骨髓间充质干细胞接种小鼠肝癌组织对小鼠生存期的影响

目的研究骨髓间充质干细胞（MSC）接种于原发性肝癌Heps荷瘤小鼠对其生存期的影响。方法体外贴壁培养法制备昆明小鼠骨髓MSC，分离培养传代扩增，流式细胞仪鉴定MSC表型。4’，6-二脒基-2-苯基吲哚（4’，6-diamidino-2-phenylindole，DAPI）对MSC进行标记备用。随机取54只8周龄昆明小鼠，建立Heps肝癌移植瘤模型。移植瘤长径达0．5～0．8cm时，随机分为MSC组、DAPI标记MSC组（DAPI组）、生理盐水（NS）对照组（NS对照组），每组18只。MSC组及DAPI组分别在小鼠Heps肝癌移植瘤内注射2×10^6 MSC及DAPI标记的MSC，NS对照组注射同体积的NS。每组各有6只荷瘤小鼠用于观察生存期。①自注射日起，隔日测量各组小鼠肿瘤长、短径，计算注射后3、7、14及28天的肿瘤体积；②记录注射当天开始至小鼠死亡的时间，计算各组小鼠平均生存时间；③于注射后3、7、14及28天，各组分批处死3只小鼠。取肿瘤组织，石蜡包埋并切片，苏木精-伊红染色后光学显微镜观察。结果①体外贴壁法成功制备MSC，流式细胞仪对第3代MSC进行鉴定，高表达CD29（97．84％），低表达CD34（5．56％）、CIM5（7．66％）。②小鼠Heps肝癌腹水瘤细胞成功制备小鼠Heps肝癌移植瘤模型，成瘤率100％。③注射MSC后1周，MSC组及DAPI组肿瘤体积增大明显，与NS对照组差异有显著性（P〈0．05）。④MSC组平均生存期为45天（95％可信区间：33～56天），DAPI组平均生存期为34天（95％可信区间：31～37天），NS对照组平均生存期为33天（95％可信区间：28～37天）；MSC组与NS对照组差异有显著性（P〈0．05），其余组间生存期差异无显著性（P〉0．05）。⑤各组小鼠肿瘤苏木精-伊红染色：28天时，MSC组及DAPI组肿瘤组织坏死明显，范围较NS对照组更大。结论MSC可在原发性肝癌Heps荷瘤小鼠肿瘤组织内定植，MSC接种后1周内肿瘤增长

Impact of inoculation of bone marrow-derived mesenchymal stem cells into hepatocellular carcinoma tissue on survival time in mice with orthotopic hepatocellular carcinoma

Objective To investigate the impact of mesenchymal stem cells （MSC） inoculated into the established model of Heps - bearing mice on survival time. Methods MSC were obtained through adherent culture method. Phenotypes of MSC were analyzed by flow cytometry. MSC were labeled with DAPI in vitro. We developed 54 mice with subcutaneously transplanted liver carcinomas. When the maximal diameters of the tumor reached 0.5 - 0.8 cm, the mice were randomly divided into three groups （n = 18 each） ： MSC group, MSC labeled with DAPI group （ DAPI group）, Normal saline （NS） control group （ NS control group）. MSC and MSC marked by DAPI were all administrated into the mouse right rear back tumor tissue of 2 × 10^6. The NS control group were administrated with the same volume of NS. 6 mice in each group were used to observe the survival time. ① The size of the implanted tumors was measured every 2 days and the tumor volume was calculated on 3th, 7th, 14th and 28th day. ② The survival time of the tumor - bearing mice was
recorded and the mean survival time was calculated. ③ 3 mice from each group were sacrificed at 3th, 7th, 14th and 28th day after inoculation respectively. Tumor tissue samples collected at above 4 time points were imbedded in paraffin for routine pathological section, hematoxylin - eosin staining and light microscopy observation. Results ① Purified MSC were successfully obtained through adherent culture method. Passage 3 cells were collected and analyzed by flow eytometry. The results showed that MSC CD29 （97.84%）was positive, whereas, CD34 （5.56%） and CD45 （7.66%） were negative.② The model of Heps - bearing mice were successfully established and the rate of tumor formation is 100%. ③ At the first week after MSC were administrated, the volume of the tumors was markedly increased in the MSC group and the DAPI group compared with the NS control group （ P 〈 0.05 ）. ④ In the MSC group, the mean survival time was 45 days （95% confidence interval： 33 -56 days?