重组pEGFP-N1-IGF-1质粒在体转染骨质疏松大鼠的实验研究
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摘要
研究背景
胰岛素样生长因子-1(IGF-1)是有效地促进骨合成代谢和生长的重要内分泌生长因子,其水平的下降被证实是骨质疏松重要的发病机制之一,目前基因治疗骨质疏松等骨代谢疾病正在成为研究的发展方向,本实验使用质粒重组体pEGFP-N1-IGF-1在体转染骨质疏松大鼠,研究提高其胰岛素样生长因子-1水平的可行性及对骨质疏松大鼠骨质量的影响。
目的
通过本实验证实质粒重组体pEGFP-N1-IGF-1在体转染骨质疏松大鼠可以获得胰岛素样生长因子-1的增高表达并对大鼠的骨质疏松症形成有效的控制,为应用该重组质粒治疗骨质疏松症做理论准备。
方法
雌性SD大鼠30只,随机分为3组：假手术组、pEGFP-N1组和pEGFP-N1-IGF-1组,各10只。假手术组予以开腹后切除少量脂肪组织,pEGFP-N1组和pEGFP-N1-IGF-1组开腹后予双侧卵巢切除。术后12周造模成功,依次给予盐水、pEGFP-N1载体以及质粒重组体pEGFP-N1-IGF-1分别复合脂质体后经尾静脉流体力学注射。注射后定期行全身荧光活体成像及肝脏切片观察转染及表达效果,并定期测定血清胰岛素样生长因子-1浓度至活体实验结束。活体测定大鼠腰4椎体的骨密度值后,处死,取大鼠L4椎体进行椎体压缩实验测定一系列骨生物力学参数,评价该质粒转染对骨质疏松大鼠骨质量的影响。
结果
1.转染后pEGFP-N1组和pEGFP-N1-IGF-1组大鼠均可见全身大部出现明显荧光表达,以肝脏及尾部为著,肝脏切片镜下观察可见明显荧光表达。
2.转染后3周内pEGFP-N1-IGF-1转染大鼠血清胰岛素样生长因子-1表达水平显著升高,6周内显著优于pEGFP-N1转染大鼠(P<0.05)。pEGFP-N1-IGF-1组骨密度、腰椎椎体骨生物力学参数均显著优于pEGFP-N1组(P<0.05),与假手术组差异无统计学意义(P>0.05)。
结论
质粒重组体pEGFP-N1-IGF-1能够在骨质疏松大鼠体内成功转染并表达胰岛素样生长因子-1蛋白,对大鼠骨质疏松症形成治疗作用,为应用胰岛素样生长因子-1基因治疗骨质疏松症奠定了坚实的理论基础。
同时,根据IGF-1降低血糖以及促进微血管生长的特性,可以预见,糖尿病骨质疏松以及糖尿病导致的局部组织缺血坏死也将成为这一基因转染方法的适应症,为其治疗提供一种新思路。
Backgroud
As an important growth factor of promote synthesis and growth metabolism of bone, the decline of Serum IGF-1 levels has been proven to be an important reason leading to osteoporosis.Gene therapy for osteoporosis and other bone metabolic diseases has becoming a direction of the research, therefore, we used the recom-binant plasmid pEGFP-N1-IGF-1 in vivo transfection of osteoporosis rats to study the feasibility of improving their serum IGF-1 levels as well as bone quality in this experiment.
Objective
To study the expression as well as therapeutic effect of recombinant plasmid pEGFP-N1-IGF-1 in the osteoporosis rats in vivo, so as to make theoretical preparation for the treatment of osteoporosis using this recombinant plasmid.
Methods
In this study, a total of 30 normal female SD rats were divided randomly into three even groups.The rats in sham group was unovariectomized while in group pEGFP-N1 and pEGFP-N1-IGF-1 was ovariectomized. After 12 weeks, the three groups were given saline, pEGFP-N1 vector and recombinant plasmid pEGFP-N1-IGF-1 complex liposomes hydrodynamic injection via the tail vein in proper order. Fluorescence in vivo imaging and liver biopsy observations, serum concentrations of IGF-1 were determined at regular intervals. Also, bone mineral density and bone biomechanical parameters were measured.
Results:
1.After transfection, rats of group pEGFP-N1 and pEGFP-N1-IGF-1 could be seen noticeable fluorescence expression not only in the whole body but also liver biopsy, which was significantly different compared with the sham group.
2. The expression level of IGF-1, lumbar bone mineral density and bone biomecha than nical parameters of group pEGFP-N1-IGF-1 were significantly better that of group pEGFP-N1 (P＜0.05), among them, the data of latter two index has no significant statistical difference with sham group (P＞0.05).
Conclusions
Recombinant plasmid pEGFP-N1-IGF-1 can be successfully transfected and expressed IGF-1 protein in osteoporosis rats and formed a definite therapeutic effect, which can be a substantial foundation for further application of IGF-1 gene therapy to osteoporosis.
At the same time, it is can be predicted that osteoporosis and ischemic necrosis of the local organization led by diabetes would become the indication for this gene transfer method.
胰岛素样生长因子-1(IGF-1)是有效地促进骨合成代谢和生长的重要内分泌生长因子,其水平的下降被证实是骨质疏松重要的发病机制之一,目前基因治疗骨质疏松等骨代谢疾病正在成为研究的发展方向,本实验使用质粒重组体pEGFP-N1-IGF-1在体转染骨质疏松大鼠,研究提高其胰岛素样生长因子-1水平的可行性及对骨质疏松大鼠骨质量的影响。
目的
通过本实验证实质粒重组体pEGFP-N1-IGF-1在体转染骨质疏松大鼠可以获得胰岛素样生长因子-1的增高表达并对大鼠的骨质疏松症形成有效的控制,为应用该重组质粒治疗骨质疏松症做理论准备。
方法
雌性SD大鼠30只,随机分为3组：假手术组、pEGFP-N1组和pEGFP-N1-IGF-1组,各10只。假手术组予以开腹后切除少量脂肪组织,pEGFP-N1组和pEGFP-N1-IGF-1组开腹后予双侧卵巢切除。术后12周造模成功,依次给予盐水、pEGFP-N1载体以及质粒重组体pEGFP-N1-IGF-1分别复合脂质体后经尾静脉流体力学注射。注射后定期行全身荧光活体成像及肝脏切片观察转染及表达效果,并定期测定血清胰岛素样生长因子-1浓度至活体实验结束。活体测定大鼠腰4椎体的骨密度值后,处死,取大鼠L4椎体进行椎体压缩实验测定一系列骨生物力学参数,评价该质粒转染对骨质疏松大鼠骨质量的影响。
结果
1.转染后pEGFP-N1组和pEGFP-N1-IGF-1组大鼠均可见全身大部出现明显荧光表达,以肝脏及尾部为著,肝脏切片镜下观察可见明显荧光表达。
2.转染后3周内pEGFP-N1-IGF-1转染大鼠血清胰岛素样生长因子-1表达水平显著升高,6周内显著优于pEGFP-N1转染大鼠(P<0.05)。pEGFP-N1-IGF-1组骨密度、腰椎椎体骨生物力学参数均显著优于pEGFP-N1组(P<0.05),与假手术组差异无统计学意义(P>0.05)。
结论
质粒重组体pEGFP-N1-IGF-1能够在骨质疏松大鼠体内成功转染并表达胰岛素样生长因子-1蛋白,对大鼠骨质疏松症形成治疗作用,为应用胰岛素样生长因子-1基因治疗骨质疏松症奠定了坚实的理论基础。
同时,根据IGF-1降低血糖以及促进微血管生长的特性,可以预见,糖尿病骨质疏松以及糖尿病导致的局部组织缺血坏死也将成为这一基因转染方法的适应症,为其治疗提供一种新思路。
Backgroud
As an important growth factor of promote synthesis and growth metabolism of bone, the decline of Serum IGF-1 levels has been proven to be an important reason leading to osteoporosis.Gene therapy for osteoporosis and other bone metabolic diseases has becoming a direction of the research, therefore, we used the recom-binant plasmid pEGFP-N1-IGF-1 in vivo transfection of osteoporosis rats to study the feasibility of improving their serum IGF-1 levels as well as bone quality in this experiment.
Objective
To study the expression as well as therapeutic effect of recombinant plasmid pEGFP-N1-IGF-1 in the osteoporosis rats in vivo, so as to make theoretical preparation for the treatment of osteoporosis using this recombinant plasmid.
Methods
In this study, a total of 30 normal female SD rats were divided randomly into three even groups.The rats in sham group was unovariectomized while in group pEGFP-N1 and pEGFP-N1-IGF-1 was ovariectomized. After 12 weeks, the three groups were given saline, pEGFP-N1 vector and recombinant plasmid pEGFP-N1-IGF-1 complex liposomes hydrodynamic injection via the tail vein in proper order. Fluorescence in vivo imaging and liver biopsy observations, serum concentrations of IGF-1 were determined at regular intervals. Also, bone mineral density and bone biomechanical parameters were measured.
Results:
1.After transfection, rats of group pEGFP-N1 and pEGFP-N1-IGF-1 could be seen noticeable fluorescence expression not only in the whole body but also liver biopsy, which was significantly different compared with the sham group.
2. The expression level of IGF-1, lumbar bone mineral density and bone biomecha than nical parameters of group pEGFP-N1-IGF-1 were significantly better that of group pEGFP-N1 (P＜0.05), among them, the data of latter two index has no significant statistical difference with sham group (P＞0.05).
Conclusions
Recombinant plasmid pEGFP-N1-IGF-1 can be successfully transfected and expressed IGF-1 protein in osteoporosis rats and formed a definite therapeutic effect, which can be a substantial foundation for further application of IGF-1 gene therapy to osteoporosis.
At the same time, it is can be predicted that osteoporosis and ischemic necrosis of the local organization led by diabetes would become the indication for this gene transfer method.
引文
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[2]QING Yong,DONG Bi-rong.Evidence-based treatment of osteoporosis in post-menopausal women[J].West China Medical Journal 2006,21(3):431-432.
[3]Yakar S,Rosen CJ,Beamer WG,et al. Circulating levels of IGF-1 directly regulate bone growth and density[J].J Clin Invest,2002,110(6):771-781.
[4]Sanjay Kumar, Selvarangan Ponnazhagan. Gene Therapy for Osteoinduction[J]. Current Gene Therapy,2004,4:287-296.
[5]Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins[J]. Growth Factors,2004,22(4):233-241.
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