| Objective: Exploring the way to get liver cancer stem-like cells and to observe the biological characteristics and expression of cancer stem cell surface markers.Method:1To collect human liver cancer cell line SMMC-7721 cells cultured with ordinary medium(adherent cultivation) and with serum-free medium(suspension cultivation).2To observe morphologic patterns of SMMC-7721 tumor cells treated with two kinds of cultivation, under inverted microscope.3To measure expressions of tumor stem cell surface markers CD90,CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds of cultivation by the flow cytometry.4To detect m RNA expressions of the tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds cultivation by Real-Time PCR.5To observe changes of the tumor volume in nude mice after subcutaneous injection of SMMC-7721 tumor cells treated with two kinds of cultivation and to compare their tumorigenic ability and capacity to proliferate.Results:1The morphology patterns of SMMC-7721 tumor cells treated with two different kinds of cultivation.Growing like typical cobblestone and arraying regularly on SMMC-7721 tumor cells in ordinary medium were observed under inverted microscope, while SMMC-7721 tumor cells in suspensive culture gathered from single cell into spherical or near spherical cells.2Comparison of expressions of tumor stem cell surface markers CD90, CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds of cultivation by the flow cytometry.The results: The expression of CD90(571.77±8.70) and CD24(233.56±10.85) in suspension culture group was significantly higher than that in adherent culture group(19.15 ± 0.54 、 17.37 ± 0.15). The difference was statistically significant(CD90 t=-109.80 P=0.00;CD24 t=-34.51 P=0.00). But there was no significant difference between the expressions of CD133 in suspension culture group(1.42±0.61) and adherent culture group(1.48±1.09)(t=0.08 P=0.94).3The change of m RNA expressions of tumor stem cell surface markers CD90,CD133 and CD24 in SMMC-7721 tumor cells treated with two kinds.3.1Identification of RNA There were two bright bands correspond to the 28 S and 18 S RNA,another a duller 5S strip observed under ultraviolet. The ratio of optical density of 28 s and 18 S bands was about(1.5~2):1 and no RNA dispersion zone, indicating that the RNA was integral.3.2The relative expression of m RNA The Real-Time PCR results showed that the gene of CD90, CD24 and CD133 were amplified. All the amplification curve had the platform, melting curve was the standard single peak, no impurity peaks, indicating that the PCR products were specificity. Assumed the m RNA expression of adherent culture group was 1, the relative expression of CD90, CD24, CD133 m RNA in suspension culture group were 1.72 ± 0.19, 1.72 ± 0.19, 1.05 ± 0.03 respectively. The m RNA expression of CD90(1.72±0.19) and CD24(1.72±0.19) in suspension culture group was significantly higher than that in adherent culture group. The difference was statistically significant(CD90t=-6.55 P=0.02, CD24 t=-6.71 P=0.00). But there was no significant difference on expressions between suspension culture group(1.05±0.03) and adherent culture group for the CD133(t=-3.41 P=0.08).4Comparison of the tumorigenic ability and capacity to proliferate in vivo of tumor cells treated with two kinds of cultivation.According to the length(a, unit: mm) and width(b, unit: mm) of the tumor in nude mice after subcutaneous injection of SMMC-7721 tumor cells treated with two kinds of culture measured every other day, the volume(V= ab2/2, unit:mm3) was calculated. The results were as follows. The volume on 2th day after subcutaneous injection in adherent culture group was 71.23±51.44,4th day: 60.08±24.33, 6th day: 45.14±15.24, 8th day: 32.56±10.16, 10 th day: 31.73 ± 7.01, 12 th day: 26.41 ± 13.95, 14 th day: 26.95 ± 14.14. The volume on 2th day after subcutaneous injection in suspension culture group was 60.17±47.93, 4th day: 41.09±19.37, 6th day: 32.90±14.13,8th day:74.87±27.54, 10 th day: 97.30±37.63, 12 th day: 209.06±138.40, 14 th day:294.69±250.65. Statistical analysis showed there was significant difference between the two groups(F=11.76,P=0.01) and their tumorigenic ability and capacity to proliferate in vivo were different. And there was interaction between the measuring time and the way of cultivation(F=6.18,P=0.03). With the extension of time measurement, the tendency of the volume change from2 th day after subcutaneous injection to 6th day in suspension culture group went down(2th day: 60.17±47.93, 4th day: 41.09±19.37, 6th day: 32.90±14.13) and then significantly increased(8th day: 74.87 ± 27.54, 10 th day:97.30±37.63, 12 th day: 209.06±138.40, 14 th day: 294.69±250.65). But, the tendency of the tumor volume changing in adherent culture group slowly decreased and then became stable(2th day: 71.23 ± 51.44, 4th day:60.08±24.33, 6th day: 45.14±15.24,8th day: 32.56±10.16, 10 th day: 31.73±7.01, 12 th day: 26.41±13.95, 14 th day: 26.95±14.14). These showed the suspension culture group’s capacity to proliferate in vivo was better than the adherent culture group. And in adherent culture group, the subcutaneous tumor volume of number 4 nude mice gradually reduced to not touch within 14 days.This showed tumorigenic ability in the suspension culture groups was higher than that in the adherent culture group.Conclusion:1Part of the human liver cancer cell line SMMC-7721 tumor cells treated with suspension culture obtained some similar characteristics of cancer stem cells were regarded as hepatocarcinoma stem-like cells.2The expressions of CD90, CD24 in SMMC-7721 tumor cells treated with suspension culture and their m RNA levels were significantly higher than that in adherent culture. But the expression of CD133 in SMMC-7721 tumor cells treated with two kinds of culture method was not significantly different.Thus some liver cancer stem cells possibly existed in the human liver cancer cell line SMMC-7721 tumor cells after treated with suspension culture. But the specific cell surface markers of cells which like liver cancer stem cell needs further screening and determination.3The tumorigenic ability and capacity to proliferate in vivo were significantly different in SMMC-7721 tumor cells treated with two kinds of cultivation. The tumorigenic ability and capacity to proliferate in vivo in suspension culture groups was better than that in the adherent culture group. It shows part of the human liver cancer cell line SMMC-7721 tumor cells treated with suspension culture possibly existed some liver cancer stem cells again.Whether suspension cultivation could be used as the preferred research method in acquisition of liver cancer stem-like cells would require further study. |
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