骨髓基质细胞体外诱导分化移植治疗大鼠脊髓损伤的研究

Study on bone marrow stromal cells transplantation after differentiation on the spinal cord injury of rats

目的探讨大鼠骨髓基质细胞（BMSCs）在体外由碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）、全反式维甲酸（RA）联合诱导下分化成的神经细胞样细胞移植治疗大鼠脊髓损伤的可行性。方法采用出生4周SD大鼠的全骨髓细胞进行培养，利用bFGF＋EGF＋RA在体外诱导BMSCs向神经细胞样细胞分化。在倒置相差显微镜下每日观察、记录BMSCs的诱导分化情况，并应用免疫组化技术对细胞进行兔抗神经元特异性烯醇化酶（NSE）单克隆抗体鉴定。实验动物分为3组：细胞移植组（A组）、模型对照组（B组）、正常对照组（C组）。A、B组采用改良Allen’s法建立大鼠急性脊髓损伤模型。A组在造模后立即将诱导后的BMSCs悬液多点缓慢注入到损伤节段的头侧和尾侧，B组同法注射等量磷酸盐缓冲液。C组不造模，于相同部位注射等量磷酸盐缓冲液。于伤后1～5周，按照BBB法观察脊髓神经功能恢复情况并处死动物取材，用HE染色观察脊髓病理变化。结果诱导7d后有部分细胞具备神经元样细胞形态，胞体呈锥形或圆形，有较长单极或多极的突起，有NSE阳性细胞表达；细胞移植后1～5周，A、B组动物运动功能均有不同程度恢复，但B组恢复较慢，BBB评分差异有统计学意义（P〈0．05）；组织形态学观察示A组损伤脊髓恢复较好。结论bFGF、EGF和RA可以联合诱导BMSCs向神经干细胞分化，并可调控神经元分化，将其移植于大鼠脊髓损伤区，可促进脊髓损伤的恢复。

Objective To observe the effects of the neuron-like cells differentiated from bone marrow stromal cells （BMSCs） which were induced by basic fibroblast growth factor （bFGF）, epidermal growth factor （EGF） and all-transretinoic acid（RA） transplantation on the spinal cord injury（SCI） of rats. Methods Adult marrow stromal cells were cultured from bone marrow of four week-old SD rats. bFGF ＋ EGF ＋ RA were used to induce the third-generation BMSCs. A phase contrast microscope was used to observe the differentiation of BM- SCs, daily. Immunohistochemistry technique was used to identify the differentiated BMSCs with neuron specific enolase （NSE）. The adult SD rats were randomly divided into three groups：SCI cured with transplantion of BMSCs to injured site （groupA）, SCI received PBS solution （group B）, control group （group C）. The induced BMSCs were transplanted into the injured site immediately after the SCI model was made by Allen device. At 1 - 5 week after injury, the neurofunction of spinal cord was evaluated by Basso, Beattie and Bresnahan （BBB） method. And at the same time, the animals were sacrificed and the lesion areas of the spinal cord were cut for morphological studies by hematoxylin and eosin staining. Results After seven days of culture, some BMSCs appeared typical morphological neuronal traits. Cell bodies appeared refractile, cone-like or spherical with long processes, NSE was expressed. Animals in group A and group B recovered their motor functions in a raring degree, the difference of BBB score was significant（P 〈 0.05 ）. Hematoxylin and eosin staining demonstrated that the lesion areas of the spinal cord in group A recovered better. Conclusion Combination of bFGF, EGF and RA can induce BMSCs to differentiate into neural stem cells and neurons, and can regulate the differentiation and promote the maturation of neurons. Transplantation of differetiated BMSCs can promote the recovery of SCI.