CAJ | 학술논문

目的观察Wnt3a转基因小鼠骨髓间充质干细胞(MSC)对T淋巴细胞增殖的影响.方法从C57BL/6小鼠骨髓中分离培养MSC,用流式细胞术检测细胞表面标志并在不同诱导条件下进行成骨和成脂分化鉴定；采用AdEasy系统将携带Wnt3a基因的重组质粒以脂质体法转染HEK293细胞,收集病毒液感染MSC,以含绿色荧光蛋白的腺病毒(Ad-GFP)作对照,荧光显微镜下观察GFP的表达,Western blot检测MSC中Wnt3a和β-catenin蛋白表达；制备BALB/c小鼠脾淋巴细胞悬液,Wnt3a转基因MSC、Ad-GFP转导MSC分别按1∶100、1∶50及1∶10比例和ConA刺激的脾淋巴细胞共培养,48 h后应用CCK-8法、ELISA法检测T淋巴细胞的相对增殖率和培养上清中细胞因子含量变化.结果成功获得MSC,细胞表型检测为CD44+、CD90.2+、CD34-、CD45-,诱导后可向成骨细胞、脂肪细胞分化；包装的腺病毒滴度达1×1010 pfu/ml,病毒液感染MSC后72 h GFP表达最强,感染效率为50％～60％.Western blot结果显示Wnt.3a转基因MSC中Wnt3a和β-catenin蛋白的表达水平明显增强；MSC对T淋巴细胞增殖的抑制作用呈剂量依赖性,在MSC与脾淋巴细胞比例为1∶10时,Ad-GFP转导MSC组T淋巴细胞增殖率为(55.41±1.75)％,IFN-γ分泌量为(326.70±14.41)pg/ml；Wnt3a转基因MSC组T淋巴细胞增殖率为(37.27±2.66)％,IFN-γ分泌量为(218.80±12.93)pg/ml,但各组MSC对IL-2的分泌无明显影响.结论 Wnt3a转基因MSC较Ad-GFP转导的MSC抑制T淋巴细胞增殖的作用更显著.
목적 관찰Wnt3a전기인소서골수간충질간세포(MSC)대T림파세포증식적영향.방법 종C57BL/6소서골수중분리배양MSC,용류식세포술검측세포표면표지병재불동유도조건하진행성골화성지분화감정；채용AdEasy계통장휴대Wnt3a기인적중조질립이지질체법전염HEK293세포,수집병독액감염MSC,이함록색형광단백적선병독(Ad-GFP)작대조,형광현미경하관찰GFP적표체,Western blot검측MSC중Wnt3a화β-catenin단백표체；제비BALB/c소서비림파세포현액,Wnt3a전기인MSC、Ad-GFP전도MSC분별안1∶100、1∶50급1∶10비례화ConA자격적비림파세포공배양,48 h후응용CCK-8법、ELISA법검측T림파세포적상대증식솔화배양상청중세포인자함량변화.결과성공획득MSC,세포표형검측위CD44+、CD90.2+、CD34-、CD45-,유도후가향성골세포、지방세포분화；포장적선병독적도체1×1010 pfu/ml,병독액감염MSC후72 h GFP표체최강,감염효솔위50％～60％.Western blot결과현시Wnt.3a전기인MSC중Wnt3a화β-catenin단백적표체수평명현증강；MSC대T림파세포증식적억제작용정제량의뢰성,재MSC여비림파세포비례위1∶10시,Ad-GFP전도MSC조T림파세포증식솔위(55.41±1.75)％,IFN-γ분비량위(326.70±14.41)pg/ml；Wnt3a전기인MSC조T림파세포증식솔위(37.27±2.66)％,IFN-γ분비량위(218.80±12.93)pg/ml,단각조MSC대IL-2적분비무명현영향.결론 Wnt3a전기인MSC교Ad-GFP전도적MSC억제T림파세포증식적작용경현저.
Objective To observe the effect of Wnt3a-transduced mouse bone marrow mesenchymal stem cells(MSC)on the proliferation of T lymphocytes.Methods MSC were isolated from C57BL/6 mouse bone marrow and expanded in vitro,then identified by flow cytometry and their differentiation capacity into osteocytes and adipocytes were determined.Recombinant plasmids containing Wnt3a gene,were transfected with lipofectamine into HEK293 cells by the AdEasy system.Viral particles were collected to infect MSC and adenovirus vector expressing GFP(Ad-GFP)was used as control.The expression of GFP in MSC was observed using fluorescence microscopy and the protein levels of Wnt3a and β-catenin were determined by Western blot.Wnt3a-transduced and Ad-GFP transduced MSC were separately cocultured with spleen lymphocytes stimulated by ConA,at the ratio of 1 ∶ 100,1 ∶ 50 or 1 ∶ 10 respectively.The proliferation rate of T lymphocytes was estimated by Cell Cout Kit-8(CCK-8)and the level of cytokine by ELISA.Results FCM analysis showed that the MSC were highly positive for CD90.2,CD44 and negative for CD34,CD45,they could differentiate into osteoblasts and adipocytes after induction ; The titer of recombinant adenoviruses was up to 1 ×1010 pfu/ml.After infected with the adenoviruses,MSC had the strongest GFP expression at 72 h and the efficiency of infection was 50％-60％.The expressions of Wnt3a and β-catenin protein in the Wnt3a-transduced MSC were significantly increased.MSC could suppress the proliferation of T lymphocytes in a dose-dependent manner.When MSC cocultured with spleen lymphocytes at 1∶10 ratio,T lymphocyte proliferation rate and the level of IFN-γwere(55.41 ± 1.75)％ and(326.70 ± 14.41)pg/ml respectively in Ad-GFP transduced MSC group,while in Wnt3a-transduced MSC group,they were(37.27 ± 2.66)％ and (218.80 ± 12.93)pg/ml respectively.There was no effect on the production of IL-2.Conclusion Compared to Ad-GFP transduced MSC,Wnt3a-transduced MSC exhibit a more potent inhibitory effect on the proliferation of T lymphocytes.