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外傷歯の歯髄再生療法の基盤となる歯髄細胞の分化誘導法の確立

牙髓细胞诱导分化方法的建立，为外伤牙牙髓再生治疗奠定基础

基本信息

批准号：
20659296
负责人：
大島勇人
金额：
$ 2.05万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2009
项目状态：
已结题

项目摘要

【目的】歯の損傷後の歯髄治癒過程における象牙芽細胞分化機構ならびに胎生期ラベリング法によりマウス歯髄組織幹細胞をブロモデオキシウリジン(BrdU)によりラベルし、歯髄におけるBrdU label-retaining cells(LRCs)の分化能を解明することを目的に、歯の移植後の顆粒球マクロファージコロニー刺激因子(GM-CSF)およびオステオポンチン(OPN)の反応、LRCsの分化能を免疫細胞化学的に検索した。【方法】妊娠後期ICRマウスに3～4日間BrdUを腹腔内投与し、生後3週後に深麻酔下で上顎第一臼歯抜去後に歯冠部を舌下部へ他家移植した。術後1日～2週後にアルデヒド系固定液で灌流固定し、EDTA脱灰後、パラフィン切片を作製し、抗GM-CSF・抗OPN・抗ネスチン抗体・抗BrdU鼎を用いた免疫染色を行った。なお無処置群の左側臼歯を対照群とした。【結果と考察】対照群歯髄では、歯髄中央部血管周囲にLRCsが局在し、咬頭頂領域を中心に歯髄・象牙質界面に弱いオズテオポンチン陽性反応が見られ、象牙芽細胞はネスチン強陽性を示したが、歯髄内はGM-CSFは陰性であった。術後に歯髄のネスチン免疫陽性反応が消失したが、3～7日後に、GM-CSF陽性細胞、OPN陽性細胞の出現に引き続き、ネスチン陽性象牙芽細胞様細胞が歯髄・象牙質界面に再配列した。14日後には歯髄腔に骨組織形成が惹起されたが、骨芽細胞がOPN陽性反応を示した。GM-CSF陽性反応産物は象牙細管内にも見られ、既存の象牙質と再生象牙質の界面にOPN陽性反応が観察された。さらに、in situ hybridizationによりOPN遺伝子発現を検索ると、免疫反応とほぼ同じ発現パターンを示した。また、LRCsは再生象牙芽細胞にコミットされていた。以上より、歯の移植後の歯髄治癒過程における象牙芽細胞の分化には、GM-CSF発現とOPN発現が重要な役割を果たし、LRCsが象牙芽細胞に分化することが明らかとなった。

[objective] the mechanism of differentiation of ivory germ cells in the process of post-natal treatment can be used to determine the purpose of differentiation of ivory germ cells. [objective] the differentiation mechanism of ivory germ cell differentiation in the postnatal period of postnatal treatment, the differentiation mechanism of ivory germ cell differentiation in fetal period, the method of cell differentiation in fetal phase, the method of cell differentiation in fetal phase, the tissue tissue in the tissue group, the cell cycle (BrdU), the cell cycle (LRCs), the differentiation of ivory bud (LRCs), the differentiation of ivory germ cells in the postnatal period, the differentiation mechanism of ivory bud differentiation, the differentiation mechanism of ivory germ differentiation, the differentiation of fetal ivory germ differentiation, the differentiation of ivory germ differentiation, the differentiation of ivory bud differentiation, the differentiation of ivory bud differentiation, the differentiation of ivory bud After transplantation, the differentiation of LRCs could immunize the cytochemistry of GM-CSF (stimulating factor GM-CSF), anti-granulocyte (OPN). [methods] during the period of 3 ~ 4 days after pregnancy, ICR was injected intraperitoneally with BrdU. After 3 weeks of birth, the first acetabulum was removed under deep anesthesia, and the sublingual part of the crown was transplanted into his home. After 1 ~ 2 days, the fixed liquid was perfused and fixed, after EDTA was removed, the sections were made into sections, and the anti-GM-CSF, anti-OPN, anti-BrdU antibodies were stained with immunostaining. There is no place for the group. The left side of the group is located on the left. [results] according to the results of the investigation, the perivascular LRCs in the central part of the blood vessel was present, the interface of the ivory in the center of the field, the interface of the ivory, the strength of the ivory germ, the strength of the ivory germ, and the GM-CSF of the ivory germ. After 3-7 days, GM-CSF sexual cells, OPN sexual cells, ivory bud cells, ivory germ cells and ivory interface were detected. 14 days later, the formation of bone tissue caused by bone formation and the sexual reaction of OPN in bone bud cells. GM-CSF sexual reflex material, ivory tube, existing ivory, regenerated ivory, interface, OPN, sexual reflection, observation. The symptoms were detected in the children of in situ hybridization and OPN, and the immune response was detected. The ivory buds were regenerated by LRCs and the ivory buds were regenerated. In the process of post-transplant treatment, ivory germ differentiation, GM-CSF OPN, LRCs ivory bud differentiation, and ivory bud differentiation were observed.