| Objective: To establish an effective method of isolation and purification of mousebone marrow mesenchymal stem cells (BMSCs) in vitro.Methods: The cells of whole bone marrow from4weeks female ICR mouse werecultured by the whole bone marrow adherence method for isolating marrowmesenchymal stem cells (BMSCs), in which BMSCs were enriched by replacingmedium on24h and diminishing the trypsin digestion time (1min) during primaryculture period. The morphological features of enriched cells and three cell surfacemarkers (CD29,CD44and CD45) were detected for identification of the character andpurity of BMSCs.Results:①Morphological observationThe adherent spindle-shaped cellsappeared individually after24hours of primary culture. The cell colonies formed with30%and80%cell conjugation after3and7days of primary culture respectively, andnon-adherent round cells in which blood cells predominated interspersed among thecolonies. The cell conjugation could reach90%on7thday after first passage，andapproximately100%conjugation with uniform size of cells arranged spirally wasfound on15thday of passage3.②Identification of BMSCs The positive rates ofCD29and CD44of enriched cells in passage3were99.24%and97.43%respectively,in which the positive rates of CD45was only0.21%.Conclusion: The primary cultured BMSCs would grow more rapidly and stably aftersubculture. Highly purified BMSCs could be enriched by replacing medium on24hand diminishing the trypsin digestion time during primary culture period. Objective: To evaluate the value of BMSCs transplantation on functionalreconstruction of mouse ovary.Methods: The mouse model of premature ovarian failure (POF) in60female ICRmice was established through abdominal injection of CTX（120mg/kg）and BUS（12mg/kg）, of those were equally divided into BMSCs transplantation group andcontrol group, meanwhile30normal female ICR mice received physiological salineintraperitoneal injection were served as blank group.14days later,0.1ml (5×106)green fluorescent protein(GFP)-transgenic BMSCs was injected through vena caudalisin BMSCs transplantation group, while equal volume physiological saline wereadministrated through vena caudalis in control group and blank group. Thebackground characteristics of mice, the level of FSH and E2, histological charactersof ovary as well as the distribution of green fluorescence in ovary were observed andcompared among three groups.Results:①Background characteristics of mice Following appearance ofanorexia， fatigue and lethargy, the weight of mice in POF model decreasedsignificantly compared with that in blank group, and was approximately only the halfof original weight on14thdays of POF model established. After BMSCstransplantation, the weight of mice in transplantation group increased graduallyfollowing the improvement of feeding, sleeping and activity.②The levels of FSHand E2There was no significant difference in blank group before and afteradministration of physiological saline. On14thdays of POF model established, thelevel of FSH raised and E2lowered significantly in both BMSCs transplantation groupand control group compared with blank group. But the change trends of FSH and E2intransplantation group were reversed, and the levels of FSH and E2were close to the blank group on28thdays of BMSCs transplantation.③Histology of ovaries Theamount of total follicles, primordial, growing and mature follicles reducedsignificantly on14thdays of POF model established，with the increase of atreticafollicles compared with blank group. On28thday of BMSCs transplantation,following the decrease of atretica follicles and interstitial fibrosis, the amount offollicles in all different stages increased significantly compared with the control group.④Fluorescence microscopy observation On28thday of BMSCs transplantation,granular green fluorescence signals were found dispersedly around follicles.Conclusion: BMSCs can localize in ovary and take part in reconstruction of damagedovarian, and BMSCs transplantation could improve ovary function. |
PDF Full Text Request