CAJ | 학술논문

目的：观察人成骨细胞在过量氟作用下未折叠蛋白反应（UPR）信号通路的差异基因表达，探索在氟中毒条件下成骨细胞内质网应激的作用。方法体外培养人成骨细胞染氟模型，用不同浓度的氟干预24 h后M T S法检测细胞存活率和流式细胞仪检测细胞凋亡百分率，用UPR信号通路PCR Array芯片检测通路中相关基因表达的情况，并用蛋白免疫印迹法（Western blot）检测蛋白表达。结果在氟化钠浓度为0、5、10、20、40、80 mg/L 时，细胞存活率分别为（100．6785±2．8303）％、（105．3934±2．5384）％、（106．1257±2．0483）％、（77．9773±2．5443）％（与对照组比较 P＜0．05）、（30．2377±0．6327）％（与对照组比较 P＜0．05）。流式细胞仪检测，凋亡率分别为5mg/L组4．8％，10mg/L组13．8％，20mg/L组37．0％，40mg/L组58．9％，80 mg/L组63．2％（P＜0．05）。PCR芯片检测发现1个基因表达下调，14个基因表达上调。Western blot结果显示， BIP、ATF4、CHOP、IRE1均呈现出不同程度的随氟剂量增加蛋白表达逐渐上调。XBP1在NaF 5～20 mg/L表达逐渐增强，在40与80 mg/L时表达减弱。结论氟化钠可以通过PERK和IRE1途径引起成骨细胞内质网应激，并且可以引起成骨细胞凋亡。
목적：관찰인성골세포재과량불작용하미절첩단백반응（UPR）신호통로적차이기인표체，탐색재불중독조건하성골세포내질망응격적작용。방법체외배양인성골세포염불모형，용불동농도적불간예24 h후M T S법검측세포존활솔화류식세포의검측세포조망백분솔，용UPR신호통로PCR Array심편검측통로중상관기인표체적정황，병용단백면역인적법（Western blot）검측단백표체。결과재불화납농도위0、5、10、20、40、80 mg/L 시，세포존활솔분별위（100．6785±2．8303）％、（105．3934±2．5384）％、（106．1257±2．0483）％、（77．9773±2．5443）％（여대조조비교 P＜0．05）、（30．2377±0．6327）％（여대조조비교 P＜0．05）。류식세포의검측，조망솔분별위5mg/L조4．8％，10mg/L조13．8％，20mg/L조37．0％，40mg/L조58．9％，80 mg/L조63．2％（P＜0．05）。PCR심편검측발현1개기인표체하조，14개기인표체상조。Western blot결과현시， BIP、ATF4、CHOP、IRE1균정현출불동정도적수불제량증가단백표체축점상조。XBP1재NaF 5～20 mg/L표체축점증강，재40여80 mg/L시표체감약。결론불화납가이통과PERK화IRE1도경인기성골세포내질망응격，병차가이인기성골세포조망。
Objective To observe gene different expression of unfolded protein response signaling pathway in human osteoblasts under the excessive fluoride ,and explore the role of endoplasmic reticulum stress in fluorosis .Methods Human osteoblasts were cultured with fluoride ,intervening for 24 h .Cell viability and apoptosis were inspected by MTS assay and flow cytometer respective‐ly .The UPR signaling pathway was examined by real time PCR array ,and protein expressions were detected by Western blot .Re‐sults T he cell survival rates w ere (100 .678 5 ± 2 .830 3 )% ,(105 .393 4 ± 2 .538 4 )% ,(106 .125 7 ± 2 .048 3 )% ,(77 .977 3 ± 2 .544 3)% (P<0 .05) ,(30 .237 7 ± 0 .632 73)% (P<0 .05) treated with sodium fluoride at the concentration 0 ,5 ,10 ,20 ,40 ,80 mg/L respectively .Apoptosis rate inspected by flow cytometer was 4 .8% in 5 mg/L group ,13 .8% in 10 mg/L group ,37 .0% in 20 mg/L group ,58 .9% in 40 mg/L group ,63 .2% in 80 mg/L group (P<0 .05) .Only 1 gene was down regulated and 14 genes were up regulated .Western blot analysis showed BIP ,ATF4 ,CHOP and IRE1 both showed their protein expression gradually up regula‐ted with fluorine dose .XBP1 expression gradually increased in NaF 5-20 mg/L ,and its expression decreased at 40 and 80 mg/L . Conclusion Sodium fluoride can cause osteoblasts endoplasmic reticulum stress pathway through PTEN and IRE1 pathway ,and at high concentrations can cause apoptosis of osteoblast .