Isolation And Identification Of Heterogenic Subpopulation From HepG2 Cell Lines By Using Rhodamine123 Extrusion Assay

The concept that a rare population of tissue stem cells may be the cellular origin of cancer has been proposed and this transform is caused by accumulation of genetic mutation. Cancer stem cells are defined as cells that have the ability to perpetuate themselves through selfrenewal and to capable of initiating new tumors following a prolonged period of remission through differentiation, which may cause the occurance of tumor and be used to prevent tumor. Similarities have been observed between cancer stem cells and stem cells with respect to their enormous capacity for proliferation and differentiation. Researchers have isolated leukaemia cells that have markers of HSCs from acute myeloid leukaemia (AML). Only these rare leukaemia cells can transfer AML from human patients to NOD/SCID mice. Recently, there are many approvals for cancer stem cells. Now the cancer stem cells have been found in many malignant tumors, for example breast cancer, pancreatic cancer, pancreatic cancer, prostatic carcinoma and human train tumor, etc. The oval cells which are originated from bile duct of the periportal regions play an important role in the process of hepatocarcinogenesis. Primary hepatocellular carcinoma is one of the most malignant tumors in our country and its mortality rate is high for its poor prognosis. In clinic, there are great differences of drug resistance between the liver cancer cells and the heterogeneity indicate the existence of cancer stem cells.The basic principle of Rho123/FCM mainly based on the dye rejection of the stem cells which is a specific functional marker of the stem cells. Now it has been known that mostly all stem cells can efflux bump the fluorescent Rho123 dye by P-gp transporter on the membrane of them. The stem cells stained with the Rho123 dye demonstrate low fluorescence. This method was firstly used to isolated HSCs from the bone marrow.Object: To establish a method to seperate different subpopulations of HepG2 cell line according to the ability of these cells to extrude the Rho123 dye.Method:1. Flow Cytometry Analysis for Rho efflux and cells sorting Aliquot the cancer cells suspension into two labeled centrifuge tube. All two tubes of cancer cells stain with the Rho123 dye, but one of tube add inhibitor of Ca(2+) channel in additionally. Both of two tubes incubate at 37℃on water bath. Then place Rho123-stained cancer cells on the cytometer, Display the gate with low fluorescence of Rho123. Low fluorescence group can be found by establish gate. Compare the low (Rho^low) and high (Rho^high) fluorescence groups of cancer cells suspension.2. Growth curve of Rho^low and Rho^high groupAfter Rho^low and Rho^high group being inoculated in 96-holes were cultured separately. Then evaluate the proliferation ability of cells by growth curve.3. Clone formation of Rho^low and Rho^high groupSort Rho^low and Rho^high group were seed them into soft agar. Observe the growth, proliferation and clone formation of two types of liver cancer cells in vivo. Calculate the rate of clone formation.4. Immunocytochemistry of Rho^low and Rho^high groupImmunocytochemical staining was performed with commercially available primary antibody and other reagents completed according to the manufacturer's protocol. Essentially, coverslips were incubated in the 4% normal horse serum for murine monoclonal antibodies blocking serum for 30 minutes at room temperature followed by incubation at 4℃overnight with the mouse monoclonal anti-AFP antibody in the dilution of 1:100. After washing in Tris buffer, species-specific secondary antibody was added and incubated for 30 minutes at room temperature. Coverslips were then stained with 3,3′-diaminobenzidine (DAB) for 10 minutes atroom temperature, rinsed in running water, and dehydrated with xylene, and mounted in DPX (BDH). Negative controls were obtained by omitting the primary antibody. A probability of less than 5% (P < 0.05) was considered statistically significant. 5. Nude mouse tumors fomations of Rho^low and Rho^high group Subcutaneously inject the 2×10⁴ Rho^low and Rho^high group into nude mouse respectively. Observe the growth of xenograft tumors.Result:1. Flow Cytometry Analysis for Rho Efflux and Cells Sorting Here we show that the Rho^low group was detected in HepG2 cell lines with flow cytometry analysis by the ability of these cells to extrude the Rho123 dye, but it part is only 1.6%.2. Growth curve of Rho^low and Rho^high groupIt was found that Rho123low group had higher proliferation ability than Rho123high group in doubing time(16.9 h vs 24.7 h) and max multiple of proliferation (15.2 vs 12.3).3. Clone formation of Rho^low and Rho^high groupThe sorted Rho^low group grow well under free serum medium in vivo, and they have very high rate of clone formation in the soft agar; In contrast, the Rho^high group shows growth retardation and have very low rate of clone formation under the same condition.4. Immunocytochemistry of Rho^low and Rho^high groupImmunocytochemical staining was performed to show the presence of AFP in Rho^low and Rho^high group, and AFP is much more expressed in Rho^low group on the protein level.5. Nude mouse tumors fomations of Rho^low and Rho^high group The result of nude mouse bearing tumor reveals that Rho^low cells have very strong tumorigenic capacity.ConclusionSubpopulations with stem cell properties of HepG2 cell line can be inriched by Rho123/FCM, indicating HCC is heterogeneous.