摘要
目的:探讨大鼠骨髓间充质细胞向肝细胞样细胞的诱导分化,观察诱导分化细胞的形态和分子生物学特性,及其对急性肝损伤大鼠的治疗作用.方法:分离、培养并纯化大鼠骨髓间充质细胞,培养基中加入肝细胞生长因子(HGF)、纤维生长因子-4(FGF-4)及上皮细胞生长因子(EGF),分别于7、14、21、28d观察细胞形态变化,采用RT-PCR方法检测细胞白蛋白(ALB)、甲种胎儿球蛋白(AFP)、细胞角蛋白-18(CK-18)的mRNA表达,将诱导分化28d的细胞经DAPI染料染色后,经门静脉注入同种异体大鼠体内,经24、48、72及168h分批处死大鼠,观察大鼠肝组织内荧光细胞的分布.大鼠腹腔注射硫代乙酰胺制作急性肝衰竭模型,将1×106及5×106个诱导分化细胞经门静脉注入大鼠体内,经48、168h采血查肝功,168h处死大鼠,取肝组织病理学检查.结果:大鼠间充质细胞经上述3种细胞因子诱导后,形态和生物学行为方面都发生向肝细胞样细胞方面的转化,经门静脉注入大鼠体内后,有大量该种细胞在肝组织内分布,24h荧光细胞最多,随时间延长逐渐减少,可持续7d.骨髓间充质细胞的诱导分化细胞经门静脉注入肝损伤模型大鼠体内后,大鼠肝功能有所好转,注入1×106细胞大鼠ALT由注入前238.0±113.5U/L,48h后降至189±68.4U/L,168h后降至149.0±54.2U/L,TBIL由注入前2.9±1.6μmol/L,48h至3.0±1.4μmol/L,168h至1.3±0.3μmol/L;注入5×106个细胞者(ALT)由注入前238.0±113.5U/L,48h后降至169.7±46.0U/L,168h后降至103.7±46.0U/L,TBIL由注入前2.9±1.6μmol/L,48h至2.9±1.3μmol/L,168h至0.9±0.3μmol/L.结论:大鼠骨髓间充质细胞可在某些细胞因子的诱导作用下发生类肝细胞样转化,将转化细胞经门静脉植入同种异体大鼠体内后可在肝组织内存活并对大鼠肝损伤有一定修复作用.
AIM： To induce the differentiation of rat bone marrow mesenchymal stem cells towards hepatocyte-like cells, observe the morphology and molecular biological feature of differentiated cells and investigate their therapeutic effects against hepatic injury.
METHODS： Rat bone marrow mesenchymal stem cells were separated, cultured and purified. Hepatocyte growth factor （HGF）, fibroblast growth factor-4 （FGF-4） and epidermal growth factor （EGF） were then added to culture medium to induce differentiation. The expression of albumin, alpha-fetoprotein （AFP） and cytokeratin 18 （CK 18） mRNAs was detected using reverse transcription-polymerase chain reaction （RT-PCR） on days 7, 14, 21 and 28 after induction. On day 28, the differentiated cells were stained with DAPI （4＇, 6-diamidino-2-phenylindole） and injected to rats via the portal vein. These rats were sacrificed 24, 48, 72 and 168 hours after injection, respectively. The liver samples were taken to examine fluorescent distribution in the liver under microscopy. Acute hepatic failure （AHF） was induced in rats by intraperitoneal injection of thioacetamide. Differentiated cells at a density of 1 × 10^6 or 5 × 10^6 cells/L were injected to AHF rats via the portal vein. Blood samples were taken from these rats to perform liver function tests 48 and 168 hours after injection. The rats were executed 168 hours after injection to take liver samples for histological examination.
RESULTS： After induction with HGF, FGF-4 and EGF, bone marrow mesenchymal stem cells differentiated towards hepatocyte-like cells. The differentiated cells exhibited the morphology and biological behavior of hepatocyte-like cells. After differentiated cells stained with DAPI were injected into rats via the portal vein, many stained cells were distributed in the liver, and some of them could live for more than 7 days. When the differentiated cells were injected into AHF rats via the portal vein, hepatic injury in these rats was improved. In AHF rats injected with differentiated cells at a density of 1 × 10^6 cells/L, serum alanine aminotransferase （ALT） changed from 238.0 ± 113.5 U/L at baseline to 189 ± 68.4 U/L at 48 hours and 149.0 ± 54.2 U/L at 168 hours, and serum total bilirubin （TBIL） from 2.9 ± 1.6 μmol/L at baseline to 3.0 ± 1.4 μmol/L at 48 hours and 1.3 ± 0.3 μmol/L at 168 hours. In AHF rats injected with differentiated cells at a density of 5 × 10^6 cells/L, serum ALT changed from 238.0 ± 113.5 U/L at baseline to 169.7 ± 46.0 U/L at 48 hours and 103.7 ± 46.0 U/L at 168 hours, and serum TBIL from 2.9 ± 1.6 μmol/L to 2.9 ± 1.3 μmol/L at 48 hours and 0.9 ± 0.3 μmol/L at 168 hours.
CONCLUSION： Rat bone marrow mesenchymal stem cells can differentiate towards hepatocyte-like cells in the presence of inducing factors. Differentiated cells can live in the liver and improve hepatic injury in rats with AHF.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第26期2654-2661,共8页
World Chinese Journal of Digestology
基金
山东省自然科学基金资助项目
No.Y2005C27~~
