Effect of Dlk1 and Jagged1 gene on the proliferation and transdifferentiation of the primary type Ⅱ alveolar epithelial cells / 中华实用儿科临床杂志

ABSTRACT

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 proliferationcompared 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 numbersF =486.73,P =0.02; cell proliferationF =37.16,P =0.02).The mRNA expressioncompared 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 ligandsDlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.