DNA증폭 번역(한국어 원본)RT-PCR
24well plate에 HA, BGS-7를 끼워 넣고 시편과 plate에 BMSC를 2x104cells/well되도록 seeding한 후 다음날 분화 media로 갈아주었다. 1주, 3주 후에 각각 easy-blue RNA isolation kit (Intron사)를 이용하여 total RNA를 얻어 Nano drop을 이용하여 total RNA를 정량한 후, RNA 2ug으로 superscript III (Invitrogen사)를 이용하여 cDNA를 합성하였다. cDNA합성 후 ALP, osteopontin, osteocalin, Runx2, GAPDH에 대하여 gene specific PCR을 진행하였다 (Table 1). 모든 PCR products는 1.5% agarose gel에 가시화 되고 gel doc에 의하여 상대적인 gene expression이 정성 분석되었다.
Western blot
24well plate에 HA, BGS-7를 끼워 넣고 시편과 plate에 BMSC를 2x104cells/well되도록 seeding한 후 다음날 분화 media로 갈아주었다. 1주, 3주 후에 각각 proprep protein isolation buffer (Intron사, korea)를 이용하여 total protein를 얻어 Nano drop을 이용하여 protien을 정량 하였다. SDS PAGE를 이용한 전기영동 후 PVDF로 transfer하고 5% skim milk blocking 1h, 1st antibody(osteocalcin, RUNx2) 1h, HRP conjugated-2nd Antibody 1h incubation 후 ECL을 이용하여 film으로 develop 하였다. 각각의 band는 Multi guage program을 이용하여 beta actin으로 normalization한 후 정량 분석하였다
The mean surface roughnesses of three different kinds of materials were not statistically different.
Discussion
Moreover, hydroxycarbonate apatite layer이 fibronectin같은 molecule의 adsorption에 유리하게 작용하고 fibronectin-mediated cell adhesion에 유리하게 작용하기 때문으로 볼 수도 있다. [26-28] 또, 이렇게 bioactive glass-ceramics가 생성한 hydroxycarbonate apatite layer가 BMPs와 다른 growth factor의 adsorption에 favorable 하게 작용함으로써 mesenchymal stem cell의 osteogenic diferentiation에 유리하게 작용하였다고 볼 수 있다 [28, 29]. |
DNA증폭 번역(영어 번역본)RT-PCR
HA, BGS-7 was inserted into the 24 well plates and after the specimen and the plates were seeded with BMSC to make 2x104cells/well, they were changed to differentiation media. After quantifying the total RNA by using the Nano drop from the total RNA obtained by easy-blue RNA isolation kit (Intron Co) after 1 week and 3 weeks respectively, the cDNA was synthesized using the superscript III (Invetrogen Co) with RNA 2ug. After synthesizing the cDNA, the gene specific PCR was performed for the ALP, osteopontin, osteocalin, Runx2, GAPDH (Table 1). All PCR products were visualized by 1.5% of agarose gel and the relative gene expression was comprehensively analyzed through gel doc.
Western blot
HA, BGS-7 was inserted into the 24 well plates and after the specimen and the plates were seeded with BMSC to make 2x104cells/well, they were changed to differentiation media on the next day. After obtaining the total protein using the proprep protein isolation buffer (Intron Co, Korea) after 1 week and 3 weeks respectively, the protein was quantified by using the Nano drop. After electrophoresis using the SDS PAGE, the protein was transferred to PVDF and was developed to film using ECL after going through 5% skim milk blocking 1h, 1st antibody(osteocalcin, RUNx2) 1h, and HRP conjugated-2nd Antibody 1h incubation respectively. Each band was normalized to beta actin using the Multi gauge program, and was analyzed in fixed quantity.
The mean surface roughness of three different kinds of materials were not statistically different.
Discussion
Moreover, it could be because hydroxycarbonate apatite layer applies positive effects on the absorption of molecules such as fibronectin and also to fibronectin-mediated cell adhesion [26-28]. In addition, it can also be regarded that because bioactive glass-ceramics generated hydroxycarbonate apatite layer affected favorably to the absorption of growth factor in contrast to BMPs, it imposed favorable influence on the osteogenic differentiation of mesenchymal stem cell [28, 29]. |
