摘要
[目的]考察腺病毒载体搭载的重组人组织金属蛋白酶抑制因子-3基因(RAdTIMP-3)转染兔椎间盘后对椎间盘内主要基质成分质量的影响,并探讨其用于椎间盘退变治疗的可行性。[方法]扩增Lac-Z基因标记的RAdTIMP-3和重组腺病毒载体RAd66,并进行纯化、鉴定及滴度测定。将30只日本大白兔随机分入5组,分别向L4、5、L5、6椎间盘内注射生理盐水(第1组)、1.0×1010OPU/mlRAd66(第2组)以及1.0×1010OPU/mlRAdTIMP-3(第3、4、5组)各25μl。各组分别于注射后2、2、1、2、4周收集标本,X-gal染色检测转染效率,RT-PCR检测TIMP-3及聚集蛋白聚糖核心蛋白的表达,TIMP-3及H型胶原免疫组化染色、藏红O-快绿染色及Tunel染色考察转染对椎间盘基质的调控作用。[结果](1)RAdTIMP-3经扩增和纯化后浓度可达1.9×10TM OPU/ml。(2)RT-PCR显示,3、4、5组TIMP-3基因表达均较1、2组明显增加,3、4、5组核心蛋白基因表达较1、2组略有增强。(3)Tunel染色显示各组凋亡细胞比例无明显差异。(4)第5组藏红O-快绿染色及II型胶原免疫组化染色染色强度均好于1、2组。[结论]RAdTIMP-3能够广泛而安全地在兔椎间盘内表达,并改善椎间盘基质成分的质和量,具备用于椎间盘退变治疗的潜力。
[ Objective] To investigate the influence of recombinant adenovirus carrying tissue inhibitor of metalloproteinase - 3 （ RAdTIMP - 3 ） on the main compositions of rabbits intervertebral discs and to assess its potential in treatment for intervertebral disc degeneration. [ Method] RadTIMP- 3 and empty adenovims vector with Lac- Z gene （Rad66） was propagated in 293 Cells and was purified, identified and tittered. Thirty Japanese white rabbits were randomly divided into 5 groups. And 25 μ1 of various reagents were injected to the L4,5 and L5,6 intervertebral discs of the rabbits as follows ： normal saline in group 1, 1.0 × 10^10 OPU/ml of RAd66 in Group 2, and 1.0 × 10^10 OPU/ml of RAdTIMP -3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, l, 2 and 4 weeks after injection respectively. Then X -gal staining, And Group 1, RT - PCR for TIMP - 3 and aggrecan core protein, TUNEL staining, immunohistochemical staining for TIMP - 3 and type Ⅰ ! collagen and Safranin O - Fast green staining was carried out to assess the effects of RadTIMP -3 transfection. [ Result] （ 1 ） concentration of RAdTIMP -3 reached 1.9 x 10^12 OPU/ml after propagation and purificatiorL （2） RT - PCR shows that the expression of TIMP - 3 was significantly raised in group 3, 4, 5, as compared with group 1 or 2. And the expression of core protein gene in group 3, 4, 5 increased slightly than in group 1 and 2. （3） TUNEL staining revealed that there was not significant difference between the positive -staining rates of any two of the groups. （4） TIMP -3 staining exhibited an obvious increase of positive - staining rates in group 3, 4 and 5 as compared with groupi or 2. The staining density of Safranin O - Fast Green staining and immunohistochemical staining for type Ⅱ collagen of group 5 was obviously higher than that of group 1 or 2. [ Conclusion ] RAdTIMP - 3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the po
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2009年第5期356-360,共5页
Orthopedic Journal of China
基金
a grant from the National Natural Sciences Foundation of China(No:30700841)
