中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2013年
5期
288-293
,共6页
董凯%柳忠豪%张晓洁%许丰伟
董凱%柳忠豪%張曉潔%許豐偉
동개%류충호%장효길%허봉위
成骨细胞%细胞骨架%富血小板纤维蛋白
成骨細胞%細胞骨架%富血小闆纖維蛋白
성골세포%세포골가%부혈소판섬유단백
Osteoblasts%Cytoskeleton%Platelet-rich fibrin
目的 探讨富血小板纤维蛋白提取液(platelet-rich fibrin extract,PRFe)对成骨细胞增殖、分化及细胞骨架的影响,以期为富血小板纤维蛋白在临床的应用提供理论基础.方法 实验组使用含PRFe的α-最低必需培养基(α-minimum essential medium,α-MEM)培养液(10％胎牛血清)培养细胞,对照组使用不含PRFe的α-MEM培养液(10％胎牛血清).甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测培养1、3、5d细胞增殖的A值；以碱性磷酸酶(alkaline phosphatase,ALP)活性检测1、3、5、7d的细胞分化情况；茜素红染色观察14、21 d细胞分泌钙结节的情况；激光共聚焦显微镜观察培养后3、6、9及12 h的细胞骨架形态；荧光实时定量PCR检测核心结合因子α1(core-binding factor α1,Cbfα1)和骨钙蛋白基因分别在培养3、7d的表达.结果 MTT检测结果显示,随培养时间延长,细胞数量逐渐增加,在培养1、3、5d实验组细胞A值(分别为0.336±0.011、0.571±0.039、0.787±0.050)均显著高于对照组(分别为0.300±0.021、0.387±0.040、0.527±0.034) (P ＜0.05).两组ALP活性均随培养时间延长而增加,实验组的增加更为显著,在培养1、3、5、7d实验组A值(分别为0.146 ±0.014、0.199±0.017、0.390±0.020及0.492 ±0.019)均显著大于对照组(分别为0.115±0.014、0.145±0.015、0.190 ±0.015及0.230±0.026)(P＜0.05).茜素红染色结果显示,两组随培养时间延长细胞分泌的钙结节均逐渐增多,培养14、21 d实验组细胞分泌的钙结节积分吸光度值(分别为22.119±3.694、31.528±3.162)均较对照组(分别为8.498±2.041、15.162 ±2.526)显著增加(P＜0.05).在各时间点,实验组细胞骨架均较对照组更加伸展；两组细胞的基因表达量也随时问逐渐增加,实验组细胞Cbfα1和骨钙蛋白基因表达量均显著高于对照组(P＜0.05).结论 PRFe能有效促进成骨细胞的增殖和分化,并对细胞骨架早期的排列和伸展有明显的促进作用.
目的 探討富血小闆纖維蛋白提取液(platelet-rich fibrin extract,PRFe)對成骨細胞增殖、分化及細胞骨架的影響,以期為富血小闆纖維蛋白在臨床的應用提供理論基礎.方法 實驗組使用含PRFe的α-最低必需培養基(α-minimum essential medium,α-MEM)培養液(10％胎牛血清)培養細胞,對照組使用不含PRFe的α-MEM培養液(10％胎牛血清).甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法檢測培養1、3、5d細胞增殖的A值；以堿性燐痠酶(alkaline phosphatase,ALP)活性檢測1、3、5、7d的細胞分化情況；茜素紅染色觀察14、21 d細胞分泌鈣結節的情況；激光共聚焦顯微鏡觀察培養後3、6、9及12 h的細胞骨架形態；熒光實時定量PCR檢測覈心結閤因子α1(core-binding factor α1,Cbfα1)和骨鈣蛋白基因分彆在培養3、7d的錶達.結果 MTT檢測結果顯示,隨培養時間延長,細胞數量逐漸增加,在培養1、3、5d實驗組細胞A值(分彆為0.336±0.011、0.571±0.039、0.787±0.050)均顯著高于對照組(分彆為0.300±0.021、0.387±0.040、0.527±0.034) (P ＜0.05).兩組ALP活性均隨培養時間延長而增加,實驗組的增加更為顯著,在培養1、3、5、7d實驗組A值(分彆為0.146 ±0.014、0.199±0.017、0.390±0.020及0.492 ±0.019)均顯著大于對照組(分彆為0.115±0.014、0.145±0.015、0.190 ±0.015及0.230±0.026)(P＜0.05).茜素紅染色結果顯示,兩組隨培養時間延長細胞分泌的鈣結節均逐漸增多,培養14、21 d實驗組細胞分泌的鈣結節積分吸光度值(分彆為22.119±3.694、31.528±3.162)均較對照組(分彆為8.498±2.041、15.162 ±2.526)顯著增加(P＜0.05).在各時間點,實驗組細胞骨架均較對照組更加伸展；兩組細胞的基因錶達量也隨時問逐漸增加,實驗組細胞Cbfα1和骨鈣蛋白基因錶達量均顯著高于對照組(P＜0.05).結論 PRFe能有效促進成骨細胞的增殖和分化,併對細胞骨架早期的排列和伸展有明顯的促進作用.
목적 탐토부혈소판섬유단백제취액(platelet-rich fibrin extract,PRFe)대성골세포증식、분화급세포골가적영향,이기위부혈소판섬유단백재림상적응용제공이론기출.방법 실험조사용함PRFe적α-최저필수배양기(α-minimum essential medium,α-MEM)배양액(10％태우혈청)배양세포,대조조사용불함PRFe적α-MEM배양액(10％태우혈청).갑기새서기사서(methyl thiazolyl tetrazolium,MTT)법검측배양1、3、5d세포증식적A치；이감성린산매(alkaline phosphatase,ALP)활성검측1、3、5、7d적세포분화정황；천소홍염색관찰14、21 d세포분비개결절적정황；격광공취초현미경관찰배양후3、6、9급12 h적세포골가형태；형광실시정량PCR검측핵심결합인자α1(core-binding factor α1,Cbfα1)화골개단백기인분별재배양3、7d적표체.결과 MTT검측결과현시,수배양시간연장,세포수량축점증가,재배양1、3、5d실험조세포A치(분별위0.336±0.011、0.571±0.039、0.787±0.050)균현저고우대조조(분별위0.300±0.021、0.387±0.040、0.527±0.034) (P ＜0.05).량조ALP활성균수배양시간연장이증가,실험조적증가경위현저,재배양1、3、5、7d실험조A치(분별위0.146 ±0.014、0.199±0.017、0.390±0.020급0.492 ±0.019)균현저대우대조조(분별위0.115±0.014、0.145±0.015、0.190 ±0.015급0.230±0.026)(P＜0.05).천소홍염색결과현시,량조수배양시간연장세포분비적개결절균축점증다,배양14、21 d실험조세포분비적개결절적분흡광도치(분별위22.119±3.694、31.528±3.162)균교대조조(분별위8.498±2.041、15.162 ±2.526)현저증가(P＜0.05).재각시간점,실험조세포골가균교대조조경가신전；량조세포적기인표체량야수시문축점증가,실험조세포Cbfα1화골개단백기인표체량균현저고우대조조(P＜0.05).결론 PRFe능유효촉진성골세포적증식화분화,병대세포골가조기적배렬화신전유명현적촉진작용.
Objective To evaluate the effect of platelet-rich fibrin extract (PRFe) on proliferation and differentiation and F-actin cytoskeleton of osteoblasts.Methods The experimental group used the α-minimum essential medium(α-MEM) containing PRFe(10％ fetal bovine serum),and the control group used the α-MEM (10％ fetal bovine serum).The number of the osteoblasts at 1st,3rd,5th d was detected by methyl thiazolyl tetrazolium(MTT) assay,and the differentiation of osteoblast at lst,3rd,5th,7 th d detected by the activity of alkaline phosphatase(ALP).The alizarin red dye was used to observe the number of calcium nodus at 14th,21st d.The F-actin cytoskeleton was evaluated by confocal laser scanning microscope (CLSM) at 3rd,6th,9th,12th h.The level of osteogenetic biomarkers osteocalcin(OCN) and core-binding factor α1 (Cbfα1) at 3rd,7th d were quantified by real-time PCR.Results A significant increase of absorbance at 1st,3rd,5th d was showed in experimental group (0.336 ± 0.011,0.571 ± 0.039,0.787 ± 0.050) compared to control group (0.300 ± 0.021,0.387 ±0.040,0.527 ±0.034) (P ＜0.05).The absorbance of experimental group at lst,3rd,5th,7th d(0.146 ± 0.014,0.199 ±0.017,0.390 ±0.020,0.492 ±0.019) was significantly higher than that of control group(0.115 ± 0.014,0.145 ± 0.015,0.190 ± 0.015,0.230 ± 0.026) (P ＜ 0.05).The integrated absorbance of the calcium nodus in experimental group at 14th,21st d (22.119 ± 3.694,31.528 ± 3.162) was significantly higher than in control group(8.498 ±2.041,15.162 ±2.526) (P ＜0.05).The Cbfα1 and OCN gene expression in experimental group was higher than in control group (P ＜ 0.05).Conclusions PRFe could enhance the proliferation and differentiation of osteoblasts and promote the spread of F-actin cytoskeleton.
