全反式维甲酸与VEGF联合应用诱导小鼠胚胎成纤维细胞定向成骨分化

目的研究全反式维甲酸(all-trans retinoic acid,ATRA)与VEGF联合应用对于小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)定向成骨分化的影响。方法取NIH孕鼠(孕12~15 d)子宫内的胎鼠,去掉头、心、肝等器官后,采用胰蛋白酶消化贴壁法分离培养MEFs。利用HEK-293细胞采用反复冻融法扩增重组腺病毒红色荧光蛋白(recombinant adenovirus-red fluorescent protein,Ad-RFP)及Ad-VEGF。采用ALP染色和ALP定量检测法检测单独ATRA或VEGF以及ATRA和VEGF联合应用培养MEFs第3、5天ALP活性变化。将第3~4代MEFs分为A、B、C、D 4组,分别加入DMSO+Ad-RFP、ATRA、Ad-VEGF、ATRA+Ad-VEGF。实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测第3、7天成骨相关标志物ALP、Ⅰ型胶原、骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)及成血管相关标志物VEGF、血管生成素1(angiopoietin 1,ANGPT1)、内皮黏蛋白(endomucin,EMCN)mRNA表达;免疫组织化学染色检测各组第3、5、7天OPN、VEGF蛋白表达;茜素红染色检测各组成骨诱导14、21 d钙盐沉积水平。取15只4~6周龄无胸腺雌性裸鼠,随机分为3组,每组5只,分别于背侧与腹侧皮下注射经ATRA、Ad-VEGF、ATRA+Ad-VEGF处理后的MEFs。植入后5周行X线片观察、大体观察及组织学染色(Masson、HE及番红O-固绿染色),观察各组裸鼠体内异位成骨情况。结果成功分离培养MEFs;扩增后的Ad-RFP及Ad-VEGF成功转染MEFs,转染效率约为50%和20%。ALP活性检测示,单独使用ATRA或VEGF均能增强MEFs中ALP活性,且ATRA作用较VEGF强;ATRA和VEGF联合使用较单独使用显著增强了MEFs中ALP活性(P<0.05)。qRT-PCR检测示,ATRA联合Ad-VEGF使用上调了早期成骨分化相关标志物ALP、OPN、Ⅰ型胶原mRNA相对表达量(P<0.05);7 d时成血管相关标志物VEGF、EMCN及ANGPT1 mRNA相对表达量有所上升(P<0.05)。免疫组织化学染色示,ATRA联合Ad-VEGF不仅增强了OPN蛋白表达,VEGF蛋白表达在第7天也有所增强。茜素红染色示,单独应用ATRA或Ad-VEGF诱导钙盐沉积作用较弱,二者联合应用明显增强了MEFs成骨晚期钙盐沉积的效应。裸鼠体内植入实验结果显示,X线片观察发现与其余两组相比,ATRA+Ad-VEGF组存在明显骨块,且骨块体积较大,组织学染色可见大量胶原及成熟骨小梁、骨基质形成以及胶原骨组织。结论ATRA与VEGF联合应用能诱导MEFs定向成骨分化。

All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts

Objective To investigate the effect of all-trans retinoic acid(ATRA)and vascular endothelial growth factor(VEGF)on the osteogenic differentiation of mouse embryonic fibroblasts(MEFs).Methods The fetal mice in the uterus of NIH pregnant mice(pregnancy 12-15 days)were collected,and the heads and hearts etc.were removed.Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture.HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein(Ad-RFP)and Ad-VEGF by repeatedly freezing and thawing.Alkaline phosphatase(ALP)staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3 rd and 5 th days.The cultured 3 rd to 4 th generation MEFs were divided into groups A,B,C,and D,and were cultured with DMSO plus Ad-RFP,ATRA,Ad-VEGF,ATRA plus Ad-VEGF,respectively.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the mRNA expressions of osteogenic markers including ALP,collagen typeⅠ,osteopontin(OPN),osteocalcin(OCN),and angiogenic markers including VEGF,angiopoietin 1(ANGPT1),and endomucin(EMCN)on the3 rd and 7 th days.Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3 rd,5 th,and 7 th days in each group.Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction.Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group.Then MEFs treated with ATRA,Ad-VEGF,and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides,respectively.X-ray observation,gross observation,and histological staining(Masson,HE,and Safranin O-fast green stainings)were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group.Results MEFs were successfully isolated and cultured.The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50%and 20%transfection rates.ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs(P<0.05),and ATRA had a stronger effect than Ad-VEGF;and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs(P<0.05).qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP,OPN,and collagen type I(P<0.05);the relative mRNA expressions of angiogenesis-related markers VEGF,EMCN,and ANGPT1 increased at 7 days(P<0.05).Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression,but also increased VEGF protein expression on 7 th day.Alizarin red staining showed that the application of ATRA or AdVEGF induced weak calcium salt deposition,and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs.The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group,and the bone was larger than that in other groups.Histological staining showed a large amount of collagen and mature bone trabeculae,bone matrix formation,and gray-green collagen bone tissue,indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo.Conclusion The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.