同种异体骨髓间充质干细胞移植治疗兔股骨头坏死的实验研究
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摘要
目的:研究同种异体骨髓间充质干细胞(BMSCs)移植治疗兔股骨头坏死(osteonecrosis of the femoral head,ONFH)中供体细胞在宿主体内的存活、分布及转归情况。
方法:采用密度梯度离心法行BMSCs分离培养并传代,取第3代细胞行1,1'-双十八烷-3,3,3',3'-四甲基吲哚羰花青-高氯酸盐(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate,DiI)荧光标记。通过股骨头内局部注射液氮制作股骨头坏死动物模型。将48只雌性新西兰大白兔随机分成3组,A组16只为单纯造模组,B组造模后注射自体DiI荧光标记BMSCs悬液,C组造模后注射雄兔异体DiI荧光标记BMSCs。术后2、4、6、8周各组分别处死4只动物,行双侧股骨头X线观察,取股骨头标本行HE染色及Masson染色,测定新生骨小梁在股骨头所占的体积百分比。取心脏、肺、肝、脾、肾组织及股骨头标本组织,采用荧光显微镜观察各组织中DiI荧光细胞的表达,采用PCR检测各组织中SRY基因的表达,以实时荧光定量PCR监测供体细胞在体内的数量变化。运用原位杂交技术对股骨头标本内的SRY基因进行检测。
结果:
1.经DiI荧光染剂标记后细胞形状及生长曲线与未标记细胞基本一致。
2.A组动物在术后3 d时,股骨头出现坏死;4 w时出现修复反应,X线可见囊性变;6 w时软骨下骨硬化,出现新月征。
3.各时间点B、C组新生骨小梁占股骨头体积百分比均高于A组,比较差异均有统计学意义(P<0.01),B、C组间比较差异无统计学意义(P>0.05)。
4.术后各时间点B、C组心脏、肺、肝、脾、肾组织均未见DiI荧光标记细胞。PCR结果显示,C组各时间点心脏、肺、肝、脾、肾组织中均未检测到SRY基因;C组各时间点股骨头标本上可见SRY基因的表达,且随时间逐渐减少, A、B组未见。
5.C组原位杂交显示DAB染色阳性细胞在植入区存活,随时间推移逐渐减少,靠近股骨颈区域的阳性细胞主要集中在坏死骨表面,股骨头软骨下区域阳性细胞主要集中于新骨骨陷凹内。
结论:
1.液氮局部注射成功制作出兔股骨头坏死模型,并可有效控制股骨头坏死的位置及大小,为相关研究提供可靠的动物模型。
2.在无免疫抑制的情况下,同种异体BMSCs和自体BMSCs移植对实验兔股骨头缺血性坏死的治疗效果相似,均成功修复股骨头坏死,两者在病理学上无明显差异。
3.移植后同种异体BMSCs可在股骨头局部存活,但数量逐渐减少,但局部并无排异反应,且不在体内再分布。
4.移植后的同种异体BMSCs转化成成骨细胞,促进成骨,且膜下成骨与软骨内成骨两种方式并存。
Objective To evaluate SRY as a engrafting track of the transplanted bone marrow mesenchymal stem cells (BMSCs) survival and new bone formation in the osteonecrosis of the femoral head. Methods Forty-nine 3-month-old New Zealand White rabbits were included, weighing 2-2.5 Kg, 48 female and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by DiI. The animal model of ONFH were established with forty eight female rabbits by injecting liquid nitrogen, and femoral head was not dislocated, which were divided into 3 groups, 16 rabbits in each group, group A only established animal model as control, autologous BMSCs marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson trichrome staining and the regenerating trabecular volume percentages were determined at 2, 4, 6 and 8 weeks after operation respectively. The examples of the heart, lung, liver, spleen and kidney were obtained. The transplanted BMSCs were traced by fluorescence microscope, the SRY gene expression was detected by polymerase chain reaction (PCR) for cells survival. Results The X-ray showed necrosis in the femoral head of group A gradually. HE and Masson trichrome staining results showed that compared with the group A, the recovery condition of the necrotic femoral head in the group B and C was better. At each time-point of group B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly. The result between group B and C was inconspicuous. Cells marked by DiI were not been founded in the tissue of the heart, lung, liver, spleen and kidney at each time point. PCR showed that the expression of SRY gene was not observed at the heart, lung, liver, spleen and kidney of three groups at each time point. The expression of SRY gene was clearly identified in the femoral head of all 16 rabbits in the group B at each time point. Conclusion Allografting of bone marrow mesenchymal stem cells transplanted into the femoral head can survive and induce new bone formation without redistribution. Both intramembranous ossification and endochondral ossification, cartilaginous ossification is showed in the femoral head by in situ hybridization for SRY.
方法:采用密度梯度离心法行BMSCs分离培养并传代,取第3代细胞行1,1'-双十八烷-3,3,3',3'-四甲基吲哚羰花青-高氯酸盐(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate,DiI)荧光标记。通过股骨头内局部注射液氮制作股骨头坏死动物模型。将48只雌性新西兰大白兔随机分成3组,A组16只为单纯造模组,B组造模后注射自体DiI荧光标记BMSCs悬液,C组造模后注射雄兔异体DiI荧光标记BMSCs。术后2、4、6、8周各组分别处死4只动物,行双侧股骨头X线观察,取股骨头标本行HE染色及Masson染色,测定新生骨小梁在股骨头所占的体积百分比。取心脏、肺、肝、脾、肾组织及股骨头标本组织,采用荧光显微镜观察各组织中DiI荧光细胞的表达,采用PCR检测各组织中SRY基因的表达,以实时荧光定量PCR监测供体细胞在体内的数量变化。运用原位杂交技术对股骨头标本内的SRY基因进行检测。
结果:
1.经DiI荧光染剂标记后细胞形状及生长曲线与未标记细胞基本一致。
2.A组动物在术后3 d时,股骨头出现坏死;4 w时出现修复反应,X线可见囊性变;6 w时软骨下骨硬化,出现新月征。
3.各时间点B、C组新生骨小梁占股骨头体积百分比均高于A组,比较差异均有统计学意义(P<0.01),B、C组间比较差异无统计学意义(P>0.05)。
4.术后各时间点B、C组心脏、肺、肝、脾、肾组织均未见DiI荧光标记细胞。PCR结果显示,C组各时间点心脏、肺、肝、脾、肾组织中均未检测到SRY基因;C组各时间点股骨头标本上可见SRY基因的表达,且随时间逐渐减少, A、B组未见。
5.C组原位杂交显示DAB染色阳性细胞在植入区存活,随时间推移逐渐减少,靠近股骨颈区域的阳性细胞主要集中在坏死骨表面,股骨头软骨下区域阳性细胞主要集中于新骨骨陷凹内。
结论:
1.液氮局部注射成功制作出兔股骨头坏死模型,并可有效控制股骨头坏死的位置及大小,为相关研究提供可靠的动物模型。
2.在无免疫抑制的情况下,同种异体BMSCs和自体BMSCs移植对实验兔股骨头缺血性坏死的治疗效果相似,均成功修复股骨头坏死,两者在病理学上无明显差异。
3.移植后同种异体BMSCs可在股骨头局部存活,但数量逐渐减少,但局部并无排异反应,且不在体内再分布。
4.移植后的同种异体BMSCs转化成成骨细胞,促进成骨,且膜下成骨与软骨内成骨两种方式并存。
Objective To evaluate SRY as a engrafting track of the transplanted bone marrow mesenchymal stem cells (BMSCs) survival and new bone formation in the osteonecrosis of the femoral head. Methods Forty-nine 3-month-old New Zealand White rabbits were included, weighing 2-2.5 Kg, 48 female and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by DiI. The animal model of ONFH were established with forty eight female rabbits by injecting liquid nitrogen, and femoral head was not dislocated, which were divided into 3 groups, 16 rabbits in each group, group A only established animal model as control, autologous BMSCs marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson trichrome staining and the regenerating trabecular volume percentages were determined at 2, 4, 6 and 8 weeks after operation respectively. The examples of the heart, lung, liver, spleen and kidney were obtained. The transplanted BMSCs were traced by fluorescence microscope, the SRY gene expression was detected by polymerase chain reaction (PCR) for cells survival. Results The X-ray showed necrosis in the femoral head of group A gradually. HE and Masson trichrome staining results showed that compared with the group A, the recovery condition of the necrotic femoral head in the group B and C was better. At each time-point of group B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly. The result between group B and C was inconspicuous. Cells marked by DiI were not been founded in the tissue of the heart, lung, liver, spleen and kidney at each time point. PCR showed that the expression of SRY gene was not observed at the heart, lung, liver, spleen and kidney of three groups at each time point. The expression of SRY gene was clearly identified in the femoral head of all 16 rabbits in the group B at each time point. Conclusion Allografting of bone marrow mesenchymal stem cells transplanted into the femoral head can survive and induce new bone formation without redistribution. Both intramembranous ossification and endochondral ossification, cartilaginous ossification is showed in the femoral head by in situ hybridization for SRY.
引文
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