大鼠骨髓间充质干细胞在视网膜色素变性大鼠体内分化的实验研究
摘要
目的研究体外培养的大鼠骨髓间充质干细胞(Mesenchymal stem cells，MSCs)视网膜下移植后在视网膜色素变性模型鼠内微环境中的存活、分化情况。
方法采用贴壁筛选法体外分离、培养Lewis大鼠MSCs，并经流式细胞仪鉴定CD90(+)CD45(-)后，用Brdu进行标记。用3％NaIO_3100mg／Kg对Lewis鼠进行腹腔注射后建立视网膜色素变性模型，并经病理切片证实视网膜色素上皮细胞(Retinal Pigment Epithelium RPE)及视网膜光感受器细胞破坏。将25只大鼠随机分为5组，每组5只。每只大鼠的右眼为实验眼，在睫状体平部穿刺，经玻璃体腔进入视网膜下腔注射MSCs悬液，左眼作为对照眼，视网膜下腔注射等量PBS液。术后1，2，3，4，5周分别处死一组动物，摘除眼球行冰冻病理切片后分别用小鼠抗人BrdU单抗、FITC标记羊抗小鼠二抗和小鼠抗大鼠Rhodopsin单抗、小鼠抗大鼠全角蛋白(Pan-cytokeratin，PCK)单抗、Cy3标记羊抗小鼠二抗双重荧光标记。观察术后1，2，3，4，5周MSCs在视网膜色素变性微环境中的存活、分化情况。
结果视网膜下移植术后第1周即可见MSCs位于视网膜色素上皮层和光感受器细胞层，第1与第2周MSCs的Rhodopsin标记和全角蛋白(Pan-cytokeratin，PCK)标记阴性，第3-5周可见MSCs Rhodopsin标记和全角蛋白(PCK)标记阳性。结果初步表明，实验组视网膜下移植的MSCs后可以表达视网膜色素上皮细胞和光感受器细胞的表面抗原。
结论MSCs移植于视网膜色素变性大鼠的视网膜下腔后，分布于视网膜色素上皮层和光感受器细胞层。第1-2周MSCs细胞不表达Rhodopsin和全角蛋白(PCK)，即尚未向RPE细胞和光感受器细胞方向分化。自第3-5周可见MSCs向RPE细胞和光感受器细胞方向分化。
Objective To observe the growth and differentiation of subretinal-transplantedrat mesenchymal stem cells (MSCs) in the microenvironment of retinitis pigmentosainduced by NaIO_3.
Methods Lewis rats MSCs cultured in-vitro were selected by adherence to theculture dish, identified by CD90(+) CD45(-) through flow cytometry and labeled withBrdu. 25 Lewis rats retinitis pigmentosa models were established by 3％NaIO_3intraperitoneal injection and subsequently confirmed by pathological section. 25 ratswere randomly distributed into 5 groups, 5 rats in every group. The right eyes wereinjected with MSCs in subretinal space, while the left eyes were injected with thesame amount of PBS. The rats were killed and globes were enucleated 1,2,3,4 or 5weeks after the surgery separately. Frozen sections were stained with Brdu primarymonoclonal antibody, FITC- labeled secondary monoclonal antibody and rhodopsin orpan- cytokeratin primary monoclonal antibody seperately combined withCy3-1abeled secondary monoclonal antibody successively. The sections wereobserved under fluorescent microscope.
Results MSCs distributed in the in the layer of RPE cells and photoreceptorcells in the 1st week after surgery. From the 3rd to 5th week, Rhodopsin (+) andpan-cytokeratin (+) MSCs were found. This indicate that the MSCs transplanted intothe subretinal space can demonstrate the surface antigen of the RPE and RPC cells.
Conclusions MSCs distributed in the layer of RPE cells and photoreceptor cellsafter transplantation. During 3rd to 5th week after transplantation, MSCs have thepotential to differentiate into photoreceptor cell and RPE cell.
方法采用贴壁筛选法体外分离、培养Lewis大鼠MSCs，并经流式细胞仪鉴定CD90(+)CD45(-)后，用Brdu进行标记。用3％NaIO_3100mg／Kg对Lewis鼠进行腹腔注射后建立视网膜色素变性模型，并经病理切片证实视网膜色素上皮细胞(Retinal Pigment Epithelium RPE)及视网膜光感受器细胞破坏。将25只大鼠随机分为5组，每组5只。每只大鼠的右眼为实验眼，在睫状体平部穿刺，经玻璃体腔进入视网膜下腔注射MSCs悬液，左眼作为对照眼，视网膜下腔注射等量PBS液。术后1，2，3，4，5周分别处死一组动物，摘除眼球行冰冻病理切片后分别用小鼠抗人BrdU单抗、FITC标记羊抗小鼠二抗和小鼠抗大鼠Rhodopsin单抗、小鼠抗大鼠全角蛋白(Pan-cytokeratin，PCK)单抗、Cy3标记羊抗小鼠二抗双重荧光标记。观察术后1，2，3，4，5周MSCs在视网膜色素变性微环境中的存活、分化情况。
结果视网膜下移植术后第1周即可见MSCs位于视网膜色素上皮层和光感受器细胞层，第1与第2周MSCs的Rhodopsin标记和全角蛋白(Pan-cytokeratin，PCK)标记阴性，第3-5周可见MSCs Rhodopsin标记和全角蛋白(PCK)标记阳性。结果初步表明，实验组视网膜下移植的MSCs后可以表达视网膜色素上皮细胞和光感受器细胞的表面抗原。
结论MSCs移植于视网膜色素变性大鼠的视网膜下腔后，分布于视网膜色素上皮层和光感受器细胞层。第1-2周MSCs细胞不表达Rhodopsin和全角蛋白(PCK)，即尚未向RPE细胞和光感受器细胞方向分化。自第3-5周可见MSCs向RPE细胞和光感受器细胞方向分化。
Objective To observe the growth and differentiation of subretinal-transplantedrat mesenchymal stem cells (MSCs) in the microenvironment of retinitis pigmentosainduced by NaIO_3.
Methods Lewis rats MSCs cultured in-vitro were selected by adherence to theculture dish, identified by CD90(+) CD45(-) through flow cytometry and labeled withBrdu. 25 Lewis rats retinitis pigmentosa models were established by 3％NaIO_3intraperitoneal injection and subsequently confirmed by pathological section. 25 ratswere randomly distributed into 5 groups, 5 rats in every group. The right eyes wereinjected with MSCs in subretinal space, while the left eyes were injected with thesame amount of PBS. The rats were killed and globes were enucleated 1,2,3,4 or 5weeks after the surgery separately. Frozen sections were stained with Brdu primarymonoclonal antibody, FITC- labeled secondary monoclonal antibody and rhodopsin orpan- cytokeratin primary monoclonal antibody seperately combined withCy3-1abeled secondary monoclonal antibody successively. The sections wereobserved under fluorescent microscope.
Results MSCs distributed in the in the layer of RPE cells and photoreceptorcells in the 1st week after surgery. From the 3rd to 5th week, Rhodopsin (+) andpan-cytokeratin (+) MSCs were found. This indicate that the MSCs transplanted intothe subretinal space can demonstrate the surface antigen of the RPE and RPC cells.
Conclusions MSCs distributed in the layer of RPE cells and photoreceptor cellsafter transplantation. During 3rd to 5th week after transplantation, MSCs have thepotential to differentiate into photoreceptor cell and RPE cell.
引文
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2. Beresford JN.Osteogenic stem cells and the stromal system of bone and marrow. Clin Orthop 1989;270-80
3. Owen M. Marrow stromal stem cells. J Cell Sci Suppl 1988;10:63-76
4. Ferrari, G et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science1998; 279:1528-30
5. Bruder SP, fink DJ, CaplanAI.Mesenchymal stem cells in bone development,bone repair,and skeletal regeneration therapy.J Cell Biochem.l994;56(3):283-94
6. Minoru Tomita, Yasushi Adachi, Haruhiko Yamada et al.Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem cell 2002;20:279-83
7. Donald O, Jan K, Stefano C et al.Bone marrow cells regenerate infracted myocardium.Nature 2001;410(5):701-5
8. Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701-705.
9. Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229-1234.
10. Shi Q, Rafii S, Wu MH-D et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362-367.
11. Krause DS, Thelse ND, Collector MI et al.Multi-orgn,multi-lineage engraftment by a single bone marrow-derived stem cell.Cell 2001; 105:369-77
12. Woodbury D, Schwarz EJ, Prockop DJ,Black IB.Adult rat and human bone marrow stromal cells differentiate into neurons.J Neurosci Res 2000;61:364-70
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