TGF-β_3修饰退变髓核细胞移植修复兔退变椎间盘的效果观察  

TRANSPLANTATION OF TRANSFORMING GROWTH FACTOR β_3 GENE-MODIFIED NUCLEUS PULPOSUS CELLS FOR INTERVERTEBRAL DISC DEGENERATION IN RABBITS

作　　者：宋希元[1] 彭圣智[1] 

机构地区：[1]济宁医学院基础学院,山东济宁272067

出　　处：《中国修复重建外科杂志》2012年第7期790-795,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：济宁医学院青年科研基金项目~~

摘　　要：目的探讨TGF-β3基因修饰后退变髓核细胞生物学效应以及植入兔退变椎间盘后对退变椎间盘的影响。方法将重组腺病毒载体Ad-TGF-β3与第2代退变髓核细胞按10∶1比例混合培养转染(Ad-TGF-β3组),待细胞融合后传代,MTT检测转染细胞增殖活性,Western blot检测TGF-β3蛋白含量,免疫细胞化学染色观察对数生长期转染细胞Ⅱ型胶原染色阳性率;采用病毒空载体转染髓核细胞(Adv组)和未经转染髓核细胞(空白组)作为对照。取30只新西兰兔,体重3.2～3.5 kg,雌雄不限,通过针刺L3、4、L4、5和L5、6椎间盘制备椎间盘退变模型。将实验动物按照随机数字法分为3组,转染细胞组(A组,n=12)、退变细胞组(B组,n=12)和空白对照组(C组,n=6)。A、B组将100μL浓度为1×105个/mL对应细胞悬液注射入退变椎间盘,C组同法注入等量PBS。注射后6、10、14周取A、B组各4只、C组2只实验动物处死,取L3、4、L4、5和L5、6椎间盘行组织学观察,RT-PCR检测Ⅱ型胶原和蛋白多糖mRNA表达。结果 Ad-TGF-β3转染后髓核细胞活性明显改善;转染后3、7、14 d,TGF-β3在髓核细胞内表达逐渐升高;Ad-TGF-β3组髓核细胞细胞质内见棕黄色Ⅱ型胶原阳性染色,阳性率显著高于Adv组及空白组(P<0.05)。组织学观察示,A组椎间盘退变程度较B、C组明显减轻。6、10、14周A组Ⅱ型胶原和蛋白多糖mRNA表达显著高于B、C组,差异均有统计学意义(P<0.05)。结论 TGF-β3基因修饰退变髓核细胞后可明显改善细胞生物活性,转染后髓核细胞植入兔体内可明显增加退变椎间盘的基质分泌。Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 （TGF-β3） gene-modified nucleus pulposus （NP） cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 （Ad-TGF-β3） gene （Ad-TGF-β3 group）, and then the cell biological features were observed by cell vitality assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected （Adv group） and the un-transfected cells （blank group） were used as controls. The model of lumbar disc degeneration was established by needling L3. 4, L4, 5, and L5. 6 in 30 New Zealand rabbits （weighing 3.2-3.5 kg, male or female）. Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells （100 μL, 1×10^5/mL, group A, n=12）, no gene-modified nucleus pulposus cells （100 μL, 1×10^5/mL, group B, n=12）, and phosphate- buffered saline （PBS, 100 μL, group C, n=6） were injected into degenerative lumbar intervertebral discs, respectively. L3.4, L4. 5, and L.6 disc were harvested from the rabbits （4 in groups A and B, 2 in group C） at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viability of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group （P 〈 0.05）. The disc degeneration in group A was slighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significa

关 键 词：髓核细胞 TGF-Β3 基因治疗 椎间盘退变 兔 

分 类 号：R681.53[医药卫生—骨科学]