课题背景：本实验系辽宁省重点实验室－医学组织工程实验室分支课题，辽宁省教育厅创新团队项目子课题，申请资金10万元。现已完成细胞培养、种子细胞体内移植、神经支架制作等前期工作，发表国家级文章2篇。

偏倚或不足：实验如果对早期移植和晚期移植结果进行比较，体内移植后细胞分化情况作出定量测定，并结合基因技术或组织工程技术就会更为完善。

相关链接：现在细胞移植策略治疗脊髓损伤主要集中在单一细胞移植，或单细胞联合营养因子移植，或经基因修饰后单细胞移植，主要目的或是通过移植细胞替代缺失的神经元细胞，或是通过移植促进髓鞘修复、神经纤维再生及分泌营养因子等。但单一细胞移植存活、发挥功能方面都不如联合细胞移植，现已有学者应用神经干细胞联合许旺细胞，或嗅鞘细胞移植，或胚胎脊髓联合嗅鞘细胞，来进行移植，取得了更好的效果。

摘要
目的：骨髓间充质干细胞和许旺细胞作为脊髓组织工程学的两个重要的种子细胞，均可进行自体移植，具有其他种子细胞无以比拟的优势。应用联合细胞移植治疗大鼠脊髓半横切模型，观察其疗效。
方法：实验于2007-03/12在辽宁医学院科学实验中心完成。①实验材料：清洁级健康成年SD雄性大鼠60只，体质量250~ 300 g ，由辽宁医学院实验动物中心提供。将大鼠按随机数字表法分为3组：联合细胞组、骨髓间充质干细胞组、磷酸盐缓冲液组，每组20只。实验过程中对动物处置符合动物伦理学标准。②实验方法：采用全骨髓培养法制备大鼠骨髓间充质干细胞，混合酶消化法制备大鼠许旺细胞。制作大鼠右侧T10脊髓半横切模型，联合细胞组注射骨髓间充质干细胞和许旺细胞，骨髓间充质干细胞组注射骨髓间充质干细胞，磷酸盐缓冲液组注射磷酸盐缓冲液。③实验评估：术后4周行网格及斜板实验，并检测大鼠损伤区脊髓诱发电位波幅及潜伏期，并行苏木精-伊红染色、免疫组织化学染色观察。
结果：纳入大鼠60只，每组术后各2只因腹胀及感染死亡，进入结果分析54只。 ①网格实验及斜板实验均显示，联合细胞组大鼠恢复情况明显优于其他两组（P < 0.01），骨髓间充质干细胞组优于磷酸盐缓冲液组（P < 0.01）。②神经电生理检测显示，联合细胞组诱发电位波幅比其他两组高（P < 0.01），诱发电位潜伏期比其他两组短（P < 0.01），骨髓间充质干细胞组优于磷酸盐缓冲液组（P < 0.01）。③联合细胞组囊性病变区面积最小，其次是骨髓间充质干细胞组，磷酸盐缓冲液组囊性变区面积最大（P < 0.01）。④联合细胞组损伤侧灰质内神经元样细胞数多于其他两组（P < 0.01）。
结论：骨髓间充质干细胞和许旺细胞可以联合移植，在体内存活良好，并互相协同促进损伤脊髓恢复，比单一细胞移植疗效更佳。
关键词：骨髓间充质干细胞；许旺细胞；联合移植；脊髓损伤

智晓东，吕刚.大鼠骨髓间充质干细胞和许旺细胞联合移植治疗脊髓损伤[J].中国组织工程研究与临床康复，2008，12(16):3015-3018 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3015(ps).pdf]

Effects of intravenous transplantation of marrow stromal cells on transforming growth factor beta 1 in brain tissues of rats with focal cerebral ischemia/reperfusion injury

Abstract

BACKGROUND：Bone marrow mesenchymal stem cells (BMSCs) and Schwann’s cells with predominance as important seed cells in spinal cord tissue engineering can be used in autologous transplantation. This study aimed to evaluate the therapeutic effect on rat spinal cord injury model of semi-transsection by their combination transplantation.
METHODS: The experiment was performed from March to December 2007 at the Scientific Experiment Center of Liaoning Medical College. ①Sixty healthy adult male SD rats (250-300 g) provided by Scientific Experiment Center of Liaoning Medical College were randomly divided into three groups, a combined cell transplantation group, a BMSC transplantation group and a control group with phosphate buffered solution (PBS) injection, twenty rats in each group. The disposal in the experiment was accorded with the animal ethical standards. ②BMSCs were cultured by whole bone marrow culture and rat Schwann’s cells were cultured by enzyme digestion. Hemi-transsection models of T10 spinal cord injury in the right side were made. BMSCs and Schwann’s cells were injected into rats of the combined cell transplantation group. BMSCs were injected into rats of the BMSC transplantation group. PBS was injected into rats of the control group. ③Four weeks after the surgery, gross observation and inclined plane tests were performed. Spinal cord evoked potential amplitude and latent phase were observed. Hematoxylin-eosin staining and immunohistochemical method were conducted.
RESULTS: Of the 60 rats, 54 were included in the final analysis, because 2 rats in each group died of abdominal distention and infection.①Gross observation and inclined plane test showed that result of the combined cell transplantation group were better than the other two groups(P < 0.01), and the control group was worst in the three groups(P < 0.01). ②Neuro-electrophysiology showed that induced potential amplitude was higher in the the combined cell transplantation group (P < 0.01), but latent phase was shorter (P < 0.01) compared to the other two groups. It was better in the BMSC transplantation group than the control group (P < 0.01). ③Area of cystic lesion regions was the smallest in the combined cell transplantation group, followed by the BMSC transplantation group and the control group in order (P < 0.01). ④Number of neuron-like cells was more in the gray substance in the combined cell transplantation group compared to the other two groups (P < 0.01).
CONCLUSION: Combination transplantation of BMSCs and Schwann’s cells can promote the recovery of spinal cord injury by interaction. Their combination can obtain better outcome.

Zhi XD, Lü G.Cotransplantation of rat bone marrow mesenchymal stem cells and Schwann’s cells for treating spinal cord injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3015-3018(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3015(ps).pdf]