转染癌基因的骨髓基质干细胞体外分化特征

Characteristics of in vitro differentiation of bone marrow mesenchymal stem cells transfected with oncogene

目的:观察转染癌基因的骨髓基质干细胞在体外分化情况,为肝细胞癌的细胞源研究提供实验依据。方法:实验于2003-05/2004-06在南方医科大学药理教研室实验室完成。①两步法获取大鼠肝细胞,梯度离心法分离大鼠骨髓基质干细胞。②单基因转染是单独将c-myc或K-ras癌基因瞬时转染大鼠骨髓基质干细胞,6孔培养板中培养,24h后荧光显微镜下观察骨髓基质干细胞转染结果。双基因转染步骤相同,只是将c-myc和K-ras癌基因同时转染大鼠骨髓基质干细胞。③c-myc癌基因转染组、K-ras癌基因转染组、双癌基因转染组常规培养,加入含体积分数为0.1胎牛血清的DMEM培养基,于37℃、体积分数为0.05的CO2孵箱培养,每24h半量更换培养液。④c-myc癌基因转染+肝细胞组、K-ras癌基因转染+肝细胞组、双癌基因转染+肝细胞组将已转染癌基因的骨髓基质干细胞,置于叠加的培养板半透膜的上方(细胞密度均为1×105个/cm2),再将肝细胞置于半透膜的下方(每孔细胞密度为3×105/cm2)进行共培养,其余步骤同常规培养。⑤通过反转录聚合酶式反应和细胞免疫组化检测骨髓基质干细胞分化情况。结果:①癌基因转染24h骨髓基质干细胞检测结果:单独转染c-myc或K-ras癌基因的细胞,其绿色荧光蛋白呈均匀一致分布;双基因转染的细胞,绿色荧光蛋白呈点片状分布。②各组骨髓基质干细胞向肿瘤细胞分化检测结果:c-myc癌基因转染组、K-ras癌基因转染组、双癌基因转染组的骨髓基质干细胞,均未向肿瘤细胞分化;c-myc癌基因转染+肝细胞组、K-ras癌基因转染+肝细胞组、双癌基因转染+肝细胞组的骨髓基质干细胞,均向肝细胞癌发展;空白对照组骨髓基质干细胞细胞均为阴性。此外,双癌基因转染+肝细胞组的骨髓基质干细胞分化增殖迅速,反转录聚合酶式反应和免疫组化检测发现,培养第7天出现甲胎蛋白表达,并迅速增加,而第7天出现的白蛋白和细胞角蛋白18表达迅速减弱,第14天消失。结论:转染癌基因的骨髓基质干细胞,在向肝细胞诱导的条件下,部分癌基因可以使干细胞分化为肝癌细胞;多癌基因转染时,更易于使干细胞分化为肝癌细胞。

AIM： To investigate the differentiation of mesenchymal stem cells （MSCs） transfected with oncogenes in vitro and provide an experimental rationale on the cell origin of hepatocellular carcinoma （HCC）. METHODS： The experimentation was completed at the laboratory of Department of Pharmacology, Southern Medical University from May 2003 to June 2004. （1）Rat MSCs were seperated through gradient centrifugatidn. （2）Monogenic transfectidn meant rat MSCs were transfected with c-myc or K-ras oncogene using transient transfectidn, while digenic transfection indicated MSCs were transfected with c-myc and K-ras oncogenes. Then MSCs were cultured on 6-well plate and examined with fluorescence microscope 24 hours later.（3）MSCs that were transfected with c-myc, K-ras, c-myc and K-ras oncogenes were conventionally cultured on DMEM containing 0.1 volume fraction of fetal bovine serum at 37 ℃ in CO2 incubator of 0.05 volume fraction. And the medium was changed for half dose every 24 hours.（4） Transfected MSCs were placed on the upper of semipermeable membrane at the density of 1×10^5 cells/cm^2 whereas hepatic cells on the lower of semipermeable membrane with the density of 3×10^5 cells/cm^2.（5）The differentiation of MSCs was detected using reverse transcription polymerase chain reaction （RT-PCR） and immuocytochemistry. RESULTS： （1） Conventionally cultured MSCs transfected with c-myc or K-ras oncogene expressed green fluorescent protein （GFP） evenly, MSCs, which were transfected with c-myc and K-ras as well as co-cultured with hepatic cells, expressed GFP in dot and piece. （2）MSCs, which were transfected with c-myc and K-ras did not differentiate into tumor cells; while those co-cultured with hepatic cells developed HCC; MSCs of control group were negative. In addition, the results of （RT-PCR） and immuocytochemistry indicated that MSCs, which were transfected with c-myc and K-ras as well as co-cultured with hepatic cells, expressed the alpha fetal protein at day 7 and the expression was enhanced rapidly, but albumin and cytokeratin-18 appeared at day 7 and then reduced rapidly until their expression disappeared at day 14. CONCLUSION： MSCs which were transfected with oncogenes, especially multiple oncogenes, can differentiate into liver cancer cells under the influence of some oncogenes,