中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2015年
3期
216-220
,共5页
多壁碳纳米管%聚乳酸-羟基乙酸共聚物%成骨细胞
多壁碳納米管%聚乳痠-羥基乙痠共聚物%成骨細胞
다벽탄납미관%취유산-간기을산공취물%성골세포
Multi-walled carbon nanotubes%Poly lactic-co-glycolic acid copolymer%Osteoblast
目的 研究鼠成骨细胞MC3T3-E1与制备的聚乳酸-羟基乙酸共聚物(PLGA)与多壁碳纳米管(MWCNT)的复合膜共培养时反应的机制.方法 使用溶剂浇铸法制备PLGA-MWCNT复合膜.把MC3T3-E1细胞在PLGA-MWCNT复合膜、PLGA膜及聚苯乙烯组织培养板(TCPS)上培养72 h,通过RT-PCR观察成骨标志基因碱性磷酸酶(ALP)、骨涎蛋白(BSP)、骨钙素(OCN)表达的变化.Western免疫印迹和免疫荧光分析观察各组细胞活性、黏着斑激酶(pFAK)的表达.应用Western免疫印迹检测各组MC3T3-E1细胞p-ERK1/2、p-JNK、p-p38促分裂原活化蛋白激酶(MAPK)蛋白的表达.各组添加ERK1/2抑制剂PD98059 (30 mmol/L),培养72 h后进行MTT、Live/Dead染色和Texas Red-labeledphalloidin染色,使用激光共聚焦显微镜观察细胞活性和肌动蛋白纤维产生的变化.结果 与PLGA组和TCPS组比较,PLGA-MWCNT组细胞的ALP、BSP、OCN的表达水平明显更高(均P＜0.05),而PLGA和TCPS组3种成骨标志基因表达差异没有统计学意义(均P＞0.05).PLGA-MWCNT组pFAK染色更加均匀、表达水平最高,MC3T3-E1细胞的pFAK表达水平PLGA-MWCNT组＞PLGA组＞TCPS组(2.3±0.3比1.4±0.2比1±0.2,均P＜0.05).PLGA-MWCNT组的MC3T3-E1细胞的p-ERK1/2的条带灰度值更高,表达水平要明显高于PLGA组及TCPS组(3.3±0.2比1.3±0.2、1±0.2,均P＜0.05),而各组p-p38 MAPK及p-JNK的条带灰度值差异则无统计学意义(均P＞0.05).各组细胞加入ERK1/2抑制剂PD98059培养72 h后,3组细胞活性和肌动蛋白纤维产生均减少(均P＜0.05),与其余两组相比,PLGA-MWCNT组的细胞活性下降更明显,肌动蛋白纤维产生减少更显著.PLGA-MWCNT复合膜的live/dead染色图像上出现较多的红染细胞,其细胞活性比未加前明显下降(0.33±0.02比0.57±0.03,P＜0.05).结论 PLGA/MWCNT复合膜能够促进MC3T3-E1细胞成骨基因的表达、FAK及ERK1/2激活,从而促进MC3T3-E1细胞分化、肌动蛋白产生.
目的 研究鼠成骨細胞MC3T3-E1與製備的聚乳痠-羥基乙痠共聚物(PLGA)與多壁碳納米管(MWCNT)的複閤膜共培養時反應的機製.方法 使用溶劑澆鑄法製備PLGA-MWCNT複閤膜.把MC3T3-E1細胞在PLGA-MWCNT複閤膜、PLGA膜及聚苯乙烯組織培養闆(TCPS)上培養72 h,通過RT-PCR觀察成骨標誌基因堿性燐痠酶(ALP)、骨涎蛋白(BSP)、骨鈣素(OCN)錶達的變化.Western免疫印跡和免疫熒光分析觀察各組細胞活性、黏著斑激酶(pFAK)的錶達.應用Western免疫印跡檢測各組MC3T3-E1細胞p-ERK1/2、p-JNK、p-p38促分裂原活化蛋白激酶(MAPK)蛋白的錶達.各組添加ERK1/2抑製劑PD98059 (30 mmol/L),培養72 h後進行MTT、Live/Dead染色和Texas Red-labeledphalloidin染色,使用激光共聚焦顯微鏡觀察細胞活性和肌動蛋白纖維產生的變化.結果 與PLGA組和TCPS組比較,PLGA-MWCNT組細胞的ALP、BSP、OCN的錶達水平明顯更高(均P＜0.05),而PLGA和TCPS組3種成骨標誌基因錶達差異沒有統計學意義(均P＞0.05).PLGA-MWCNT組pFAK染色更加均勻、錶達水平最高,MC3T3-E1細胞的pFAK錶達水平PLGA-MWCNT組＞PLGA組＞TCPS組(2.3±0.3比1.4±0.2比1±0.2,均P＜0.05).PLGA-MWCNT組的MC3T3-E1細胞的p-ERK1/2的條帶灰度值更高,錶達水平要明顯高于PLGA組及TCPS組(3.3±0.2比1.3±0.2、1±0.2,均P＜0.05),而各組p-p38 MAPK及p-JNK的條帶灰度值差異則無統計學意義(均P＞0.05).各組細胞加入ERK1/2抑製劑PD98059培養72 h後,3組細胞活性和肌動蛋白纖維產生均減少(均P＜0.05),與其餘兩組相比,PLGA-MWCNT組的細胞活性下降更明顯,肌動蛋白纖維產生減少更顯著.PLGA-MWCNT複閤膜的live/dead染色圖像上齣現較多的紅染細胞,其細胞活性比未加前明顯下降(0.33±0.02比0.57±0.03,P＜0.05).結論 PLGA/MWCNT複閤膜能夠促進MC3T3-E1細胞成骨基因的錶達、FAK及ERK1/2激活,從而促進MC3T3-E1細胞分化、肌動蛋白產生.
목적 연구서성골세포MC3T3-E1여제비적취유산-간기을산공취물(PLGA)여다벽탄납미관(MWCNT)적복합막공배양시반응적궤제.방법 사용용제요주법제비PLGA-MWCNT복합막.파MC3T3-E1세포재PLGA-MWCNT복합막、PLGA막급취분을희조직배양판(TCPS)상배양72 h,통과RT-PCR관찰성골표지기인감성린산매(ALP)、골연단백(BSP)、골개소(OCN)표체적변화.Western면역인적화면역형광분석관찰각조세포활성、점착반격매(pFAK)적표체.응용Western면역인적검측각조MC3T3-E1세포p-ERK1/2、p-JNK、p-p38촉분렬원활화단백격매(MAPK)단백적표체.각조첨가ERK1/2억제제PD98059 (30 mmol/L),배양72 h후진행MTT、Live/Dead염색화Texas Red-labeledphalloidin염색,사용격광공취초현미경관찰세포활성화기동단백섬유산생적변화.결과 여PLGA조화TCPS조비교,PLGA-MWCNT조세포적ALP、BSP、OCN적표체수평명현경고(균P＜0.05),이PLGA화TCPS조3충성골표지기인표체차이몰유통계학의의(균P＞0.05).PLGA-MWCNT조pFAK염색경가균균、표체수평최고,MC3T3-E1세포적pFAK표체수평PLGA-MWCNT조＞PLGA조＞TCPS조(2.3±0.3비1.4±0.2비1±0.2,균P＜0.05).PLGA-MWCNT조적MC3T3-E1세포적p-ERK1/2적조대회도치경고,표체수평요명현고우PLGA조급TCPS조(3.3±0.2비1.3±0.2、1±0.2,균P＜0.05),이각조p-p38 MAPK급p-JNK적조대회도치차이칙무통계학의의(균P＞0.05).각조세포가입ERK1/2억제제PD98059배양72 h후,3조세포활성화기동단백섬유산생균감소(균P＜0.05),여기여량조상비,PLGA-MWCNT조적세포활성하강경명현,기동단백섬유산생감소경현저.PLGA-MWCNT복합막적live/dead염색도상상출현교다적홍염세포,기세포활성비미가전명현하강(0.33±0.02비0.57±0.03,P＜0.05).결론 PLGA/MWCNT복합막능구촉진MC3T3-E1세포성골기인적표체、FAK급ERK1/2격활,종이촉진MC3T3-E1세포분화、기동단백산생.
Objective To investigate the mechanism underlying the response of osteoblast cell line MC3T3-E1 cultured on poly-(lactic-co-glycolic acid) and carboxylated multiwall (PLGA-MWCNTs) nanocomposite films.Methods After the MC3T3-E1 cells were cultured on PLGA-MWCNTs,poly-(lacticco-glycolic acid) film (PLGA) and tissue culture polystyrene (TCPS) for 72 h,RT-PCR was performed to determine the expression of bone-specific genes,including the alkaline phosphatase (ALP),bone sialoprotein (BSP) and osteocalcin (OCN).Western blotting,and immunofluorescence analysis was used to examine the cell viability and the expression of focal adhesion kinase (FAK).The expression of p-ERK1/2,pJNK and p-p38 MAPK proteins in each group of MC3T3-E1 cells was determined by Western blotting.Then,the three groups of MC3T3-E1 cells were added with 30 mmol/L PD98059 (a specific ERK1/2 inhibitor).After culture for 72 h,stained with MTT,Live/Dead and Texas Red-labeled phalloidin,the cells were observed for cell viability and production of actin fiber under confocal laser scanning microscope.Results The mRNA levels of ALP,BSP,and OCN in MC3T3-E1 cells on PLGA-MWCNTs films were higher compared with those on PLGA or TCPS (all P＜0.05),whereas these levels did not differ significantly between cells on PLGA and on TCPS (P＞0.05).The staining pattern of pFAK for MC3T3-T1 cells cultured on PLGA-MWCNTs films were more evenly distributed when compared with those on PLGA or TCPS,and the expression level of pFAK in cells on PLGA-MWCNTs films (2.3±0.3) was much higher than that in cells on PLGA (1.4±0.2) or TCPS (1.0±0.2) (all P＜0.05).The expression level of p-ERK 1/2 in MC3T3-E1 cells on PLGA-MWCNTs films (3.3±0.2) were nearly two times higher than that in the cells on PLGA (1.3±0.2) or TCPS (1.0±0.2) (all P＜0.05).There was no statistical difference in expression of p-JNK or p-p38 MAPK among groups (all P＞0.05).After culture with the ERK1/2 inhibitor PD98059 for 72 h,the cell viability and actin fiber production were reduced in all groups (all P＜0.05).The MC3T3-T1 cells on PLGA-MWCNTs films showed significantly reduced viability and actin fiber production compared with the cells on PLGA or TCPS.Live/dead staining revealed that the cells on PLGA-MWCNTs films were more readily to be red-stained,and these cells appeared less viable compared with those not added with PD98059 (0.33±0.02 vs 0.57±0.03,P＜0.05).Conclusion PLGA-MWCNTs films can promote cell differentiation and actin fiber formation in MC3T3-E1 cells by inducing the expression of bone-specific genes and activating the FAK and ERK 1/2.
