CAJ | 학술논문

背景:目前所报道的脐带间充质干细胞体外培养条件及培养效率不尽相同,尚缺乏统一标准.而且由于不同来源的间充质干细胞生物学特征尚有一定差异,因此建立脐带间充质干细胞简便、高效的培养体系十分必要.目的:观察人脐带来源的间充质干细胞在体外不同培养体系中的生长状态,以及不同腺病毒感染的效率.方法:采用胶原酶消化法从正常足月新生儿脐带中分离出间充质干细胞,贴壁法纯化培养,细胞贴壁后利用低糖DMEM,MesenPRO RS~(TM) Medium和STEMPRO~(R) MSC SFM这3种培养体系进行体外扩增.取对数生长期的第3～5脐带间充质干细胞,应用腺病毒Ad5-EGFP,Ad5/11-EGFP,Ad5/35-EGFP分别以感染复数=1,10,100进行感染,分别于感染后24,56,72 h倒置荧光显微镜观察病毒感染及绿色荧光表达情况.结果与结论:使用低糖DMEM培养的细胞初期融合时间长,STEMPRO~(R) MSC SFM培养的细胞虽然连接紧密,但消化传代后不易贴壁,而MesenPRO RS~(TM) Medium培养的细胞在相同时间内能达到较高的细胞密度,更适于脐带间充质干细胞的体外扩增.Ad5/35-EGFP感染脐带间充质干细胞的效率明显高于其他两种腺病毒,但可导致细胞凋亡;腺病毒Ad5/11-EGFP对脐带间充质干细胞的感染效率较佳,随着感染复数的升高,所表达的荧光强度也逐渐增大.
배경:목전소보도적제대간충질간세포체외배양조건급배양효솔불진상동,상결핍통일표준.이차유우불동래원적간충질간세포생물학특정상유일정차이,인차건립제대간충질간세포간편、고효적배양체계십분필요.목적:관찰인제대래원적간충질간세포재체외불동배양체계중적생장상태,이급불동선병독감염적효솔.방법:채용효원매소화법종정상족월신생인제대중분리출간충질간세포,첩벽법순화배양,세포첩벽후이용저당DMEM,MesenPRO RS~(TM) Medium화STEMPRO~(R) MSC SFM저3충배양체계진행체외확증.취대수생장기적제3～5제대간충질간세포,응용선병독Ad5-EGFP,Ad5/11-EGFP,Ad5/35-EGFP분별이감염복수=1,10,100진행감염,분별우감염후24,56,72 h도치형광현미경관찰병독감염급록색형광표체정황.결과여결론:사용저당DMEM배양적세포초기융합시간장,STEMPRO~(R) MSC SFM배양적세포수연련접긴밀,단소화전대후불역첩벽,이MesenPRO RS~(TM) Medium배양적세포재상동시간내능체도교고적세포밀도,경괄우제대간충질간세포적체외확증.Ad5/35-EGFP감염제대간충질간세포적효솔명현고우기타량충선병독,단가도치세포조망;선병독Ad5/11-EGFP대제대간충질간세포적감염효솔교가,수착감염복수적승고,소표체적형광강도야축점증대.
BACKGROUND: In vitro culture condition and culture efficiency are different in reported umbilical cord-derived mesenchymal stem cells, and lacked of unified standards. Different derived mesenchymal stem cells have different biological properties. Therefore, it is very necessary to establish a simple and high-performance culture system for umbilical cord-derived mesenchymal stem cells. OBJECTIVE: To observe the growth state of human umbilical cord-derived mesenchymal stem cells in different culture systems in vitro and adenovirus infection efficiency. METHODS: Mesenchymal stem cells were separated from healthy full-termed delivery fetus using collagenase digestion method and purified by adherent culture. These cells were cultured and amplified in DMEM (low glucose), MesenPRO RS~(TM) Medium and STEMPRO~(R) MSC SFM in vitro. The 3-5 passage mesenchymal stem cells were infected by the Ad5-EGFP, Ad5/11-EGFP, Ad5/35-EGFP as multiplicity of infection (MOI)=1, 10, 100. Viral infection and green fluorescence expression were observed at post-infection 24, 56 and 72 hours using inverted fluorescence microscope. RESULTS AND CONCLUSION: The cell morphology in STEMPR~(R) MSC SFM was different from other two culture system and these cells were not easy to adherent after trypsin digestion. Cell doubling time in the MesenPRO RS~(TM) Medium was shorter than other two groups. Mesenchymal stem cells were infected by Ad5/35-EGFP with higher efficiency than other two kinds of adenovirus, but part of cells appeared apoptosis. The infection efficiency of Ad5/11-EGFP was highest. The fluorescence intensity was gradually increased with increased MOI.