基于CNA35靶向液态氟碳纳米粒的超声分子成像检测糖尿病肾病大鼠肾脏纤维化的实验研究

Experimental study of CNA35-targeted fluorocarbon nanoparticles-based ultrasound molecular imaging for the detection of renal fibrosis in rats with diabetic nephropathy

目的制备一种由胶原结合蛋白35(CNA35)介导的靶向液态氟碳纳米粒(PFP-NPs),探讨其在超声分子成像检测糖尿病肾病(DN)大鼠肾脏纤维化中的应用价值。方法制备经荧光标记的非靶向PFP-NPs和CNA35靶向PFP-NPs,经转化生长因子-β诱导人肾小管上皮HK-2细胞间质转化,发生胶原沉积,然后分别将非靶向PFP-NPs和CNA35靶向PFP-NPs与细胞共同孵育,于倒置荧光显微镜下观察细胞上PFP-NPs荧光信号。建立DN大鼠模型,分为非靶向组和靶向组,每组各10只,分别静脉注射非靶向PFP-NPs与CNA35靶向PFP-NPs,应用超声分子成像检测两组DN大鼠静脉注射PFP-NPs后10 min、30 min肾脏超声分子成像信号强度;然后处死DN大鼠并取肾脏组织,检测肾脏组织PFP-NPs荧光信号强度及Ⅰ型胶原荧光信号强度;免疫组织化学检测肾脏组织Ⅰ型胶原染色面积占比,分析肾脏超声分子成像信号强度与Ⅰ型胶原染色面积占比的相关性。结果CNA35靶向PFP-NPs孵育的人肾小管上皮HK-2细胞上的荧光信号强度高于非靶向PFP-NPs孵育的细胞(388.21±15.28 vs.25.79±3.62),差异有统计学意义(P<0.05)。靶向组DN大鼠肾脏组织PFP-NPs荧光信号强度高于非靶向组(946.02±83.55 vs.73.69±21.72),差异有统计学意义(P<0.05);靶向组Ⅰ型胶原荧光信号强度与非靶向组比较差异无统计学意义(969.07±96.67 vs.943.39±106.18),且CNA35靶向PFP-NPs荧光信号与Ⅰ型胶原荧光信号区域重合。体内超声分子成像显示,靶向组DN大鼠静脉注射PFPNPs后10 min、30 min肾脏组织超声分子成像信号强度均高于非靶向组(8.37±1.27 vs.1.92±0.25、9.73±1.25 vs.2.08±1.32),差异均有统计学意义(均P<0.05)。相关性分析显示,DN大鼠肾脏超声分子成像信号强度与Ⅰ型胶原染色面积占比呈正相关(r=0.838,P<0.001)。结论成功制备了CNA35靶向PFP-NPs,其能够靶向结合大鼠肾脏胶原高表达部位,实现了对DN大鼠肾脏纤维化的靶向超声分子成像。

Objective To prepare targeted fluorocarbon nanoparticles(PFP-NPs)labeled with the collagen marker CNA35,and to explore application value for ultrasound molecular imaging in detecting renal fibrosis in rats with diabetic nephropathy(DN).Methods Non-targeted PFP-NPs and CNA35-targeted PFP-NPs labeled with fluorescent markers were prepared.The induction of human renal tubular epithelial cell HK-2 mesenchymal transformation and collagen deposition were mediated by transforming growth factor-β.The non-targeted PFP-NPs and targeted PFP-NPs were subsequently co-incubated with cells,followed by observation of the cellular fluorescence signal using a fluorescence microscope.The DN rats model were established and divided into a non-targeted group and a targeted group,10 rats in each group.The non-targeted PFP-NPs and CNA35-targeted PFP-NPs were intravenously administered to the two groups,respectively.Ultrasound molecular imaging was used to detect the signal intensity of renal ultrasound molecular imaging in DN rats in two groups at 10 min and 30 min after intravenous administration.The DN rats were put to death,then the kidney tissues were collected for detecting the fluorescence signals emitted by PFP-NPs and typeⅠcollagen.The expression and percentage of staining area for typeⅠcollagen in renal tissue were analyzed by immunohistochemistry,and the correlation between the signal intensity of renal ultrasound molecular imaging and the distribution percentage of typeⅠcollagen was analyzed.Results The fluorescence signal intensity of human renal tubular epithelial HK-2 cells incubated with CNA35-targeted PFP-NPs was higher than that of cells incubated with non-targeted PFP-NPs(388.21±15.28 vs.25.79±3.62),with a statistically significant difference(P<0.05).The fluorescence signal intensity of PFP-NPs in the renal tissue of DN rats in the targeted group was higher than that of non-targeted group(946.02±83.55 vs.73.69±21.72),with a statistically significant difference(P<0.05).There was no significant difference of fluorescence signal intensity of typeⅠcollagen between the targeted group and the non-targeted group(969.07±96.67 vs.943.39±106.18).The in vivo ultrasound molecular imaging demonstrated that the signal intensity of renal ultrasound molecular imaging of DN rats in the targeted group was higher than that of the non-targeted group at both 10 min and 30 min after intravenous administration(8.37±1.27 vs.9.73±1.25,1.92±0.25 vs.2.08±1.32),with statistically significant differences(both P<0.05).Correlation analysis showed a positive correlation between the signal intensity of renal ultrasound molecular imaging and the distribution percentage of typeⅠcollagen(r=0.838,P<0.001).Conclusion CNA35-targeted PFP-NPs were successfully prepared and demonstrated specific binding to collagen-enriched renal regions,enabling targeted ultrasound molecular imaging of renal fibrosis in DN rats.