中国实验诊断学
中國實驗診斷學
중국실험진단학
Chinese Journal of Laboratory Diagnosis
2015年
11期
1813-1817
,共5页
张聪%刘洪美%李庆伟%陈国武%梁啸%孟纯阳
張聰%劉洪美%李慶偉%陳國武%樑嘯%孟純暘
장총%류홍미%리경위%진국무%량소%맹순양
骨形态发生蛋白 2%血管内皮生长因子 165,腺病毒载体%细胞增殖
骨形態髮生蛋白 2%血管內皮生長因子 165,腺病毒載體%細胞增殖
골형태발생단백 2%혈관내피생장인자 165,선병독재체%세포증식
Bone morphogenetic protein 2%Vascular endothelial growth factor 165%Adenovirus vectors%Cell prolif-eration
目的：探讨携带骨形态发生蛋白2（bone morphogenetic protein 2，BMP-2）与血管内皮生长因子165（vascu-lar endothelial growth factor 165，VEGF-165）双基因的腺病毒载体转染骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）后对细胞形态特征及增殖能力的影响。方法选取4只新西兰大白兔抽取骨髓液，分离鉴定获得第3代 BMSCs 做为实验对象，实验分成4组：Ad-BMP-2-VEGF-165转染组（A 组）；Ad-BMP-2转染组（B 组）；Ad-VEGF-165转染组（C 组）；BMSCs 对照组（D 组），每组6个复孔，连测3次。首先，将腺病毒载体转染 BMSCs，获得最佳感染复数（multiplicity of infection，MOI）；其次，转染后1-3 d 观察转染各组细胞与 D 组形态和贴壁状况差异变化；再次，ELISA 法检测转染各组目的蛋白表达量与 D 组的差异；最后，MTT 法测定1-7 d 各组细胞增殖曲线，并进行组间比较。结果BMSCs 分离鉴定成功，第3代细胞高表达 CD29（99．82％），CD44（94．14％）抗原。腺病毒转染组最佳 MOI 值分别为 A 组 MOI＝80，B 组 MOI＝80，C 组 MOI＝100。腺病毒转染后各组细胞形态和贴壁特性未见明显改变，边界清晰，细胞呈梭形，细胞核较大呈圆形或椭圆形，生长旺盛。转染组细胞高表达目的蛋白，在第5d 目的蛋白浓度到高峰，A 组 BMP-2浓度为1525．47±58．16 pg／ml，B 组为1506．48±92．65 pg／ml；A 组 VEGF-165浓度为1732．14±126．25 pg／ml，C 组为1733．59±127．07 pg／ml，转染组在不同时间点的目的蛋白表达量与 D 组差异均具有统计学意义（P ＜0．05）。MTT 法显示第1 d、第2 d BMSCs 未见明显增长，第3 d 开始呈指数增长，第5 d 达高峰；第3 d，A 组、B 组吸光度（optical density，OD）值分别为0．113±0．004、0．112±0．003，均高于 C 组、D 组（P ＜0．05）；第4 d， A、B、C 各组 OD 值分别为0．113±0．004、0．110±0．003、0．105±0．001，均高于 D 组0．102±0．004，且差异有统计学意义（P ＜0．05）。结论BMP-2与 VEGF-165对 BMSCs 形态和贴壁性无明显影响，但是均可以促进 BMSCs 增殖，且两者对细胞的增殖无明显叠加作用。
目的：探討攜帶骨形態髮生蛋白2（bone morphogenetic protein 2，BMP-2）與血管內皮生長因子165（vascu-lar endothelial growth factor 165，VEGF-165）雙基因的腺病毒載體轉染骨髓間充質榦細胞（bone marrow mesenchymal stem cells，BMSCs）後對細胞形態特徵及增殖能力的影響。方法選取4隻新西蘭大白兔抽取骨髓液，分離鑒定穫得第3代 BMSCs 做為實驗對象，實驗分成4組：Ad-BMP-2-VEGF-165轉染組（A 組）；Ad-BMP-2轉染組（B 組）；Ad-VEGF-165轉染組（C 組）；BMSCs 對照組（D 組），每組6箇複孔，連測3次。首先，將腺病毒載體轉染 BMSCs，穫得最佳感染複數（multiplicity of infection，MOI）；其次，轉染後1-3 d 觀察轉染各組細胞與 D 組形態和貼壁狀況差異變化；再次，ELISA 法檢測轉染各組目的蛋白錶達量與 D 組的差異；最後，MTT 法測定1-7 d 各組細胞增殖麯線，併進行組間比較。結果BMSCs 分離鑒定成功，第3代細胞高錶達 CD29（99．82％），CD44（94．14％）抗原。腺病毒轉染組最佳 MOI 值分彆為 A 組 MOI＝80，B 組 MOI＝80，C 組 MOI＝100。腺病毒轉染後各組細胞形態和貼壁特性未見明顯改變，邊界清晰，細胞呈梭形，細胞覈較大呈圓形或橢圓形，生長旺盛。轉染組細胞高錶達目的蛋白，在第5d 目的蛋白濃度到高峰，A 組 BMP-2濃度為1525．47±58．16 pg／ml，B 組為1506．48±92．65 pg／ml；A 組 VEGF-165濃度為1732．14±126．25 pg／ml，C 組為1733．59±127．07 pg／ml，轉染組在不同時間點的目的蛋白錶達量與 D 組差異均具有統計學意義（P ＜0．05）。MTT 法顯示第1 d、第2 d BMSCs 未見明顯增長，第3 d 開始呈指數增長，第5 d 達高峰；第3 d，A 組、B 組吸光度（optical density，OD）值分彆為0．113±0．004、0．112±0．003，均高于 C 組、D 組（P ＜0．05）；第4 d， A、B、C 各組 OD 值分彆為0．113±0．004、0．110±0．003、0．105±0．001，均高于 D 組0．102±0．004，且差異有統計學意義（P ＜0．05）。結論BMP-2與 VEGF-165對 BMSCs 形態和貼壁性無明顯影響，但是均可以促進 BMSCs 增殖，且兩者對細胞的增殖無明顯疊加作用。
목적：탐토휴대골형태발생단백2（bone morphogenetic protein 2，BMP-2）여혈관내피생장인자165（vascu-lar endothelial growth factor 165，VEGF-165）쌍기인적선병독재체전염골수간충질간세포（bone marrow mesenchymal stem cells，BMSCs）후대세포형태특정급증식능력적영향。방법선취4지신서란대백토추취골수액，분리감정획득제3대 BMSCs 주위실험대상，실험분성4조：Ad-BMP-2-VEGF-165전염조（A 조）；Ad-BMP-2전염조（B 조）；Ad-VEGF-165전염조（C 조）；BMSCs 대조조（D 조），매조6개복공，련측3차。수선，장선병독재체전염 BMSCs，획득최가감염복수（multiplicity of infection，MOI）；기차，전염후1-3 d 관찰전염각조세포여 D 조형태화첩벽상황차이변화；재차，ELISA 법검측전염각조목적단백표체량여 D 조적차이；최후，MTT 법측정1-7 d 각조세포증식곡선，병진행조간비교。결과BMSCs 분리감정성공，제3대세포고표체 CD29（99．82％），CD44（94．14％）항원。선병독전염조최가 MOI 치분별위 A 조 MOI＝80，B 조 MOI＝80，C 조 MOI＝100。선병독전염후각조세포형태화첩벽특성미견명현개변，변계청석，세포정사형，세포핵교대정원형혹타원형，생장왕성。전염조세포고표체목적단백，재제5d 목적단백농도도고봉，A 조 BMP-2농도위1525．47±58．16 pg／ml，B 조위1506．48±92．65 pg／ml；A 조 VEGF-165농도위1732．14±126．25 pg／ml，C 조위1733．59±127．07 pg／ml，전염조재불동시간점적목적단백표체량여 D 조차이균구유통계학의의（P ＜0．05）。MTT 법현시제1 d、제2 d BMSCs 미견명현증장，제3 d 개시정지수증장，제5 d 체고봉；제3 d，A 조、B 조흡광도（optical density，OD）치분별위0．113±0．004、0．112±0．003，균고우 C 조、D 조（P ＜0．05）；제4 d， A、B、C 각조 OD 치분별위0．113±0．004、0．110±0．003、0．105±0．001，균고우 D 조0．102±0．004，차차이유통계학의의（P ＜0．05）。결론BMP-2여 VEGF-165대 BMSCs 형태화첩벽성무명현영향，단시균가이촉진 BMSCs 증식，차량자대세포적증식무명현첩가작용。
Objective To explore the effect of BMSCs morphology and proliferation transfected by adenovirus vec-tors co-expressing BMP-2 and VEGF-165.Methods Four New Zealand white rabbits were selected to extract marrow, and the third generation of BMSCs was been experimental subjects after separation and identification.The experiment subjects were divided into four groups,i.e.,Ad-BMP-2VEGF-165 transfect group (group A),AdBMP-2 transfect group (group B),Ad-VEGF-165 transfect group (group C),the control group of BMSCs (group D).First,Appling ad-enovirus vector to transfect BMSCs,we observe the multiplicity of infection.Second,the morphology of each groups will be observed when they were transfected at 1-3 days.Third,the ELISA was selected to detect interest proteins expres-sion and the differences in the group D.In the end,each cells proliferation curve determined by MTT method at 1-7 days and compared the difference of OD between groups.Results BMSCs were successfully isolated and identified and the third generation cells high expressed antigen of CD29 (99.82%)and CD44 (94.14%).The good MOI of adenovirus transfection group is group A (MOI = 80),group B (MOI = 80),group C(MOI= 100),respectively.The morpholo-gy and adherence of transfect cells features were not seen obvious change,clear boundary,spindle morphology,larger nucleus,higher proliferation.The transfect groups expressed higher concentration of interest proteins and protein con-centration goes to the peak at the 5 d.The BMP-2 concentration was 1525.47± 58.16 pg/ml (group A),1506.48± 92.65 pg/ml (group B),respectively.The VEGF-165 concentration was 1732.14±126.25 pg/ml (group A),1733.59 ±127.07 pg/ml (group C),respectively.There were statistical difference between transfect groups and D group of in-terest proteins expression at different time (P <0.05).At 4 days,there were significant statistical difference between OD values of groups A (0.113±0.004),B (0.110±0.003)and C (0.105±0.001)and group D (0.102±0.004)(P <0.05).Conclusion The interest proteins of BMP-2 and VEGF-165 have not significantly affected on the morphology and adherence of BMSCs,but they are able to promote BMSCs proliferation and the effect of proliferation on cells does not accumulate in the dual-proteins culture medium.
