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【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25 μl。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。
【关键词】 组织金属蛋白酶抑制因子-3; 椎间盘; 聚集蛋白聚糖;Ⅱ型胶原
Recombinant adenovirus carrying tissue inhibitor ofmetalloproteinase-3 gene regulates the matrix ofrabbit intervertebral disc in vivo
XIONG Li-ming熊蠡茗,GUO Bing郭兵, SHAO Zeng-wu邵增务,YANG Shu-hua杨述华, XIE Mao谢卯, WANG He-zhong王河忠
Department of Orthopedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, 430022
Abstract：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.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 μl of various reagents were injected to the L4、5 and L5、6 intervertebral discs of the rabbits as follows:normal saline in group 1, 1.0×1010 OPU/ml of RAd66 in Group 2, and 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, 1, 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.(1)concentration of RAdTIMP-3 reached 1.9×1012 OPU/ml after propagation and purification. (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.RAdTIMP-3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the potential to be used in treatment for intervertabral disc degeneration.
Key words:tissue inhibitor of metalloproteinases-3; intervertebral disc; aggrecan; type Ⅱ collagen
Chinese Library Classification Number：R681Document Code：AArticle ID：1005-8478(2009)05-0356-05
Degeneration of intervertebral disc(IVD) is the main cause of the common spiuae diseases such as prolapse of intervertebral disc and spinal stenosis etc.It weakens the patients’ working ability and the quality of lives,it also wastes a lot of medical resources.IVD is a matrix-based tissue,the degeneration of the main matrix composition:aggrecan is the key point and the first important performance of oeegeneratin of IVD.So to control and protect the matrix is a significant direction in the degeueratiwe IVD therapy research.Aggrecanase is the most important protease in the degradation process of aggrecan.The tissue inhibitor of metalloproteinase (TIMPs) family keeping the important balance of matrix metabolism,are the natural inhibitors of aggrecanase and MMPs.TIMP-3 of the family shows the strongest inhibition effect to aggrecanase and this effect if were ten times more than TIMP-1[1] used in the recent research of gene therapy of degeuorative IVD.Theoretically,it can play a therapy role in raising the quality and amount of the intervertebral matrix in order to improve the living environment of the intervertebral cells and inhibit the degeneration of IVD.In this research we intends to transfect the recombinant adenovirus vector carrying TIMP-3(RAdTIPMP-3) into the rabbits’ IVDs in order to continue to increase the TIMP-3 content of the IVDs,improve the matrix quality of the IVD and evaluate the possibility of degeneration IVD therapy by using RAdTIMP-3.
1 MATERIALS AND METHODS
1.1 MATERIALS:RAdTIMP-3 marked with Lac-Z gene and empty adenovims vector RAd66 for control were supplied by Pro. Anderew H Baker, Bristol University,the United Kingdom. Human embryonic kidney cells were purchased from Cell Collection Center of Wuhan University. Japanese white rabbits were purchased from Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, License Number: SCXK (E) 2004-2007. Trizol was purchased from Invitrogen Company. RT-PCR related agents were purchased from MBI Fermentas Company and Primers from TaKaRa TIMP-3 and Type II collagenase were purchased from Boster Biological Technology of Wuhan. SP agent of immunohistochemistry was purchased from Zhongshan Company. TUNEL Dyeing kit was purchased from Precinorm Company in Germany. X-gal Dyeing kit was purchased from Beyotime Institute of Biotechnology.
1.2 METHODS:
1.2.1 Amplification, titer measurement and identification of the recombinant adenovirus.
RAdTIMP-3 and RAd66 were amplified by 293 cell culture in DMEM/F-12 with 5% fetal bovine serum (FBS) at 37℃～5%CO2. The cells were collected after the cytopathie effect (CPE) appeared, then three freeze-thaw cycles are used at -20℃/37℃, and the surface clear liquid was collected for the next amplification. After three times of amplification, virus suspension was purified through CsC1 density gradient centrifugation and then storaged at -70℃. The concentration of purified virus was measured by the spectrophotometer method at 260nm wavelength. 100/xl viruses were treated by proteinase K, phenol extraction and PCR identification after ethanol precipitation. The amplification conditions were 94℃ for 45s; 58℃ for 30s; 30 cycles of 94℃. The upstream primers and downstream primers of TIMP-3 were respectively 5′-TCTGCAACTCCGACATCGTG-3′ and 5′-CGGATGCAGGCGTAGTGTT-3′[2]. The upstream primer and downstream primers of adenovirus vector were 5′-TCGTTTCTCAGCAGCTTGG-3′' and 5′-CATCTGAACTCAAAGCGTGG-3′[3] separately.
1.2.2 Animal grouping and disc injection
Totally 30 Japanese white rabbits aged 4 months, of either sex, (provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, Certification No. SYKK (e) 2002-0028) were randomly divided into five groups. With the above-mentioned design[4], 25 μl of various reagents were injected to the L4、5 and L5、6 IVDs of the rabbits as follow: normalsaline in group 1;1.0×1010 OPU/ml of RAd66 in Group 2; 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The IVDs of each group were collected respectively after 2, 2, 1, 2 and 4 weeks after the injection.
1.2.3 X-gal staining
The remaining IVDs of each group were fixed by 10% formalin for 24 h, embedded with paraffin routinely and then made into specific staining slice of 5 μm. After the specimens were dewaxed the sections to water, they were stained for 24h according to the direction of β-galactosidase in situ dyeing kit and observed under Olympus light microscope. The blue stained cells were positive cells with Lac-Z gene expression.Ten visual fields under the microscope were taken as a unit of enumeration and the transfection efficiency of adenovims= number of positive cells/total cell count×100%.
1.2.4 TIMP-3 and aggrecan core protein gene expression detection
Six intervetebral discs were randomly selected in each group, one-step Trizol were used to extract total RNA and the reverse transcription kit was used for reverse transcription. Then 2 μg cDNA was subjected to PCR reaction. The PCR primers of TIMP-3 and the reaction conditions were as above-mentioned. The primers and reaction conditions as our team previously reported were used in PCR of aggrecan core protein and β-actin[5]. PCR products were treated by 2% agarose gel electrophoresis and the result of protein band intensity being was calculated by HMIAS22000 analyzer. The ratio r of target gene and β-actin protein bands intensity being was used as the relative gene expression.
1.2.5 Extracellular matrix and cell apoptosis detection
TIMP-3 and type II collagen were stained by immunohistochemistry S-P method:after the specimens were dewaxed the sections to warer, they then were treated by heat-induced antigen retrieval and the 1:80 diluted rabbit anti-human of TIMP-3 and type II collagen polyclonal antibody were incubated for 30min at 37℃. According to the instructions of goat anti-rabbit immunohistochemistry S-P kit, added the second antibody etc. until the hematoxylin was restained; sealed slices; detected the content and distribution of aggrecan by Safranin O-Fast Green Staining[5]. Cell apotosis was reflected by TUN-EL staining and the staining was completed by ultrafine pathological staffroom in Tongji Medical College, Huazhong University of Science Technology.The photograghs were taken by HMIAS-2000 auto image analysis system and ten visual fields under the microscope were taken as a unit of enumeration to calculate the positive stained cells rate.
1.2.6 Statistic analysis
SPSS 12.0 was used for statistics analysis. The result of RT-PCR was analyzed by independent sample t test. Positive cell rate of X-gal staining, TIMP-3 immunohistochemical staining and Tunel staining were denoted by x-±S. After the positive and negative cell number were cumulated, then x2 test was used for analyzing the difference of positive cell rate in each group.
2 RESULT
2.1 Purification and identification for adenovirus vector
The concentration of the purified virus suspension was 1.9×1012 OPU/ml. The adenovirus and TIMP-3 amplified band were obviously detected by PCR.
2.2 X-gal staining
Positive cell rate of X-gal staining from group 1 to group 5 were 0, (81.72±3.88)%, (81.51±4.43) %, (81.85±2.69) %, and (64±3.64) % respectively. Positive cell rate of Group 4 was the highest in the three RAdTIMP-3 transfection groups(Fig,1),but there were no significant differences of positive cell rate between group 4 and group 3 (x2=0.345, P>0.05 ), while compared with that of group 4, the group 5 decreased markedly (x2=26.183,P<0.001) .
2.3 TIMP-3 and aggrecan core protein gene expression by PCR
The r value of TIMP-3 gene in each group were 0.385±0.017, 0.375±0.055,1.022±0.089, 0.941±0.046 and 0.865±0.070 respectively. The weak expression of TIMP-3 between group 1 and group 2 were similar, there was no statistical difference (t=0.950, P>0.05) ; the expression of TIMP-3 gene in group 3 and group 4 were enhanced significantly with contrast to that of group 1 (t=12.195,P<0.01; t=19.546,P<0.01 );compared with the expression of group 3 and 4, that of group 5 decreased slightly, but was still stronger than group 1 (t=11.556, P<0.01) (Fig.2B).
The r value of aggrecan core protein gene in each group were 0.731±0.020,0.729±0.020,0.793±0.020, 0.835±0.018, and 0.827±0.027 respectively. The expression of aggrecan core protein gene between group 1 and group 2 were similar, (t=0.101, P>0.05);the expression of that in group 3,4, and 5 were enhanced slightly in contrast to that of group 1, especially for group 4 (t=6.588,P<0.05 ) (Fig.2B).
2.3.1 TUNEL staining
Positive cell rate of TUNEL staining in each group were (10.1±1.89)%, (10.52±1.28) %, (10.17±1.38)%, (9.74±1.6)% and (10.25±1.22)% respectively. There was no statistical difference between group 1 and group 2,3,4,5.(x2=0.045, P>0.05;x2=0.000, P>0.05;x2=0.074,P>0.05;x2=0.000, P>0.05) (Fig.3A、B).Fig.1X-gal staining 100× Group 3 Positive cells rate of X-gal staining reaching to 82.4% Fig.2aRCR bands of β-actin in Group 1～5 Fig.2bPRC bands of TIMP-3 and aggrecan,314bp for the core protein band,and 545bp for TIMP-3 band2.3.2 TIMP-3 staining, Safranin O-Fast Green Staining and type II collagen staining
Positive cell rate of TIMP-3 staining in each group were (20.53±4.69)%,(21.75±2.81)% ,(82.19±4.30) % ,(81.59±4.31)% and (70.85±4.34)% separately.Positive cell rate of group 1 and 2 were similar (x2=0.263, P>0.05);those of group 3,4,and 5 were obviously higher than group 1 (x2=252.006, P<0.01;x2=252.108,P<0.01;x2=170.919, P<0.01) (Fig.4a、b); positive cell rate of group 3 and 4 were similar (x2=0.038, P>0.05) ; that of group 5 was decreased significantly ther gvoup3 (x2=13.142, P<0.01).
Safranin O Staining mainly reflected the content ofglycosaminoglycan chains in aggrecan, while the cell nuclears were stained by Fast Green. Safranin O Staining showed that the structures of nucleus pulposus and annulus fibrosus were all clear and intact from group 2 to 5. The concentration of jacinth in group 5(Fig.5b) was the most brilliant with contrast to group 1 (Fig.5a) , which showed the content and quality of aggrecan in group 5 were improved obviously.
Type II collagen staining showed: the structure of nucleus pulposus and annulus fibrosus were all clear and intact in each group: type II collagen was distributed along the fibers (Fig.6a) and the positive staining was all abundant.Especially in group 5 (Fig.6b) , compared with group 1, the positive staining was more obvious.Fig.3Tunel staining 200× Tunel staining of nucleus pulposus of intervertebral disc in group5(Fig.3b) showed there were not obvious increase of apoptosis cells in group 5 in contrast to those of group 1(Fig.3a) Fig.4TIMP-3 immunohistochemistry staining 200× TIMP-3 immunohistochemistry staining of inner annulus fibrosus showed most cells were positive stained and the positive rate Safranin O-Fast Green Staining 200× Safranin O staining of nucleus pulposus tissue was relatively light,but the structure of fibers was clear and of group 3 was higher than that of group 1(Fig.4a) Fig.5 the lacune-like structure was complete(Fig.5a) in group 1.Compared with the staining of group 1,that of group 5 was enhanced significantly and the tissue structures wer good (Fig.5b) Fig.6Type Ⅱ collagen staining 200× Type Ⅱ collagen staining of inner annulus fibrosus in group 1 showed that the lamellar structures of fibers were clear(Fig.6a),while the brownish yellow staining zones in group 5 were obviously more than those of group 1 and the tissue structures were good(Fig.6b)3 DISCUSSION
The structure of IVD is characterized by main matrix, in which cells are scattering distribution, and there was complicated interaction between matrix and cells. Degeneration of matrix resulting in deterioration of cell biomechanical environment and diffusion barrier of nutrient etc., thus further decrease the capacity of matrix repairing in IVD cells, and then will lead to irreversible degeneration of IVD tissue. Various aggrecan-based proteoglycans are the important structural basis for matrix to keep its viscoelasticity and bear biomechanical loading. Loss of aggrecan is our important early change in the degeneration of intervetebral disc and continuously exists in the process of degeneration. It is the key point raat shoued be improved for curing the degeneration of IVD. Increasing the content and quality of aggrecan will help to improve the living environment of IVD cells [6], reduce the cell damage caused by external factors such as mechanical factors and inflammatory .factors etc., and recover the balance between injury and reparation of IVD tissue.
On the one hand, loss of aggrecan is caused by the decrease of cell synthesis. On another hand, the increased activities of metalloproteinases headed by aggrecanase and MMPs is a significant factor leading to the decrease of quality and content of matrix[7]. Restriction enzyme digestion of aggrecanase between G1 and G2 zone of aggrecan core protein chain directly results in the abscission of core protein from hyaluronate, and paves the way for further enzyme digestion of MMPs to aggrecan.With the decrease of aggrecan quality, the effect of aggrecan protecting collagen against enzyme digestion of MMPs gradually losses, and the whole structure of matrix was damaged. TIMPs family are the powerful natural inhibitors of metalloproteinases. Many scholars at home and abroad have reported that TIMP-1 gene therapy can inhibit MMPs to improve the quality of IVD matrix, but TIMP-1 mainly inhibits the restriction enzyme digestion of MMPs, and it can' efficiently inhibit the severe damage of core protein produced by aggrecanase to inhibit the degeneration of matrix from headater. TIMP-3 is a powerful aggrecanase inhibitor, which is at least ten times more than MMPs, and the decrease of TIMP-3 content is also an important biological event in the degeneration of IVD[8].Theoretically, TIMP-3 can provide aggrecan with better protection than TIMP-1,which will lead to the effect of improving IVD matrix. Therefore, this research used the classic vector of intervetebral disc gene therapy-adenovirus vector carrying TIMP-3 gene to transfect intervetebral disc tissue in order to continuously increase the concentration of TIMP-3 in intervetebral disc.
In this research, positive cell rates of X-gal staining of group 2,3, and 4 were similar. As the transfection time prolonged, positive staining rate of group 5 decreased somewhat, but still kept a relative higher rate. Consistent with the related reports[9] about adenovirus vector transfecting IVD tissue, the result reflected the efficient transfection ability of RAdTIMP-3. RT-PCR and immunohistochemical staining showed the high expression of TIMP-3 gene in IVD after transfection, and the timeliness of expression could last for more than 4 weeks. The assumption of increasing the concentration of TIMP-3 in a long time could be basically satisfied.In each group the result of TUNEL staining showed that neither the transfection of RAdTIMP-3 and RAd66 nor the high concentration of TIMP-3 could increase the cells apoptosis ratio in IVD, although there were related reports about TIMP-3 inducing apoptosis in some cells. Seen from this study, the cell transfection of TIMP-3 didn′t result in the obvious damage of cells.
Core protein, PCR, safranin O staining, and type II collagen staining reflected that matrix of IVD gradually improved after the transfection of RAdTIMP-3. TIMP-3 didn′t directly increase the secretion of aggrecan and type II collagen, so in the early stage of transfection it wasn't like TGF-β1 gene therapy appearing the rapid increase of the expression of aggrecan and type II collagen, and after 2-weeks transfection, the peak contents of aggrecan and type II collagen gradually appeared. With the transfection time prolonged, maybe the cell state were improved by the increased quality of matrix[6],and the expression of aggrecan also had a small increase. After 4-weeks transfection, although the expression of TIMP-3 began to decrease, but the expression of core protein gene was still stronger than the control group, which showed that the damage/repair balance of IVD may incline to the direction of repair.
In conclusion, the essence of degenerative disc is their own cell functional deterioration, apotosis and necrosis[10], this research showed RAdTIMP-3 could transfect the cells of IVDs efficiently and increase the concentration of TIMP-3 in discs for a long time to improve the quality of matrix in IVDs. So this gene has the potential for the therapy of degeneration of IVD. Although the only improvement of matrix quality may not reverse the degeneration completely, TIMP-3 is doubtlessly a beneficial complement for the early and comprehensive therapy of IVD degeneration.On the other hand, the problems of unsatisfactory longterm results or further degeneration of IVDs, faced by tissue engineering of intervertebral, may be solved by means of the regulatory effect of TIMP-3 to matrix of the discs.
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作者单位：华中科技大学同济医学院附属协和医院骨科，中国武汉市 430002