hBMP-7基因修饰的骨髓基质细胞修复骨质疏松大鼠牙周组织缺损的实验研究
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摘要
目的:用人骨形成蛋白-7(human bone morphogenetic protein-7, hBMP-7)基因转染SD大鼠骨髓基质细胞(bone marrow stromal cells,BMSCs),并复合胶原膜BME-10X在体外构建组织工程化复合物,移植修复骨质疏松症SD大鼠牙周组织缺损,探讨hBMP-7基因修饰的BMSCs在促进骨质疏松大鼠牙周组织再生中的作用,为基因治疗与组织工程技术联合在牙周组织再生治疗中的进一步应用奠定基础。
方法:1、利用切除SD大鼠双侧卵巢的方法建立骨质疏松大鼠模型,通过体重检测、骨密度测量、血清生化检测以及标本组织学观察确定绝经后骨质疏松症模型建立成功;2、全骨髓贴壁法培养SD大鼠骨髓基质细胞,观察去卵巢(OVX)大鼠和假手术大鼠(SHAM)BMSCs的增殖情况及碱性磷酸酶(alkaline phosphatase, ALP)表达的差异。体外通过重组腺病毒转染hBMP-7基因,通过荧光倒置相差显微镜观察、流式细胞仪、RealTime-PCR、免疫细胞化学染色、ELISA以及Western Blot检测目的基因的转录和表达。3、体外hBMP-7基因转染后,观察细胞形态变化,MTT法描绘细胞的生长曲线,流式细胞技术检测细胞的周期变化、ALP、及钙化结节等成骨细胞表型。4、将转染hBMP-7基因的BMSCs与胶原膜BME-10X复合,通过荧光显微镜、激光共聚焦扫描显微镜、扫描电镜及组织学进行观察,分析细胞和胶原膜载体的复合情况。5、人工构建OVX大鼠牙周组织缺损模型,将转染hBMP-7基因的BMSCs与未转染的BMSCs接种于胶原膜BME-10X后,植入牙周缺损内。并以空白胶原膜植入缺损部位和缺损处只覆盖e-PTFE膜为对照组,4周后取材作病理切片,H.E染色观察牙周组织再生情况,测量新生牙槽骨面积、新生牙骨质面积以及新生牙周膜宽度,结果进行统计学分析。
结果:1、去卵巢手术8周后对大鼠进行相关检测证实大鼠骨质疏松模型构建成功。2、通过重组腺病毒AD5-hBMP7-EGFP对OVX和SHAM大鼠BMSCs成功进行瞬时转染。腺病毒转染72h后转染效率为达到70%以上,转染的细胞发出较强的绿色荧光。RT-PCR、免疫细胞化学染色、ELISA检测和Western Blot检测表明,hBMP-7在OVX和SHAM大鼠BMSCs中得到了有效表达。3、hBMP-7基因转染后细胞形态略有变化,增殖能力无明显改变,细胞周期无明显变化,ALP活性明显增高,钙化结节略有增大。4、转染细胞复合胶原膜后生长良好,24h后细胞在胶原膜中贴附、伸展,并复层生长。在激光共聚焦显微镜下,可见发出绿色荧光的细胞附着于胶原膜,分层扫描可见各层胶原膜均有不同数量的BMSCs附着。5、动物实验结果发现:各实验组和对照组均可见不同程度的牙周组织再生;各实验组新生牙槽骨面积与对照组相比有明显增加(P<0.05);转染基因组的新生牙槽骨量与未转染基因组和空白BME-10X组相比差异有显著性(P<0.05);而OVX大鼠BMSCs转染组和SHAM大鼠BMSCs转染组组间比较则无显著性差异(P>0.05);OVX大鼠BMSCs组和SHAM大鼠BMSCs组组间比较也无显著性差异(P>0.05);各组新生牙骨质面积之间以及新生牙周膜宽度之间无显著性差异(P>0.05)。
结论:1、通过去除大鼠双侧卵巢可成功构建骨质疏松大鼠模型。2、hBMP-7基因可在骨质疏松大鼠BMSCs中有效表达,并可加速骨质疏松大鼠BMSCs向成骨细胞表型转化。3、hBMP-7基因转染的骨质疏松大鼠BMSCs在胶原膜BME-10X上生长良好,hBMP-7基因转染的BMSCs与胶原膜复合物可促进骨质疏松大鼠牙周组织再生,具有很好的临床应用前景。
Objective:This study is to assess the potential effects of human bone morphogenetic protein-7 (hBMP-7) gene transfected bone marrow stromal cells (BMSCs) in periodontal regeneration of osteoporosis Sprague Dawley rats. hBMP-7 gene was transfected into BMSCs, which was then inplanted in a collagen membrane (BME-10X) and transplanted into periodontal fenestration defects of osteoporosis Sprague Dawley rats.
Methods: 1 Osteoporosis models of the female Sprague Dawley rats were induced by ovariectomy. Eight weeks after the operation, the body weight, the bone mineral density (BMD), histological examination and serum levels of biochemical indices were investigated to confirm the osteoporosis models were successful. 2. BMSCs from osteoporosis rats and the sham control were isolated and cultured in DMEM. The morphology, growth feature and alkaline phosphatase (ALP) of BMSCs were monitored. Then BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus and observed by fluorescence microscope. RT-PCR, immunohistochemical straining,FCM,ELISA, as well as Western Blot were employed to observe the transcription and expression of hBMP-7. 3. The morphology and growth feature of the transfected cells were detected and FCM were used to test the cell cycle. Alkaline phosphatase (ALP) and Von Kossa were tested to determine osteogenic property of osteoblast. 4. The hBMP-7 transfected BMSCs were cultured with BME-10X in vitro. Histological examination was performed with light microscopy and H.E stained. The adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and scanning electron microscope. 5. BMSCs with different treatment were seeded on the collagen membranes. The complex and blank collagen membranes without cells were implanted into the acute periodontal defects in osteoporosis rats, and then the defects were covered with e-PTFE membranes. Defects only covered with e-PTFE membranes were served as empty controls. Histometric measurements with H&E staining were performed after 4 weeks, the formation of new alveolar bone area, cementum area and the width of periodontal ligament were measured and compared.
Results: 1. Eight weeks after ovariectomy, osteoporosis models of the female Sprague Dawley rats were successful according to the criteria. 2. The BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus showed a successful transfection. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection and the transfection efficiency was over 70% by calculating the ratio of luminescent cells. Using RT-PCR, immunocytochemistry, ELISA and Western Blot, it was convinced that effective expression of hBMP-7 in all transfected cells. 3. Compared to the control group, morphological feature of the transfected cells showed little alterations, however, there were no significant differences of cell proliferation and apoptosis rate compared with the controls. ALP activity was increased significantly, as well as the size of mineralization nodule formation in transfected cells. 4. The transfected cells adhered to BME-10X and stretched well after 24h of culture. By tomographic scanning with confocal laser scanning confocal microscope, it was showed that cell and material intergrated together, cell attached to BME-10X with different amount. 5. Animal study showed various degrees of periodontal tissue regeneration in the experimental and control groups. The area of newly formed alveolar bone was significantly larger in all experimental groups compared with the empty control group (P<0.05). The area of new alveolar bone from the transfected BMSCs was significantly higher than that of the non-transfected BMSCs and the control (P<0.05). When the defect healing was compared between the OVX rats and SHAM rats, it was noted that there was no significant difference (P>0.05) in both transfected BMSCs and non-transfected BMSCs.
Conclusion: 1. Osteoporosis models of the female Sprague Dawley rats can be made by ovariectomy. 2. The transfected BMSCs can highly express hBMP-7. The transfection of hBMP-7 gene promoted the differentiation of BMSCs to osteoblasts. BMSCs transfected by hBMP-7 gene may serve as an ideal cell source for periodontal tissue engineering. 3. The transfected cells adhered to BME-10X and stretched well. hBMP-7 gene enhanced periodontal defect healing, which shows a potential in clinical application.
方法:1、利用切除SD大鼠双侧卵巢的方法建立骨质疏松大鼠模型,通过体重检测、骨密度测量、血清生化检测以及标本组织学观察确定绝经后骨质疏松症模型建立成功;2、全骨髓贴壁法培养SD大鼠骨髓基质细胞,观察去卵巢(OVX)大鼠和假手术大鼠(SHAM)BMSCs的增殖情况及碱性磷酸酶(alkaline phosphatase, ALP)表达的差异。体外通过重组腺病毒转染hBMP-7基因,通过荧光倒置相差显微镜观察、流式细胞仪、RealTime-PCR、免疫细胞化学染色、ELISA以及Western Blot检测目的基因的转录和表达。3、体外hBMP-7基因转染后,观察细胞形态变化,MTT法描绘细胞的生长曲线,流式细胞技术检测细胞的周期变化、ALP、及钙化结节等成骨细胞表型。4、将转染hBMP-7基因的BMSCs与胶原膜BME-10X复合,通过荧光显微镜、激光共聚焦扫描显微镜、扫描电镜及组织学进行观察,分析细胞和胶原膜载体的复合情况。5、人工构建OVX大鼠牙周组织缺损模型,将转染hBMP-7基因的BMSCs与未转染的BMSCs接种于胶原膜BME-10X后,植入牙周缺损内。并以空白胶原膜植入缺损部位和缺损处只覆盖e-PTFE膜为对照组,4周后取材作病理切片,H.E染色观察牙周组织再生情况,测量新生牙槽骨面积、新生牙骨质面积以及新生牙周膜宽度,结果进行统计学分析。
结果:1、去卵巢手术8周后对大鼠进行相关检测证实大鼠骨质疏松模型构建成功。2、通过重组腺病毒AD5-hBMP7-EGFP对OVX和SHAM大鼠BMSCs成功进行瞬时转染。腺病毒转染72h后转染效率为达到70%以上,转染的细胞发出较强的绿色荧光。RT-PCR、免疫细胞化学染色、ELISA检测和Western Blot检测表明,hBMP-7在OVX和SHAM大鼠BMSCs中得到了有效表达。3、hBMP-7基因转染后细胞形态略有变化,增殖能力无明显改变,细胞周期无明显变化,ALP活性明显增高,钙化结节略有增大。4、转染细胞复合胶原膜后生长良好,24h后细胞在胶原膜中贴附、伸展,并复层生长。在激光共聚焦显微镜下,可见发出绿色荧光的细胞附着于胶原膜,分层扫描可见各层胶原膜均有不同数量的BMSCs附着。5、动物实验结果发现:各实验组和对照组均可见不同程度的牙周组织再生;各实验组新生牙槽骨面积与对照组相比有明显增加(P<0.05);转染基因组的新生牙槽骨量与未转染基因组和空白BME-10X组相比差异有显著性(P<0.05);而OVX大鼠BMSCs转染组和SHAM大鼠BMSCs转染组组间比较则无显著性差异(P>0.05);OVX大鼠BMSCs组和SHAM大鼠BMSCs组组间比较也无显著性差异(P>0.05);各组新生牙骨质面积之间以及新生牙周膜宽度之间无显著性差异(P>0.05)。
结论:1、通过去除大鼠双侧卵巢可成功构建骨质疏松大鼠模型。2、hBMP-7基因可在骨质疏松大鼠BMSCs中有效表达,并可加速骨质疏松大鼠BMSCs向成骨细胞表型转化。3、hBMP-7基因转染的骨质疏松大鼠BMSCs在胶原膜BME-10X上生长良好,hBMP-7基因转染的BMSCs与胶原膜复合物可促进骨质疏松大鼠牙周组织再生,具有很好的临床应用前景。
Objective:This study is to assess the potential effects of human bone morphogenetic protein-7 (hBMP-7) gene transfected bone marrow stromal cells (BMSCs) in periodontal regeneration of osteoporosis Sprague Dawley rats. hBMP-7 gene was transfected into BMSCs, which was then inplanted in a collagen membrane (BME-10X) and transplanted into periodontal fenestration defects of osteoporosis Sprague Dawley rats.
Methods: 1 Osteoporosis models of the female Sprague Dawley rats were induced by ovariectomy. Eight weeks after the operation, the body weight, the bone mineral density (BMD), histological examination and serum levels of biochemical indices were investigated to confirm the osteoporosis models were successful. 2. BMSCs from osteoporosis rats and the sham control were isolated and cultured in DMEM. The morphology, growth feature and alkaline phosphatase (ALP) of BMSCs were monitored. Then BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus and observed by fluorescence microscope. RT-PCR, immunohistochemical straining,FCM,ELISA, as well as Western Blot were employed to observe the transcription and expression of hBMP-7. 3. The morphology and growth feature of the transfected cells were detected and FCM were used to test the cell cycle. Alkaline phosphatase (ALP) and Von Kossa were tested to determine osteogenic property of osteoblast. 4. The hBMP-7 transfected BMSCs were cultured with BME-10X in vitro. Histological examination was performed with light microscopy and H.E stained. The adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and scanning electron microscope. 5. BMSCs with different treatment were seeded on the collagen membranes. The complex and blank collagen membranes without cells were implanted into the acute periodontal defects in osteoporosis rats, and then the defects were covered with e-PTFE membranes. Defects only covered with e-PTFE membranes were served as empty controls. Histometric measurements with H&E staining were performed after 4 weeks, the formation of new alveolar bone area, cementum area and the width of periodontal ligament were measured and compared.
Results: 1. Eight weeks after ovariectomy, osteoporosis models of the female Sprague Dawley rats were successful according to the criteria. 2. The BMSCs were transfected with recombinant AD5-hBMP7-EGFP virus showed a successful transfection. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection and the transfection efficiency was over 70% by calculating the ratio of luminescent cells. Using RT-PCR, immunocytochemistry, ELISA and Western Blot, it was convinced that effective expression of hBMP-7 in all transfected cells. 3. Compared to the control group, morphological feature of the transfected cells showed little alterations, however, there were no significant differences of cell proliferation and apoptosis rate compared with the controls. ALP activity was increased significantly, as well as the size of mineralization nodule formation in transfected cells. 4. The transfected cells adhered to BME-10X and stretched well after 24h of culture. By tomographic scanning with confocal laser scanning confocal microscope, it was showed that cell and material intergrated together, cell attached to BME-10X with different amount. 5. Animal study showed various degrees of periodontal tissue regeneration in the experimental and control groups. The area of newly formed alveolar bone was significantly larger in all experimental groups compared with the empty control group (P<0.05). The area of new alveolar bone from the transfected BMSCs was significantly higher than that of the non-transfected BMSCs and the control (P<0.05). When the defect healing was compared between the OVX rats and SHAM rats, it was noted that there was no significant difference (P>0.05) in both transfected BMSCs and non-transfected BMSCs.
Conclusion: 1. Osteoporosis models of the female Sprague Dawley rats can be made by ovariectomy. 2. The transfected BMSCs can highly express hBMP-7. The transfection of hBMP-7 gene promoted the differentiation of BMSCs to osteoblasts. BMSCs transfected by hBMP-7 gene may serve as an ideal cell source for periodontal tissue engineering. 3. The transfected cells adhered to BME-10X and stretched well. hBMP-7 gene enhanced periodontal defect healing, which shows a potential in clinical application.
引文
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