Dlkl、Jaggedl基因在大鼠肺泡Ⅱ型上皮细胞增殖及转分化中的作用

Effect of Dlkl and Jaggedl gene on the proliferation and transdifferentiation of the primary type Ⅱ alveolar epithelial cells

目的研究Notch的配体Dlkl和Jaggedl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Dlkl和重组人核转录因子-κB（rhNF-κB）（Jaggedl激活剂），正常组加入含120mL／L胎牛血清的DMEM培养基作为对照，光镜和电镜下观察48、72、96h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（M1Tr）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（AECI）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Dlkl和Jaggedl、通路活化标志Notchl、靶基因Hesl及转分化标志物SP-C、AQP5mRNA的表达变化。结果细胞数及增殖能力：与正常组比较，Dlkl组细胞数及增殖能力明显提高[细胞数（×10^9／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-t-O．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（×10^9／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，Dlkl组SP-CmRNA表达明显增多（P〈0．05），AQPSmRNA表达延迟且明显减少（P〈0．05），JaggedlmRNA始终呈弱表达或不表达，Dlkl、NotchlmRNA表达增多（P〈0．05），HeslmRNA表达减少（P〈0．05）；rhNF-KB组SP—CmRNA表达明显降低（P〈0．05），AQP5mRNA表达提前，且增多（P〈0．05），DlklmRNA弱表达或不表达，Jag-gedl、Hesl、NotchlmRNA表达增强（P〈0．05）。单因素方差分析3组细胞之间SP-C、AQP5、Dlkl、Jaggedl、Hesl、NotchlmRNA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Noteh信号通路后，对AECⅡ转分化和增殖有不同影响，Dlkl能促进增殖，抑制转分化，而Jaggedl则抑制增殖，促进转分化。

Objective To study the effects of the Notch ligands Dlkl and recombinant human nucleu factor- κB（Jaggedl ） on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signa- ling pathway activated. Methods The primary type Ⅱ alveolar epithelial cells （ AEC Ⅱ ） cultured with recombinant protein Dlkl and recombinant human nueleu faetor-κB （ rhNF-κB ） （ activator of Jaggedl ）, respectively, and then cul- tured 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） ;Immunofluo- rescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C （SP-C） and AEC I spe- cific protein aquaporin5 （ AQP5 ） ; the expression of SP-C, AQPS, Dlkl, Jagged1, Notchl and Hesl mRNA were detected by real time-PCR. Results The cell population and proliferation ： compared with control group, AEC Ⅱ proliferation was promoted in the Dlkl group Ⅰ cell numbers （ ×10^9/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 AECⅠ ;while AEC Ⅱ proliferation was inhibited in rhNF-KB group [ cell numbers （ ×10^9/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 resuhs （ 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 acceleratedAEC 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 Dlkl group was significantly higher（ P 〈 0.05 ） while the expression of AQP5 mRNA was remarkably lower and delayed（ P 〈 0.05 ） ,the expression of Jaggedl mRNA was weak or little, Dlkl and Notchl mRNA were up-regulated （ P 〈 0.05 ）, and the Hesl mRNA was reduced（ P 〈 0.05 ） ; the ex- pression of SP-C mRNA of rhNF-KB group was significantly reduced （ P 〈 0.05 ）, while the AQP5 mRNA expressed ahead of time and increased （ P 〈 0.05 ）, Jaggedl, Hesl and Notchl mRNA were higher（ P 〈 0.05 ）, and the Dlkl mRNA was weak. One-Way ANOVA showed that the difference in the expressions of SP-C, AQPS, Dlkl ,Jaggedl, Hesl and Notchl mRNA among the 3 groups had statistical 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：Dlkl promoted proliferation and inhibited differentiation, while Jaggedl inhibited proliferation and promoted transdifferentiation.