筛选鉴定Thy-1标记阳性肝癌细胞系中的卵圆细胞样干细胞:可能是肝肿瘤干细胞吗?

Screening and identification of oval cells-like stem cells of Thy-1-labeled positive hepatoma carcinoma cells:Are they tumor stem cells?

背景:肝癌的转移复发被认为可能存在有肿瘤干细胞,后者在肿瘤细胞中数目极少,使得临床鉴别存在一定困难。Thy-1是卵圆细胞的表面标记蛋白,通过特异性标记物间接证明肿瘤中存在肿瘤干细胞是目前研究的重点。目的:探讨肝癌细胞中是否存在Thy-1标记阳性的卵圆细胞,并进行筛选鉴定。方法:流式细胞仪检测卵圆细胞标记蛋白Thy-1在肝癌细胞HepG2,BEL7402及正常肝细胞QSG7701的阳性表达情况,免疫磁珠分选Thy-1标记阳性的肝癌细胞,免疫荧光鉴定分选效率。裸鼠皮下接种实验设立3组,Thy-1+细胞组裸鼠于背部皮下接种分选的Thy-1+细胞5×105/只,HepG2细胞阳性对照组同法接种HepG2细胞0.5×107/只,Thy-1-细胞阴性对照组同法接种分选的Thy-1-细胞0.5×107/只,1个月后观察各组成瘤性。结果与结论:卵圆细胞表面标记蛋白Thy-1在肝癌细胞HepG2,BEL7402中的表达明显高于正常肝细胞QSG7701(P<0.05)。Thy-1+细胞组免疫荧光染色阳性率为85%,HepG2细胞阳性对照组为1%,Thy-1-细胞阴性对照组几乎无免疫荧光表达。HepG2细胞阳性对照组、Thy-1+细胞组5只裸鼠全部成瘤,且瘤体体积相对较大;而Thy-1-细胞阴性对照组仅有1只裸鼠成瘤,瘤体体积明显小于前2组(F=144.568,P<0.05),提示肝癌细胞中存在Thy-1标记阳性的卵圆细胞样干细胞,其可能是肝癌细胞中的肿瘤干细胞。

BACKGROUND： There may be tumor stem cells during metastasis and recurrence of liver cancer. The number of tumor stem cells is very few, so it is difficult to be identified. Thy-1 is a surface marking protein of oval cells. Specific marker can indirectly confirm the existence of tumor stem cells in the tumor, which is a focus in recent studies. OBJECTIVE： To study whether Thy-1 positive marked oval cells was expressed in hepatoma carcinoma cells, and investigate its screening and identification. METHODS： Flow cytometry was used to identify that Thy-1 positive expression in hepatoma carcinoma cells HepG2 and BEL-7402 and normal hepatocytes QSG-7701. Immunomagnetic bead sorting was utilized to select hepatoma carcinoma cells with Thy-1. Immunofluorescence was used to identify selected efficiency. Naked mouse inoculated with hepatoma carcinoma cells in the skin test was assigned to three groups. In the Thy-1＋ cells group, naked mice were inoculated with Thy-1＋ cells （5×105/mouse） underneath the skin of the back. In the HepG2 cell positive control group, naked mice were inoculated with HepG2 cells （0.5×107/mouse） underneath the skin of the back. In the Thy-1-cells negative control group, naked mice were inoculated with Thy-1-cells （0.5×107/mouse） underneath the skin of the back. Tumorigenicity in each group was observed one month later. RESULTS AND CONCLUSION： Thy-1 expression in hepatoma carcinoma cells HepG2 and BEL7402 was significantly greater compared with normal hepatocytes QSG7701 （P 0.05）. Positive rate of immunofluorescence staining was 85% in the Thy-1＋ cell group, 1% in the HepG2 cell positive control group, 0% in the Thy-1-cell negative control group. There were nubble of five mice in HepG2 cell positive control and Thy-1＋ groups, and the volume was large. However, there was one nubble in Thy-1-group, and the volume was significantly small （F=144.568, P 0.05）. Results indicated that hepatoma carcinoma cells contained Thy-1 labeled positive oval cells-like stem cells, which may be tumor stem cells in hepatoma carcinoma cells.