共同培养骨髓基质干细胞对髓核细胞增殖分化的影响

Effects of nucleus pulposus cells co-cultured with bone marrow-derived stroma cells on the proliferation and differentiation of nucleus cells

目的:针对椎间盘退行性变的组织修复目前集中于细胞移植治疗,实验在体外联合培养条件下观察骨髓基质干细胞对髓核细胞增殖分化的影响。方法:实验于2005-12/2006-09在华中科技大学同济医学院附属同济医院骨科实验室和同济医院卫生部重点实验室进行。①实验方法:4月龄健康新西兰大耳白兔3只,麻醉后于髂嵴处作骨髓腔穿刺,抽吸6mL骨髓,分离骨髓基质干细胞;相同实验兔抽取骨髓后立即处死,取腰骶部脊柱之椎间盘,切开纤维环,取出髓核组织,分离椎间盘髓核细胞,同时设立单独髓核细胞培养组。②实验评估:在相同的培养条件下,定期观察两组髓核细胞在光镜下形态学变化及电镜下细胞超微结构的改变;于诱导培养7,14d通过RT-PCR法、Western blot法和免疫组化法法检测两组髓核细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达。结果:①髓核细胞大体形态学及超微结构变化:髓核细胞与骨髓基质干细胞共同培养后,髓核细胞很快就呈现为圆形或多角形,且细胞形体变大、折光性好,贴壁时间早,数量增殖快,超微结构细胞表面可见许多短而粗的微绒毛突起,胞质内有大量扩张的粗面内质网、丰富的线粒体和高尔基体,细胞合成代谢旺盛。单独培养的髓核细胞胞体小,贴壁、增殖慢,细胞器不丰富。②髓核细胞Ⅱ型胶原蛋白与聚集蛋白聚糖检测结果:RT-PCR和Western blot检测显示诱导培养7,14d骨髓基质干细胞与髓核细胞共同培养组的Ⅱ型胶原蛋白、聚集蛋白聚糖条带均明显亮于单独髓核细胞培养组,吸光度值比较差异有显著性意义(P<0.01)。免疫组化检测显示骨髓基质干细胞与髓核细胞共同培养组中Ⅱ型胶原蛋白和聚集蛋白聚糖在细胞胞浆中的表达随时间延长而逐渐增加,2周达到高峰,单独髓核细胞培养组中此两种蛋白表达很低,且培养第7,14天无明显变化。结论:骨髓基质干细胞在体外与髓核细胞共同培养时,能激活髓核细胞并促进其增殖分化,增加髓核细胞外基质的合成。提示骨髓基质干细胞与髓核细胞联合培养有利于退变椎间盘的髓核细胞修复、再生。

AIM： On benefit of the reparation of the intervertebral disc degeneration with cell transplantation therapy, the aim of this experiment should investigated the effect of nucleus pulposus ceils in co-culture with bone marrow-aenvea stroma cells （BMSCs） on proliferation and differentiation.METHODS： The experiment was performed at Laboratory of Department of Orthopaedics, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology and Key Laboratory of Health Department of Tongji Hospital from December 2005 to September 2006.①Three healthy flap-eared New Zealand rabbits aged 4 months were aspirated on iliac crest under anesthesia for 6 mL bone marrow from which BMSCs were separated, then sacrificed and excised the lumbar intervertebral disc from which the nucleus pulposus cells were separated. Simultaneously, simple nucleus pulposus cell culture group was established. ②The nucleus pulposus＇s morphology in the same culture condition were observed regularly under light microscope and transmission electron microscope （TEM）. RT-PCR, Western blot and immunohistochemical method were applied to detect the expression of collagen Ⅱ and aggrecan in nucleus pulposus cells after 7 days and 14 days of co-culture with BMSCs. RESULTS： ①General morphology and ultrastructure of nucleus pulposus cells： The nucleus pulposus cells co-cultured with BMSCs showed bigger and round or polygon shape, early adherence time and quickly double quantity. Moreover, they displayed higher anabolic metabolism with more microvillus and abundant organelle. However, the nucleus pulposus cells of the control group became small, slowly adherence, a little organelle and low anabolic metabolism. ②Expressions of collagen Ⅱ and aggrecan in nucleus pulposus cells： The results of RT-PCR and Western blot of collagen Ⅱ and aggrecan in the experiment group showed better than the control after co-culture for 7 days and 14 days, and there were a significant difference of absorbance （A） between the two groups （P〈0.01）, The results of immunohistochemical method of collagen Ⅱ and aggrecan also showed more expressions in the experimental group and it was on the peak after 2 weeks. The collagen 11 and aggrecan expressions were low in the only nucleus pulposus cell culture group and no change was found at the 7^th and 14^th days. CONCLUSION： The nucleus pulposus cells co-cultured with BMSCs should become higher differentiation activity and showed long-term growth and quickly proliferation. It can be served as optimal cell source for therapy of the degeneration disc disease.