壳聚糖/β-甘油磷酸钠/胶原水凝胶包裹IR-780碘化物标记肌腱干细胞的体内示踪研究  

In vivo tracing study of IR-780 labeled tendon stem cells wrapped with C/GP/Co hydrogel

作　　者：杨志金 徐江[1] 盛晓飞 陈康[1] 段少飞 李斌[1] 唐康来 Yang Zhijin;Xu Jiang;Sheng Xiaofei;Chen Kang;Duan Shaofei;Li Bin;Tang Kanglai(Department of Rehabilitation Medicine,920th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army,Kunming 650032,China;Department of Orthopedic Surgery/Sports Medicine Center,The First Affiliated Hospital of Army Medical University,Chongqing 400038,China)

机构地区：[1]中国人民解放军联勤保障部队第九二O医院康复医学科,昆明650032 [2]陆军军医大学第一附属医院骨科运动医学中心,重庆400038

出　　处：《创伤外科杂志》2021年第10期781-787,共7页Journal of Traumatic Surgery

基　　金：国家自然科学基金重点项目(81230040)。

摘　　要：目的探讨肌腱干细胞(TSCs)用IR-780碘化物标记之后的生物学特性以及壳聚糖(C)/β-甘油磷酸钠(GP)/胶原(Co)水凝胶包裹IR-780碘化物标记TSCs的体内示踪情况。方法 14只雄性SD大鼠(4周龄,体重约100g),2只用于提取大鼠TSCs,4只用于活体成像观察,8只用于组织学分析。用酶消化法提取大鼠跟腱来源TSCs,用IR-780碘化物标记TSCs,激光共聚焦显微镜下观察IR-780碘化物标记TSCs的细胞内定位,用流式细胞术检测其标记阳性率及对TSCs细胞周期的影响,用CCK-8法检测IR-780碘化物标记TSCs对其增殖的影响,用C/GP/Co水凝胶包裹IR-780碘化物标记的TSCs移植至大鼠跟腱损伤部位。通过全身近红外成像及组织学观察,检测TSCs在大鼠体内的存活及分布情况。结果成功提取大鼠跟腱来源TSCs,并成功制作C/GP/Co水凝胶。IR-780碘化物可成功标记TSCs,其标记阳性率为95.75%,荧光强度显著高于对照组,且对TSCs的细胞周期及增殖无明显不良影响。通过全身近红外成像及组织学观察,很容易检测到大鼠跟腱损伤区域IR-780碘化物标记的TSCs,荧光信号在大鼠身体其他部位几乎检测不到。从1~28d,IR-780碘化物的荧光信号逐渐减弱,但28d时仍能检测到明显荧光。结论 IR-780碘化物可有效标记TSCs,其标记阳性率为95.75%,且对TSCs的细胞周期及增殖无明显不良影响;IR-780碘化物标记的TSCs即使在28d时也有明显可检测的荧光;TSCs包裹在C/GP/Co水凝胶内移植至大鼠跟腱损伤部位,至少存活了4周。C/GP/Co水凝胶包裹TSCs可用于大鼠跟腱损伤的体内实验研究。Objective To investigate the biological characteristics of tendon stem cells(TSCs)labeled with IR-780 iodide,and the in vivo tracking of IR-780 labeled TSCs wrapped in(chitosan)C/(β-glycerophosphate)GP/(collagen)Co hydrogel.Methods Fourteen male SD rats(4 weeks old,weighing about 100g)were selected for this study.Among them,two rats were used to extract TSCs,four were used for in vivo imaging observation,and eight were used for histological analysis.The TSCs derived from rat Achilles tendon were extracted by enzyme digestion,the TSCs were labeled with IR-780 iodide,and the intracellular localization of IR-780 iodide labeled TSCs was observed under a laser confocal microscope.Flow cytometry was used to detect the positive rate of the label and its influence on the cell cycle of TSCs.CCK-8 method was used to detect the effect of IR-780 iodide-labeled TSCs on their proliferation.The IR-780 iodide-labeled TSCs were wrapped with C/GP/Co hydrogel and transplanted to the injury site of the rat Achilles tendon.Through whole-body near-infrared imaging and histological observation,the survival and distribution of TSCs in the rat were detected.Results The TSCs from rat Achilles tendons were successfully extracted and C/GP/Co hydrogels were successfully produced.IR-780 iodide can successfully label TSCs with a positive rate of 95.75%.The fluorescence intensity was significantly higher than that of the control group,and it had no obvious adverse effects on the cell cycle and proliferation of TSCs.Through whole-body near-infrared imaging and histological observation,it was easy to detect the IR-780 iodide-labeled TSCs in the rat Achilles tendon injury area,and the fluorescence signal was almost undetectable in other parts of the rat body.From 1 to 28 days,the fluorescence signal of IR-780 iodide gradually weakened,but obvious fluorescence was still detected at 28 days.Conclusion IR-780 iodide can effectively label TSCs with a positive rate of 95.75%,and has no obvious adverse effects on the cell cycle and proliferation of TSC

关 键 词：肌腱损伤 肌腱干细胞 肌腱组织工程 IR-780碘化物 示踪技术 大鼠 

分 类 号：R685[医药卫生—骨科学]