人脐带间充质干细胞移植对急性肾小管坏死再生修复的实验研究
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摘要
目的：观察人脐带间充质干细胞在急性肾小管坏死模型的体内的分布、归巢及再生修复的作用。
方法：
1、人脐带间充质干细胞体外培养、扩增和分化,移植前均用DAPI标记；
2、急性肾小管坏死模型的建立：肌注新鲜配制的0.2%二氯化汞溶液7ml/kg建立急性肾小管坏死的家犬模型；将总量900mg/kg庆大霉素,首次300mg/kg,12h后200mg/kg,余400mg/kg分两次每隔24h皮下注射,共3天,建立急性肾小管坏死的大鼠模型,
3、人脐带间充质干细胞移植：采用经外周静脉注射法移植和股动脉介入经左肾动脉直接注射方法移植
4、指标测定：①细胞定位测定：采用荧光显微镜观察肾脏DAPI阳性细胞。②肾小管功能检测：BUN、SCr、尿蛋白等。③肾脏病理检测：HE, PAS染色；④相关因子的检测：免疫组化法检测肾组织iNOS的表达
结果：
1、二氯化汞溶液和庆大霉素可以成功制造急性肾小管坏死模型
2、对照组实验动物移植人脐带间充质干细胞后各个时间点均可在肾小管检测DAPI阴性
3、造模成功的实验动物移植人脐带间充质干细胞后各个时间点均可在肾小管检测DAPI阳性,而在心脏、肝脏及胰腺检测DAPI阴性；移植后实验动物死亡率明显降低；肾功能明显改善；肾脏病理明显减轻；肾脏局部iNOS促炎症因子下调。
结论：
1、人脐带间充质干细胞移植后可归巢到损伤的肾小管,降低动物死亡率,减轻肾脏病理损害及改善肾功能,对正常肾脏无影响；
2、对于急性肾小管坏死模型动物,人脐带间充质干细胞选择性存在于肾脏内,对心脏、肝脏及胰腺无影响。
3、人脐带间充质干细胞对肾小管的修复通过下调iNOS表达。
Objective:To observe the distribution, chemo taxis and repairation role of human umbilical cord mesenchymal stem cells in the animals of acute tubular necrosis.
Methods:
1.Cultured and expanded the human umbilical cord mesenchymal stem cells in vitro,labeled the cells with DAPI before transplantation.
2.Made ATN models:given 0.2% HgCl2,7mg/kg by intramuscular injection to establish model of Acute tubular necrosis; injected the total dose of 900mg/kg gentamicin, for the first time injected 300mg/kg,200mg/kg was injected after 12h, and injected 200mg/kg subcutaneous twice at intervals of 24h, within 3 days.
3.Transplanted human umbilical cord mesenchymal stem cells:By peripheral veins injection and by femoral artery intervention.
4.Detected following parameters:①Traced the cells homing:observed the DAPI positive cells under fluorescence microscope.②Detectde the function of renal tubule:BUN、SCr、urine protein etc.③observed the renal pathologic Change by HE and PAS staining.④Detected some relative cytokines:iNOS by immunohistochemistry.
Results:
1.Gentamicin and HgCl2 could make ATN model successfully.
2.The control group did not have any umbilical cord mesenchymal stem cells in the kidneys.
3.For animals with acute tubular necrosis,DAPI positive cells could be detected in the tubule at each time of 5 points after transplantaion, liver and pancreas had no human umbilical cord mesenchymal stem cells existed;The mortality rate lowered obviously;The renal function ameliorated visiblely;The renal pathologic change relieved significantly;proinflammatory cytokine iNOS was down-regulated.
Conclusion:
1.The transplanted human umbilical cord mesenchymal stem cells can survive in the kidneys which experienced the development of acute renal tubular necrosis, the mortality rate lowered obviously,the renal function ameliorated visiblely,the renal pathologic change relieved significantly, and do not affect the normal kidney.
2.For dogs with acute tubular necrosis, human umbilical cord mesenchymal stem cells selectively can survived in the kidney, and have no effect on heart, liver and pancreas.
3.The possible mechanism of regeneration was that human umbilical cord mesenchymal stem cells could down-regulate iNOS expression.
方法：
1、人脐带间充质干细胞体外培养、扩增和分化,移植前均用DAPI标记；
2、急性肾小管坏死模型的建立：肌注新鲜配制的0.2%二氯化汞溶液7ml/kg建立急性肾小管坏死的家犬模型；将总量900mg/kg庆大霉素,首次300mg/kg,12h后200mg/kg,余400mg/kg分两次每隔24h皮下注射,共3天,建立急性肾小管坏死的大鼠模型,
3、人脐带间充质干细胞移植：采用经外周静脉注射法移植和股动脉介入经左肾动脉直接注射方法移植
4、指标测定：①细胞定位测定：采用荧光显微镜观察肾脏DAPI阳性细胞。②肾小管功能检测：BUN、SCr、尿蛋白等。③肾脏病理检测：HE, PAS染色；④相关因子的检测：免疫组化法检测肾组织iNOS的表达
结果：
1、二氯化汞溶液和庆大霉素可以成功制造急性肾小管坏死模型
2、对照组实验动物移植人脐带间充质干细胞后各个时间点均可在肾小管检测DAPI阴性
3、造模成功的实验动物移植人脐带间充质干细胞后各个时间点均可在肾小管检测DAPI阳性,而在心脏、肝脏及胰腺检测DAPI阴性；移植后实验动物死亡率明显降低；肾功能明显改善；肾脏病理明显减轻；肾脏局部iNOS促炎症因子下调。
结论：
1、人脐带间充质干细胞移植后可归巢到损伤的肾小管,降低动物死亡率,减轻肾脏病理损害及改善肾功能,对正常肾脏无影响；
2、对于急性肾小管坏死模型动物,人脐带间充质干细胞选择性存在于肾脏内,对心脏、肝脏及胰腺无影响。
3、人脐带间充质干细胞对肾小管的修复通过下调iNOS表达。
Objective:To observe the distribution, chemo taxis and repairation role of human umbilical cord mesenchymal stem cells in the animals of acute tubular necrosis.
Methods:
1.Cultured and expanded the human umbilical cord mesenchymal stem cells in vitro,labeled the cells with DAPI before transplantation.
2.Made ATN models:given 0.2% HgCl2,7mg/kg by intramuscular injection to establish model of Acute tubular necrosis; injected the total dose of 900mg/kg gentamicin, for the first time injected 300mg/kg,200mg/kg was injected after 12h, and injected 200mg/kg subcutaneous twice at intervals of 24h, within 3 days.
3.Transplanted human umbilical cord mesenchymal stem cells:By peripheral veins injection and by femoral artery intervention.
4.Detected following parameters:①Traced the cells homing:observed the DAPI positive cells under fluorescence microscope.②Detectde the function of renal tubule:BUN、SCr、urine protein etc.③observed the renal pathologic Change by HE and PAS staining.④Detected some relative cytokines:iNOS by immunohistochemistry.
Results:
1.Gentamicin and HgCl2 could make ATN model successfully.
2.The control group did not have any umbilical cord mesenchymal stem cells in the kidneys.
3.For animals with acute tubular necrosis,DAPI positive cells could be detected in the tubule at each time of 5 points after transplantaion, liver and pancreas had no human umbilical cord mesenchymal stem cells existed;The mortality rate lowered obviously;The renal function ameliorated visiblely;The renal pathologic change relieved significantly;proinflammatory cytokine iNOS was down-regulated.
Conclusion:
1.The transplanted human umbilical cord mesenchymal stem cells can survive in the kidneys which experienced the development of acute renal tubular necrosis, the mortality rate lowered obviously,the renal function ameliorated visiblely,the renal pathologic change relieved significantly, and do not affect the normal kidney.
2.For dogs with acute tubular necrosis, human umbilical cord mesenchymal stem cells selectively can survived in the kidney, and have no effect on heart, liver and pancreas.
3.The possible mechanism of regeneration was that human umbilical cord mesenchymal stem cells could down-regulate iNOS expression.
引文
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