Isolation Of Rat Primary Hepatic Side Population And Analysis Of Its Telomerase Activity

Objectives:Hoechst33342/ fluorescence activated cell sorting method is used in this study to analyze whether the rat primary hepatic cells can be devided into the side population cells and the non-side population cells, which possess different efflux ability of the DNA-binding dye Hoechst33342. We detect these two populations'telomerase activity, which is used to identify if the SP cells own one of the stem cells'characteristics, the better self-renewal ability. At the same time it elucidates whether this Hoechst/FACS method can be applied to the isolation of the hepatic stem cells.Methods:1. Hrimary hepatic cells from adult rat are isolated by two-step in situ collagenase perfusion. The cells are washed with Percoll isodensity gradient centrifugation. The number and viability of the cells are counted by the trypan blue dye exclusion. The hepatic cells are seeded with 1×108 cells/L in the DMEM/F-12 medium and the attachment efficiency is calculated after 24 hours.2. The fresh isolated cells are divided into two aliquots. Both are stained with the Hoechst33342, but one is added with inhibitor of Ca2+ channel additionally. Both are incubated at 37℃on water bath for 90min. Then the Hoechst fluorescence in the hepatocytes is detected by the flow cytometry. The Hoechst fluorescence is excited (activated) at 355nm and is collected at two different wavelengths. One is the Hoechst blue fluorescence at 450nm and the other is the Hoechst red fluorescence at 675nm. Display the histogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). SP and non-SP are distinguished by setting the"gate".3. The SP and non-SP cells'telomerase activity are detected according to the telomerase activity detection kit manual. After pretreation, the productions are followed with PCR and detected by the fluorospectrophotometer. Then the fluorescence unit is changed into the TMA unit according to the specification.Results:1. After being washed with Percoll, (1.8±0.3)×107 cells are collected from every gram of rat liver. The viability of the fresh cells is more than 98%. The attachment efficiency after 24 hours is (67±7) %.2. The side population can be observed on the fluorescence profile when the emission of Hoechst is analyzed by dual-wavelength. The percentage of the SP cells is about 2%. When the inhibitor of Ca2+ channel is used, the side population eliminates mostly because of inhibition of efflux mediated by the ABC transporter. The SP cells enter into the main population on the fluorescence profile because of the enhancement of the fluorescence. 3. The TMA of the SP cells is (28.1±1.3) TPG and that of non-SP cells is (2.0±0.2) TPG. TMA is much more expressed in SP cells (t=50.440, P<0.01).Conclusion:1. The SP and non-SP cells which possess different DNA-binding dye efflux characteristics can be obtained from the primary rat hepatic cells by using the Hoechst/FACS method.2. The TMA is much more expressed in SP cells than that in non-SP cells, which reveals that the SP cells have better self-renewal ability and anti-aging ability and demonstrate partial characteristics of stem cells.