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
2013年
2期
89-95
,共7页
区彩文%陈国钦%张建武%邓菊%吴智业%陈敏生
區綵文%陳國欽%張建武%鄧菊%吳智業%陳敏生
구채문%진국흠%장건무%산국%오지업%진민생
小分子水凝胶%腺病毒%肝细胞生长因子%骨髓%间质干细胞
小分子水凝膠%腺病毒%肝細胞生長因子%骨髓%間質榦細胞
소분자수응효%선병독%간세포생장인자%골수%간질간세포
Small molecular hydrogel%Adenovirus%Hepatocyte growth factor%Bone marrow%Mesenchymal stem cells
目的 观察小分子水凝胶(SMH)结合腺病毒(Ad)介导的肝细胞生长因子(HGF)基因修饰对大鼠骨髓间充质干细胞(MSC)生物学特性的影响.方法 取第9代MSC株进行实验,分为普通培养下MSC对照组(MSC组)、普通培养下转染Ad-HGF的MSC组(Ad-HGF+MSC组)、在SMH中培养的转染Ad-HGF的MSC组(SMH+Ad-HGF+MSC组)、在SMH中培养的MSC组(SMH+MSC组).将Ad-HGF转染MSC,48 h后流式细胞仪检测转染率.采用实时定量PCR检测MSC组、Ad-HGF+MSC组、SMH+Ad-HGF+MSC组细胞转染48 h后的mRNA水平.采用ELISA法测定Ad-HGF+MSC、MSC、Ad-EGFP+MSC、SMH+Ad-HGF+MSC组细胞上清液中HGF蛋白的含量,持续至转染后23 d.普通显微镜观察比较MSC组、SMH+MSC组、Ad-HGF+MSC组、SMH+Ad-HGF+MSC组细胞在转染继续培养7d后的形态.MTT法检测各组细胞1～7d的生长情况,绘制生长曲线.流式细胞仪检测转染2周后各组细胞表面标志物和细胞周期.各组细胞在5-aza诱导24 h后继续培养3周,免疫荧光化学检测细胞向心肌样细胞分化的能力情况.结果 Ad-HGF转染MSC 48 h后,转染率为74.7％.转染后48 h,MSC组Ct值为14.99;SMH+Ad-HGF+MSC组Ct值为8.38;Ad-HGF+MSC组Ct值为8.51;转染两组均出现明确的HGF基因表达.Ad-HGF+MSC组、SMH+Ad-HGF+MSC两组上清中均有HGF蛋白分泌,Ad-HGF+MSC组转染后48 h含量达最高峰,为121 μg/L,持续至23 d.SMH+Ad-HGF+MSC组HGF含量高峰在第3天,达118μg/L,持续至23 d.MSC组、Ad-HGF+MSC组细胞均呈成纤维细胞样生长,而转染或未转染Ad-HGF的MSC细胞在SMH水凝胶中三维培养时,细胞多为棒状,一些成球形,细胞之间的分支连接紧密,有聚集生长的趋势.Ad-HGF+MSC组与MSC组细胞生长曲线相似,各时间点吸光度A值差异无统计学意义,生长趋势吻合.SMH+MSC组与SMH+Ad-HGF+MSC组细胞生长速度前4d与MSC、Ad-HGF+MSC组比较,吸光度A值差异均无统计学意义;4d后细胞的吸光度A值则较Ad-HGF+MSC组、MSC组细胞明显升高(均P＜0.05).SMH培养的两组细胞生长趋势吻合,各时间点吸光度A值比较差异无统计学意义.2周后SMH+MSC、Ad-HGF+MSC、SMH+Ad-HGF+MSC、MSC组细胞的CD44、CD90、CD49的阳性表达率均较高,而CD45、CD34、CD31的阳性表达率均较低,相同抗原各组之间表达率差异均无统计学意义.4组细胞的G0-G1期、S期、G2-M期的细胞比例差异均无统计学意义.经5-aza诱导MSC 24 h后并继续培养3周,SMH+Ad-HGF+MSC、Ad-HGF+MSC、SMH+MSC组细胞cTnT阳性表达率较MSC组明显增高[(27.07±6.49)％、(21.43±6.75)％、(28.12±7.26)％比(20.09±4.17)％,均P＜0.05],SMH+Ad-HGF+MSC组细胞cTnT阳性表达率较Ad-HGF+MSC、MSC组明显增高,而与SMH+MSC组差异无统计学意义.结论 SMH作为三维培养载体有利于细胞的生长增殖.MSC经Ad-HGF基因修饰后结合SMH培养并没有改变其细胞表面标志物及细胞周期.SMH结合Ad-HGF基因修饰有可能促进5-aza对MSC的诱导分化作用.
目的 觀察小分子水凝膠(SMH)結閤腺病毒(Ad)介導的肝細胞生長因子(HGF)基因脩飾對大鼠骨髓間充質榦細胞(MSC)生物學特性的影響.方法 取第9代MSC株進行實驗,分為普通培養下MSC對照組(MSC組)、普通培養下轉染Ad-HGF的MSC組(Ad-HGF+MSC組)、在SMH中培養的轉染Ad-HGF的MSC組(SMH+Ad-HGF+MSC組)、在SMH中培養的MSC組(SMH+MSC組).將Ad-HGF轉染MSC,48 h後流式細胞儀檢測轉染率.採用實時定量PCR檢測MSC組、Ad-HGF+MSC組、SMH+Ad-HGF+MSC組細胞轉染48 h後的mRNA水平.採用ELISA法測定Ad-HGF+MSC、MSC、Ad-EGFP+MSC、SMH+Ad-HGF+MSC組細胞上清液中HGF蛋白的含量,持續至轉染後23 d.普通顯微鏡觀察比較MSC組、SMH+MSC組、Ad-HGF+MSC組、SMH+Ad-HGF+MSC組細胞在轉染繼續培養7d後的形態.MTT法檢測各組細胞1～7d的生長情況,繪製生長麯線.流式細胞儀檢測轉染2週後各組細胞錶麵標誌物和細胞週期.各組細胞在5-aza誘導24 h後繼續培養3週,免疫熒光化學檢測細胞嚮心肌樣細胞分化的能力情況.結果 Ad-HGF轉染MSC 48 h後,轉染率為74.7％.轉染後48 h,MSC組Ct值為14.99;SMH+Ad-HGF+MSC組Ct值為8.38;Ad-HGF+MSC組Ct值為8.51;轉染兩組均齣現明確的HGF基因錶達.Ad-HGF+MSC組、SMH+Ad-HGF+MSC兩組上清中均有HGF蛋白分泌,Ad-HGF+MSC組轉染後48 h含量達最高峰,為121 μg/L,持續至23 d.SMH+Ad-HGF+MSC組HGF含量高峰在第3天,達118μg/L,持續至23 d.MSC組、Ad-HGF+MSC組細胞均呈成纖維細胞樣生長,而轉染或未轉染Ad-HGF的MSC細胞在SMH水凝膠中三維培養時,細胞多為棒狀,一些成毬形,細胞之間的分支連接緊密,有聚集生長的趨勢.Ad-HGF+MSC組與MSC組細胞生長麯線相似,各時間點吸光度A值差異無統計學意義,生長趨勢吻閤.SMH+MSC組與SMH+Ad-HGF+MSC組細胞生長速度前4d與MSC、Ad-HGF+MSC組比較,吸光度A值差異均無統計學意義;4d後細胞的吸光度A值則較Ad-HGF+MSC組、MSC組細胞明顯升高(均P＜0.05).SMH培養的兩組細胞生長趨勢吻閤,各時間點吸光度A值比較差異無統計學意義.2週後SMH+MSC、Ad-HGF+MSC、SMH+Ad-HGF+MSC、MSC組細胞的CD44、CD90、CD49的暘性錶達率均較高,而CD45、CD34、CD31的暘性錶達率均較低,相同抗原各組之間錶達率差異均無統計學意義.4組細胞的G0-G1期、S期、G2-M期的細胞比例差異均無統計學意義.經5-aza誘導MSC 24 h後併繼續培養3週,SMH+Ad-HGF+MSC、Ad-HGF+MSC、SMH+MSC組細胞cTnT暘性錶達率較MSC組明顯增高[(27.07±6.49)％、(21.43±6.75)％、(28.12±7.26)％比(20.09±4.17)％,均P＜0.05],SMH+Ad-HGF+MSC組細胞cTnT暘性錶達率較Ad-HGF+MSC、MSC組明顯增高,而與SMH+MSC組差異無統計學意義.結論 SMH作為三維培養載體有利于細胞的生長增殖.MSC經Ad-HGF基因脩飾後結閤SMH培養併沒有改變其細胞錶麵標誌物及細胞週期.SMH結閤Ad-HGF基因脩飾有可能促進5-aza對MSC的誘導分化作用.
목적 관찰소분자수응효(SMH)결합선병독(Ad)개도적간세포생장인자(HGF)기인수식대대서골수간충질간세포(MSC)생물학특성적영향.방법 취제9대MSC주진행실험,분위보통배양하MSC대조조(MSC조)、보통배양하전염Ad-HGF적MSC조(Ad-HGF+MSC조)、재SMH중배양적전염Ad-HGF적MSC조(SMH+Ad-HGF+MSC조)、재SMH중배양적MSC조(SMH+MSC조).장Ad-HGF전염MSC,48 h후류식세포의검측전염솔.채용실시정량PCR검측MSC조、Ad-HGF+MSC조、SMH+Ad-HGF+MSC조세포전염48 h후적mRNA수평.채용ELISA법측정Ad-HGF+MSC、MSC、Ad-EGFP+MSC、SMH+Ad-HGF+MSC조세포상청액중HGF단백적함량,지속지전염후23 d.보통현미경관찰비교MSC조、SMH+MSC조、Ad-HGF+MSC조、SMH+Ad-HGF+MSC조세포재전염계속배양7d후적형태.MTT법검측각조세포1～7d적생장정황,회제생장곡선.류식세포의검측전염2주후각조세포표면표지물화세포주기.각조세포재5-aza유도24 h후계속배양3주,면역형광화학검측세포향심기양세포분화적능력정황.결과 Ad-HGF전염MSC 48 h후,전염솔위74.7％.전염후48 h,MSC조Ct치위14.99;SMH+Ad-HGF+MSC조Ct치위8.38;Ad-HGF+MSC조Ct치위8.51;전염량조균출현명학적HGF기인표체.Ad-HGF+MSC조、SMH+Ad-HGF+MSC량조상청중균유HGF단백분비,Ad-HGF+MSC조전염후48 h함량체최고봉,위121 μg/L,지속지23 d.SMH+Ad-HGF+MSC조HGF함량고봉재제3천,체118μg/L,지속지23 d.MSC조、Ad-HGF+MSC조세포균정성섬유세포양생장,이전염혹미전염Ad-HGF적MSC세포재SMH수응효중삼유배양시,세포다위봉상,일사성구형,세포지간적분지련접긴밀,유취집생장적추세.Ad-HGF+MSC조여MSC조세포생장곡선상사,각시간점흡광도A치차이무통계학의의,생장추세문합.SMH+MSC조여SMH+Ad-HGF+MSC조세포생장속도전4d여MSC、Ad-HGF+MSC조비교,흡광도A치차이균무통계학의의;4d후세포적흡광도A치칙교Ad-HGF+MSC조、MSC조세포명현승고(균P＜0.05).SMH배양적량조세포생장추세문합,각시간점흡광도A치비교차이무통계학의의.2주후SMH+MSC、Ad-HGF+MSC、SMH+Ad-HGF+MSC、MSC조세포적CD44、CD90、CD49적양성표체솔균교고,이CD45、CD34、CD31적양성표체솔균교저,상동항원각조지간표체솔차이균무통계학의의.4조세포적G0-G1기、S기、G2-M기적세포비례차이균무통계학의의.경5-aza유도MSC 24 h후병계속배양3주,SMH+Ad-HGF+MSC、Ad-HGF+MSC、SMH+MSC조세포cTnT양성표체솔교MSC조명현증고[(27.07±6.49)％、(21.43±6.75)％、(28.12±7.26)％비(20.09±4.17)％,균P＜0.05],SMH+Ad-HGF+MSC조세포cTnT양성표체솔교Ad-HGF+MSC、MSC조명현증고,이여SMH+MSC조차이무통계학의의.결론 SMH작위삼유배양재체유리우세포적생장증식.MSC경Ad-HGF기인수식후결합SMH배양병몰유개변기세포표면표지물급세포주기.SMH결합Ad-HGF기인수식유가능촉진5-aza대MSC적유도분화작용.
Objective To investigate the effects of adenovirus transfection and small molecular hydrogels (SMHs) mediated HGF on the biological characteristics of bone marrow mesenchymal stem cells (MSCs) in rats.Methods The MSCs of the 9th passage were assigned to be treated with common culture media (group MSC),Ad-HGF transfection followed by culture in common media (group Ad-HGF+ MSC),incubation with SMHs alone (group SMH + MSC),and Ad-HGF transfection followed by incubation with SMHs (group SMH+Ad-HGF+MSC).MSCs were transfected with Ad-HGF and the assessment of transfection rate was carried out at 48 h by using flow cytometry.Expressions of HGF mRNA in groups MSC,Ad-HGF+ MSC and SMH+Ad-HGF+MSC were examined by RT-PCR.The supernatant HGF protein of MSCs in groups MSC,Ad-EGFP+MSC,Ad-HGF+MSC and SMH+Ad-HGF+MSC were detected by ELISA till day 23 following transfection.The phase-contrast microscope was employed to compare the MSCs morphology of groups MSC,Ad-HGF+MSC and SMH+Ad-HGF+MSC.MTT approach was adopted to determine the growth of cells in all groups at days 1 to 7 for depiction of growth plots,and flow cytometry was used to assay the cellular surface markers and cell cycles at week 2 following transfection.After 24-h incubation with 5-aza,MSCs of 4 groups were cultured for 3 weeks,and assessment of the capacity of differentiation to cardiac cells was carried out by immune cell fluorescence assay.Results Incubation of MSCs for 48 h yielded a transfection rate of 74.7％.At 48 h after transfection,Ct value was 14.99 in group MSC,8.38 in group SMH+ Ad-HGF+MSC,8.51 in group Ad-HGF+MSC.There was conclusive HGF gene expression in two transfection groups.Apart from group SMH +Ad-HGF+ MSC,supematant HGF protein was noted in group Ad-HGF+ MSC,in which the level peaked at 48 h (121 μg/L) following transfection and sustained to day 23.The supernatant HGF protein peaked at day 3 (118 μg/L) following transfection and sustained to day 23 in group SMH+ Ad-HGF+ MSC.The cells in groups MSC and Ad-HGF+ MSC grew mimicking fibroblasts,whilst those in groups SMH+ Ad-HGF+ MSC and SMH+MSC appeared mostly rod-or partly spherical-shaped,with tight junction and clustered proliferation.Groups MSC and Ad-HGF+ MSC were characterized by similar growth plots,in which the light absorption at each time point did not differ statistically (all P＞0.05),suggesting a close pattern of cellular growth.These two groups,when compared with groups SMH+MSC and SMH+Ad-HGF+MSC,showed similar cellular growth trends without marked difference in absorption at all time points,and had no significant difference in the absorption at days 1 to 4 (all P＞0.05) but had considerably elevated level thereafter (all P＜0.05).At week 2,groups MSC,SMH+MSC,Ad-HGF+ MSC and SMH+Ad-HGF+MSC all had higher expressions of CD44,CD90 and CD49,and lower expressions of CD45,CD34 and CD31.There were no significant differences of uniform antigens in all the 4 groups.The differences in the distribution of Go-G1,S stage and G2-M were unremarkable among all the 4 groups.At week 3 following incubation of MSC with 5-aza for 24 h,groups SMH+Ad-HGF+MSC,Ad-HGF+MSC and SMH+ MSC had increased cellular cTnT expression compared with group MSC [(27.07±6.49)％,(21.43±6.75)％and (28.12±7.26)％ vs (20.09±4.17)％,all P＜0.05].Group SMH+Ad-HGF+MSC had a higher rate of cTnT expression than groups Ad-HGF+MSC and MSC,but was similar to group SMH+MSC.Conclusions The SMH may act as a three-dimensional culture medium to promote cellular proliferation.This is evidenced by the fact that the addition of SMH to Ad-HGF modified MSCs does not alter the cellular surface markers nor the cell cycles but may facilitate the differentiation of MSCs induced by 5-aza.
