Capacity of colonizing to the liver after allografting of mesenchymal stem cells in rats / 中国组织工程研究

ABSTRACT

BACKGROUND: Meseuchymal stem cells (MSCs) have extremely strong self-duplication ability and multidirectional ifferentiation potential. When bone marrow stromal cells (BMSC) are isolated and cultured in vitro, implanted in vivo, the distribution and colonization are still unclear, which is concerned with whether BMSC can be usedas target cells in clinic.OBJECTIVE: To explore the capacity of colonizing to the liver after allografting of green fluorescent protein (GFP) labeled MSCs of rats by different approaches.DESIGN: Factorial design.SETTING: Department of General Surgery, Second People's Hospital of Guangdong Province, Postdoctoral Workstation of Sun Yat-Sen University;Department of General Surgery, Zhujiang Hospital, Southern Medical University; Department of Organ Transplantation, Third Hospital Affiliated to Sun Yat-Sen University.MATERIALS: The experiment was performed at the Staff Room of Pharmacology, Basic Department, First Military Medical University of Chinese PLA from January 2003 to December 2004. A total of 36 clean adult SD rats were selected and randomly assigned into 5 groups: CCL4 plus portal vein transplantation group (n=6), portal vein transplantation control group (n=6), CCL4 plus caudal vein transplantation group (n=6), caudal vein transplantation control group (n=6) and mixed group (n=12).METHODS: ① MSCs were obtained from rat marrow and labeled with GFP. After amplifying in vitro, MSCs suspension was implanted with thin needle, with the volume of 0.5 mL/100 g. ②CCL4 plus portal vein transplantation group: In 3 days before MSCs transplantation, the rats were administrated with 20 g/L CCL4 2.5 mL/kg by gastric perfusion every day. The dose was double at the first time. Labeled MSCs were implanted from portal vein. Portal vein transplantation control group: Before transplantation the MSCs were bred commonly, and the labeled MSCs were implanted from portal vein. CCL4 plus caudal vein transplantation group: In 3 days before MSCs transplantation, the rats were administrated with 20 g/L CCL42.5 mL/kg by gastric perfusion every day. The dose was double at the first time. Labeled MSCs were implanted from caudal vein. Caudal vein transplantation control group: Before transplantation the MSCs were bred commonly, and the labeled MSCs were implanted from caudal vein. Mixed group: On the basis of the former 4 groups, 2 rats were implanted with non-labeled MSCs; Another 2 rats fed with CCL4 for 3 days and normal feed were established, without MSCs transplantation. ③At days 3 and 7 after transplantation expression of transplanted MSCs in liver of rats of each group were examined with fluorescent quantitative PCR.MAIN OUTCOME MEASURES: ①Results of MSCs isolation, purification, in vitro amplification and phenotype identification, ②result of GFP-labeled MSCs, ③observation of growth of rats following allografting of MSCs, and ④result of quantitative identification of GFP positive DNA amount in hepatic tissues of each group.RESULTS: Totally 36 experimental SD rats were involved in the result analysis. ①Percoll gradient separating medium was applied to isolate bone marrow of rats. The obtained cells were transferred and amplified,and then mostly showed coincident shuttle shape. Cells did not express CD34 and CD45, but CD29, CD44 and CD90 of MSCs, which were noncommitted stem cells in non-differentiating status that were different from hemopoietic stem cells in bone marrow. ②The green fluorescent cells appeared 24 hours after MSCs transfection. From hour 48 to 72 the number of positive cells significantly increased, with strong intensity.The transfection efficiency was 20%-30% under high-power field, and most of the cells were with green fluorescence. But green fluorescent cells did not appear in the MSCs cells as control. ③After allografting of labeled or non-labeled MSCs of rats with different approaches, at day 1 the rats were listless with bad food appetite, less mobilization; At day 2mostly of them had normal diet and mood, but there was no significant difference in rats of each group. ④The rats in each group with the exception of mixed group had green fluorescent protein positive cells in liver at days 3 and 7. The number of green fluorescent protein positive DNA was higher in liver tissues in the CCL4 plus portal vein transplantation group and CCL4 plus caudal vein transplantation group than in the portal vein transplantation control group and caudal vein transplantation control group (P＜0.05).CONCLUSION: Duration and amount of stem cells colonizing in liver may be associated with liver injury, while not related to the implantation approach. In normal animals with uninjured liver the stem cells can colonize in liver, and the amount is associated with transplantation approach and post-transplantation duration.