CAJ | 학술논문

目的观察Notch信号通路激活或抑制对表皮干细胞增殖和分化的影响.方法体外分离培养并纯化大鼠表皮干细胞,分组加入Notch信号激活剂Jagged1/FC蛋白(1000 μg/L)和抑制剂γ-分泌酶抑制剂(DAPT,16 μmol/L),空白对照加磷酸盐缓冲液(PBS),共培养48 h后,通过观察各组细胞形态学变化、克隆计数、噻唑蓝(MTT)法检测吸光度(A)值,比较各组表皮干细胞的增殖能力；通过流式细胞仪及免疫细胞组化法检测细胞表面标记物β1整合素(CD29)及角蛋白CK19、CK10的阳性细胞百分比,比较各组表皮干细胞的分化.结果 3组细胞形态无明显差异.分组培养第5天,Jagged1/FC组贴壁细胞的克隆计数为(45.7±3.8)个,显著高于DAPT组[(16.9±2.1)个]及对照组[(33.4±3.1)个,P＜0.05]；MTT法检测细胞增殖能力,Jagged1/FC组明显高于对照组,DAPT组明显低于对照组(P＜0.05)；流式细胞仪检测Jagged1/FC组的β1整合素表达率(96.42±2.57)％显著高于DAPT组[(72.58±3.87)％]及阴性对照组[(8 8.75±3.14)％,P＜0.05].免疫组织化学CK19阳性细胞百分比Jagged1/FC组明显高于对照组,DAPT组明显低于对照组(P＜0.05)；而CK10阳性细胞百分比Jagged1/FC组明显低于对照组,DAPT组明显高于对照组(P＜0.05).结论激活Notch信号通路能促进表皮干细胞增殖并维持低分化状态,而抑制Notch信号通路能促进表皮干细胞向表皮细胞分化.
목적 관찰Notch신호통로격활혹억제대표피간세포증식화분화적영향.방법 체외분리배양병순화대서표피간세포,분조가입Notch신호격활제Jagged1/FC단백(1000 μg/L)화억제제γ-분비매억제제(DAPT,16 μmol/L),공백대조가린산염완충액(PBS),공배양48 h후,통과관찰각조세포형태학변화、극륭계수、새서람(MTT)법검측흡광도(A)치,비교각조표피간세포적증식능력；통과류식세포의급면역세포조화법검측세포표면표기물β1정합소(CD29)급각단백CK19、CK10적양성세포백분비,비교각조표피간세포적분화.결과 3조세포형태무명현차이.분조배양제5천,Jagged1/FC조첩벽세포적극륭계수위(45.7±3.8)개,현저고우DAPT조[(16.9±2.1)개]급대조조[(33.4±3.1)개,P＜0.05]；MTT법검측세포증식능력,Jagged1/FC조명현고우대조조,DAPT조명현저우대조조(P＜0.05)；류식세포의검측Jagged1/FC조적β1정합소표체솔(96.42±2.57)％현저고우DAPT조[(72.58±3.87)％]급음성대조조[(8 8.75±3.14)％,P＜0.05].면역조직화학CK19양성세포백분비Jagged1/FC조명현고우대조조,DAPT조명현저우대조조(P＜0.05)；이CK10양성세포백분비Jagged1/FC조명현저우대조조,DAPT조명현고우대조조(P＜0.05).결론 격활Notch신호통로능촉진표피간세포증식병유지저분화상태,이억제Notch신호통로능촉진표피간세포향표피세포분화.
Objective To observe the effects of activating and inhibiting Notch signaling pathway on epidermal stem cell (ESC) proliferation and differentiation.Methods ESCs of SD rats were isolated,cultured and purified.Recombinant rat Jagged1/FC chimera (1000 μg/L),an activator of the Notch signaling pathway,and DAPT (16 μmol/L),an inhibitor of the Notch signaling system,were added into the culture medium respectively.The cells in the medium added with phosphate buffered saline were used as control group.After co-culture for 48 h,the proliferation of ESCs was observed.The morphological changes and clone counting in each group were determined after culture.Methyl thiazol tetrazolium (MTT) assay was used to calculate the growth curve.Integrinβ1 (CD29) was identified by flow cytometry.The expression level of cytokeratin (CK) 19 and CK10 in each group was detected by using immunohistochemistry.Results There was no significant difference in morphological changes.After culture for 5 days,the number of colony formations in Jaggedl/FC,DAPT and control groups was 45.7 ± 3.8,16.9 ± 2.1 and 33.4 ± 3.1 respectiely.There was significant difference in every two groups (P ＜ 0.05).MTT assay showed that activated Notch signaling significantly promoted the proliferation of the ESCs (P ＜0.05).The percentage of CD29 positive cells in Jaggedl/FC group was significantly higher than that in DAPT group and control group[(96.42±2.57)％ vs.(72.58 ±3.87)％ and (88.75 ±3.14)％,both P＜0.05].The expression of CK19 was significantly higher in Jaggedl/FC group than in control group (P ＜ 0.05).The expression of CK19 in DAPT group was significantly lower than in control group (P ＜0.05).On the other hand,the expression of CK10 was significantly lower in Jaggedl/FC group than in control group (P ＜0.05).The expression of CK10 in DAPT group was significantly higher than in control group (P ＜ 0.05).Conclusion Notch signaling pathway may promote the ESC proliferation and maintain the undifferentiated state.When the Notch signaling pathway is inhibited,ESCs have the tendency to differentiate into epithelial cells.