【作者】 郭建斌；

【导师】 闫福华； 陈江；

【作者基本信息】 福建医科大学 ， 口腔临床医学， 2010， 博士

【摘要】 目的将携带人白细胞介素-10 (human interleukin-10, hIL-10)基因的真核表达质粒经尾静脉高压注射的方法导入大鼠体内,探讨hIL-10基因治疗对去卵巢大鼠骨代谢及种植体骨结合的影响,为hIL-10基因治疗骨代谢性疾病及促进种植体骨结合奠定基础。方法1.扩增、鉴定携带hIL-10基因的质粒EX-A0007-M03,利用脂质体将其转入293T及RAW264.7细胞;荧光显微镜及ELISA检测目的基因的表达,破骨细胞形成及骨吸收实验鉴定hIL-10蛋白的生物学功能。2.以hIL-10为报告基因,通过尾静脉高压注射的方法将hIL-10质粒导入大鼠体内,观察不同注射条件对hIL-10在体内表达的影响,通过冰冻切片、ELISA及免疫组化等方法验证目的基因的表达。3.切除3月龄雌性SD大鼠双侧卵巢建立绝经后骨质疏松症模型,通过体重监测、子宫病理、血清ALP、Ca、P浓度、双能X线、骨组织病理及生物力学检测等方法验证绝经后骨质疏松症模型建立成功。4.在大鼠绝经后骨质疏松症模型建立成功的基础上,每周经尾静脉高压注射导入hIL-10质粒,于2w、4w、8w处死动物,通过体重监测、血清ALP、Ca、P、TRAP、IL-1β浓度、双能X线、骨组织病理及生物力学检测等方法观察hIL-10基因治疗对骨质疏松症大鼠骨代谢的影响。5.大鼠绝经后骨质疏松症模型建立成功后在其胫骨植入纯钛种植体,每周经尾静脉高压注射导入hIL-10质粒,于2w、4w、8w处死动物,通过X线、显微CT、制作带种植体硬组织磨片等方法观察hIL-10基因治疗对骨质疏松症大鼠种植体骨结合的影响。结果1.凝胶电泳和DNA测序鉴定证实EX-A0007-M03上携带的外源基因为hIL-10;EX-A0007-M03脂质体法瞬时转染293T及RAW264.7细胞获得成功,部分细胞发出绿色荧光,ELISA检测表明目的基因获得表达,转染后产生的hIL-10蛋白在体外具有抑制破骨细胞形成和骨吸收的生物学功能。2.冰冻切片、ELISA和免疫组化检测表明经尾静脉高压注射后hIL-10在大鼠体内得到高效的表达。3.去卵巢后12w大鼠体重较SHAM组明显增加,子宫萎缩,骨量丢失明显,血清ALP浓度升高,骨力学性能减退,呈现高转换型骨质疏松状态。4. hIL-10基因治疗可抑制OVX所致的TNF-α和TRAP 5b的升高(P<0.05),但对骨质疏松症大鼠和正常大鼠的骨代谢无明显影响。5. hIL-10基因治疗对骨质疏松症大鼠和正常大鼠的骨结合无显著性差异(P>0.05)。结论1. hIL-10质粒经转染后在体外具有抑制破骨细胞形成及骨吸收的生物学作用。2.尾静脉高压注射法可以使hIL-10基因在大鼠体内简单、安全、有效地得以表达。3. 3月龄雌性SD大鼠切除双侧卵巢后12w可以较好地模拟绝经后骨质疏松症。4. hIL-10基因治疗对骨质疏松症大鼠骨代谢及种植体骨结合无明显影响;IL-10对骨代谢性疾病的作用尚需要进一步的研究。更多还原

【Abstract】 Objective This study aimed to explore the effects of hIL-10 gene therapy on bone metabolism and implant osseointegration in ovariectomized rats by delivering the plasmids (EX-A0007-M03) carried with human interleukin-10 (IL-10) gene into rats via hydrodynamic tail vein injection. Thus it provided evidence for hIL-10 gene therapy on bone metabolism diseases and improvement of osseointegration.Methods 1. After amplification and identification of the plasmids (EX-A0007-M03) carried with human interleukin-10 (IL-10) gene, the plasmids were transfected into 293T and RAW264.7 cells with liposome. Fluorescence microscopy and ELISA were used to detect the expression of target gene.Osteoclast formation and bone absorption experiments were carried out to identify the biological function of hIL-10. 2. Using hIL-10 as a reporter gene, this study transfered the plasmids into rats by hydrodynamic tail vein injection, and observed the effects of different injection conditions on the hIL-10 expression in vivo, which were verificated through the frozen section, ELISA and immunohistochemistry. 3. The ovaries were completely excised bilaterally in 3 months old female SD rats and factors like the body weight, uterine pathology, serum ALP, Ca, P concentration, dual energy X ray, pathology and biomechanics of bone tissue were used to validate that the model of postmenopausal osteoporosis had been successfully established. 4. In the basis of the successful establishment of postmenopausal osteoporosis, the rats were transferred with hIL-10 plasmids by hydrodynamic tail vein injection weekly. In 2, 4, 8 weeks after injection, the animals were sacrificed, with body weight, serum ALP, Ca, P, TRAP, IL-1βconcentration, dual energy X ray, bone pathology and biomechanical testing to detect the effects of hIL-10 gene therapy on bone metabolism. 5. After the successful establishment of postmenopausal osteoporosis, the titanium implants were placed in the tibia and the rats were transferred with hIL-10 plasmids by hydrodynamic tail vein injection weekly. In 2, 4, 8 weeks after injection, the animals were sacrificed, with the effects of hIL-10 gene therapy on the osseointegraton of implants detected by methods like X-ray, micro-CT, bone-grinding slice techniques.Results 1. EX-A0007-M03 was confirmed to carry the foreign gene of hIL-10 by gel electrophoresis and DNA sequencing. EX-A0007-M03 was transiently transfected into 293T and RAW264.7 cells successfully by liposome, with some cells emitting green fluorescence. ELISA detection confirmed that the target gene was expressed; the hIL-10 protein had the biological function to inhibit osteoclast formation and resorption. 2. It was demonstrated from the frozen section, ELISA and immunohistochemistry that hIL-10 were highly expressed in rats after hydrodynamic tail vein injection. 3. In 12 weeks after ovariectomy, the rats developed the features of high turnover osteoporosis with the increased body weight and the serum ALP concentration, atrophy of the uterus, significant loss and diminished mechanical properties of bone. 4. The increase of TNF-αand TRAP 5b induced by OVX could be significantly inhibited after hIL-10 gene therapy (P<0.05), but the hIL-10 gene therapy had no effect on bone metabolism in osteoporosis or normal rats. 5.The hIL-10 gene therapy had no effect on osseointegration in osteoporosis or normal rats (P>0.05).Conclusion 1. hIL-10 plasmids have the biological function to inhibit osteoclast formation and bone resorption after transfection. 2. It is a simple, safe and effective approach to express hIL-10 gene in rats. 3. Female SD rats ovariectomized at the age of 3 months could be an ideal animal model for postmenopausal osteoporosis 12 weeks later. 4. hIL-10 gene therapy had no significant effect on bone metabolism and osseointegration in osteoporosis or normal rats. For a better understanding of IL-10 on metabolic bone disease, further studies are required.更多还原