不同剂量骨髓间充质干细胞对阿霉素肾病大鼠肾纤维化形成的影响

Effect of different doses of bone marrow mesenchymal stem cells on renal fibrosis in experimental rats with adriamycin-induced nephropathy

目的探讨不同剂量骨髓间充质干细胞(BMSC)对阿霉素慢性肾病大鼠肾纤维化形成的影响及其作用机制。方法 SD大鼠尾静脉注射阿霉素造模,首次剂量3 mg/kg,2周后相同剂量再次注射。造模成功后将大鼠随机分为M组(模型组,肾动脉注射磷酸缓冲液),A组、B组、C组(肾动脉分别移植1×10^(6)个/mL、2×10^(6)个/mL、3×10^(6)个/mL BMSC,细胞悬液均为0.5 mL),每组各9只。另取9只正常大鼠作为对照组(N组,尾静脉输注等量0.9%氯化钠溶液)。术后第4周测定各组大鼠体重,Masson染色评估肾纤维化程度,过碘酸六胺银(PASM)染色观察肾小球和肾小管基膜改变,免疫组化染色分析转化生长因子(TGF)-β1、纤维连接蛋白(FN)表达。结果 BMSC移植4周后,A、B、C组大鼠一般情况均较M组改善,体重改变最明显,体重增长变化为C组>B组>A组,但C组与B组差异无统计学意义(P>0.05)。Masson染色显示M组表现为典型的肾小球和肾小管纤维化改变,肾组织大面积蓝染,A、B、C组虽也有不同程度肾纤维化改变,但程度和范围均较M组减少(P<0.05)。PASM染色显示M组肾小球和肾小管基膜明显增厚,A、B、C组肾小球和肾小管基膜增厚程度下降(P<0.05)。免疫组化染色显示A、B、C组肾组织不同部位TGF-β1、FN表达与M组比较均下降,尤其是C组下降最明显,仅在部分肾小球和肾小管上皮细胞见少量弱阳性表达。结论 BMSC移植治疗可延缓阿霉素慢性肾病大鼠早期肾纤维化形成,且该作用随细胞移植数量增加而增强。其机制可能是通过下调TGF-β1表达抑制细胞外基质过度沉积实现的。

Objective To explore the effect of different doses of bone marrow mesenchymal stem cells(BMSC) on renal fibrosis in experimental rats with adriamycin-induced nephropathy and to discuss its mechanism. Methods A total of 36 adriamycin-induced nephropathy models of SD rat were established by injecting adriamycin via caudal vein, the initial dose of adriamycin was 3 mg/kg and the same dose was injected again after 2 weeks. After successful modeling, the experimental rats were randomly divided into model group(group M, n=9, renal injection of phosphate buffer solution), group A(n=9, renal injection 0.5 mL of 1×10^(6) BMSC), group B(n=9, renal injection 0.5 mL of 2×10^(6) BMSC) and group C(n=9, renal injection 0.5 mL of 3×10^(6) BMSC). Other 9 healthy rats were used as control group(group N, injection 0.5 mL of saline via caudal vein). Four weeks after the treatment, the body weight of rats in all groups was measured, Masson staining was adopted to assess the degree of renal fibrosis, PASM staining was employed to observe the changes of renal glomeruli and tubular basement membrane, and immunohistochemical staining was used to test the expressions of transforming growth factor-β1(TGF-β1) and fibronectin(FN). Results Four weeks after BMSC transplantation, the general condition of experimental rats in group A, B and C was more improved than that in group M, and the change of body weight was the most obvious. The order of weight gain was group C >group B >group A, but the difference in body weight between group C and group B was not statistically significant(P>0.05). Masson staining demonstrated that renal glomeruli and tubules showed typical fibrosis changes with large area blue staining of renal tissues in group M, although different degrees of renal fibrosis were observed in group A, group B and group C, their degree and extent were less prominent when compared with group M(P<0.05). PASM staining showed that in group M both renal glomeruli and tubular basement membrane were remarkably thickened, while the degree of which in group A, group B and group C was much less(P<0.05). Immunohistochemical staining revealed that the expression levels of TGF-β1 and FN in different parts of the kidney in group A, group B and group C were lower than those in group M, which was more obvious in group C as in the rats of group C only slightly and weakly positive expression could be observed in some renal glomeruli and tubular epithelial cells. Conclusion BMSC transplantation can delay the formation of early renal fibrosis in experimental rats with adriamycin-induced chronic nephropathy, and this effect is enhanced with the increase of the amount of transplanted BMSC. This mechanism may be achieved by down-regulating TGF-β1 expression, resulting in inhibition of excessive deposition of extracellular matrix.