【作者】 廖继东；

【导师】 张洹；

【作者基本信息】 暨南大学 ， 内科血液病， 2004， 博士

【摘要】 目的 探讨胎肝Sca-1⁺细胞横向分化的能力、特点和在肾损伤修复中的作用。方法取14.5天C57BL／6j小鼠的胎肝，制成单细胞悬液；用快速PCR方法鉴定细胞的性别；收集雄性细胞，用MACS（magnetic cell sorting）技术分离雄性胎肝Sca-1⁺（Stem Cell Antigen-1）细胞；用同样方法分离骨髓Sca-1⁺细胞；将2×10⁴的雄性胎肝Sca-1⁺细胞移植给受致死量γ照射的同系成年雌性小鼠；2～6月后取受体小鼠的肾、肝、肺、胃和小肠组织，制作3-5μm石蜡切片；用原位杂交技术追踪Y染色体来确定细胞的来源，同时用特异性组织化学技术显示组织特征。为了研究胎肝Sca-1⁺细胞修复能力，用甘油诱导小鼠急性肾功能衰竭（acute renal failure．ARF）的模型；3h后，腹膜下注射10μg／kg的G-CSF连续5天，监测血清肌酐和尿素氮水平以及肾组织病理形态。为了消除内源雌性干细胞的作用，用致死量射线清除小鼠体内造血干细胞，移植雄性胎肝Sca-1⁺细胞，8周后雌性小鼠骨髓变为雄性骨髓；用甘油诱导小鼠急性肾功能衰竭，72h后再次输入2×10⁴雄性胎肝Sca-1⁺细胞，8周后制取受体肾组织切片。用原位杂交技术追踪Y染色体来确定细胞的来源，并计算横向分化的频率。最后，比较了胎肝和骨髓的Sca-1⁺细胞向。肾组织细胞分化的频率。 结果 在移植雄性胎肝Sca-1⁺细胞的雌性小鼠的肾、肝、肺、胃和小肠组织切片中出现雄性细胞，各组织雄性细胞的百分数分别为（4.5±0.5）％、（0.9±0.1）％、（1.9±0.6）％、（6.1±0.5）％和（7.61±2.3）％，呈现小肠＞胃＞肾＞肺＞肝的特征；肾组织切片中的雄性细胞分别为RCA⁺、CYP_1A2⁺、Vimentin⁺、CD₄₅^-和F_4／80^-；急性肾损伤使雌性受体小鼠肾组织中的雄性细胞数增加（8.58±1.34）％，在肾小管周围出现成对排列的雄性细胞；雄性胎肝Sca-1⁺细胞输注使急性肾组织损伤雌性受体小鼠肾脏组织中的雄性细胞增多（18.13±1.91）％，在肾小管周围出现成环状排列的雄性细胞；在急性肾功能衰竭的肾组织中，雄性细胞频率高于骨髓；结论 小鼠胎肝Sca-1⁺细胞具有向肾、肝、肺、胃和小肠等组织细胞分化的潜力和修复损伤肾组织细胞的能力；小鼠胎肝Sca-1⁺细胞向肾组织细胞分化的能力优于骨髓。更多还原

【Abstract】 Object: To explore the potential and behavior of transdifferentiation of Sca-l+ cells from murine fetal liver as well as their repairing capacity in acute renal failure. Method: The single cell suspension was prepared of murine fetal liver of pregnancy of 14.5day; cell sex was determined by quick PCR and male cells were collected, and Sca-l+ cells of them were separated with MACS technology. 2x104 cells of male Sca-l+ cells were transplanted into female syngeneic mouse irradiated with lethally dose of Y ray by tail vein. During 2-6 months, the kidneys, livers, lungs, stomach and small intestinal tissue were taken out and paraffin slices of them were made. The Y-chromosome cells were traced by fluorescence in situ hybridization （FISH）, at the same time; the tissue characteristics of them were detected by immunochemistry. In order to detect the repairing capacity of Sca-l+ cells from fetal liver, the model of acute renal failure was made with glycerol. The urea nitrogen and creatine in serum were monitored after 10 H g/kg of G-CSF was injected into model mice. Furthermore, In order to reduce the interference of female hematopoietic stem cells in vivo, female bone marrow would be changed into male one with elimination of female bone marrow by radiation and transplantation of male Sca-l+ cells from fetal liver, which needed time of 8 weeks. The acute renal failure model of female mice with this male bone marrow was made again, and 72 hours later, Sca-l+ cells from fetal liver were transplanted into them. 8 weeks later, the mice were killed and kidneys were took out to be made tissue slices for detecting of FISH and immunochemistry, the transdifferentiation frequency of Sca-l+ cells from fetal liver and bone marrow were calculated and compared. Results: The male cells were found on sections of kidneys, liver, lung, gastric and smallintestinal tissue of female mice irradiated with lethally Y ray and transplanted with male sca-l+ cells, frequency of differentiation were （4.5+0.5）%, （0.9+0.1）%, （1.9+0.6）%, （6.1 0.5）% and （7.61 2.3）% respectively, phenotype characteristics of male renal tissue was RCA+/CYP1A2+/Vimentin+/CD457F4/80". There was a big increase of frequency of male cells from （4.5+₀.5）% to （8.58 1.34）% on sections of female mouse of acute renal failure compared with single dose of radiation, and there appeared a double male cell arrangement on area of renal tubules on section. There was much bigger increase of frequency of male on section of female mouse （18.13+₁.91）%, bone marrow sex of which had become male, of acute renal failure, and there appeared ring arrangement of several male cells on area of renal tubule on sections. The transdifferentiation frequency of Sca-l+ cells from fetal liver was higher than bone marrow on model of acute renal failure, as follows （3.55+0.51）% and （1.35 0.09）% respect.Conclusion: The Sca-l+ cells from fetal liver have the capacity differentiated into kidneys, liver, and lung, gastric and small intestinal tissue. The higher frequency of transdifferntiation would be obtained in acute renal failure. The transdifferentiation potential of Sca-l+ cells from fetal liver was much bigger than bone marrow.更多还原