CAJ | 학술논문

目的在永生化的小鼠肝干（祖）细胞(HP)模型上筛选并优化高效的肝细胞定向分化诱导方法，探讨HP向肝细胞定向分化过程及分子机制。方法分别采用含人白血病抑制因子(LIF)、骨形态发生蛋白(BMP)2和BMP9基因的重组腺病毒感染HP，在病毒感染后第4天、第7天和第10天用糖原染色和吲哚花青绿(ICG)摄取实验观察HP的分化成熟度，并在第4、7、10、14天通过检测白蛋白启动子调控的荧光素酶报告基因活性，观察细胞合成白蛋白情况。计量资料比较用t检验。结果 BMP2和BMP9对HP的诱导作用最强，荧光素酶活性、PAS染色和ICG摄取细胞阳性率随诱导时间的延长明显上升，在诱导后第7天最高，HP对BMP9的诱导应答最强，与对照组相比，酶活性增加了近9倍（t=17.30，P＜0.01），BMP2处理组增加了5倍（t=16.41,P＜0.01），LIF处理组增加了3倍（t=6.04,P＜0.01）。诱导第7天时，PAS染色细胞阳性率在BMP2和BMP9组分别为30％和45％; ICG细胞阳性率在BMP2和BMP9组分别为40％和30％。LIF诱导后，PAS染色、ICG摄取细胞阳性率以及荧光素酶活性有一定增加。结论 BMP2、BMP9和LIF能够诱导HP向发育晚期肝细胞分化，并初步具备成熟肝细胞的一些功能。
목적 재영생화적소서간간（조）세포(HP)모형상사선병우화고효적간세포정향분화유도방법，탐토HP향간세포정향분화과정급분자궤제。 방법분별채용함인백혈병억제인자(LIF)、골형태발생단백(BMP)2화BMP9기인적중조선병독감염HP，재병독감염후제4천、제7천화제10천용당원염색화신타화청록(ICG)섭취실험관찰HP적분화성숙도，병재제4、7、10、14천통과검측백단백계동자조공적형광소매보고기인활성，관찰세포합성백단백정황。계량자료비교용t검험。결과 BMP2화BMP9대HP적유도작용최강，형광소매활성、PAS염색화ICG섭취세포양성솔수유도시간적연장명현상승，재유도후제7천최고，HP대BMP9적유도응답최강，여대조조상비，매활성증가료근9배（t=17.30，P＜0.01），BMP2처리조증가료5배（t=16.41,P＜0.01），LIF처리조증가료3배（t=6.04,P＜0.01）。유도제7천시，PAS염색세포양성솔재BMP2화BMP9조분별위30％화45％; ICG세포양성솔재BMP2화BMP9조분별위40％화30％。LIF유도후，PAS염색、ICG섭취세포양성솔이급형광소매활성유일정증가。결론 BMP2、BMP9화LIF능구유도HP향발육만기간세포분화，병초보구비성숙간세포적일사공능。
Objective To search for the optimal approach for hepatocyte-directed differentiation of hepatic progenitor cells and investigate the molecular mechanism of the hepatic differentiation. Methods Hepatic progenitor cells were infected with recombinant adnovirus which containing human LIF, BMP2 or BMP9 gene. The maturation and differentiation of progenitor cells were examined by PAS sraining and ICG uptake methods at 4, 7 and 10 days post infection. The production of Albumin (Alb) was measured by luciferase activity at day 4, 7, 10 and 14. Results PAS staining assay revealed that BMP2 and BMP9 enhanced glycogen storage in hepatic progenitor cells most obviously at day 7. The percentages of positive cells were 30％ and 45％ respectively at 7 days post-infection. Meanwhile, 40％ and 30％ cells were positive by ICG uptake assay after BMP2 and BMP9 induction. Luciferase activity indicated that BMP9 induced ALB- Luc activity most significantly at day 7. However, less inductive activity was found in LIF-treated group. Conclusion These results indicated tuat hepatic progenitor cells were differentiated into hepatucyte-like cells by BMPs and LIF induction.