Repopulation of vascularized bone allotransplants with recipient-derived cells: detection by laser capture microdissection and real-time PCR.
Repopulation of vascularized bone allotransplants with recipient-derived cells: detection by laser capture microdissection and real-time PCR.
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DOI：
10.1002/jor.20915
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发表时间：
2009-11
影响因子：
2.8
通讯作者：
Bishop, Allen T.
中科院分区：
文献类型：
--
作者：
Pelzer, Michael;Larsen, Mikko;Friedrich, Patricia F.;Aleff, Ross A.;Bishop, Allen T.
成功的复合组织移植的机制必须包括移植嵌合体的分析，这是很少研究，特别是在钙化组织。我们已经开发了一种新的方法，使确定谱系的选定细胞在我们的模型血管化骨同种异体移植。从雌性黑琼脂（DA）供体大鼠到雄性花斑病毒-葛兰素（PVG）受体进行血管化股骨同种异体移植，代表主要组织相容性不匹配。4组在使用免疫抑制剂（+/- 2周他克莫司）和手术血运重建方面存在差异，通过植入通畅或结扎的隐动静脉（AV）束。在18周时评估结果。通过氢冲洗技术测量骨血流量，并制备横向标本进行组织学检查。对来自激光捕获显微切割的皮质骨区域的DNA进行实时PCR以确定嵌合的程度。为此，我们分析了性别决定区Y（Sry）基因的相对表达比，具体只为受体雄性大鼠DNA，亲环素管家基因。在所有组的皮质骨中均观察到大量移植嵌合体（范围77-97%）。大鼠无免疫抑制和专利AV束显示显着较高的嵌合体比那些免疫抑制和结扎AV束，维持移植细胞的活力。我们描述了一种新的方法来研究大鼠血管化骨同种异体移植物中嵌合体的程度，包括性别不匹配的移植模型，选定的骨区域的激光捕获显微切割，以及相对表达率的计算。
Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. 4 groups differed in use of immunosuppression (+/- 2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.
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影响因子：
2.8
作者：
Pelzer, Michael;Larsen, Mikko;Bishop, Allen T.
通讯作者：
Bishop, Allen T.
影响因子：
2.8
作者：
Muramatsu, K;Valenzuela, RG;Bishop, AT
通讯作者：
Bishop, AT
影响因子：
5.7
作者：
Csete, Marie
通讯作者：
Csete, Marie
影响因子：
6.2
作者：
Foster, RD;Ascher, NL;Mathes, SJ
通讯作者：
Mathes, SJ
影响因子：
2.8
作者：
Pelzer, Michael;Larsen, Mikko;Bishop, Allen T.
通讯作者：
Bishop, Allen T.