CAJ | 학술논문

背景:课题利用实验室自主研制、已获国家SFDA批准临床试验的重组肝细胞生长因子腺病毒感染骨髓间充质干细胞,结合肝细胞生长因子的促血管新生和抗细胞凋亡以及间充质干细胞的成骨功能,通过细胞治疗的手段进行骨损伤修复.目的:探讨肝细胞生长因子基因修饰的骨髓间充质干细胞移植对兔股骨头坏死骨缺损的治疗效果.设计、时间及地点:细胞-材料学体内实验,于2007-09/12在北京放射与辐射医学研究所和解放军66400部队骨病专科医院完成.材料:清洁级26～28周龄新西兰大白兔18只,由北京开源兔业养殖场提供.骨基质明胶为上海骁博科技发展有限公司产品.方法:采用贴壁法分离培养兔自体骨髓间充质干细胞,体外鉴定成骨和成脂肪能力.18只兔均建立双侧股骨头坏死骨缺损模型,随机分为3组,6只,组,单纯材料组缺损区仅填塞骨基质明胶,细胞-材料组填塞复合骨髓间充质干细胞的骨基质明胶,基因修饰细胞-材料组填寒复合Ad-HGF感染的骨髓间充质干细胞的骨基质明胶,各组细胞用量为107个/缺损,骨基质明胶用量为27 mm3/缺损,修复3个月后取材进行组织学检查.骨髓闻充质干细胞和Ad-HGF感染的骨髓间充质干细胞经荧光染料二醋酸琥珀酰酯羟基荧光素标记后,氯化钴处理72 h,流式细胞学分析细胞增殖情况.主要观察指标:骨髓间充质干细胞的诱导分化,骨髓间充质干细胞对兔股骨头坏死的修复效果,Ad-HGF感染的骨髓间充质干细胞抗低氧损伤能力.结果:培养的细胞在适当诱导条件下可分化为成骨和成脂肪细胞.修复后3个月组织学分析显示,单纯材料组仅见疏松的纤维肉芽组织填充;细胞-材料组可见致密的纤维肉芽组织填充,其间有较多的新生血管,但未见新生骨组织;基因修饰细胞-材料组多为软骨样组织填充,周围可见新骨带形成.各组Lane-Sandhu骨组织学评分比较差异有非常显著性意义(P<0.01),其中基因修饰细胞-材料组评分最高,即Ad-HGF感染的骨髓间充质干细胞具备最佳的骨修复能力.培养体系中加入氯化钴后,已增殖的Ad-HGF感染的骨髓间充质干细胞比例显著高于未感染骨髓间充质干细胞(P<0.001).结论:在骨缺损模型中Ad-HGF感染的骨髓间充质干细胞具有更强的抗低氧损伤能力,其在体内成骨能力优于未感染的骨髓间充质干细胞,提示肝细胞生长因子基因修饰的骨髓间充质干细胞移植是应用于缺血性骨坏死的有效治疗途径. 揹景:課題利用實驗室自主研製、已穫國傢SFDA批準臨床試驗的重組肝細胞生長因子腺病毒感染骨髓間充質榦細胞,結閤肝細胞生長因子的促血管新生和抗細胞凋亡以及間充質榦細胞的成骨功能,通過細胞治療的手段進行骨損傷脩複.目的:探討肝細胞生長因子基因脩飾的骨髓間充質榦細胞移植對兔股骨頭壞死骨缺損的治療效果.設計、時間及地點:細胞-材料學體內實驗,于2007-09/12在北京放射與輻射醫學研究所和解放軍66400部隊骨病專科醫院完成.材料:清潔級26～28週齡新西蘭大白兔18隻,由北京開源兔業養殖場提供.骨基質明膠為上海驍博科技髮展有限公司產品.方法:採用貼壁法分離培養兔自體骨髓間充質榦細胞,體外鑒定成骨和成脂肪能力.18隻兔均建立雙側股骨頭壞死骨缺損模型,隨機分為3組,6隻,組,單純材料組缺損區僅填塞骨基質明膠,細胞-材料組填塞複閤骨髓間充質榦細胞的骨基質明膠,基因脩飾細胞-材料組填寒複閤Ad-HGF感染的骨髓間充質榦細胞的骨基質明膠,各組細胞用量為107箇/缺損,骨基質明膠用量為27 mm3/缺損,脩複3箇月後取材進行組織學檢查.骨髓聞充質榦細胞和Ad-HGF感染的骨髓間充質榦細胞經熒光染料二醋痠琥珀酰酯羥基熒光素標記後,氯化鈷處理72 h,流式細胞學分析細胞增殖情況.主要觀察指標:骨髓間充質榦細胞的誘導分化,骨髓間充質榦細胞對兔股骨頭壞死的脩複效果,Ad-HGF感染的骨髓間充質榦細胞抗低氧損傷能力.結果:培養的細胞在適噹誘導條件下可分化為成骨和成脂肪細胞.脩複後3箇月組織學分析顯示,單純材料組僅見疏鬆的纖維肉芽組織填充;細胞-材料組可見緻密的纖維肉芽組織填充,其間有較多的新生血管,但未見新生骨組織;基因脩飾細胞-材料組多為軟骨樣組織填充,週圍可見新骨帶形成.各組Lane-Sandhu骨組織學評分比較差異有非常顯著性意義(P<0.01),其中基因脩飾細胞-材料組評分最高,即Ad-HGF感染的骨髓間充質榦細胞具備最佳的骨脩複能力.培養體繫中加入氯化鈷後,已增殖的Ad-HGF感染的骨髓間充質榦細胞比例顯著高于未感染骨髓間充質榦細胞(P<0.001).結論:在骨缺損模型中Ad-HGF感染的骨髓間充質榦細胞具有更彊的抗低氧損傷能力,其在體內成骨能力優于未感染的骨髓間充質榦細胞,提示肝細胞生長因子基因脩飾的骨髓間充質榦細胞移植是應用于缺血性骨壞死的有效治療途徑.
배경:과제이용실험실자주연제、이획국가SFDA비준림상시험적중조간세포생장인자선병독감염골수간충질간세포,결합간세포생장인자적촉혈관신생화항세포조망이급간충질간세포적성골공능,통과세포치료적수단진행골손상수복.목적:탐토간세포생장인자기인수식적골수간충질간세포이식대토고골두배사골결손적치료효과.설계、시간급지점:세포-재료학체내실험,우2007-09/12재북경방사여복사의학연구소화해방군66400부대골병전과의원완성.재료:청길급26～28주령신서란대백토18지,유북경개원토업양식장제공.골기질명효위상해효박과기발전유한공사산품.방법:채용첩벽법분리배양토자체골수간충질간세포,체외감정성골화성지방능력.18지토균건립쌍측고골두배사골결손모형,수궤분위3조,6지,조,단순재료조결손구부전새골기질명효,세포-재료조전새복합골수간충질간세포적골기질명효,기인수식세포-재료조전한복합Ad-HGF감염적골수간충질간세포적골기질명효,각조세포용량위107개/결손,골기질명효용량위27 mm3/결손,수복3개월후취재진행조직학검사.골수문충질간세포화Ad-HGF감염적골수간충질간세포경형광염료이작산호박선지간기형광소표기후,록화고처리72 h,류식세포학분석세포증식정황.주요관찰지표:골수간충질간세포적유도분화,골수간충질간세포대토고골두배사적수복효과,Ad-HGF감염적골수간충질간세포항저양손상능력.결과:배양적세포재괄당유도조건하가분화위성골화성지방세포.수복후3개월조직학분석현시,단순재료조부견소송적섬유육아조직전충;세포-재료조가견치밀적섬유육아조직전충,기간유교다적신생혈관,단미견신생골조직;기인수식세포-재료조다위연골양조직전충,주위가견신골대형성.각조Lane-Sandhu골조직학평분비교차이유비상현저성의의(P<0.01),기중기인수식세포-재료조평분최고,즉Ad-HGF감염적골수간충질간세포구비최가적골수복능력.배양체계중가입록화고후,이증식적Ad-HGF감염적골수간충질간세포비례현저고우미감염골수간충질간세포(P<0.001).결론:재골결손모형중Ad-HGF감염적골수간충질간세포구유경강적항저양손상능력,기재체내성골능력우우미감염적골수간충질간세포,제시간세포생장인자기인수식적골수간충질간세포이식시응용우결혈성골배사적유효치료도경.
BACKGROUND: Using bone marrow mesenchymal stem cells (MSCs) infected by an adenoviral vector cen-ying human hepatocyte growth factor (HGF) gene, which was independently developed by our laboratory and has been approved by SFDA in clinical trial to repair bone defects via cell therapy.OBJECTIVE: To investigate the bone restoration effect of mssenchymal stem cell and hepatocyte growth factor in the pathophysiological course of femoral head osteonecresis defects.DESIGN, TIME AND SETTING: An in vivo experiment of ceUular-rnatedal science. The experiment was performed at the Beijing Institute of Radiation Medicine and the 66400 Orthopaedic Hospital of Chinese PLA from September to December 2007. MATERIALS: Eighteen New Zealand rabbits with clean grade, aged 26-28 weeks, were supplied by Beijing Kaiyuan rabbit livestock farm. Bone matrix gelatin was purchased from Shanghai Xiaobo Technological Development Limited Co., Ltd. METHODS: MSCs from New Zealand white rabbit were isolated and culture-expanded by adhesion method and their in vitro ostengenesis and adipogenesis were identified. Eighteen New Zealand white rabbits were created femoral heads defect models and randomly divided into 3 groups, with 6 animals in each group. Animals in each group were treated with scaffold of bone matrix gelatin (27 mm3 per defect) only, scaffolds seeded with MSCs (1×107 per defect) or MSCs infected by an adenoviral vector carrying human hepatocyte growth factor gene (MSC/HGF, 1×107 per defect), respectively. Histological examination was conducted at 3 months post operation. MSCs or MSC/HGF were labeled with carboxyfluorescein diacetate succinmidyl ester dye, treated with cobalt chloride for 72 hours and their proliferative status was evaluated and compared by flow cytomatric techniques. MAIN OUTCOME MEASURES: The differentiation of MSCs, therapeutic effect of MSCs in treating femoral head necrosis, and potent ability of MSCs/HGF against hypoxia.RESULTS: The adherent cells could differentiate into osteoblasts and adipocytes under in vitro inductive condition. Histological examination revealed that the bone defects from both control and MSCs-treated groups were fille:d with fibrous tissue, though blood vessels were evident in MSCs-treated group, whereas new bony tissues were obvious in MSC/HGF group. The result was further confirmed by Lane-Sandhu scaling, which indicated that new bone formation was more evident in MSC/HGF-treated group compared with MSCs-treated or control group (P< 0.01). Flow cytometry analysis showed that the proportion of MSC/HGF-treated group that had experienced cell division was significantly higher than that of MSCs-treated group after cobalt treatment (P < 0.001).CONCLUSION: MSC/HGF exhibit greater osteogeneeis in vivo in this model compared their counterparts, which might be attributed to their resistance to hypoxic injury. The results here suggest that HGF gene modification might be an optional strategy for the application of MSCs in the management of avascular osteonecrosis.