Ectopic bone formation by electroporatic transfer of bone morphogenetic protein-4 gene

Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.     

Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo.

This study reports that recombinant adenovirus-mediated human bone morphogenetic protein-2 gene transfer can induce mesenchymal progenitor cell differentiation and bone formation. The recombinant adenovirus with the human bone morphogenetic protein-2 gene was constructed, and mature human bone morphogenetic protein-2 expression mediated by adenovirus gene transfer was detected by specific antibody. Under adenovirus-mediated bone morphogenetic-protein gene transfer, mesenchymal progenitor cell line C3H/10T 1/2 showed cell proliferation dependent on adenovirus bone morphogenetic-protein dose. The C3H/10T 1/2 cells transduced by adenovirus bone morphogenetic protein also exhibited differentiation to osteoblast phenotype, which indicates alkaline phosphatase activity. Injection of the C3H/10T 1/2 cells into the thigh muscles of nude mice led to ossicle development detectable on radiographs. Histological analysis indicated that the new ossicles that developed in the thigh muscles of the mice had different osseous components including bone trabeculae, bone marrow, and chondrified tissue. The results of this study demonstrate the potential for gene therapy by adenovirus-mediated bone morphogenetic-protein gene transfer.     

聚乙二醇化骨形态发生蛋白-2基因转染兔骨髓间充质干细胞及其表达测定

目的：聚乙二醇化骨形态发生蛋白-2基因（PEG/BMP-2）纳米颗粒转染兔骨髓间充质干细胞（rBMSCs）,检测骨形态发生蛋白-2（BMP-2）在靶细胞中的表达。方法：原代分离培养rBMSCs,分别采用PEG/BMP-2、脂质体/BMP-2转染细胞,采用流式细胞仪检测转染效率,采用Western Blot和real time RT-PCR方法检测BMP-2表达。结果：成功制备出PEG/BMP-2纳米颗粒并将PEG/BMP-2转染至rBMSCs,转染细胞中BMP-2呈高表达,且与脂质体转染法相比,具有更高的转染效率。结论：PEG/BMP-2纳米颗粒转染rBMSCs可高表达BMP-2,为骨缺损治疗修复提供新的治疗方法。     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice

onemorphogenetic proteins (BMPs)haveapowerfulcapacitytoelicitnewboneformation .ThereareseveraldeliverymethodsofBMPsintreatingbonedefects ,oneofwhichisgenetherapy .Retrovirus,adenovirusandadeno associatedvirushavebeenutilizedtodeliverBMPgene .1,2 Sincethedirectuseofthesevectorshasseveraldisadvantages ,wehavedevelopedexvivo genetherapytechniquewhichinvolvestheisolationandcultivationofautologousbonemarrow derivedmesenchymalstemcells (MSCs) ,transfectionofthecellsinvitroandimplantationofthesec…     

Expression of osterix inhibits bone morphogenetic protein-induced chondrogenic differentiation of mesenchymal progenitor cells

Osteoblasts and chondrocytes arise from common bipotential mesenchymal progenitor cells. Although the differentiation of these two cell lineages can be induced by treatment with bone morphogenetic proteins (BMPs), the responses of mesenchymal progenitors to BMP differ from cell line to cell line. Here we demonstrate that C3H/10T1/2 cells preferred chondrogenic differentiation, primary bone marrow stroma cells (MSCs) tended to convert to osteoblasts, and ST-2 cells differentiated into both the osteoblastic and chondrocytic lineages simultaneously, suggesting that a molecular switch functions to select cell fate. Osterix, the secondary master regulator of osteoblastogenesis, was induced by BMP at high and low levels in MSCs and ST-2 cells, respectively; in contrast, C3H/10T1/2 cells demonstrated only faint expression. As osterix has been suggested as a negative regulator of chondrogenesis, we hypothesized that the intense chondrocyte differentiation of C3H/10T1/2 cells may have resulted from an absence of osterix. We therefore restored osterix gene expression in C3H/10T1/2 cells using an adenovirus vector. Following BMP treatment, infection with an osterix-encoding virus dramatically inhibited the chondrocytic differentiation of C3H/10T1/2 cells, resulting instead in prominent osteoblast differentiation. These results indicate the chondrogenic potential of C3H/10T1/2 cells was abrogated by osterix expression. Chondrocyte differentiation of MSCs, however, was not enhanced by silencing the osterix gene using lentivirus-mediated shRNA, despite successful suppression of osteoblast differentiation. These results suggest that the low levels of osterix expression remaining after knockdown are sufficient to block chondrogenesis, whereas higher expression may be required to promote osteoblastic differentiation.     

Effect of bone morphogenetic protein-7 transfected bone marrow mesenchymal stem cells on renal injury in rats with chronic renal failure

Objective To observe the bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein-7 (BMP-7) gene on the expression of renal BMP-7, transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), further to explore its protective mechanism on renal injury in rats with chronic renal failure (CRF). Methods BMSCs with high expression of BMP-7 gene (BMSCs-BMP-7) and empty vector-BMSCs (BMSCs-EV) were obtained by lentiviral-mediated gene transfection. Thirty male Sprague-Dawley (SD) rats were randomly divided into 5 groups, 6 in each group: normal control (CON) group; PBS intervention (CRF with PBS infusion, CRF+PBS) group; BMSCs intervention (CRF with BMSCs infusion, CRF+BMSCs) group; BMSCs-EV intervention (CRF with BMSCs-empty vector infusion, CRF+BMSCs-EV) group and BMSCs-BMP-7 intervention (CRF with BMSCs-BMP-7 infusion, CRF+BMSCs-BMP-7) group. The CRF model was established by 5/6 nephrectomy. The CON group was a sham operation group. The corresponding 12-weeks interventions of each experimental group were performed after 2 weeks of modeling, the rats in the CON group and the CRF+PBS group were injected with 1 ml of PBS through the tail vein, and the other three groups were injected with 1 ml of the corresponding cell suspension once a week. At the time of sacrifice, blood and renal tissue samples were reserved. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of BMP-7, VEGF, TGF-β1 in kidney was assayed by Western blotting. Results At the time of sacrifice, the levels of Scr and BUN in the CRF+PBS group were significantly higher than those in the CON group (all P＜0.01); Compared with the CRF+PBS group, the Scr and BUN of the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were decreased to different extents, the differences were statistically significant (all P＜0.01); the Scr and BUN of the CRF+BMSCs-BMP-7 group were significantly lower than CRF+BMSCs group and CRF+BMSCs-EV group (all P＜0.05). The expression of BMP-7 and VEGF were the lowest in the CRF+PBS group. Compared with the CRF+PBS group, the expression of BMP-7 and VEGF in the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were significantly increased respectively (all P＜0.05). The expression of the BMP-7 and VEGF in the CRF+BMSCs-BMP-7 group were higher than those in the CRF+BMSCs group and CRF+BMSCs-EV group (P＜0.01). Compared with the CON group, the expression of TGF-β1 in the CRF+PBS group was significantly increased (P＜0.01); compared with the CRF+PBS group, the expression of TGF-β1 in the CRF+BMSCs group, CRF+BMSCs-EV and CRF+BMSCs-BMP-7 group was significantly decreased (all P＜0.01); the expression of TGF-β1 in the CRF+BMSCs-BMP-7 group was lower than the CRF+BMSCs and CRF+BMSCs-EV group (both P＜0.01). Conclusions BMSCs modified by BMP-7 has a protective effect on CRF rats; its protective mechanism may be related to antagonizing TGF-β1 and up-regulation of renal VEGF expression.     

重组BMP-4对兔骨髓基质干细胞生物学行为的影响

目的应用原核细胞基因工程方法生产出有活性的骨形态发生蛋白-4(bonemorphoge-neticprotein-4,BMP-4),观察其对骨髓基质干细胞生物学行为的影响。方法利用RT-PCR技术,从成熟的人胎盘组织中扩增出长0.34kb编码人BMP-4成熟肽的基因序列,装入表达载体pET-22b( ),转化大肠杆菌BL-21菌株,并诱导目的蛋白表达,SDS-PAGE检测表达蛋白。获得的蛋白制品经小鼠异位成骨测活证实后,诱导培养的兔骨髓基质干细胞,观测细胞形态变化、碱性磷酸酶和骨钙素的含量。结果大肠杆菌中目的蛋白表达量达菌体蛋白的15%,该蛋白可诱导骨髓基质干细胞向成骨细胞分化,形成钙结节,碱性磷酸酶和骨钙素的含量也明显增加。结论大肠杆菌可表达出有活性的BMP-4,该蛋白可诱导骨髓基质干细胞向成骨细胞分化。     

Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats

BACKGROUND: Bone morphogenetic proteins (BMPs) are now being used as bone-graft substitutes to enhance spinal fusion. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins should be improved. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells, and these autologous cells were found to induce a posterolateral fusion of the spine in syngeneic rats. METHODS: Intertransverse spinal arthrodesis (L4 and L5) was attempted in ten groups of Lewis rats with 5 x 10 (6) BMP-2-producing rat bone marrow cells (Ad-BMP-2 cells), created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group I); 5 x 10 (6) Ad-BMP-2 cells on a collagen sponge carrier (Group II); 10 micro g of recombinant BMP-2 (rhBMP-2) in a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group III); 10 micro g of rhBMP-2 in a collagen sponge carrier (Group IV); autogenous iliac crest bone-grafting (Group V); 5 x 10 (6) beta-galactosidase-producing rat bone marrow cells, created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group VI); decortication of the transverse processes alone (Group VII); 5 x 10 (6) uninfected rat bone marrow cells with a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group VIII); guanidine hydrochloride-extracted demineralized bone matrix only (Group IX); or a collagen sponge alone (Group X). Each specimen underwent plain radiography, manual palpation, and histological analysis. RESULTS: All spines in Groups I and II (BMP-2-producing bone marrow cells) and all spines in Groups III and IV were fused at four weeks postoperatively. In contrast, none of the spines in the other groups had fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that the spines that had received BMP-2-producing bone marrow cells (Groups I and II) were filled with coarse trabecular bone postoperatively, whereas those that had received rhBMP-2 (Groups III and IV) were filled with thin, lace-like trabecular bone. All of the other spines, including those that had been treated with autogenous iliac crest bone-grafting (Group V), produced little or no new bone. CONCLUSION: BMP-2-producing bone marrow cells, created by adenoviral gene transfer, produce sufficient BMP to induce an intertransverse fusion in the rat spine model.     

Ex vivo bone morphogenetic protein-9 gene therapy using human mesenchymal stem cells induces spinal fusion in rodents

OBJECTIVE: Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat. METHODS: Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the beta-galactosidase (Ad-beta-gal) gene. Ad-beta-gal served as the control. Each animal received a percutaneous, paraspinal injection of 10(6) hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-beta-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically. RESULTS: Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-beta-gal-transduced hMSCs showed no evidence of osteogenesis. CONCLUSION: The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.     

骨形态发生蛋白-2对人脐带间充质干细胞增殖和分化的影响

目的 观察骨形态发生蛋白-2(BMP-2)对人脐带间充质干细胞(hUCMSCs)增殖和分化的影响.方法 体外培养hUCMSCs,在培养基中加入20 mg/L BMP-2,噻唑蓝(MTT)比色法观察BMP-2对hUCMSCs的增殖效果,流式细胞术检测BMP-2作用后细胞表面STRO-1的表达,逆转录-聚合酶链反应(RT-PCR)测量BMP-2作用后hUCMSCs骨桥蛋白(OPN)、碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(COL1)的mRNA表达变化,碱性磷酸酶染色观察hUCMSCs在BMP-2培养基作用下ALP染色变化,Von kossa染色实验观察BMP-2对hUCMSCs钙结节的形成.结果 细胞在未加BMP-2和加BMP-2培养基中培养1、3、5、7 d,虽然细胞的增殖率上升,但各组间比较差异无统计学意义(P＞0.05),同时发现在2%血清的培养基中培养7 d后,hUCMSCs的增殖促进约10%左右.BMP-2培养7 d后细胞表面STRO-1阳性细胞比例上升明显,由25.1±4.0上升至51.1±6.4,差异有统计学意义(P＜0.01).BMP-2培养条件下COL1 mRNA的表达增强,差异有统计学意义(P＜0.05),OPN mRNA出现表达,ALP mRNA比无BMP-2培养明显增强,差异有统计学意义(P＜0.05).在BMP-2培养条件下ALP染色出现大片细胞阳性染色.在培养28 d,Von kossa染色出现明显的钙结节.结论 BMP-2对hUCMSCs成骨诱导分化作用明显,而增殖作用很弱.     

骨形态发生蛋白-2对兔骨髓基质细胞向软骨细胞分化基因表达的影响

目的 观察骨形态发生蛋白-2(BMP-2)基因对兔骨髓基质细胞(MSCs)向软骨细胞分化mRNA表达的影响.方法 从兔骨髓细胞中分离提取BMP-2 mRNA,构建重组BMP-2真核表达质粒,并转染兔MSCs,采用荧光定量逆转录-聚合酶链反应(RT-PCR)方法检测各组MSCs内Ⅱ型胶原和蛋白多糖mRNA表达水平.结果 转染后2、4 d实验组MSCs内的Ⅱ型胶原和蛋白多糖mRNA表达量28.7±4.0、236.7±48.5及26.9±4.3、208.2±36.7,均明显高于转染后2、4 d的空白对照组(16.1±2.8、99.2±24.8及14.6±2.7、111.1±18.9)和空载体对照组(14.6±2.6、85.4±24.7及16.1±2.8、98.0±22.5)(P＜0.05).结论 重组真核表达载体BMP-2质粒能够诱导MSCs向软骨细胞分化.

Abstract：

Objective To observe the effect of bone morphogenetic protein-2 (BMP-2) on gene expression during the differentiation of marrow stromal cells (MSCs) into chondrocytes. Methods The BMP-2 mRNA was extracted from the rabbit bone marrow cells. The recombinant BMP-2 eukaryotic expression plasmids were constructed and transfected into MSCs. The mRNA expression of type Ⅱ collagen and proteoglycan was detected by using quantitative polymerase chain reaction (PGR) technique. Results At 2nd, and 4th day after transfection of plasmid pEGFP-C3-BMP-2 into MSCs, the mRNA expression levels of type Ⅱ collagen and proteoglycan in MSCs of experimental group were significantly higher than controls (P ＜ 0. 05 ). Conclusion Recombinant eukaryotic expression plastmid pEGFP-C3-BMP-2 can induce MSCs differentiation towards chondrocytes.     

骨形态发生蛋白2基因转染的兔骨髓间充质干细胞复合聚乳酸-聚己内酯支架体外构建载基因仿生骨的实验研究

[目的] 构建聚乳酸-聚己内酯(PLA/PCL)吸附骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)基因的聚乙二醇(PEG)纳米复合物形成生物可降解仿生骨材料,接种兔骨髓间充质干细胞(rabbit bone mes-enchymal stem cells,rBMSCs),检测BMP-2基因的转染情况及其对细胞增殖、分化的影响.[方法] 通过溶液共混法制备PLA/PCL载基因仿生骨,接种rBMSCs细胞于仿生骨之上,培养48 h;Real-time PCR检测转染后细胞BMP-2及骨钙素mRNA表达水平;应用Western Blot及免疫组化检测rBMSCs转染后BMP-2蛋白表达;免疫荧光检测rBMSCs细胞Ⅰ型胶原蛋白表达;ELISA检测转染后细胞培养上清中分泌BMP-2浓度,同时检测细胞内碱性磷酸酶活性,从而分析细胞分化情况;流式细胞检测转染BMP-2后细胞周期的变化.[结果] 以未转染细胞作为对照,免疫组化表明转染后细胞内BMP-2表达量明显上调(P＜0.05);Real-time PCR检测结果表明BMP-2与骨钙素mR-NA表达显著增加(P＜0.05);同时Western Blot结果同免疫组化结果相似,BMP-2也有明显上调(P＜0.01);免疫荧光检测Ⅰ型胶原表达量显著上升(P＜0.01);ELISA检测细胞培养上清中BMP-2分泌量也有增加(P＜0.05);碱性磷酸酶较对照组相比活性显著增强(P＜0.05),而流式细胞检测S期细胞无明显变化(P＞0.05).[结论] 载基因仿生骨具有稳定而安全的转染性能,其转染后对rBMSCs细胞的分化效果是明显的,而对于细胞周期变化的影响并不显著.     

rhPTHl-34对成骨细胞增殖及BMP-7、BMP-9基因表达的影响

目的 研究重组人甲状旁腺激素1-34(rhPTH1-34)对成骨细胞增殖及BMP-7、BMP-9基因表达的影响.方法 通过不同剂量的重组人甲状旁腺素(rhPTH1-34)(0、10-11、10-10、10-9、10-8、10-7mol/L)间歇性(24 h/周期,前12 h rhPTH1-34干预)刺激体外培养的成骨细胞,用噻唑蓝(MTT)法检测细胞的增殖能力,RT-PCR法检测成骨细胞BMP-7、BMP-9基因的表达.结果 间歇性小剂量rhPTH1-34可明显促进成骨细胞的增殖能力及增强BMP-7、BMP-9基因的表达.结论 间歇性小剂量rhPTH1-34可促进成骨细胞增殖,可能与BMP-7、BMP-9基因表达相关.     

基因治疗对兔下颌骨牵引过程中骨形成蛋白-2表达的影响

目的 探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中骨形成蛋白-2(bone morphogenetic protein-2,BMP2)表达的影响.方法 新西兰大白兔双侧下颌骨截骨,术后3d开始牵引,连续牵引7d后,将实验动物分为A、B、C、D、E5组,A、B、C组分别在牵引区注射2 μg(0.1 μg/μl)重组质粒pIRES-hVEGF165-hBMP2、pIRES-hBMP2、pIRES-hVEGF165.D组与E组分别注射相同剂量的空质粒(pIRES)和生理盐水(NS).各组分别于固定期第7、14、28天处死动物,取材行免疫组织化学检测BMP2的表达情况,并利用病理图像分析系统进行分析.结果 BMP2主要在肉芽组织中的炎细胞及新生幼稚骨小梁表面的细胞组织中表达.固定7d时表达最强烈,各时点基因治疗组明显强于对照组.结论 电脉冲介导的基因治疗能使BMP2在牵引区的表达增强和表达时限延长并促进细胞的分裂增殖与分化,促进牵引区细胞基质的形成和新骨生成.     

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (β-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.