摘要
目的探讨BMSCs移植治疗兔股骨头坏死(osteonecrosis of the femoral head,ONFH)后细胞在体内的存活情况及坏死股骨头内新骨形成情况。方法4～5月龄新西兰大白兔49只,雌性48只,雄性1只,体重2.0～2.5kg。取兔股骨及胫骨内骨髓,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,制备浓度为2.5×108个/mL细胞悬液。采用局部液氮注射冷冻法制备兔右侧ONFH模型。将雌兔随机分为3组,每组16只,A组单纯造模为对照,B组造模后注射自体DiI荧光标记BMSCs悬液4μL,C组造模后注射雄兔异体DiI荧光标记BMSCs悬液4μL。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,荧光显微镜下观察各组织中DiI荧光标记细胞的表达,PCR检测各组织中Y染色性别决定基因(sex determining region of the Y,Sry)的表达。结果术后动物均存活至实验完成。X线片示A组股骨头随时间延长逐渐出现骨坏死;B、C组股骨头骨密度逐渐增加,未出现骨坏死。组织学观察示B、C组随时间延长骨组织修复明显,A组无明显变化。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)。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,3组各时间点心脏、肺、肝、脾、肾组织中均未见Sry基因表达;C组各时间点股骨头标本可见Sry基因的表达,A、B组未见。结论异体BMSCs移植后细胞可在股骨头局部存活并诱导新骨形成,并未见体内其他组织中再分布。
Objective To evaluate sex determining region of the Y （Sry） as a engrafting track of the transplanted BMSCs survival and new bone formation in the osteonecrosis of the femoral head （ONFH） of rabbit. Methods Fortynine 4-5-month-old New Zealand White rabbits were included, weighing 2.0-2.5 kg, 48 females and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by 1, 1＇-dioctadecyl-3, 3, Y, 3＇-tetramethylindocarbocyanine perchlorate （DiI） and the concentration of cell suspension was 2.5 × 10^8/mL. The animal model of ONFH were established with 48 female rabbits by injecting liquid nitrogen, and femoral head was not dislocated. The animal model were divided into 3 groups, 16 rabbits in each group. Group A only established animal model as control. Autologous BMSCs （4 μL） marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs （4 μL） marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson staining, and the regenerating trabecular volume percentages was 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 PCR for ceils survival. Results All rabbits survived till the end of experiment. The X-ray showed gradual necrosis in the femoral head of group A. HE and Masson staining results indicated that compared with the group A, the recovery condition of the necrotic femoral head in the groups B and C was better. At each time of groups B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly （P 〈 0.01）. There was no significant difference between groups B and C （P 〉 0.05）. The cells marked by DiI were not founded in the tissues of the heart, lung, liver, spleen and kidney in groups B and C at each
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第9期1118-1123,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
四川省卫生厅自然科学基金资助项目(080002)~~
关键词
BMSCS
股骨头坏死
Y染色体性别决定基因
兔
BMSCs Osteonecrosis of the femoral head Sex determining region of the Y Rabbit
