CAJ | 학술논문

目的研究肝星状细胞(HSC)在体外培养的卵圆细胞向成熟肝细胞分化过程中所起的作用.方法 (1)将卵圆细胞分别与原代培养的HSC混合共培养(混合共培养组),采用Millicell小室不接触共培养(不接触共培养组),卵圆细胞单独培养作为对照组.在共培养后第7、14、21天观测,采用Western blot和荧光定量PCR检测肝细胞核因子4 α(HNF-4 α)、白蛋白和甲胎蛋白(AFP)、细胞角蛋白19(CK-19)的表达量;(2)电子透射显微镜观察卵圆细胞超微结构的变化;(3)过碘酸希夫染色检测各时间点卵圆细胞内糖原颗粒的含量;(4)酶联免疫吸附法检测各时间点卵圆细胞分泌白蛋白的含量.统计学处理采用重复测量资料的多因素方差分析和LSD-t检验.结果 (1)卵圆细胞第7、14、21天表达HNF-4 α和白蛋白的mRNA的量(相对与共培养之前的表达量的倍数):混合共培养组HNF-4 α分别为(1.9±0.2)倍、(10.7±1.2)倍、(12.0±1.3)倍;白蛋白mRNA的量分别为(5.7±1.6)倍、(110.7±13.7)倍、(173.6±22.3)倍;不接触共培养组HNF-4 α分别为(1.4±0.1)倍、(3.2±0.6)倍、(8.9±1.4)倍;白蛋白mRNA的量分别为(2.9±1.4)倍、(22.3±8.5)倍、(96.3±16.3)倍.共培养组(混合共培养与不接触共培养组)的表达量均高于对照组,LSD-t值分别为32.98,10.08;13.38,7.96; P值均<0.01,差异有统计学意义.第7、14、21天AFP、CK-19的表达量:混合共培养组AFP分别为(1.1±0.2)倍、(0.2±0.0)倍、(0.0±0.0)倍;CK-19分别为(0.2±0.1)倍、(0.0±0.0)倍、(0.0±0.0)倍;不接触共培养组AFP分别为(1.0±0.2)倍、(0.2±0.1)倍、(0.1±0.0)倍;CK-19分别为(0.6±0.1)倍、(0.1±0.0)倍,(0.0±0.0)倍.共培养组(混合共培养与不接触共培养组)的表达量均低于对照组.LSD-t值分别为37.99,34.50;13.59,22.46;P值均<0.01,差异有统计学意义.(2)卵圆细胞共培养后白蛋白分泌量明显增加:混合共培养组:14 d为(15.30±0.09)ng/ml、21 d(20.98±0.12)ng/ml,不接触共培养组14 d为(11.41±0.13)ng/ml,21 d为(15.12±0.17)ng/ml,混合共培养组高于不接触共培养组,LSD-t=251.94,P<0.01,差异有统计学意义.(3)随着共培养时间的延长,卵圆细胞内质网、线粒体和高尔基体等细胞器逐渐丰富,且细胞间形成毛细胆管结构.(4)过碘酸希夫染色显示共培养后卵圆细胞内出现大量红色糖原颗粒.结论 HSC可以诱导卵圆细胞向成熟肝细胞分化,HSC除了通过细胞因子等可溶性因子途径发挥作用外,与卵圆细胞的直接接触也有着重要作用. 目的研究肝星狀細胞(HSC)在體外培養的卵圓細胞嚮成熟肝細胞分化過程中所起的作用.方法 (1)將卵圓細胞分彆與原代培養的HSC混閤共培養(混閤共培養組),採用Millicell小室不接觸共培養(不接觸共培養組),卵圓細胞單獨培養作為對照組.在共培養後第7、14、21天觀測,採用Western blot和熒光定量PCR檢測肝細胞覈因子4 α(HNF-4 α)、白蛋白和甲胎蛋白(AFP)、細胞角蛋白19(CK-19)的錶達量;(2)電子透射顯微鏡觀察卵圓細胞超微結構的變化;(3)過碘痠希伕染色檢測各時間點卵圓細胞內糖原顆粒的含量;(4)酶聯免疫吸附法檢測各時間點卵圓細胞分泌白蛋白的含量.統計學處理採用重複測量資料的多因素方差分析和LSD-t檢驗.結果 (1)卵圓細胞第7、14、21天錶達HNF-4 α和白蛋白的mRNA的量(相對與共培養之前的錶達量的倍數):混閤共培養組HNF-4 α分彆為(1.9±0.2)倍、(10.7±1.2)倍、(12.0±1.3)倍;白蛋白mRNA的量分彆為(5.7±1.6)倍、(110.7±13.7)倍、(173.6±22.3)倍;不接觸共培養組HNF-4 α分彆為(1.4±0.1)倍、(3.2±0.6)倍、(8.9±1.4)倍;白蛋白mRNA的量分彆為(2.9±1.4)倍、(22.3±8.5)倍、(96.3±16.3)倍.共培養組(混閤共培養與不接觸共培養組)的錶達量均高于對照組,LSD-t值分彆為32.98,10.08;13.38,7.96; P值均<0.01,差異有統計學意義.第7、14、21天AFP、CK-19的錶達量:混閤共培養組AFP分彆為(1.1±0.2)倍、(0.2±0.0)倍、(0.0±0.0)倍;CK-19分彆為(0.2±0.1)倍、(0.0±0.0)倍、(0.0±0.0)倍;不接觸共培養組AFP分彆為(1.0±0.2)倍、(0.2±0.1)倍、(0.1±0.0)倍;CK-19分彆為(0.6±0.1)倍、(0.1±0.0)倍,(0.0±0.0)倍.共培養組(混閤共培養與不接觸共培養組)的錶達量均低于對照組.LSD-t值分彆為37.99,34.50;13.59,22.46;P值均<0.01,差異有統計學意義.(2)卵圓細胞共培養後白蛋白分泌量明顯增加:混閤共培養組:14 d為(15.30±0.09)ng/ml、21 d(20.98±0.12)ng/ml,不接觸共培養組14 d為(11.41±0.13)ng/ml,21 d為(15.12±0.17)ng/ml,混閤共培養組高于不接觸共培養組,LSD-t=251.94,P<0.01,差異有統計學意義.(3)隨著共培養時間的延長,卵圓細胞內質網、線粒體和高爾基體等細胞器逐漸豐富,且細胞間形成毛細膽管結構.(4)過碘痠希伕染色顯示共培養後卵圓細胞內齣現大量紅色糖原顆粒.結論 HSC可以誘導卵圓細胞嚮成熟肝細胞分化,HSC除瞭通過細胞因子等可溶性因子途徑髮揮作用外,與卵圓細胞的直接接觸也有著重要作用.
목적 연구간성상세포(HSC)재체외배양적란원세포향성숙간세포분화과정중소기적작용.방법 (1)장란원세포분별여원대배양적HSC혼합공배양(혼합공배양조),채용Millicell소실불접촉공배양(불접촉공배양조),란원세포단독배양작위대조조.재공배양후제7、14、21천관측,채용Western blot화형광정량PCR검측간세포핵인자4 α(HNF-4 α)、백단백화갑태단백(AFP)、세포각단백19(CK-19)적표체량;(2)전자투사현미경관찰란원세포초미결구적변화;(3)과전산희부염색검측각시간점란원세포내당원과립적함량;(4)매련면역흡부법검측각시간점란원세포분비백단백적함량.통계학처리채용중복측량자료적다인소방차분석화LSD-t검험.결과 (1)란원세포제7、14、21천표체HNF-4 α화백단백적mRNA적량(상대여공배양지전적표체량적배수):혼합공배양조HNF-4 α분별위(1.9±0.2)배、(10.7±1.2)배、(12.0±1.3)배;백단백mRNA적량분별위(5.7±1.6)배、(110.7±13.7)배、(173.6±22.3)배;불접촉공배양조HNF-4 α분별위(1.4±0.1)배、(3.2±0.6)배、(8.9±1.4)배;백단백mRNA적량분별위(2.9±1.4)배、(22.3±8.5)배、(96.3±16.3)배.공배양조(혼합공배양여불접촉공배양조)적표체량균고우대조조,LSD-t치분별위32.98,10.08;13.38,7.96; P치균<0.01,차이유통계학의의.제7、14、21천AFP、CK-19적표체량:혼합공배양조AFP분별위(1.1±0.2)배、(0.2±0.0)배、(0.0±0.0)배;CK-19분별위(0.2±0.1)배、(0.0±0.0)배、(0.0±0.0)배;불접촉공배양조AFP분별위(1.0±0.2)배、(0.2±0.1)배、(0.1±0.0)배;CK-19분별위(0.6±0.1)배、(0.1±0.0)배,(0.0±0.0)배.공배양조(혼합공배양여불접촉공배양조)적표체량균저우대조조.LSD-t치분별위37.99,34.50;13.59,22.46;P치균<0.01,차이유통계학의의.(2)란원세포공배양후백단백분비량명현증가:혼합공배양조:14 d위(15.30±0.09)ng/ml、21 d(20.98±0.12)ng/ml,불접촉공배양조14 d위(11.41±0.13)ng/ml,21 d위(15.12±0.17)ng/ml,혼합공배양조고우불접촉공배양조,LSD-t=251.94,P<0.01,차이유통계학의의.(3)수착공배양시간적연장,란원세포내질망、선립체화고이기체등세포기축점봉부,차세포간형성모세담관결구.(4)과전산희부염색현시공배양후란원세포내출현대량홍색당원과립.결론 HSC가이유도란원세포향성숙간세포분화,HSC제료통과세포인자등가용성인자도경발휘작용외,여란원세포적직접접촉야유착중요작용.
Objective To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte. Methods The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by milllcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-41A, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit. Result (1) The relative level of HNF-4 α and albumin mRNA expression compared with pre-coculture: M-coculture:HNF-4 α: 1.9±0.2, 10.7±1.2, 12.0±1.3; albumin: 5.7±1.6, 110.7±13.7, 173.6±22.3. S-coculture: 1.4±0.1, 3.2±0.6, 8.9±1.4 times; albumin: 2.9±1.4, 22.3±8.5, 96.3±16.3. The relative level of HNF-4α and albumin mRNA expression in coculture group (M- and S-colculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P<0.01); and a higher level of HNF-4α and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P<0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1±0.2, 0.2±0.0, 0.0± 0.0; S-coculture group: AFP: 1.0±0.2, 0.2±0.1, 0.1±0.0; CK-19:0.6±0.1, 0.1±0.0, 0.0±0.0; control group: AFP: 1.0±0.1, 1.0±0.1, 1.1±0.1, CK-19:1.0±0.1, 1.1±0.1, 1.0±0.1. The rela-tive level of AFP and CK-19 mRNA expression in cocuiture group(M- and S-colculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P<0.01). (2) The albumin secretion was detected in M-coculture: 14day: (15.30±0.09) ng/ml, 21:(20.98±0.12) ng/ml; S-coculture: 14 day: (11.41±0.13) ng/ ml, 21day:(15.12±0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochon-drion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared be-tween oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups. Conclusion HSC can induce differentiation of oval cell into mature hepatocyte.