骨形成蛋白-7基因修饰的骨髓间充质干细胞对肾缺血再灌注损伤修复作用的实验研究
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摘要
目的
观察骨形成蛋白-7(BMP-7)基因修饰的骨髓间充质干细胞(MSCs)对肾缺血再灌注损伤修复的影响,明确体内BMP-7对MSCs向肾细胞定向分化的影响。
方法
密度梯度离心法分离纯化大鼠MSCs。慢病毒介导BMP-7基因体外转染大鼠MSCs。PCR法检测BMP7-MSCs细胞上清BMP-7 mRNA的表达。建立大鼠肾脏缺血再灌注损伤(IRI)模型,通过下腔静脉注射将BMP7-MSCs移植入大鼠肾IRI模型,同时设立对照组。分别于细胞移植术后第3、5、7、30d取大鼠双侧肾检测各项指标(取肾前4h予腹腔注射BrdU,50mg/kg):BrdU免疫组织化学染色检测细胞分裂活跃程度;上皮钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、a-平滑肌肌动蛋白(a-SMA)的免疫组织化学染色检测分析细胞的类型;胶原纤维染色检测分析肾脏纤维化情况;绿色荧光蛋白的荧光显微镜检测分析细胞是否来源于供体; PCR检测分析波形蛋白、a-平滑肌肌动蛋白、BMP-7蛋白、上皮钙粘蛋白表达量。
结果
1密度梯度离心法成功分离纯化大鼠MSCs。
2 BMP-7修饰的重组MSCs构建成功,重组MSCs表达BMP-7。
3 160例肾缺血再灌注损伤大鼠模型建立成功。
4与对照组相比较,MSCs移植组在缺血再灌注肾损伤早期的Scr和BUN下降得更快(P<0.05)
5与MSCs移植组比较,BMP7-MSCs移植组对大鼠缺血再灌注肾损伤早期Scr和BUN的升高的抑制作用更显著(P<0.05)。
6与对照组相比,MSCs移植组的BrdU阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)
7与MSCs移植组相比, BMP7-MSCs移植组的BrdU阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)。
8.与MSCs移植组相比,BMP7-MSCs移植组的E-cadherin+/GFP+阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)。
9与对照组相比,MSCs移植组大鼠E-cadherin的mRNA明显增多,两者差异有统计学意义(P<0.05)。
10与MSCs移植组相比,BMP7-MSCs移植组大鼠E-cadherin的mRNA明显增多,两者差异具有统计学意义(P<0.05)。
11与空白对照组相比,MSCs移植组α-SMA的表达更低,两者差异有统计学意义(P<0.05)。
12与MSCs移植组相比,BMP7-MSCs移植组α-SMA的表达更低,两者差异有统计学意义(P<0.05)。
13与空白对照组相比,MSCs移植组Vimentin的表达更低,两者差异有统计学意义(P<0.05)。
14与MSCs移植组相比,BMP7-MSCs移植组Vimentin的表达更低,差异有统计学意义(P<0.05)。
15与对照组相比,MSCs移植组大鼠BMP-7的mRNA明显增多,两者差异有统计学意义(P<0.05)。
16与MSCs移植组相比,BMP7-MSCs移植组大鼠BMP-7的mRNA明显增多,两者差异具有统计学意义(P<0.05)。
结论
密度梯度离心法能成功地分离纯化大鼠MSCs。通过慢病毒载体能成功高效地将BMP-7转染MSCs。外源MSCs和BMP7-MSCs对大鼠缺血再灌注肾损伤早期Scr和BUN的升高具有一定的抑制作用,能够促进缺血再灌注后肾损伤的肾脏细胞的增殖,能够定向分化为肾小管细胞而修复肾损伤,能够减轻肾IRI后肾间质纤维化程度;BMP7能够促进MSCs对肾损伤的修复。
Objective
To observe the impact of the MSCs carrying BMP-7 gene on the reconstruction of Ischemia reperfusion injury kidney and reveal the effects of BMP-7 on the directional differentiation of the kidney mesenchymal cells to the epithelial cells in vivo.
Methods
Rat MSCs were separated and purified by density gradient centrifugation.In vitro, rat MSCs were transfected with BMP-7 gene by slow virus vectors. The expression of the BMP-7 in the supernatant was detected by PCR. MSCs expressing BMP-7 gene were isolated and continued to be cultured for proliferation. Rat models of kidney ischemia reperfusion injury were established, then injected with BMP7-MSCs through inferior vena cava.At the same time,the controlling groups were set up.In the scheduled time (3,5,7 and 30 days after transplantation), rats in each group were kindly sacrificed 4 hours after injection of BrdU(50mg/kg)transabdominally. Bilateral kidneys of the rats were removed, then prepared for tissue slices. Analysis of the cell proliferation were taken through BrdU immunohistochemical staining, and cell types were identified through E-cadherin, vimentin and a-SMA immunohistochemical assay. Renal fibrosis was revealed through collagen staining, and the source of the donor cells was confirmed by the expression of green fluorescent proteins through a fluorescence microscope. Quantitative analysis of the expression of vementin, a-SMA, BMP-7 protein and E-cadherin were conducted through PCR methods.
Results
1. Rat MSCs had been separated and purified by density gradient centrifugation successfully.
2. Rat MSCs had been transfected with BMP-7 gene by slow virus vectors successfully. The expression of the BMP-7 in the supernatant had been detected by PCR.
3. 160 rat models of kidney ischemia reperfusion injury had been established successfully.
4. Early increase in serum Cr and BUN could be suppressed by the MSCs transplantation. Compared with the controlling groups, serum Cr and BUN levels decreased more rapidly in MSCs transplantation groups(P<0.05).
5. Synergistic inhibiting effects on the serum Cr and BUN levels between exogenous MSCs and BMP-7 could be observed in the early stage of the ischemic reperfusion injured rat kidneys. Compared with the MSCs transplantation groups, early inhibiting effects in the BMP7-MSCs transplanting groups were more significantly (P<0.05).
6. BrdU positive cells were more in the MSCs transplantation groups than that in the controlling groups (P<0.05).
7. BrdU positive cells were more in the BMP7-MSCs transplantation groups than that in the MSCs transplantation groups (P<0.05).
8. Compared with the MSCs transplantation groups, E-cadherin+/GFP+ positive cells increased in the BMP7-MSCs transplantation groups (P<0.05).
9. Post operation increasing expression of E-cadherin mRNA could be seen in each group with significant differences between every two time points (P<0.05). Increasing expression of E-cadherin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups.
10. Expression of SMA was similar between the MSCs and BMP7-MSCs transplantation groups, with their peak at the 3 days post- transplantation, and decreased thereafter. However, at each time point, there was a more decreased expression of SMA in the C groups (BMP7-MSCs transplantation group) than that in the B groups (MSCs transplantation group) (P<0.05).
11. Post operation decreased expression of Vimentin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups.
12. Post operation increasing expression of BMP-7 mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups(P<0.05).
Conclusion
Rat MSCs could be separated and purified by density gradient centrifugation successfully.Rat MSCs could be transfected with BMP-7 gene by slow virus vectors efficiently.Exogenous MSCs and BMP7-MSCs could suppress the early increase of serum Cr and BUN, promote the regeneration of the renal cells and differentiate into the renal tubular epithelium, and also alleviate the fibrosis of the renal interstitial in the ischemic reperfusion injured kidney in the rat models. BMP-7 has a promoting effect on reparation of the injured kidney via MSCs.
观察骨形成蛋白-7(BMP-7)基因修饰的骨髓间充质干细胞(MSCs)对肾缺血再灌注损伤修复的影响,明确体内BMP-7对MSCs向肾细胞定向分化的影响。
方法
密度梯度离心法分离纯化大鼠MSCs。慢病毒介导BMP-7基因体外转染大鼠MSCs。PCR法检测BMP7-MSCs细胞上清BMP-7 mRNA的表达。建立大鼠肾脏缺血再灌注损伤(IRI)模型,通过下腔静脉注射将BMP7-MSCs移植入大鼠肾IRI模型,同时设立对照组。分别于细胞移植术后第3、5、7、30d取大鼠双侧肾检测各项指标(取肾前4h予腹腔注射BrdU,50mg/kg):BrdU免疫组织化学染色检测细胞分裂活跃程度;上皮钙粘蛋白(E-cadherin)、波形蛋白(Vimentin)、a-平滑肌肌动蛋白(a-SMA)的免疫组织化学染色检测分析细胞的类型;胶原纤维染色检测分析肾脏纤维化情况;绿色荧光蛋白的荧光显微镜检测分析细胞是否来源于供体; PCR检测分析波形蛋白、a-平滑肌肌动蛋白、BMP-7蛋白、上皮钙粘蛋白表达量。
结果
1密度梯度离心法成功分离纯化大鼠MSCs。
2 BMP-7修饰的重组MSCs构建成功,重组MSCs表达BMP-7。
3 160例肾缺血再灌注损伤大鼠模型建立成功。
4与对照组相比较,MSCs移植组在缺血再灌注肾损伤早期的Scr和BUN下降得更快(P<0.05)
5与MSCs移植组比较,BMP7-MSCs移植组对大鼠缺血再灌注肾损伤早期Scr和BUN的升高的抑制作用更显著(P<0.05)。
6与对照组相比,MSCs移植组的BrdU阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)
7与MSCs移植组相比, BMP7-MSCs移植组的BrdU阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)。
8.与MSCs移植组相比,BMP7-MSCs移植组的E-cadherin+/GFP+阳性细胞个数明显增多,两者差异有统计学意义(P<0.05)。
9与对照组相比,MSCs移植组大鼠E-cadherin的mRNA明显增多,两者差异有统计学意义(P<0.05)。
10与MSCs移植组相比,BMP7-MSCs移植组大鼠E-cadherin的mRNA明显增多,两者差异具有统计学意义(P<0.05)。
11与空白对照组相比,MSCs移植组α-SMA的表达更低,两者差异有统计学意义(P<0.05)。
12与MSCs移植组相比,BMP7-MSCs移植组α-SMA的表达更低,两者差异有统计学意义(P<0.05)。
13与空白对照组相比,MSCs移植组Vimentin的表达更低,两者差异有统计学意义(P<0.05)。
14与MSCs移植组相比,BMP7-MSCs移植组Vimentin的表达更低,差异有统计学意义(P<0.05)。
15与对照组相比,MSCs移植组大鼠BMP-7的mRNA明显增多,两者差异有统计学意义(P<0.05)。
16与MSCs移植组相比,BMP7-MSCs移植组大鼠BMP-7的mRNA明显增多,两者差异具有统计学意义(P<0.05)。
结论
密度梯度离心法能成功地分离纯化大鼠MSCs。通过慢病毒载体能成功高效地将BMP-7转染MSCs。外源MSCs和BMP7-MSCs对大鼠缺血再灌注肾损伤早期Scr和BUN的升高具有一定的抑制作用,能够促进缺血再灌注后肾损伤的肾脏细胞的增殖,能够定向分化为肾小管细胞而修复肾损伤,能够减轻肾IRI后肾间质纤维化程度;BMP7能够促进MSCs对肾损伤的修复。
Objective
To observe the impact of the MSCs carrying BMP-7 gene on the reconstruction of Ischemia reperfusion injury kidney and reveal the effects of BMP-7 on the directional differentiation of the kidney mesenchymal cells to the epithelial cells in vivo.
Methods
Rat MSCs were separated and purified by density gradient centrifugation.In vitro, rat MSCs were transfected with BMP-7 gene by slow virus vectors. The expression of the BMP-7 in the supernatant was detected by PCR. MSCs expressing BMP-7 gene were isolated and continued to be cultured for proliferation. Rat models of kidney ischemia reperfusion injury were established, then injected with BMP7-MSCs through inferior vena cava.At the same time,the controlling groups were set up.In the scheduled time (3,5,7 and 30 days after transplantation), rats in each group were kindly sacrificed 4 hours after injection of BrdU(50mg/kg)transabdominally. Bilateral kidneys of the rats were removed, then prepared for tissue slices. Analysis of the cell proliferation were taken through BrdU immunohistochemical staining, and cell types were identified through E-cadherin, vimentin and a-SMA immunohistochemical assay. Renal fibrosis was revealed through collagen staining, and the source of the donor cells was confirmed by the expression of green fluorescent proteins through a fluorescence microscope. Quantitative analysis of the expression of vementin, a-SMA, BMP-7 protein and E-cadherin were conducted through PCR methods.
Results
1. Rat MSCs had been separated and purified by density gradient centrifugation successfully.
2. Rat MSCs had been transfected with BMP-7 gene by slow virus vectors successfully. The expression of the BMP-7 in the supernatant had been detected by PCR.
3. 160 rat models of kidney ischemia reperfusion injury had been established successfully.
4. Early increase in serum Cr and BUN could be suppressed by the MSCs transplantation. Compared with the controlling groups, serum Cr and BUN levels decreased more rapidly in MSCs transplantation groups(P<0.05).
5. Synergistic inhibiting effects on the serum Cr and BUN levels between exogenous MSCs and BMP-7 could be observed in the early stage of the ischemic reperfusion injured rat kidneys. Compared with the MSCs transplantation groups, early inhibiting effects in the BMP7-MSCs transplanting groups were more significantly (P<0.05).
6. BrdU positive cells were more in the MSCs transplantation groups than that in the controlling groups (P<0.05).
7. BrdU positive cells were more in the BMP7-MSCs transplantation groups than that in the MSCs transplantation groups (P<0.05).
8. Compared with the MSCs transplantation groups, E-cadherin+/GFP+ positive cells increased in the BMP7-MSCs transplantation groups (P<0.05).
9. Post operation increasing expression of E-cadherin mRNA could be seen in each group with significant differences between every two time points (P<0.05). Increasing expression of E-cadherin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups.
10. Expression of SMA was similar between the MSCs and BMP7-MSCs transplantation groups, with their peak at the 3 days post- transplantation, and decreased thereafter. However, at each time point, there was a more decreased expression of SMA in the C groups (BMP7-MSCs transplantation group) than that in the B groups (MSCs transplantation group) (P<0.05).
11. Post operation decreased expression of Vimentin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups.
12. Post operation increasing expression of BMP-7 mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups(P<0.05).
Conclusion
Rat MSCs could be separated and purified by density gradient centrifugation successfully.Rat MSCs could be transfected with BMP-7 gene by slow virus vectors efficiently.Exogenous MSCs and BMP7-MSCs could suppress the early increase of serum Cr and BUN, promote the regeneration of the renal cells and differentiate into the renal tubular epithelium, and also alleviate the fibrosis of the renal interstitial in the ischemic reperfusion injured kidney in the rat models. BMP-7 has a promoting effect on reparation of the injured kidney via MSCs.
引文
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