CAJ | 학술논문

滑膜间质干细胞（SMSCs）与其他来源的间质干细胞相比，具有更强的生长能力和更大的软骨分化潜力。但由于SMSCs固有地表达I型胶原，这将会大大损害再生软骨的整体质量。为解决此问题，文中首先设计了可抑制I型胶原表达的shRNA，并将其构建到腺病毒载体上。接着通过PacI酶切反应，使重组腺病毒载体线性化并通过脂质体转染293细胞进行病毒包装。收集的病毒初液进行滴度测定后，再次通过293细胞进行病毒扩增，经过纯化后，最终获得高滴度重组腺病毒。将重组腺病毒分别感染成纤维细胞和成骨细胞，通过荧光显微镜观察，检测转染效率，利用实时定量PCR实验验证siRNA的干扰效果。实验结果表明，包装后的重组腺病毒具有高的转染效率，能成功编译siRNA，并具有抑制I型胶原表达的能力。
활막간질간세포（SMSCs）여기타래원적간질간세포상비，구유경강적생장능력화경대적연골분화잠력。단유우SMSCs고유지표체I형효원，저장회대대손해재생연골적정체질량。위해결차문제，문중수선설계료가억제I형효원표체적shRNA，병장기구건도선병독재체상。접착통과PacI매절반응，사중조선병독재체선성화병통과지질체전염293세포진행병독포장。수집적병독초액진행적도측정후，재차통과293세포진행병독확증，경과순화후，최종획득고적도중조선병독。장중조선병독분별감염성섬유세포화성골세포，통과형광현미경관찰，검측전염효솔，이용실시정량PCR실험험증siRNA적간우효과。실험결과표명，포장후적중조선병독구유고적전염효솔，능성공편역siRNA，병구유억제I형효원표체적능력。
As compared with the stem cells from other sources, synovium-derived mesenchymal stem cells (SMSCs)are of higher proliferative and chondrogenic ability.However,SMSCs express Collagen I inherently, which may impair the overall quality of the regenerated counterpart.In order to solve this problem,first,shRNA against Collagen I was designed and constructed in the adenoviral vector.Next,linear recombinant adenoviral vector was generated by the digestion with enzyme PacI and was used to transfect 293 cells for package.Then,the titration of recombinant adenovirus was determined,and the corresponding amplification and purification were performed to obtain recombinant adenovirus with high titration.Moreover,fibroblasts and osteoblasts were respectively infected by the recombinant adenovirus,and fluorescence microscopy was adopted to detect the transfection efficiency.Fi-nally,real-time quantitative PCR experiments were carried out to verify the targeting effect of siRNA.Experimental results show that the packaged recombinant adenoviruses are of high transfection efficiency and can successfully en-code siRNA against Collagen I.