別在6h、6h和12h達(dá)到高峰,隨后逐漸降低,至48-72h降至正常水平。 4、間接免疫熒光檢測5-FU作用后大鼠氣管上皮和Jagged1、Notch3、ABCG2和CK19的表達(dá) Jagged1為膜表達(dá),多表達(dá)于上皮基底側(cè)。打擊后Oh,氣管上皮可見少量Jagged1陽性細(xì)胞,隨氣管上皮的修復(fù)逐漸增多,至6h Jagged1陽性細(xì)胞最多,隨后Jagged1蛋白表達(dá)逐漸減少,至48h,接近恢復(fù)正常水平。正常氣管上皮Notch3絕大部分為膜陽性,5-FU打擊后Oh,Notch3核陽性細(xì)胞增多,至6h達(dá)到高峰,隨后降低,至48h僅見極少的核陽性細(xì)胞。大部分Jagged-1陽性細(xì)胞與Notch3核陽性細(xì)胞相鄰,并分布與氣管上皮的相似區(qū)域。ABCG2和CK19均為膜表達(dá),表達(dá)于氣管上皮不同的細(xì)胞,變化規(guī)律與Western blot結(jié)果一致,絕大部分ABCG2陽性細(xì)胞為Notch3核陽性細(xì)胞。 5、DAPT阻斷和Jag-1持續(xù)激活Notch信號通路對大鼠氣管上皮損傷修復(fù)的影響 免疫印跡結(jié)果顯示,DAPT處理后6小時Notch通路活性明顯低于對照組,24h時Notch通路幾乎完全失活。DAPT阻斷后,ABCG2增高幅度下降,并于6h后顯著低于對照組;K19表達(dá)持續(xù)增高,并于6h后顯著高于對照組,至24h時基本達(dá)到正常水平,并持續(xù)高表達(dá);PCNA增高幅度下降,6h后顯著低于對照組,至24h時已幾乎檢測不多PCNA的表達(dá)。Jag-1持續(xù)激活Notch通路,ABCG2增高幅度升高,并于6h后顯著高于對照組;CK19表達(dá)降低,并于6h后顯著低于對照組;PCNA增高幅度上升,6h后顯著高于對照組。 HE染色顯示,5-FU誘導(dǎo)損傷后,用DAPT阻斷Notch通路,與單純5-FU作用的對照組相比,氣管上皮的形態(tài)于6h開始出現(xiàn)差異。此時,大部分氣管上皮細(xì)胞已呈立方狀;12h,可見纖毛出現(xiàn)在氣管上皮;12h—48h細(xì)胞沒有明顯增加,氣管上皮呈單層立法上皮,被覆纖毛。用Jag-1持續(xù)激活Ntoch通路的情況下,細(xì)胞增加迅速,但始終未見纖毛出現(xiàn),48h后,氣管上皮沒有恢復(fù)假覆層纖毛柱狀上皮的結(jié)構(gòu)。 6、DAPT及Jag-1預(yù)處理對大鼠氣管上皮損傷修復(fù)的影響 正常氣管上皮經(jīng)DAPT預(yù)處理后,細(xì)胞數(shù)量明顯減少。再經(jīng)5-FU作用后,氣管上皮細(xì)胞幾乎全部脫落,沒有細(xì)胞殘留。經(jīng)48h后,氣管上皮仍沒有修復(fù)的跡象。正常大鼠氣管上皮經(jīng)Jag-1預(yù)處理后,纖毛細(xì)胞明顯減少。再經(jīng)5-FU作用后,氣管基底膜上殘留細(xì)胞數(shù)量增加,并于24h時基本恢復(fù)了假覆層纖毛柱狀上皮的結(jié)構(gòu)。 免疫熒光顯示,正常氣管上皮ABCG2(+)細(xì)胞數(shù)量稀少;經(jīng)DAPT預(yù)處理后,氣管上皮未見ABCG2陽性細(xì)胞。經(jīng)Jag-1預(yù)處理后,氣管上皮ABCG2陽性細(xì)胞數(shù)量增加。 結(jié)論 1、5-FU作用后48h,氣管上皮形態(tài)及增殖分化狀態(tài)基本恢復(fù)正常水平; 2、Notch-1、Notch-3、Jagged-1、Hes1、Hey1和Hey2可能參與了正常大鼠氣管上皮穩(wěn)態(tài)的維持; 3、Notch-3、Jagged-1和Hey1可能在氣管干細(xì)胞增殖分化的過程中發(fā)揮了作用; 4、Notch信號對氣管干細(xì)胞未分化狀態(tài)的維持及增殖起著重要的作用,是氣管上皮損傷修復(fù)所不可缺少的。
[Abstract]:Preface
The number of adult stem cells is scarce, but it is vital to the body. They are an important source of injury repair and self renewal after birth. Tracheal stem cells exist in the tracheal epithelium. It is of great significance for the repair of injury and self renewal of tracheal epithelium and a variety of respiratory diseases. The research progress of tracheal stem cells is very slow due to the particularity of the environment.
Using the classical 5-FU strike method, we have established a model of regenerative repair of tracheal epithelium in vitro. This model successfully reproduces the proliferation and differentiation of the tracheal stem cells and makes the preliminary study of the mechanism of the proliferation and differentiation of the tracheal stem cells. The isolated trachea falls off in the proliferative phase and only remains on the basement membrane under the action of 5-FU. In the distributed GO phase cells; after the removal of 5-FU, the normal structure of the false stratified ciliated epithelium of the tracheal epithelium is basically restored when the tracheal epithelium is exposed to nude nucleus like cells, flat cells, and cubic cells to 48-72h. It is visible that the GO cells after the action of 5-FU contain tracheal stem cells, and the repair process of the tracheal epithelium is through the proliferation of the tracheal stem cells. Differentiation has been confirmed by previous studies, but little is known about the regulatory mechanism of this process.
Notch is a very important family of signal receptor protein in the development of vertebrates and invertebrates. It regulates the differentiation, proliferation and apoptosis of various lineages through the interaction with adjacent cells, and then determines the cell fate and development process. Its expression and function are tissue specific. In recent years, the study has been reported. Notch signaling pathway plays an important role in the proliferation and differentiation of stem cells. The role of Notch signaling in the proliferation and differentiation of tracheal stem cells has not yet been elucidated.
In this study, the 5-FU mediated damage repair model of rat tracheal epithelium was used to detect the expression and significance of Notch signal molecules during the proliferation and differentiation of rat tracheal stem cells.
Materials and methods
1, preparation of rat model of tracheal injury in vitro and DAPT and Jag-1 treatment.
About 200 grams of Wistar rats were taken, both male and female, anaesthetized with chloral hydrate, under the aseptic condition of chloral anaesthesia, taken out of the trachea under aseptic conditions, PBS flushed and placed in the DMEM/F12 culture (containing 10% fetal bovine serum). The normal trachea was taken, a tube ring was cut, the paraffin section was cut, the proteinase IV (0.5mg/ml) was poured into the other end, the digestive juice was digested at 4 degrees, and the digestive juice was collected, and FCS was collected. The terminal concentration was 2.5% terminating enzyme reaction collection positive, often tracheal epithelial cells in the 2mlEppendorf tube, -70 centigrade cryopreservation, remaining mRNA. other trachea tissues to be divided into DAPT preconditioning group, Jag-1 preconditioning group, control group, DAPT treatment group and Jag-1 treatment group.DAPT and Jag-1 preconditioning group first added 5 mu M DAPT or 40 micron M to incubate. After 12 hours, the above culture solution was abandoned, and DMEM/F12 (containing 10% fetal bovine serum) was added to the medium of 120mg/ml, 37 and 5%CO_2 for 12 hours. The above culture solution was abandoned and replaced with fresh DMEM/F12 solution (containing 10% fetal bovine serum); the control group was pure 5-FU; the DAPT treatment group and Jag-1 treatment group changed to 5 mu M DAPT after 5-FU. Or 40 M Jag-1 of DMEM/F12 (containing 10% fetal bovine serum) continue to culture, the method is the same, respectively, after the exchange of liquid 0,3,6,12,24,48 hours removed trachea tissue to be fixed, left for paraffin section, digestion, collected cells in the 2mlEppendorf tube, - 70 C preservation, in order to prepare RNA and protein extraction.
2, RT-PCR detection
According to the Trizol~ (TM) reagent, the total RNA of tracheal epithelial cells was extracted, and its concentration, purity and integrity were detected. The first chain cDNA was synthesized by using Oligo dT as primers according to the operation of the reverse transcriptase kit. The reverse transcriptase conditions were: 30 C 10min, 40 C 40min, 99 C 5min, 5 C 5min, and the PCR reaction conditions were 94 degrees C after 2min, annealing and annealing. At 72 1min, 30 cycles were carried out. Finally, the 7min. amplification products were extended by 2% agarose gel electrophoresis, and the expression results were analyzed with ethidium bromide staining, and the gray value was measured. Under the same condition, the beta -actin was used as the internal control.
3, Western blot detection
According to the protein extraction kit, the total protein of tracheal epithelial cells was extracted, SDS-PAGE electrophoresis, 50V constant pressure wet 120mins, BSA closed 2hrs at room temperature, one anti 4 centigrade incubation for night, two anti 37 C for incubating 2hrs, DAB coloration. All the steps after the transfer of membrane were washed with membrane solution for 3 times, and every time 5mins. phase was used as internal control with beta -actin.
4, indirect immunofluorescence was used to detect the expression of ABCG2, CK19, Notch3 and Jagged1 in tracheal epithelium.
Continuous section, paraffin section dewaxing to water, antigen repair, non immune animal sera closed, one resistance was Goat anti-ABCG2, Rabbit anti-CK19, Rabbit anti-Notch3 and Goat anti-Jagged1; two anti TRITC labeled Rabbit anti Goat. Negative control experiments were carried out under the microscope OlympasBX51. The equivalent 0.01mol/L PBS was used instead of the first antibody and the rest steps were the same as before.
5, statistical analysis
All RT-PCR experiments and Western blot experiments were repeated three times independently. The value of the obtained data was expressed with the mean number (means) + standard deviation (SD). The data were analyzed with SPSS 11.5 software for single factor analysis of variance, when P < 0.05 thought there was statistical significance.
Result
Expression changes of ABCG2, CK19 and PCNA in 1,5-FU induced injury repair process
There was almost no expression of ABCG2 in normal tracheal epithelium. After the removal of 5-FU, Oh, ABCG2 was expressed in a small amount, and then the expression amount increased gradually, to the peak value to 6h, and then gradually decreased to 48h to the normal level. The normal tracheal epithelium was highly expressed, and the expression of 5-FU was very small after removal of 5-FU, and then the expression gradually increased, to 12h CK19 expression. Rapid rise, to 48h almost normal level, the normal tracheal epithelium PCNA micro expression, the removal of 5-FU after the removal of the Oh expression is still very low.3h PCNA began to express and gradually increase, to the peak to the peak, then decreased, to 48h, basically restore normal level.
2, expression of Notch signal molecules in the tracheal epithelium of rats
RT-PCR results showed that Notch-1, Notch-3, Jagged-1, Hes1, Hey1 and Hey2 were expressed in the normal rats' tracheal epithelium, and Notch-2, Jagged-2, Hes3 and Hes5 were not clearly expressed during the proliferation and differentiation of tracheal stem cells after the normal tracheal epithelium and the removal of 5-FU. Difference.
Expression changes of N3ICD, Jagged1 and Hey1 in 3,5-FU induced injury repair process
The protein level of N3ICD, Jagged1 and Hey1 in the repair of tracheal epithelium in rats was detected by Western blot. It was found that the normal tracheal epithelium, N3ICD, Jagged-1 and Heyl only expressed.5-FU after the attack of Oh, its expression increased, and gradually increased with the repair of the tracheal epithelium, and reached the peak at 6h, 6h and 12h, and then gradually decreased to 48-72h to decrease. Normal level.
4, indirect immunofluorescence was used to detect the expression of Jagged1, Notch3, ABCG2 and CK19 in rat tracheal epithelium after 5-FU treatment.
The expression of Jagged1 was more expressed on the basal side of the epithelium. After Oh, a small amount of Jagged1 positive cells were found in the tracheal epithelium. With the repair of the tracheal epithelium, the number of 6h Jagged1 positive cells was the most, and the expression of Jagged1 protein decreased gradually, to 48h, close to the normal level. Most of the normal tracheal epithelium was membrane positive, 5-FU beating. After the attack, Oh, Notch3 nuclear positive cells increased, and reached the peak to 6h, then decreased, and only a few nuclear positive cells were found at 48h. Most of the Jagged-1 positive cells were adjacent to Notch3 nuclear positive cells, and the distribution of.ABCG2 and CK19 in the similar regions of the tracheal epithelium were all membrane expressions, expressed in different cells of the tracheal epithelium, and the regularity of change and Western blot The results showed that the majority of ABCG2 positive cells were Notch3 positive cells.
5, the effects of DAPT blocking and Jag-1 continuously activated Notch signaling pathway on the repair of rat tracheal epithelium.
The results of immunoblotting showed that the activity of Notch pathway at 6 hours after DAPT treatment was significantly lower than that of the control group. When the Notch pathway was almost completely inactivated by.DAPT, the ABCG2 increased significantly and was significantly lower than the control group after 6h. The expression of K19 was significantly higher than that of the control group. After 6h, the expression of K19 was significantly higher than that of the control group. The level of K19 was higher than that of the control group after 6h, and it was basically reached the normal level and continued high expression at 24h. The increase of PCNA was significantly lower than that of the control group after 6h. At the time of 24h, almost no PCNA expression was detected by.Jag-1 to continue to activate Notch pathway, ABCG2 increased significantly, and significantly higher than that of the control group after 6h, and the expression of CK19 decreased significantly after 6h, and was significantly lower than the control group after 6h; PCNA increase was increased, after 6h was significantly higher than the control group.
HE staining showed that after 5-FU induced injury, the Notch pathway was blocked by DAPT. Compared with the control group with simple 5-FU, the morphology of the tracheal epithelium began to differ from 6h. At this time, most of the tracheal epithelial cells were cubic; 12h, cilia appeared in the trachea epithelium, 12h 48h cells did not increase obviously, and the tracheal epithelium was single layer legislative epithelium. Under the condition of continuous activation of the Ntoch pathway with Jag-1, the cells increased rapidly, but no cilia appeared. After 48h, the tracheal epithelium did not restore the structure of the false cladding ciliated columnar epithelium.
6, the effects of DAPT and Jag-1 pretreatment on the repair of rat tracheal epithelium.
After DAPT preconditioning, the number of cells in the normal tracheal epithelium decreased obviously. After 5-FU action, the tracheal epithelial cells almost all fell off and no cells remained. After 48h, the tracheal epithelium was still no sign of repair. The tracheal epithelium of normal rats was reduced obviously after Jag-1 preconditioning. After 5-FU action, the tracheal basal membrane was disabled. The number of retained cells increased, and the structure of the ciliated columnar epithelium was basically restored at 24h.
Immunofluorescence showed that the number of ABCG2 (+) cells in normal tracheal epithelium was rare. After DAPT preconditioning, no ABCG2 positive cells were found in the tracheal epithelium. The number of ABCG2 positive cells in the tracheal epithelium increased after Jag-1 preconditioning.
conclusion
After 1,5-FU, the morphology, proliferation and differentiation of tracheal epithelium in 48h basically returned to normal level.
2, Notch-1, Notch-3, Jagged-1, Hes1, Hey1 and Hey2 may be involved in maintaining the homeostasis of tracheal epithelium in normal rats.
3, Notch-3, Jagged-1 and Hey1 may play an important role in the proliferation and differentiation of tracheal stem cells.
4, Notch signaling plays an important role in the maintenance and proliferation of tracheal stem cells, and is indispensable for the repair of tracheal epithelium.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 史冀鵬;離心運(yùn)動對骨骼肌生肌調(diào)節(jié)因子的影響及針刺干預(yù)研究[D];北京體育大學(xué);2011年

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本文編號：2051925