人脐带间充质干细胞生物学特性及治疗大鼠骨质疏松的初步研究
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摘要
研究目的：
1.建立从人脐带中分离间充质干细胞(MSC)、体外传代培养的方法。
2.研究其在体外的生物学特性及表型特征,并对其体外向脂肪细胞和成骨细胞定向分化进行研究。
3.尾静脉注射移植MSC,评价其对大鼠骨质疏松(OP)的治疗效果。
材料和方法：
1.用组织块培养法或酶消化法分离人间充质干细胞。
2.流式细胞术(FCM)检测细胞表面标志；观察细胞的形态学特性；MTT法检测细胞增殖能力并绘制生长曲线,描述体外培养的hUC-MSCs的生物学特性。
3.应用诱导因子诱导hUC-MSCs定向分化为脂肪细胞和成骨细胞,并通过组织化学染色的方法对分化后的细胞进行鉴定。
4.hUC-MSCs对大鼠OP的治疗作用：采用SD大鼠,按体重随机分为4组：A组正常组、B组模型组、C组实验组和D组仙灵骨葆组,除A组灌胃CMC-Na,其余各组均连续14天灌胃RA；同时C组静脉注射hUC-MSCs,D组灌胃仙灵骨葆；21天后行血清学指标检测、生物力学性能和骨微观结构观察。
结果：
1.用组织块培养法或酶消化法均能在14-20天内获得具有MSC特征的hUC-MSCs；体外实验证实,hUC-MSCs能被诱导分化为脂肪细胞和成骨细胞。
2.hUC-MSCs对大鼠骨质疏松的治疗作用
(1)血清生化指标检测结果显示,C组大鼠血清Ca2+、ALP和StrACP水平均显著低于B组(p<0.05或p<0.01),但血清Pi水平无显著性差异；组(p<0.05或p<0.01),但血清Pi水平无显著性差异；
(2)右侧股骨骨密度(BMD)测定显示,C组BMD明显高于B组(p<0.05)；
(3)右侧股骨生物力学测定发现C组最大载荷、弹性载荷、刚度和最大挠度均显著大于B组(p<0.05或p<0.01)。
(4)左侧股骨扫描电镜观察结果显示C组与B组相比,骨胶原纤维排列呈束状,走向一致,排列规则、紧密,恢复到与A组骨胶原纤维相近形态和排列。
结论：
1.成功建立从人脐带中分离培养和扩增MSC经济、简单的方法,分离的细胞具有hUC-MSCs的基本生物学特性和多向分化的潜能；
2.RA复制的OP模型为高转换型,较好的模拟了人原发性OP；
3.hUC-MSCs能明显降低OP大鼠的血清Ca2+、ALP及StrACP水平,从而抑制骨吸收,减少骨量的丢失；改善OP大鼠的股骨组织形态结构、增加骨密度,增强股骨生物力学性能；
4.hUC-MSCs对OP的修复作用,可能与抑制骨吸收、促进骨的形成,调整二者间的偶联平衡,从而改善骨的重建有关。
OBJECTIVE:
1. To establish the methods of isolation and expansion of human umbilical cord derived mesenchymal stem cells (hUC-MSCs).
2. Studying the biological characteristics of hUC-MSCs and its capacity of adipogenic and osteogenic induction in vitro.
3. To find out the feasibility of healing the osteoporosis (OP) rats by transplanting the hUC-MSCs by intravenous injection.
METHOD:
1. Obtain the term infant umbilical cord samples and apply the enzymatic or the tissue culture method to separate and purify hUC-MSCs.
2. Detecct the cell surface markers by flow cytometry; Observe the morphology; Describe the growth curve with MTT method, describe the biological characteristics of hUC-MSCs in vitro.
3. Induce the hUC-MSCs differentiation into adipocytes and osteoblasts and detect the induced cells by histochemistry staining.
4. The treatment of hUC-MSCs on RA induced rats:the female SD rats were randomly divided into four groups (control group (A), model control group (B), hUC-MSCs transplantation group (C) and Xianling Gubao group (D).Except A group administrated with 0.5%CMC-Na, other groups administrated with RA.At the same time, Xianling Gubao and hUC-MSCs were administrated; C group were adiministrated with 2X 106 hUC-MSCs A and B group were administrated with distilled water. The serum biochemical indicators, biomechanics characteristics were measured and microstructure of bone were also observed.
RESULTS:
1. We can get 80%fusion hUC-MSCs during 14-20 days both by tissue culture and enzymatic methods The proliferation became faster after passage and the cells after passage three(P3) presented a homogeneous population of spindle fibroblast-like cells. More than 90%of P3 cells expressed the CD44, CD29, CD 105, but the CD28, CD31, CD34, CD45, CD40, CD86 and HLA-DR were negative.80% of the cells were in G0/G1. The assays in vitro demonstrated the P3 cells exhibited osteogenic and adipogenic cells differentiation potential.
2. Effect of hUC-MSCs on RA simulated OP rats:
(1). In bone biochemical measurement of the serum:The serum Ca2+, ALP and StrACP of C group were significantly higher than B group (p<0.05 or p<0.01); but no found significant difference between D and B group.
(2). In BMD measurements of the right femur group C were significantly higher than in group B (p<0.05);
(3). In right femoral shaft three-point bending test, the max load,the elastic load, the rigidity and max deformation of group C were significantly higher than in group B (p< 0.05 orp<0.01);
(4). In SEM observation, collagenous fiber with regular arrangement in group A; collagen fibers displaced by absorption laeuna in group B; Collagenous fibers were much more regular in group C.
CONCLUSION:
1. The hUC-MSCs were obtained from the human umbilical cord by tissue or enzymic method; the cells shared the same characteristics MSC. The hUC-MSCs were multipotential cells.
2. Bone formation and bone mass decreased, bone microstructure destructive in RA administrated rats. RA simulated OP were a high turnover OP. The rats OP model were successfully established.
3. The hUC-MSCs reduced the serum Ca2+, ALP and StrACP level of OP rats, improved the femur microstructure. The BMD and biomechanic function also enhanced.
4. The hUC-MSCs has a significant effect on RA simulated OP rats. The cells can promote bone formation and reduce bone loss, and improve the microstructure.
5.
1.建立从人脐带中分离间充质干细胞(MSC)、体外传代培养的方法。
2.研究其在体外的生物学特性及表型特征,并对其体外向脂肪细胞和成骨细胞定向分化进行研究。
3.尾静脉注射移植MSC,评价其对大鼠骨质疏松(OP)的治疗效果。
材料和方法：
1.用组织块培养法或酶消化法分离人间充质干细胞。
2.流式细胞术(FCM)检测细胞表面标志；观察细胞的形态学特性；MTT法检测细胞增殖能力并绘制生长曲线,描述体外培养的hUC-MSCs的生物学特性。
3.应用诱导因子诱导hUC-MSCs定向分化为脂肪细胞和成骨细胞,并通过组织化学染色的方法对分化后的细胞进行鉴定。
4.hUC-MSCs对大鼠OP的治疗作用：采用SD大鼠,按体重随机分为4组：A组正常组、B组模型组、C组实验组和D组仙灵骨葆组,除A组灌胃CMC-Na,其余各组均连续14天灌胃RA；同时C组静脉注射hUC-MSCs,D组灌胃仙灵骨葆；21天后行血清学指标检测、生物力学性能和骨微观结构观察。
结果：
1.用组织块培养法或酶消化法均能在14-20天内获得具有MSC特征的hUC-MSCs；体外实验证实,hUC-MSCs能被诱导分化为脂肪细胞和成骨细胞。
2.hUC-MSCs对大鼠骨质疏松的治疗作用
(1)血清生化指标检测结果显示,C组大鼠血清Ca2+、ALP和StrACP水平均显著低于B组(p<0.05或p<0.01),但血清Pi水平无显著性差异；组(p<0.05或p<0.01),但血清Pi水平无显著性差异；
(2)右侧股骨骨密度(BMD)测定显示,C组BMD明显高于B组(p<0.05)；
(3)右侧股骨生物力学测定发现C组最大载荷、弹性载荷、刚度和最大挠度均显著大于B组(p<0.05或p<0.01)。
(4)左侧股骨扫描电镜观察结果显示C组与B组相比,骨胶原纤维排列呈束状,走向一致,排列规则、紧密,恢复到与A组骨胶原纤维相近形态和排列。
结论：
1.成功建立从人脐带中分离培养和扩增MSC经济、简单的方法,分离的细胞具有hUC-MSCs的基本生物学特性和多向分化的潜能；
2.RA复制的OP模型为高转换型,较好的模拟了人原发性OP；
3.hUC-MSCs能明显降低OP大鼠的血清Ca2+、ALP及StrACP水平,从而抑制骨吸收,减少骨量的丢失；改善OP大鼠的股骨组织形态结构、增加骨密度,增强股骨生物力学性能；
4.hUC-MSCs对OP的修复作用,可能与抑制骨吸收、促进骨的形成,调整二者间的偶联平衡,从而改善骨的重建有关。
OBJECTIVE:
1. To establish the methods of isolation and expansion of human umbilical cord derived mesenchymal stem cells (hUC-MSCs).
2. Studying the biological characteristics of hUC-MSCs and its capacity of adipogenic and osteogenic induction in vitro.
3. To find out the feasibility of healing the osteoporosis (OP) rats by transplanting the hUC-MSCs by intravenous injection.
METHOD:
1. Obtain the term infant umbilical cord samples and apply the enzymatic or the tissue culture method to separate and purify hUC-MSCs.
2. Detecct the cell surface markers by flow cytometry; Observe the morphology; Describe the growth curve with MTT method, describe the biological characteristics of hUC-MSCs in vitro.
3. Induce the hUC-MSCs differentiation into adipocytes and osteoblasts and detect the induced cells by histochemistry staining.
4. The treatment of hUC-MSCs on RA induced rats:the female SD rats were randomly divided into four groups (control group (A), model control group (B), hUC-MSCs transplantation group (C) and Xianling Gubao group (D).Except A group administrated with 0.5%CMC-Na, other groups administrated with RA.At the same time, Xianling Gubao and hUC-MSCs were administrated; C group were adiministrated with 2X 106 hUC-MSCs A and B group were administrated with distilled water. The serum biochemical indicators, biomechanics characteristics were measured and microstructure of bone were also observed.
RESULTS:
1. We can get 80%fusion hUC-MSCs during 14-20 days both by tissue culture and enzymatic methods The proliferation became faster after passage and the cells after passage three(P3) presented a homogeneous population of spindle fibroblast-like cells. More than 90%of P3 cells expressed the CD44, CD29, CD 105, but the CD28, CD31, CD34, CD45, CD40, CD86 and HLA-DR were negative.80% of the cells were in G0/G1. The assays in vitro demonstrated the P3 cells exhibited osteogenic and adipogenic cells differentiation potential.
2. Effect of hUC-MSCs on RA simulated OP rats:
(1). In bone biochemical measurement of the serum:The serum Ca2+, ALP and StrACP of C group were significantly higher than B group (p<0.05 or p<0.01); but no found significant difference between D and B group.
(2). In BMD measurements of the right femur group C were significantly higher than in group B (p<0.05);
(3). In right femoral shaft three-point bending test, the max load,the elastic load, the rigidity and max deformation of group C were significantly higher than in group B (p< 0.05 orp<0.01);
(4). In SEM observation, collagenous fiber with regular arrangement in group A; collagen fibers displaced by absorption laeuna in group B; Collagenous fibers were much more regular in group C.
CONCLUSION:
1. The hUC-MSCs were obtained from the human umbilical cord by tissue or enzymic method; the cells shared the same characteristics MSC. The hUC-MSCs were multipotential cells.
2. Bone formation and bone mass decreased, bone microstructure destructive in RA administrated rats. RA simulated OP were a high turnover OP. The rats OP model were successfully established.
3. The hUC-MSCs reduced the serum Ca2+, ALP and StrACP level of OP rats, improved the femur microstructure. The BMD and biomechanic function also enhanced.
4. The hUC-MSCs has a significant effect on RA simulated OP rats. The cells can promote bone formation and reduce bone loss, and improve the microstructure.
5.
引文
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