Characteristics and feasibility of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine / 中国组织工程研究

ABSTRACT

BACKGROUND: The indicant of successful transplanted bone marrow stem cells is that the labeled transplanted ceils can survive in the target organs and can their biological functions. Up to date, at cell level, there are several labeled methods such as enzyme-linked, 3H-TdR, fluorescent and 5-bromodexyuridine method, etc.OBJECTIVE: To observe the biological characteristics of bone marrowderived mesenchymal stem cells labeled with 5-bromodexyuridine. DESIGN: Observations in single kind of sample SETTING: Department of Thoracic Cardiac Surgery, Affiliated Hospital of North China Coal Medical College MATERIALS The experiment was carried out in the Experimental Center of North China Coal Medical College from July, 2003 to November,2004 on six Japanese long-eared rabbits ,with the age of 3 months , of either gender and the body mass of (3.00±0.25)kg.METHODS:①Monocytes were isolated from the bone marrow with Percoll reagent of the concentration of 1 073 g/L. The mesenchymal stem cells were cultured and proliferated with Eagle's medium, which was improved by adding 10% low sugar Dulbecco's to fetal bovine serum, so that their purity could reach about 95%. Secondly, in the experimental groups, the percentage of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine was detected after 24 hours, 48 hours, 72 hours and 96hours, respectively. The negative-controlled group was composed of bone marrow-derived mesenchymal stem cells non-labeled with 5-bromodexyuridine while the blank-controlled group was made by substituting phosphate buffer or normal mice serum for primary antibody.MAIN OUTCOME MEASURES: In three different groups, 200 cells of each group were detected and observed at different time. Outcomes of the immunochemical stauning and counting of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were also determined.RESULTS: The positive bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were observed in all the experimental groups. The positive materials were brown, granular and dfiffusedly scattered in the nucleus while no positive cell was observed in the controlled groups. After 24 hours, 48 hours, 72 hours and 96 hours, the numbers of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were 48±2, 100±4, 173t2, 178±3 respectively. The labeling percentage was raised gradually with the elongation of time. 72 hours later,the labeling percentage is above 85%. The numbers in negative-controlled and blank-controlled groups were all zero.CONCLUSION: The appropriate time of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuriding is 72 hours. The sensibility of post-labeling detection is high due to the results being observed in the low power microscope, which makes this method suitable to quantitative analysis in massive tissue. These results show that the method of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine is feasible.