目的 研究Notch的配体Dlk1和Jagged1分别激活Notch信号通路后对大鼠肺泡Ⅱ型上皮细胞(AECⅡ)转分化及增殖的影响.方法 在细胞培养体系中,分别加入重组蛋白Dlk1和重组人核转录因子-κB(rhNF-κB) (Jagged1激活剂),止常组加入含120 mL/L胎牛血清的DMEM培养基作为对照,光镜和电镜下观察48、72、96 h时间点AECⅡ增殖和转分化状况.血细胞计数器计数细胞;四甲基偶氮唑盐(MTT)比色法检测细胞增殖能力;免疫荧光双标法检测AECⅡ特异性肺泡表面活性蛋白C(SP-C)及肺泡Ⅰ型上皮细胞(AEC Ⅰ)特异性水通道蛋白5 (AQP5)的表达;时实荧光定量PCR检测转分化前后各时间点Notch配体Dlk1和Jagged1、通路活化标志Notch1、靶基因Hesl及转分化标志物SP-C、AQP5 mRNA的表达变化.结果 细胞数及增殖能力:与正常组比较,Dlk1组细胞数及增殖能力明显提高[细胞数(×109/L) 9.05±0.45比7.95±0.65,11.68±0.43比8.68±0.52,11.55 ±0.17比8.73±0.48,P均＜0.05;MTT结果(A值)0.699±0.050比0.462±0.080,0.912±0.080比0.535±0.040,0.726±0.050比0.540±0.020,P均＜0.05],转分化速度减慢;rhNF-κB组细胞数及增殖能力降低[细胞数(×109/L) 4.95±0.33比7.95 ±0.65,4.73±0.71比8.68±0.52,4.04 ±0.11比8.73±0.48,P均＜0.05;细胞增殖能力0.398±0.030比0.462±0.080,0.402±0.070比0.535±0.040,0.380±0.110比0.540±0.020,P均＜0.05],转分化速度加快;单因素方差分析3组细胞之间的差别有统计学意义(细胞数:F=486.73,P=0.02;细胞增殖能力:F=37.16,P=0.02).mRNA表达:与正常组比较,Dlk1组SP-C mRNA表达明显增多(P＜0.05),AQP5 mRNA表达延迟且明显减少(P＜0.05),Jagged1 mRNA始终呈弱表达或不表达,Dlk1、Notch1 mRNA表达增多(P＜0.05),Hes1 mRNA表达减少(P＜ 0.05);rhNF-κB组SP-CmR NA表达明显降低(P＜0.05),AQP5 mRNA表达提前,且增多(P＜0.05),Dlk1 mRNA弱表达或不表达,Jag-ged1、Hes1、Notch1 mRNA表达增强(P＜0.05).单因素方差分析3组细胞之间SP-C、AQP5、Dlk1、Jagged1、Hes1、Notch1 mRNA的表达差异统计学意义(F=96.80,P=0.01;F=82.55,P=0.01;F =269.80,P=0.00;F=312.34,P=0.00;F=169.17,P=0.01;F=19.85,P=0.02).结论 不同配体激活Notch信号通路后,对AECⅡ转分化和增殖有不同影响,Dlk1能促进增殖,抑制转分化,而Jagged1则抑制增殖,促进转分化.
目的 研究Notch的配體Dlk1和Jagged1分彆激活Notch信號通路後對大鼠肺泡Ⅱ型上皮細胞(AECⅡ)轉分化及增殖的影響.方法 在細胞培養體繫中,分彆加入重組蛋白Dlk1和重組人覈轉錄因子-κB(rhNF-κB) (Jagged1激活劑),止常組加入含120 mL/L胎牛血清的DMEM培養基作為對照,光鏡和電鏡下觀察48、72、96 h時間點AECⅡ增殖和轉分化狀況.血細胞計數器計數細胞;四甲基偶氮唑鹽(MTT)比色法檢測細胞增殖能力;免疫熒光雙標法檢測AECⅡ特異性肺泡錶麵活性蛋白C(SP-C)及肺泡Ⅰ型上皮細胞(AEC Ⅰ)特異性水通道蛋白5 (AQP5)的錶達;時實熒光定量PCR檢測轉分化前後各時間點Notch配體Dlk1和Jagged1、通路活化標誌Notch1、靶基因Hesl及轉分化標誌物SP-C、AQP5 mRNA的錶達變化.結果 細胞數及增殖能力:與正常組比較,Dlk1組細胞數及增殖能力明顯提高[細胞數(×109/L) 9.05±0.45比7.95±0.65,11.68±0.43比8.68±0.52,11.55 ±0.17比8.73±0.48,P均＜0.05;MTT結果(A值)0.699±0.050比0.462±0.080,0.912±0.080比0.535±0.040,0.726±0.050比0.540±0.020,P均＜0.05],轉分化速度減慢;rhNF-κB組細胞數及增殖能力降低[細胞數(×109/L) 4.95±0.33比7.95 ±0.65,4.73±0.71比8.68±0.52,4.04 ±0.11比8.73±0.48,P均＜0.05;細胞增殖能力0.398±0.030比0.462±0.080,0.402±0.070比0.535±0.040,0.380±0.110比0.540±0.020,P均＜0.05],轉分化速度加快;單因素方差分析3組細胞之間的差彆有統計學意義(細胞數:F=486.73,P=0.02;細胞增殖能力:F=37.16,P=0.02).mRNA錶達:與正常組比較,Dlk1組SP-C mRNA錶達明顯增多(P＜0.05),AQP5 mRNA錶達延遲且明顯減少(P＜0.05),Jagged1 mRNA始終呈弱錶達或不錶達,Dlk1、Notch1 mRNA錶達增多(P＜0.05),Hes1 mRNA錶達減少(P＜ 0.05);rhNF-κB組SP-CmR NA錶達明顯降低(P＜0.05),AQP5 mRNA錶達提前,且增多(P＜0.05),Dlk1 mRNA弱錶達或不錶達,Jag-ged1、Hes1、Notch1 mRNA錶達增彊(P＜0.05).單因素方差分析3組細胞之間SP-C、AQP5、Dlk1、Jagged1、Hes1、Notch1 mRNA的錶達差異統計學意義(F=96.80,P=0.01;F=82.55,P=0.01;F =269.80,P=0.00;F=312.34,P=0.00;F=169.17,P=0.01;F=19.85,P=0.02).結論 不同配體激活Notch信號通路後,對AECⅡ轉分化和增殖有不同影響,Dlk1能促進增殖,抑製轉分化,而Jagged1則抑製增殖,促進轉分化.
목적 연구Notch적배체Dlk1화Jagged1분별격활Notch신호통로후대대서폐포Ⅱ형상피세포(AECⅡ)전분화급증식적영향.방법 재세포배양체계중,분별가입중조단백Dlk1화중조인핵전록인자-κB(rhNF-κB) (Jagged1격활제),지상조가입함120 mL/L태우혈청적DMEM배양기작위대조,광경화전경하관찰48、72、96 h시간점AECⅡ증식화전분화상황.혈세포계수기계수세포;사갑기우담서염(MTT)비색법검측세포증식능력;면역형광쌍표법검측AECⅡ특이성폐포표면활성단백C(SP-C)급폐포Ⅰ형상피세포(AEC Ⅰ)특이성수통도단백5 (AQP5)적표체;시실형광정량PCR검측전분화전후각시간점Notch배체Dlk1화Jagged1、통로활화표지Notch1、파기인Hesl급전분화표지물SP-C、AQP5 mRNA적표체변화.결과 세포수급증식능력:여정상조비교,Dlk1조세포수급증식능력명현제고[세포수(×109/L) 9.05±0.45비7.95±0.65,11.68±0.43비8.68±0.52,11.55 ±0.17비8.73±0.48,P균＜0.05;MTT결과(A치)0.699±0.050비0.462±0.080,0.912±0.080비0.535±0.040,0.726±0.050비0.540±0.020,P균＜0.05],전분화속도감만;rhNF-κB조세포수급증식능력강저[세포수(×109/L) 4.95±0.33비7.95 ±0.65,4.73±0.71비8.68±0.52,4.04 ±0.11비8.73±0.48,P균＜0.05;세포증식능력0.398±0.030비0.462±0.080,0.402±0.070비0.535±0.040,0.380±0.110비0.540±0.020,P균＜0.05],전분화속도가쾌;단인소방차분석3조세포지간적차별유통계학의의(세포수:F=486.73,P=0.02;세포증식능력:F=37.16,P=0.02).mRNA표체:여정상조비교,Dlk1조SP-C mRNA표체명현증다(P＜0.05),AQP5 mRNA표체연지차명현감소(P＜0.05),Jagged1 mRNA시종정약표체혹불표체,Dlk1、Notch1 mRNA표체증다(P＜0.05),Hes1 mRNA표체감소(P＜ 0.05);rhNF-κB조SP-CmR NA표체명현강저(P＜0.05),AQP5 mRNA표체제전,차증다(P＜0.05),Dlk1 mRNA약표체혹불표체,Jag-ged1、Hes1、Notch1 mRNA표체증강(P＜0.05).단인소방차분석3조세포지간SP-C、AQP5、Dlk1、Jagged1、Hes1、Notch1 mRNA적표체차이통계학의의(F=96.80,P=0.01;F=82.55,P=0.01;F =269.80,P=0.00;F=312.34,P=0.00;F=169.17,P=0.01;F=19.85,P=0.02).결론 불동배체격활Notch신호통로후,대AECⅡ전분화화증식유불동영향,Dlk1능촉진증식,억제전분화,이Jagged1칙억제증식,촉진전분화.
Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.
