CAJ | 학술논문

目的：探讨慢性肾衰竭模型中透明质酸（ HA）与CD44的相互作用对骨髓间充质干细胞（ MSC）归巢和修复受损肾脏能力的影响。方法根据随机数字表法将32只雄性SD大鼠分为：假手术组（ n＝8）、模型组（ n＝8）、MSC组（n＝8）和MSC/抗CD44组（n＝8）。采用间隔1周的两步法建立大鼠5/6肾切除模型，假手术组只进行麻醉和肾被膜剥离，而不进行肾切除术。第二次术后第8周分别给予尾静脉注射1×107/ml MSC、1 ml 磷酸盐缓冲液（ PBS）、1×107/ml MSC、1×107/ml抗CD44抗体OX50预孵的MSC。治疗4周后处死大鼠，留取血标本和残肾组织。检测血肌酐和尿素氮水平；过碘酸雪夫（ PAS）染色和Masson染色后观察病理改变，应用肾小球硬化指数和肾小管间质损伤指数来判定肾小球和肾小管间质损伤程度；免疫组化法观察残肾组织中巨噬细胞和肌成纤维细胞的变化；免疫荧光法追踪归巢的MSC；免疫组化法和免疫印迹法观察HA的表达；双荧光法观察MSC与HA的共定位关系。结果与假手术组相比，模型组大鼠的血肌酐、尿素氮、肾小球硬化指数、肾小管间质损伤指数升高（ P＜0.05），肾脏病理改变主要为肾小球肥大、局灶节段性肾小球硬化，炎性细胞浸润和肾间质纤维化，包括巨噬细胞和肌成纤维细胞数量明显增加；与模型组相比，MSC组、MSC/抗CD44组肌酐、尿素氮、肾小球硬化指数、肾小管间质损伤指数均降低（ P＜0.05）。假手术组肾皮质几乎不表达HA，而模型组的肾皮质大量表达HA。免疫荧光法显示，治疗4周后，假手术组未发现有归巢的MSC，MSC组的残肾组织中有较多的MSC，MSC/抗CD44组归巢的MSC减少。双免疫荧光结果显示较多MSC归巢至HA表达丰富的区域。结论 HA和CD44的相互作用影响MSC的归巢能力，但不影响MSC修复受损肾脏的能力。
목적：탐토만성신쇠갈모형중투명질산（ HA）여CD44적상호작용대골수간충질간세포（ MSC）귀소화수복수손신장능력적영향。방법근거수궤수자표법장32지웅성SD대서분위：가수술조（ n＝8）、모형조（ n＝8）、MSC조（n＝8）화MSC/항CD44조（n＝8）。채용간격1주적량보법건립대서5/6신절제모형，가수술조지진행마취화신피막박리，이불진행신절제술。제이차술후제8주분별급여미정맥주사1×107/ml MSC、1 ml 린산염완충액（ PBS）、1×107/ml MSC、1×107/ml항CD44항체OX50예부적MSC。치료4주후처사대서，류취혈표본화잔신조직。검측혈기항화뇨소담수평；과전산설부（ PAS）염색화Masson염색후관찰병리개변，응용신소구경화지수화신소관간질손상지수래판정신소구화신소관간질손상정도；면역조화법관찰잔신조직중거서세포화기성섬유세포적변화；면역형광법추종귀소적MSC；면역조화법화면역인적법관찰HA적표체；쌍형광법관찰MSC여HA적공정위관계。결과여가수술조상비，모형조대서적혈기항、뇨소담、신소구경화지수、신소관간질손상지수승고（ P＜0.05），신장병리개변주요위신소구비대、국조절단성신소구경화，염성세포침윤화신간질섬유화，포괄거서세포화기성섬유세포수량명현증가；여모형조상비，MSC조、MSC/항CD44조기항、뇨소담、신소구경화지수、신소관간질손상지수균강저（ P＜0.05）。가수술조신피질궤호불표체HA，이모형조적신피질대량표체HA。면역형광법현시，치료4주후，가수술조미발현유귀소적MSC，MSC조적잔신조직중유교다적MSC，MSC/항CD44조귀소적MSC감소。쌍면역형광결과현시교다MSC귀소지HA표체봉부적구역。결론 HA화CD44적상호작용영향MSC적귀소능력，단불영향MSC수복수손신장적능력。
Objective To explore the effect of hyaluronic acid( HA)and CD44 on the homing and improving renal function of Mesenchymal Stem Cells( MSC)in a rat chronic renal failure model. Methods 32 grade clean adult male SD rats were randomly divided into 4 groups:sham operation group,renal failure group,MSC group,MSC/anti -CD44 group,8 in each group. Establishment of rat remnant kidney model:after one week adaptive breeding,all cut right kidney first,then cut 2/3 left kidney 1 week later. But in sham operation group,after anesthesia,rats were only given renal capsule stripping. 8 weeks af-ter model establishment,1 ml MSC(1 × 107/ml),1 ml PBS,1 ml MSC(1 × 107/ml)and 1 ml MSC(1 × 107/ml)incuba-ted with OX50(anti CD44 antibody)were injected via tail vein in every group respectively. After treatment of 4 weeks,all rats were killed and blood sample and kidney tissue were collected. Serum Cr and BUN were tested. Pathological changes of kidney were assessed by PAS and Masson stain. Glomerulosclerosis index and renal tubular mesenchymal damage index were calculated by semi-quantitative method. Tracing MSC using immunofluoresent assay. Double fluorescence immunostain of MSC and HA was used in kidney sections of rat. Expression of HA was assayed by immunohistochemical staining and western blot. Results Com-pared with sham operation group,renal failure group displayed significantly elevated Cr,BUN,glomerulosclerosis index and renal tubular mesenchymal damage index,and the difference was significant(P<0. 05). The results of PAS and Masson staining in-dicated that compared to sham operation group major pathological changes in residule kidney tissues were glomerular hypertrophy, cloudy swelling of renal epithelial cells,focal mesangial cell proliferation,widened mesangial matrix,focal glassy degeneration of glomerular capillary,renal interstitial inflammatory cell infiltration,focal interstitial fibrosis. MSC and MSC/anti-CD44 treat-ments could alleviate the above pathological changes in MSC group and MSC/anti-CD44 group(P<0. 05). After treatment of 4 weeks,there was no MSC homing in sham operation group,increased MSC homing was detected in residule kidney tissues after MSC administration,whereas the number of MSC homing was decreased in MSC/anti-CD44 group. There was no expression HA in sham operation group,but HA was significantly observed in injured cortical renal tissue. MSC was likely home to the area con-tained abundant HA in renal tissue. Conclusion Interaction between CD44 and HA has the potential to influnce MSCs homing to remnant kidney. This impact might supress MSC homing,but the ability of MSC in recovery of renal function was unchanged.