大鼠气管干细胞增殖分化过程中Notch信号分子的表达及意义

发布时间：2018-06-22 06:27

本文选题：气管干细胞 + 5-FU　；参考：《中国医科大学》2008年博士论文

【摘要】： 前言成体干细胞数量稀少但对机体却至关重要,它们是生后机体损伤修复、自我更新的重要来源。气管上皮存在着气管干细胞,它对气管上皮的损伤修复和自我更新及多种呼吸系统疾病具有重要意义,但由于气管上皮结构的复杂性及所处微环境的特殊性,气管干细胞的研究进展极为缓慢。利用经典的5-FU打击的方法,我们在体外建立了气管上皮再生修复的模型,这一模型成功再现了气管干细胞的增殖分化过程,使气管干细胞增殖分化机制的初步研究成为可能。离体气管在5-FU的作用下,增殖期细胞脱落,基底膜上仅残余散在分布的GO期细胞;去除5-FU作用后,气管上皮经裸核样细胞、扁平细胞、立方细胞,至48-72h时,基本恢复气管上皮的假复层纤毛上皮正常结构。可见,5-FU作用后的GO期细胞中含有气管干细胞,气管上皮的修复过程正是通过气管干细胞的增殖分化完成的,先前的研究已经证实了这一点。但我们对这一过程的调控机制还所知甚少。 Notch是脊椎动物和无脊椎动物发育过程中一类十分重要的信号受体蛋白家族,它通过与邻近细胞间的相互作用来精确调控各谱系细胞的分化、增殖和凋亡,进而决定细胞命运和发育过程,其表达与功能具有组织特异性。近年来研究报道,Notch信号通路在干细胞增殖分化过程中起着重要的作用,Notch信号对气管干细胞增殖分化的作用尚未阐明。本研究利用5-FU介导的大鼠气管上皮损伤修复模型,检测了大鼠气管干细胞增殖分化过程中Notch信号分子的表达及意义。材料和方法 1、离体大鼠气管损伤修复模型的制备及DAPT和Jag-1处理取约200克左右的Wistar大鼠,雌雄不限,水合氯醛麻醉,无菌条件下取出气管,PBS冲洗,置于DMEM/F12培养液中(含10%胎牛血清)。取正常气管,剪取一气管环固定,留做石蜡切片,灌入蛋白酶ⅩⅣ(0.5mg/ml),结扎另一端,4℃消化过夜,收集消化液,加FCS至终浓度为2.5%终止酶反应收集正,常气管上皮细胞于2mlEppendorf管中,-70℃冻存,留做mRNA。其余气管组织分为DAPT预处理组、Jag-1预处理组、对照组、DAPT处理组和Jag-1处理组。DAPT及Jag-1预处理组先在培养液中加入5μM DAPT或40μM Jag-1,37℃,5%CO_2孵育12小时,弃去上述培养液,于DMEM/F12(含10%胎牛血清)培养液中加入终浓度为120mg/ml的5-FU,37℃,5%CO_2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);对照组单纯5-FU作用;DAPT处理组和Jag-1处理组在5-FU作用后换为含有5μM DAPT或40μM Jag-1的DMEM/F12(含10%胎牛血清)继续培养,方法同上,分别于换液后0、3、6、12、24、48小时取出气管组织分别固定,留做石蜡切片;消化,收集细胞于2mlEppendorf管中,—70℃保存,以备RNA和蛋白提取。 2、RT-PCR检测按Trizol~(TM)试剂使用说明提取气管上皮细胞总RNA,检测它的浓度,纯度和完整性。按逆转录试剂盒使用说明操作,选用Oligo—dT做引物合成第一链cDNA,逆转录条件为:30℃10min,40℃40min,99℃5min,5℃5min;PCR反应条件为:94℃变性2min后,94℃30s,退火,72℃1min,进行30个循环,最后于72℃延伸7min。扩增产物使用2%琼脂糖凝胶电泳,溴化乙啶染色分析表达结果,测灰度值,统计。相同条件下用β-actin作为内对照。 3、蛋白印迹检测按蛋白抽提试剂盒说明抽提气管上皮细胞总蛋白,SDS-PAGE电泳,50V恒压湿转120mins,BSA室温封闭2hrs,一抗4℃孵育过夜,二抗37℃孵育2hrs,DAB显色。转膜后各步之间均用洗膜液洗膜3次,每次5mins。相同条件下用β-actin作为内对照。 4、间接免疫荧光检测气管上皮ABCG2,CK19,Notch3和Jagged1的表达连续切片,石蜡切片脱蜡至水,抗原修复,非免疫动物血清封闭,一抗分别为Goat anti-ABCG2,Rabbit anti-CK19,Rabbit anti-Notch3和Goat anti-Jagged1;二抗分别TRITC标记Rabbit anti Goat IgG和FITC标记Goat anti Rabbit IgG。DAPI复染细胞核。50%缓冲甘油封片,荧光显微镜OlympasBX51下观察并照相。阴性对照实验:用等量的0.01mol/L PBS代替一抗,其余步骤同前。 5、统计分析所有的RT-PCR实验和蛋白印迹实验均独立重复三次,所得数据的值用均数(means)±标准偏差(SD)来表示。用SPSS 11.5软件对所得数据进行单因素方差分析,当P＜0.05认为有统计学意义。结果 1、5-FU诱导损伤修复过程中ABCG2、CK19和PCNA的表达变化正常气管上皮几乎没有ABCG2表达,去除5-FU作用后Oh,ABCG2少量表达,随后表达量逐渐增高,至6h达到峰值,随后逐渐降低,至48h几乎恢复正常水平;正常气管上皮CK19高表达,去除5-FU作用后Oh,CK19表达极微量,之后表达逐渐增加,至12h后CK19表达量迅速上升,至48h几乎恢复正常水平;正常气管上皮PCNA微量表达,去除5-FU作用后Oh表达量仍然很低。3h后PCNA开始表达并逐渐增高,至6h达到峰值,随后下降,至48h时,基本恢复正常水平。 2、Notch信号分子在大鼠气管上皮的表达 RT-PCR结果显示,正常大鼠气管上皮中Notch-1、Notch-3、Jagged-1、Hes1、Hey1和Hey2均有表达;Notch-2、Jagged-2、Hes3和Hes5在正常气管上皮及去除5-FU作用后气管干细胞增殖分化过程中均没有明显表达。Notch-3、Jagged-1和Hey1在5-FU打击修复过程中表达量有显著差异。 3、5-FU诱导损伤修复过程中N3ICD,Jagged1及Hey1的表达变化蛋白印迹检测大鼠气管上皮损伤修复过程中N3ICD,Jagged1及Hey1的蛋白水平发现,正常气管上皮N3ICD、Jagged-1和Heyl仅有微量表达。5-FU打击后Oh,其表达增高,并随着气管上皮的修复逐渐增加,分别在6h、6h和12h达到高峰,随后逐渐降低,至48-72h降至正常水平。 4、间接免疫荧光检测5-FU作用后大鼠气管上皮和Jagged1、Notch3、ABCG2和CK19的表达 Jagged1为膜表达,多表达于上皮基底侧。打击后Oh,气管上皮可见少量Jagged1阳性细胞,随气管上皮的修复逐渐增多,至6h Jagged1阳性细胞最多,随后Jagged1蛋白表达逐渐减少,至48h,接近恢复正常水平。正常气管上皮Notch3绝大部分为膜阳性,5-FU打击后Oh,Notch3核阳性细胞增多,至6h达到高峰,随后降低,至48h仅见极少的核阳性细胞。大部分Jagged-1阳性细胞与Notch3核阳性细胞相邻,并分布与气管上皮的相似区域。ABCG2和CK19均为膜表达,表达于气管上皮不同的细胞,变化规律与Western blot结果一致,绝大部分ABCG2阳性细胞为Notch3核阳性细胞。 5、DAPT阻断和Jag-1持续激活Notch信号通路对大鼠气管上皮损伤修复的影响免疫印迹结果显示,DAPT处理后6小时Notch通路活性明显低于对照组,24h时Notch通路几乎完全失活。DAPT阻断后,ABCG2增高幅度下降,并于6h后显著低于对照组;K19表达持续增高,并于6h后显著高于对照组,至24h时基本达到正常水平,并持续高表达;PCNA增高幅度下降,6h后显著低于对照组,至24h时已几乎检测不多PCNA的表达。Jag-1持续激活Notch通路,ABCG2增高幅度升高,并于6h后显著高于对照组;CK19表达降低,并于6h后显著低于对照组;PCNA增高幅度上升,6h后显著高于对照组。 HE染色显示,5-FU诱导损伤后,用DAPT阻断Notch通路,与单纯5-FU作用的对照组相比,气管上皮的形态于6h开始出现差异。此时,大部分气管上皮细胞已呈立方状;12h,可见纤毛出现在气管上皮;12h—48h细胞没有明显增加,气管上皮呈单层立法上皮,被覆纤毛。用Jag-1持续激活Ntoch通路的情况下,细胞增加迅速,但始终未见纤毛出现,48h后,气管上皮没有恢复假覆层纤毛柱状上皮的结构。 6、DAPT及Jag-1预处理对大鼠气管上皮损伤修复的影响正常气管上皮经DAPT预处理后,细胞数量明显减少。再经5-FU作用后,气管上皮细胞几乎全部脱落,没有细胞残留。经48h后,气管上皮仍没有修复的迹象。正常大鼠气管上皮经Jag-1预处理后,纤毛细胞明显减少。再经5-FU作用后,气管基底膜上残留细胞数量增加,并于24h时基本恢复了假覆层纤毛柱状上皮的结构。免疫荧光显示,正常气管上皮ABCG2(+)细胞数量稀少;经DAPT预处理后,气管上皮未见ABCG2阳性细胞。经Jag-1预处理后,气管上皮ABCG2阳性细胞数量增加。结论 1、5-FU作用后48h,气管上皮形态及增殖分化状态基本恢复正常水平; 2、Notch-1、Notch-3、Jagged-1、Hes1、Hey1和Hey2可能参与了正常大鼠气管上皮稳态的维持; 3、Notch-3、Jagged-1和Hey1可能在气管干细胞增殖分化的过程中发挥了作用; 4、Notch信号对气管干细胞未分化状态的维持及增殖起着重要的作用,是气管上皮损伤修复所不可缺少的。
[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.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【引证文献】