Experimental study of heterotopic bone formation by in vivo and in vitro gene transfer of bone morphogenetic protein(BMP) and sonic hedgehog(SHH) in the maxillofacial resions.

颌面部骨形态发生蛋白（BMP）和音刺猬（SHH）体内外基因转移异位骨形成的实验研究。

• 批准号: 13470430
• 负责人: UEDA Minoru
• 金额: $ 6.14万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470430/
• 关键词: BMP(Bone Morphoaenetic Protein); SHH(Sonic hedgehog); Bone; Cartilage; plasmid vector; muscle flap; 骨形成; 骨軟骨形成; アデノウィルスベクター

cDNA encoding the NH2-terminal domain of human and mouse sonic hedgehog(Shh) was amplified by PCR from the full length human and mouse Shh cDNA. It was inserted into the multiple cloning site of the pCR3.1(+) expression vector. The complete coding resion of human and mouse BMP-2 was inserted into the multiple cloning site of the pCR3.1(+) expression vector as well as BMP-4. Human and mouse fibroblasts derived from oral tissue were transfected with human and mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each by calcium phosphate precipitation. Selected storongly positive clones were mouse fibroblast-clones by transfected with mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each. Selected positive clone was grown to confluence in large scale flasks and cells were enclosed in gelatin capsules and implanted into the thigh muscles of nude mice. 14days after implantation, a radiopaque area was observed in cells-implanted area on the soft X-ray films and light microscopic examination disclo … More sed new bone formation by endochondral ossification. with toluidine bulue staining.Vascularized muscle flaps in mice cervical, shoulder and limbs were elevated to examine longterm-viability and the following results were obtained that ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap were useful for gene-transfer and cells-implant experiments.In the A9 cells, C3H10T1/2 cells, KMS-6 cells and fibroblasts derived from mouse oral tissues transfected with mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each, BMP and Shh-positive cells were detected in C3H10T1/2 cells and fibroblasts derived from mouse oral tissues. Though BMP and Shh protein secretion was stable by 12 passages, these protein secretions and gene expressions were not detected soon and therefore, these gene expressions were transient as we, had expected.Storongly positive cells transfected with mouse BMP-2, BMP4, and Shh cDNA plasmid vector each were enclosed in gelatin capsules and implanted into ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap and a radiopaque area was observed in only cells-implanted area on the soft X-ray films and light microscopic examination disclosed new bone formation.Optison solution(200μl) containing mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each were injected into ectopectoralis flap, superficial gluteal muscles flap and biceps femoris muscle flap with ultrasound for 2 minutes(2.5 W/cm2). 14days after injection, a small radiopaque area was observed in only solution-injected area on the soft X-ray films and light microscopic examination disclosed new bone formation. Less

通过PCR从全长人和小鼠Shh cDNA扩增编码人和小鼠音刺猬（Shh）的NH 2-末端结构域的cDNA。将其插入pCR3.1（+）表达载体的多克隆位点。将人和小鼠BMP-2的完整编码区插入pCR3.1（+）表达载体的多克隆位点，以及BMP-4。通过磷酸钙沉淀，用人和小鼠BMP-2、BMP-4和Shh cDNA质粒载体分别转染源自口腔组织的人和小鼠成纤维细胞。筛选出的强阳性克隆为分别用小鼠BMP-2、BMP-4和Shh cDNA质粒载体转染的小鼠成纤维细胞克隆。将选择的阳性克隆在大规模烧瓶中生长至汇合，并将细胞封闭在明胶胶囊中并植入裸鼠的大腿肌肉中。植入14天后，软X线片及光镜检查可见细胞植入区有不透射线区， ...更多信息 通过软骨内骨化形成新骨。将带血管蒂的小鼠颈、肩、四肢肌瓣移植于体外，观察其长期存活情况，结果表明：胸大肌瓣、臀浅肌瓣和股二头肌瓣可用于基因转移和细胞移植实验，A9细胞、C3 H10 T1/2细胞、分别用小鼠BMP-2、BMP-4和Shh cDNA质粒载体转染KMS-6细胞和小鼠口腔组织来源的成纤维细胞，在C3 H10 T1/2细胞和小鼠口腔组织来源的成纤维细胞中检测到BMP和Shh阳性细胞。虽然BMP和Shh蛋白的分泌在12代时是稳定的，但这些蛋白的分泌和基因的表达并没有很快被检测到，因此，这些基因的表达是短暂的，正如我们所预期的。软X线片和光镜检查显示，仅在细胞植入区域观察到不透射线区域，并发现新骨形成。含小鼠BMP-2的Optison溶液（200μl），将BMP-4和Shh cDNA质粒载体分别注入胸大肌皮瓣、臀浅肌皮瓣和股二头肌肌皮瓣，超声（2.5W/cm 2）2 min。注射后14天，软X线片上仅在溶液注射区观察到一个小的不透射线区域，光镜检查显示新骨形成。少

项目成果

Ueda M: "Clinical application of tissue engineering concept andits industrialization"Surgery Frontier. Vol.10(3). 49-54 (2003)

上田男：《组织工程概念的临床应用及其产业化》外科前沿。

Hattori H, Mizutani H, Ueda M: "Sonic Hedgehog"CLINICAL CALCIUM. 12・2. 55-59 (2002)

服部 H、水谷 H、上田 M：“音速刺猬”临床钙 12・2（2002 年）。

上田実: "再生医療の実用化と産業移転"Surgery Frontier. 10・3. 49-54 (2003)

上田稔：“再生医学的实际应用和产业转移”《外科前沿》10・3（2003）。

服部 宇: "Sonic hedgehog"CLINICAL CALCIUM. 12・2. 233-237 (2002)

服部 U：“音速刺猬”临床钙 12・2（2002 年）。

Ueda M: "Maxillofacial bone regeneration using tissue engineering concepts."Dentistry in Japan. 39. 199-207 (2003)

Ueda M：“利用组织工程概念进行颌面骨再生。”日本牙科。