华西口腔医学杂志
華西口腔醫學雜誌
화서구강의학잡지
WEST CHINA JOURNAL OF STOMATOLOGY
2015年
4期
347-351
,共5页
徐智云%刘鑫%黄兴%杨德琴
徐智雲%劉鑫%黃興%楊德琴
서지운%류흠%황흥%양덕금
细胞间隙连接蛋白43基因%原位杂交%斑马鱼
細胞間隙連接蛋白43基因%原位雜交%斑馬魚
세포간극련접단백43기인%원위잡교%반마어
connexin 43 gene%in situ hybridization%zebra fish
目的:探讨细胞间隙连接蛋白43(cx43)基因在斑马鱼早期胚胎中的表达模式,为进一步研究其在牙齿发育过程中的功能奠定基础。方法提取受精后72?h的斑马鱼胚胎总RNA,逆转录合成cDNA,特异性扩增cx43基因片段,连接到PGEMT载体并判断连接方向,选择相应的RNA聚合酶体外转录,合成地高辛标记的cx43基因反义mRNA探针。运用全时相胚胎原位杂交技术检测斑马鱼胚胎发育早期cx43基因在牙齿发育部位的表达分布,茜素红染色法比较cx43原位杂交表达区域与咽齿解剖部位的关系。结果获得了cx43基因的反义mRNA探针。cx43基因在斑马鱼受精后各个时期咽弓牙齿相应部位均可见到强棕褐色阳性信号。受精后9?d时cx43原位杂交表达部位与咽齿解剖部位基本重叠一致。结论 cx43基因参与牙胚发育和矿化过程,且主要在矿化晚期发挥关键调控作用。
目的:探討細胞間隙連接蛋白43(cx43)基因在斑馬魚早期胚胎中的錶達模式,為進一步研究其在牙齒髮育過程中的功能奠定基礎。方法提取受精後72?h的斑馬魚胚胎總RNA,逆轉錄閤成cDNA,特異性擴增cx43基因片段,連接到PGEMT載體併判斷連接方嚮,選擇相應的RNA聚閤酶體外轉錄,閤成地高辛標記的cx43基因反義mRNA探針。運用全時相胚胎原位雜交技術檢測斑馬魚胚胎髮育早期cx43基因在牙齒髮育部位的錶達分佈,茜素紅染色法比較cx43原位雜交錶達區域與嚥齒解剖部位的關繫。結果穫得瞭cx43基因的反義mRNA探針。cx43基因在斑馬魚受精後各箇時期嚥弓牙齒相應部位均可見到彊棕褐色暘性信號。受精後9?d時cx43原位雜交錶達部位與嚥齒解剖部位基本重疊一緻。結論 cx43基因參與牙胚髮育和礦化過程,且主要在礦化晚期髮揮關鍵調控作用。
목적:탐토세포간극련접단백43(cx43)기인재반마어조기배태중적표체모식,위진일보연구기재아치발육과정중적공능전정기출。방법제취수정후72?h적반마어배태총RNA,역전록합성cDNA,특이성확증cx43기인편단,련접도PGEMT재체병판단련접방향,선택상응적RNA취합매체외전록,합성지고신표기적cx43기인반의mRNA탐침。운용전시상배태원위잡교기술검측반마어배태발육조기cx43기인재아치발육부위적표체분포,천소홍염색법비교cx43원위잡교표체구역여인치해부부위적관계。결과획득료cx43기인적반의mRNA탐침。cx43기인재반마어수정후각개시기인궁아치상응부위균가견도강종갈색양성신호。수정후9?d시cx43원위잡교표체부위여인치해부부위기본중첩일치。결론 cx43기인삼여아배발육화광화과정,차주요재광화만기발휘관건조공작용。
Objective This?study?aims?to?investigate?the?expression?of?connexin?43?(cx43)?gene?during?early?development?in?zebra?fish?and?provide?a?foundation?for?further?research?of?cx43?gene?in?tooth?development.?Methods???Total?RNA?was?extracted?within?72?h?after?fertilization?of?zebra?fish?embryos?and?then?reversed?transcribed?to?generate?the?cDNA?library.?The?specific?fragments?of?the?cx43?gene?were?then?cloned?and?connected?to?the?PGEMT?vector.?After?confirming?the?constructed?plasmid,?the?corresponding?RNA?polymerase?was?chosen,?and?the?digoxin-labeled?anti-sense?mRNA?probe?of?cx43?was?syn-thesized?in vitro.?The?cx43?gene?expression?of?zebra?fish?indifferent?stages?was?carried?out?by?in?situ?hybridization.?The?rela-tionship?of?the?cx43?gene?expression?and?anatomy?of?the?pharyngeal?teeth?were?compared?by?alizarin?red?staining.?Results?The?mRNA?antisense?probe?of?cx43?was?acquired.?The?positive?signal?of?sepia?was?observed?in?the?different?stages?of?zebra?fish?pharyngeal?teeth?after?fertilization.?After?fertilization?for?9?days,?the?expression?site?of?cx43?in?situ?hybridization?was?over-lapped?in?accordance?with?the?anatomical?site?of?the?pharyngeal?teeth.?Conclusion???cx43?gene?participates?in?tooth?develop-ment?and?mineralization?process?and?plays?a?crucial?role?in?later?mineralization.