hPDGF-B基因修饰的组织工程化复合物修复牙周组织缺损的实验研究
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摘要
目的用携带人血小板源性生长因子-B (platelet-derived growth factor-B, PDGF-B)基因的真核表达质粒EX-A0380-M03转染Beagle犬牙龈成纤维细胞,复合脱细胞真皮基质(acellular dermal matrix, ADM)体外构建组织工程化复合物,移植修复犬慢性Ⅱ。根分叉病变,探讨hPDGF-B基因修饰的牙龈成纤维细胞在牙周组织再生中的作用,为基因治疗与组织工程联合应用于牙周组织缺损再生治疗奠定基础。
方法1.借助改良组织块法+半消化法分离培养Beagle犬牙龈成纤维细胞,手术、免疫细胞化学以及碱性磷酸酶法确定细胞来源;2.观察DMEM、RPMI1640两种培养液对Beagle犬牙龈成纤维细胞体外生物学活性的影响;3.转化、扩增、鉴定携带hPDGF-B基因的质粒EX-A0380-M03,并利用脂质体将其转入Beagle犬牙龈成纤维细胞;RT-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。4.体外基因转染后,观察细胞形态变化,MTT及流式细胞仪检测细胞增殖和凋亡的情况。Realtime PCR法检测转染细胞I型胶原、XII型胶原以及α-SMA表达的变化。5.组织学及扫描电镜观察细胞与ADM复合情况;将基因修饰的Beagle犬牙龈成纤维细胞与ADM复合,并将复合的组织工程化复合物植入裸鼠皮下,于2、4、8周了解基因修饰的工程化复合物体内再生情况。6.人工构建5条Beagle犬慢性II。根分叉病变模型,将转染目的基因的牙龈成纤维细胞与未转染细胞分别以5×10~6个/mL接种于ADM上,覆盖于根分叉缺损外侧,12周后处死动物,观察牙龈再生的情况。制备颊舌向病理切片,HE染色观察牙周组织再生情况。
结果1.分离培养的细胞为来源于中胚层的成纤维样细胞。2.DMEM和RPMI1640对Beagle犬牙龈成纤维细胞的游出成功率、增殖以及I型胶原的表达无显著性差异。3.酶切和DNA测序鉴定证实EX-A0380-M03上携带的外源基因为hPDGF-B;EX-A0380-M03脂质体法瞬时转染牙龈成纤维细胞获得成功,部分细胞发出绿色荧光,转染效率为18%-38%。RT-PCR、免疫细胞化学染色和ELISA检测表明,hPDGF-B在牙龈成纤维细胞中得到了有效表达。Western Blot证实转染细胞合成的蛋白为PDGF-BB—eGFP融合蛋白。4.目的基因转染后细胞形态部分发生变化,增殖显著增强,凋亡率无明显变化。转染细胞I胶原表达上调,而XII型胶原和α-SMA表达下调。5.ADM与牙龈成纤维细胞复合后,在其表面和内部生长良好。植入裸鼠皮下后,未转染细胞在ADM上形成类结缔组织,而转染细胞则与ADM形成类骨质样结构。6.基因强化的组织工程化复合物治疗后,转染细胞组与未转染细胞组在龈退缩上无显著性差异(P>0.05),但均优与单纯膜组(P<0.05)。转染细胞组颊侧牙周膜、牙骨质及牙槽骨的质量优于未转染组和单纯膜组。
结论1.体外成功分离培养Beagle犬牙龈成纤维细胞;2.DMEM和RPMI1640两种培养液均适合Beagle犬牙龈成纤维细胞的培养;3.hPDGF–B基因可在牙龈成纤维细胞内有效表达;4.hPDGF-B基因转染可提高牙龈成纤维细胞的增殖速率,并通过胶原堆积促进牙周组织的再生;5.ADM生物相容性良好,既可作为支架、也可作为屏障膜用于引导组织再生术;6.hPDGF-B基因强化的组织工程化技术是修复牙周组织缺损的一种较好方法,具有良好的临床应用前景。
Objective To transfect the plasmid(EX-A0380-M03) carried human platelet-derived growth factor-B(hPDGF-B) gene into gingival fibroblasts(GFs). To repair the experimental class II furcation defects of canine by a tissue-engineered compound which was constructed by transfected cells and acellular dermal matrix(ADM). To estimate the potential effects of hPDGF-B gene-modified GFs on periodontal regeneration and attain a new clew based on restoring the periodontal defects.
Methods 1.The GFs of Beagle dog were isolated, cultured and purified by half-digestion and improved tissue block culture. The origination of tissue block was verified by operation and histology. Cells were identified by immunocytochemistry and detection of alkaline phosphatase. 2.The effects of two media, DMEM and RPMI1640, acting upon the GFs, were investigated by MTT, flow cytometry and immunocytochemistry. 3.The plasmid(EX-A0380-M03) was transformed, amplified and identified by a series of molecular biological methods.Subsequently, the plasmid identified correctly was transfected into GFs by liposome-mediated gene transfer method. RT-PCR, immunocytochemistry, ELISA as well as Western Blot were employed to observe the expression of hPDGF. 4.Morphological changes of transfected cells were recorded by optical and fluorescent microscope. Then, MTT and flow cytometry were used to estimate the proliferation and apoptosis of gene-modified cells.The expression of collagen I and XII, as well as theα-SMA was detected by Realtime Quantitative PCR. 5.GFs cultured on the surface of ADM were observed by histology and scanning electron microscope.The gene-modified tissue-engineered compound which was contructed by transfected GFs and ADM was implanted into subcutaneous tissue of nude mice. After 2, 4 or 8 weeks, animals were sacrificed and the regeneration of tissue-engineered compound was evaluated by histology. 6.Thirty experimental class II furcation defects in five Beagle dogs were prepared artificially.The ADM on which was seeded by transfected or non-transfected GFs at initial concentration of 5×106/mL was covered on the buccal side of experimental class II furcation defects.The animals were sacrificed at 12 weeks postoperatively and gingival regeneration was estimated by comparing the position of the gingival margin. Histological section prepared buccolingually was used to evaluate the regeneration of periodontal tissue.
Results 1.The cells isolated was a kind of fibroblast-like cell originating from mesoderm. 2.There was no significant difference between DMEM and RPMI1640 in successful rate of emigration, proliferation and expression of collagen I. 3.Exogenous gene inserted in the plamid was hPDGF-B, which was confirmed by restriction enzyme digestion and DNA sequencing. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection,and the transfection efficiency was 18-38% by calculating the ratio of luminescent cells. RT-PCR, immunocytochemistry and ELISA convinced effective expression of hPDGF-B. Fusion protein(PDGF-BB—eGFP) expressed by transfected cells was detected by Western Blot. 4.Compared to the normal cells, morphological feature of partial transfected cells changed. Proliferation was enhanced after gene interference, but apoptosis has no obvious change.At transcription level, the expression of collagen I was up-regulated, while collagen XII andα-SMA was down-regulated. 5.After seeded on the ADM, GFs exhibited good growth. Under the subcutaneous tissue of nude mice, non-transfected group formed connective-like tissue, while transfected group formed osteoid at last.6.There was on significant difference between the transfected and non-transfected groups in gingival recession (P>0.05) after using the tissue-engineered compound to cure the class II furcation defects.However, the group using simple membrane(ADM and BME-10X) had worse recession(P<0.05) than two groups mentioned above. Buccal periodontal tissue regeneration of transfected group was the best one of the three experimental objects. Conclusion 1.Gingival fibroblasts were isolated and cultured successfully in vitro. 2.The two media, DMEM and RPMI1640 were both fit for the culture of gingival fibroblasts. 3.hPDGF-B was expressed efficiently in gingival fibroblasts. 4.The proliferation was increased after transfection and regeneration might be attained by collagen accumulation. 5. ADM exhibited excellent biocompatibility and was suitable for guided tissue regeneration as a kind of barrier membrane, also as a scaffold. 6.hPDGF-B gene enhanced tissue engineering might have a favorable application prospect in repairing the periodontal defects.
方法1.借助改良组织块法+半消化法分离培养Beagle犬牙龈成纤维细胞,手术、免疫细胞化学以及碱性磷酸酶法确定细胞来源;2.观察DMEM、RPMI1640两种培养液对Beagle犬牙龈成纤维细胞体外生物学活性的影响;3.转化、扩增、鉴定携带hPDGF-B基因的质粒EX-A0380-M03,并利用脂质体将其转入Beagle犬牙龈成纤维细胞;RT-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。4.体外基因转染后,观察细胞形态变化,MTT及流式细胞仪检测细胞增殖和凋亡的情况。Realtime PCR法检测转染细胞I型胶原、XII型胶原以及α-SMA表达的变化。5.组织学及扫描电镜观察细胞与ADM复合情况;将基因修饰的Beagle犬牙龈成纤维细胞与ADM复合,并将复合的组织工程化复合物植入裸鼠皮下,于2、4、8周了解基因修饰的工程化复合物体内再生情况。6.人工构建5条Beagle犬慢性II。根分叉病变模型,将转染目的基因的牙龈成纤维细胞与未转染细胞分别以5×10~6个/mL接种于ADM上,覆盖于根分叉缺损外侧,12周后处死动物,观察牙龈再生的情况。制备颊舌向病理切片,HE染色观察牙周组织再生情况。
结果1.分离培养的细胞为来源于中胚层的成纤维样细胞。2.DMEM和RPMI1640对Beagle犬牙龈成纤维细胞的游出成功率、增殖以及I型胶原的表达无显著性差异。3.酶切和DNA测序鉴定证实EX-A0380-M03上携带的外源基因为hPDGF-B;EX-A0380-M03脂质体法瞬时转染牙龈成纤维细胞获得成功,部分细胞发出绿色荧光,转染效率为18%-38%。RT-PCR、免疫细胞化学染色和ELISA检测表明,hPDGF-B在牙龈成纤维细胞中得到了有效表达。Western Blot证实转染细胞合成的蛋白为PDGF-BB—eGFP融合蛋白。4.目的基因转染后细胞形态部分发生变化,增殖显著增强,凋亡率无明显变化。转染细胞I胶原表达上调,而XII型胶原和α-SMA表达下调。5.ADM与牙龈成纤维细胞复合后,在其表面和内部生长良好。植入裸鼠皮下后,未转染细胞在ADM上形成类结缔组织,而转染细胞则与ADM形成类骨质样结构。6.基因强化的组织工程化复合物治疗后,转染细胞组与未转染细胞组在龈退缩上无显著性差异(P>0.05),但均优与单纯膜组(P<0.05)。转染细胞组颊侧牙周膜、牙骨质及牙槽骨的质量优于未转染组和单纯膜组。
结论1.体外成功分离培养Beagle犬牙龈成纤维细胞;2.DMEM和RPMI1640两种培养液均适合Beagle犬牙龈成纤维细胞的培养;3.hPDGF–B基因可在牙龈成纤维细胞内有效表达;4.hPDGF-B基因转染可提高牙龈成纤维细胞的增殖速率,并通过胶原堆积促进牙周组织的再生;5.ADM生物相容性良好,既可作为支架、也可作为屏障膜用于引导组织再生术;6.hPDGF-B基因强化的组织工程化技术是修复牙周组织缺损的一种较好方法,具有良好的临床应用前景。
Objective To transfect the plasmid(EX-A0380-M03) carried human platelet-derived growth factor-B(hPDGF-B) gene into gingival fibroblasts(GFs). To repair the experimental class II furcation defects of canine by a tissue-engineered compound which was constructed by transfected cells and acellular dermal matrix(ADM). To estimate the potential effects of hPDGF-B gene-modified GFs on periodontal regeneration and attain a new clew based on restoring the periodontal defects.
Methods 1.The GFs of Beagle dog were isolated, cultured and purified by half-digestion and improved tissue block culture. The origination of tissue block was verified by operation and histology. Cells were identified by immunocytochemistry and detection of alkaline phosphatase. 2.The effects of two media, DMEM and RPMI1640, acting upon the GFs, were investigated by MTT, flow cytometry and immunocytochemistry. 3.The plasmid(EX-A0380-M03) was transformed, amplified and identified by a series of molecular biological methods.Subsequently, the plasmid identified correctly was transfected into GFs by liposome-mediated gene transfer method. RT-PCR, immunocytochemistry, ELISA as well as Western Blot were employed to observe the expression of hPDGF. 4.Morphological changes of transfected cells were recorded by optical and fluorescent microscope. Then, MTT and flow cytometry were used to estimate the proliferation and apoptosis of gene-modified cells.The expression of collagen I and XII, as well as theα-SMA was detected by Realtime Quantitative PCR. 5.GFs cultured on the surface of ADM were observed by histology and scanning electron microscope.The gene-modified tissue-engineered compound which was contructed by transfected GFs and ADM was implanted into subcutaneous tissue of nude mice. After 2, 4 or 8 weeks, animals were sacrificed and the regeneration of tissue-engineered compound was evaluated by histology. 6.Thirty experimental class II furcation defects in five Beagle dogs were prepared artificially.The ADM on which was seeded by transfected or non-transfected GFs at initial concentration of 5×106/mL was covered on the buccal side of experimental class II furcation defects.The animals were sacrificed at 12 weeks postoperatively and gingival regeneration was estimated by comparing the position of the gingival margin. Histological section prepared buccolingually was used to evaluate the regeneration of periodontal tissue.
Results 1.The cells isolated was a kind of fibroblast-like cell originating from mesoderm. 2.There was no significant difference between DMEM and RPMI1640 in successful rate of emigration, proliferation and expression of collagen I. 3.Exogenous gene inserted in the plamid was hPDGF-B, which was confirmed by restriction enzyme digestion and DNA sequencing. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection,and the transfection efficiency was 18-38% by calculating the ratio of luminescent cells. RT-PCR, immunocytochemistry and ELISA convinced effective expression of hPDGF-B. Fusion protein(PDGF-BB—eGFP) expressed by transfected cells was detected by Western Blot. 4.Compared to the normal cells, morphological feature of partial transfected cells changed. Proliferation was enhanced after gene interference, but apoptosis has no obvious change.At transcription level, the expression of collagen I was up-regulated, while collagen XII andα-SMA was down-regulated. 5.After seeded on the ADM, GFs exhibited good growth. Under the subcutaneous tissue of nude mice, non-transfected group formed connective-like tissue, while transfected group formed osteoid at last.6.There was on significant difference between the transfected and non-transfected groups in gingival recession (P>0.05) after using the tissue-engineered compound to cure the class II furcation defects.However, the group using simple membrane(ADM and BME-10X) had worse recession(P<0.05) than two groups mentioned above. Buccal periodontal tissue regeneration of transfected group was the best one of the three experimental objects. Conclusion 1.Gingival fibroblasts were isolated and cultured successfully in vitro. 2.The two media, DMEM and RPMI1640 were both fit for the culture of gingival fibroblasts. 3.hPDGF-B was expressed efficiently in gingival fibroblasts. 4.The proliferation was increased after transfection and regeneration might be attained by collagen accumulation. 5. ADM exhibited excellent biocompatibility and was suitable for guided tissue regeneration as a kind of barrier membrane, also as a scaffold. 6.hPDGF-B gene enhanced tissue engineering might have a favorable application prospect in repairing the periodontal defects.
引文
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