PET-MRI分子示踪结合组织免疫荧光定位双相成像研究移植BMSCs在犬股骨头坏死病灶中的分布与转归
结题报告
批准号:
81201439
项目类别:
青年科学基金项目
资助金额:
23.0 万元
负责人:
杭栋华
依托单位:
学科分类:
H0611.运动系统疾病研究新技术与新方法
结题年份:
2015
批准年份:
2012
项目状态:
已结题
项目参与者:
易诚青、王建东、朱宗昊、杨庆、徐乔龙
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中文摘要
已经明确股骨头坏死(ONFH)与病灶内间充质干细胞数量及分化能力下降有关,因此近来使用骨髓间充质干细胞(BMSCs)移植治疗股骨头坏死(ONFH)的报道潮涌。ONFH的病理特征是坏死区缺血,散在死骨和纤维结缔组织,由此难以解释在病灶中直接移植BMSCs就可以获得类似于心血管和神经退行性疾病的干细胞治疗效果,关键矛盾在于ONFH中移植BMSCs的存活、分布和分化的确切状态和机制尚属未知。.2009年申请者课题组首先报道ONFH病灶植入BMSCs可存活12周并部分分化为成骨细胞,但使用的是离体标本,而非活体连续示踪与整体成像。本申请是上述课题的延续,将HSV1-sr39tk- - -mRFP1- - -SPION共标记BMSCs,使用PET-MRI连续示踪与组织免疫荧光双相定位相结合的方法。希望以此明确BMSCs在ONFH病灶中的存活与分布分化的具体机制,为股骨头坏死的干细胞治疗提供基础科学支持。
英文摘要
Decrease of BMSC pool and reduced ability in proliferation of BMSCs in osteonecrosis of femoral head (ONFH) in human patients has been confirmed, which have put the stem cell therapy for ONFH into a boom recently.The characteristic change in pathology of ONFH is ischemia, bone sequestrum and dense granulation tissue scattered. So, it is hard to explain that the excellent repair progress in ONFH could be obtained simply with the transplanted BMSCs to the necrotic area, which has been confirmed in cytotherapy for the treatment of heart and neural system diseases however.We think he key obstacle is that the mechanism of the survival status, distribution and differentiation of BMSCs in the repair process has not been clearly addressed yet. . Our research team has reported for the first time that the transplanted BMSCs could survival in the necrotic area for at least 12 weeks and differentiated to osteoblast finally. However, there was a defect in this research that it was not an in vivo noninvasive cell tracking experiment and the specimens had to be obtained by killing the animals. This application is actually a modified protocol of the previous research. We designed a new marker of HSV1-sr39tk- - -mRFP1- - -SPION for the purpose of noninvasive and continuous multimodality imaging in vivo. Autologous BMSCs will be transfected with this marker. The rapid two-stage immunofluorescence technique with double immunofluorescence labeling will be used for the detection of the final differentiation of transplanted BMSCs, which may develop to osteoblast, adipocyte, fibroblast or vascular endothelial cell. In short, this application puts focus on the survival, distribution and differentiation status of BMSCs transplanted in a traumatic ONFH to elucidate the possible mechanisms involved in the repair and provide fundamentally scientific support for the cytotherapy of ONFH.
本研究使用股骨颈基底部截骨后内固定联合结扎阻断旋股内外侧动脉的方法制作ONFH动物模型;将HSV1-sr39tk—mRFP1—SPION共标记BMSCs植入股骨头坏死病灶,使用PET-MRI连续示踪与组织免疫荧光双相定位相结合的方法研究BMSCs在ONFH病灶中的存活与分布分化的具体机制。本研究的成果有:1. 确认股骨颈截骨结合旋骨内外侧动脉切断可以稳定构建并维持犬股骨头坏死动物模型。同时首次使用MRI检查方法确认动物模型ONFH,并证实即便是股骨头坏死发生后1年,犬股骨头也不会出现塌陷,这在既往文献中未有报道;2. 确认HSV1-sr39tk-mRFP1可以共标记BMSCs,SPION可以后续标记上述转染的细胞且维持干细胞表型不变,这既往未见报道;3. 证实PET-MRI可以活体追踪HSV1-sr39tk—mRFP1—SPION共标记BMSCs,其特异性均提示植入后2周细胞仍有显著存活。这是目前关于BMSCs在股骨头坏死病灶中可以存活的最强证据,也是唯一活体连续追踪的证据,目前未见其它报道。遗憾的是,组织免疫荧光双相定位部分研究由于脱钙与抗原保留的矛盾问题目前面临困难,目前仍在反复探索实验条件,预期结果乐观,能够按照申请书中计划,发表2篇SCI文章。
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