重组腺相关病毒2介导hTGF-β_1基因体内转染兔退变髓核细胞对蛋白多糖含量的影响

BIOLOGICAL EFFECTS OF RECOMBINANT ADENO-ASSOCIATED VIRUS 2 MEDIATED HUMAN TRANSFORMING GROWTH FACTOR β_1 ENCODING GENE TRANSFER TO RABBIT DEGENERATIVE NUCLEUS PULPOSUS CELLS ON PROTEOGLYCAN LEVEL

目的应用重组腺相关病毒2(recombinant adeno-associated virus2,rAAV2)介导hTGF-β1基因体内转染兔退变椎间盘髓核细胞,观察基因产物的表达及其对退变髓核细胞蛋白多糖合成的生物调节作用。方法于24只成年新西兰大白兔,雌雄不限,体重1.7～2.2kg,L1、2、L2、3、L3、4、L4、5椎间盘注射25μL浓度为1mmol/L的纤维结合素片段(fi bronectin fragment,Fn-f)制备椎间盘退变模型,注射Fn-f4周时的造模椎间盘模拟早期退变的椎间盘。将24只兔随机分为3组(n=8),A、B、C组分别于造模椎间盘髓核内注射25μL rAAV2-hTGF-β1(1×1012vg/mL)、rAAV2-增强绿色荧光蛋白(enhanced green?uorescent protein,EGFP,rAAV2-EGFP)、PBS。术后1周A、C组各处死2只兔,取髓核组织采用免疫组织化学染色观察hTGF-β1的表达;术后4、8、12周每组取2只兔髓核组织,35S整合分析法检测新合成蛋白多糖的含量;术后12周处死B组2只兔,荧光显微镜观察髓核组织中EGFP的表达。结果术后1周,免疫组织化学染色示A组髓核细胞及基质中广泛存在强阳性染色颗粒,C组仅存在少量阳性颗粒。35S整合法检测示,各时间点A组35S蛋白多糖合成率均高于B、C组,比较差异有统计学意义(P<0.05);B、C组间比较差异无统计学意义(P>0.05)。术后12周,荧光显微镜下观察B组盘髓核组织可见大量绿色荧光表达。结论新型基因转导载体rAAV2可有效介导hTGF-β1基因体内转染兔退变髓核细胞,基因产物可持续表达超过12周,hTGF-β1可有效促进退变髓核细胞蛋白多糖的合成。

Objective To verify the potential of the recombinant adeno-associated virus 2 （rAAV2） vector as a strategy for human transforming growth factor β1 （hTGF-β1） gene transfer in degenerative intervertebral discs of rabbit,to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. MethodsRabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment （Fn-f,1 mmol/ L）,4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits （male or female and weighing 1.7-2.2 kg） which were divided into 3 groups （n=8）. Group A received the 25 μL rAAV2-hTGF-β1 （1 × 10^12 vg/mL）; group B received rAAV2-enhanced green fluorescent protein （rAAV2-EGFP）; and group C received PBS. Two rabbits of groups A,C were killed 1 week after injection,the immunohistochemical staining for hTGF-β1 was performed on the slices of nucleus pulposus （NP） tissues. At 4,8,and 12 weeks after gene transferring,NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using ^35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan,which was significantly more than that from groups B and C （P〈 0.05）,and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks,it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.