中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
23期
3627-3632
,共6页
郭辉%张于娟%魏晓光%徐彪%陈永珍
郭輝%張于娟%魏曉光%徐彪%陳永珍
곽휘%장우연%위효광%서표%진영진
干细胞%骨髓干细胞%骨髓间充质干细胞%低氧%缺氧诱导因子1α%血管内皮生长因子%国家自然科学基金
榦細胞%骨髓榦細胞%骨髓間充質榦細胞%低氧%缺氧誘導因子1α%血管內皮生長因子%國傢自然科學基金
간세포%골수간세포%골수간충질간세포%저양%결양유도인자1α%혈관내피생장인자%국가자연과학기금
bone marrow%mesenchymal stem cells%anoxia%hypoxia-inducible factor%alpha subunit%vascular endothelial growth factors
背景：作为种子细胞来源的骨髓间充质干细胞移植后能否在相对缺氧的体内环境下生存，是移植成功与否的重要环节。因此研究体外低氧环境下骨髓间充质干细胞的生长状态，可以为体内细胞移植提供实验依据。目的：观察体外低氧环境对大鼠骨髓间充质干细胞生长及血管内皮生长因子表达的影响。方法：分离培养大鼠骨髓间充质干细胞，光镜观察细胞的形态；取第3代骨髓间充质干细胞，按氧浓度分为两组，分别在常氧条件下(体积分数为21%氧气)和低氧条件下(体积分数为3%氧气)培养72 h。用CCK-8法和流式细胞术检测两组细胞的增殖情况，Western-blot印迹法检测常氧组和低氧组细胞中缺氧诱导因子1α与血管内皮生长因子蛋白的表达。结果与结论：①成功分离和培养了骨髓间充质干细胞，光镜下细胞呈长梭形，形态均一。②CCK-8法结果显示各时间点低氧组的细胞数目均高于常氧组，在36，48 h时活力增加显著(P <0.05)。③流式细胞术结果显示，与常氧组比较，低氧组处于S期的细胞数量增加，且细胞增殖指数也显著增加(P<0.05)。④W estern-blot印迹法结果显示，常氧组细胞缺氧诱导因子1α及血管内皮生长因子蛋白仅有少量的表达，而低氧组两蛋白表达量均呈时间依赖性上调(P<0.05)。以上结果说明低氧可诱导体外培养的大鼠骨髓间充质干细胞的增殖，又上调了缺氧诱导因子1α与血管内皮生长因子蛋白的表达，随着低氧时间的延长呈升高趋势。
揹景：作為種子細胞來源的骨髓間充質榦細胞移植後能否在相對缺氧的體內環境下生存，是移植成功與否的重要環節。因此研究體外低氧環境下骨髓間充質榦細胞的生長狀態，可以為體內細胞移植提供實驗依據。目的：觀察體外低氧環境對大鼠骨髓間充質榦細胞生長及血管內皮生長因子錶達的影響。方法：分離培養大鼠骨髓間充質榦細胞，光鏡觀察細胞的形態；取第3代骨髓間充質榦細胞，按氧濃度分為兩組，分彆在常氧條件下(體積分數為21%氧氣)和低氧條件下(體積分數為3%氧氣)培養72 h。用CCK-8法和流式細胞術檢測兩組細胞的增殖情況，Western-blot印跡法檢測常氧組和低氧組細胞中缺氧誘導因子1α與血管內皮生長因子蛋白的錶達。結果與結論：①成功分離和培養瞭骨髓間充質榦細胞，光鏡下細胞呈長梭形，形態均一。②CCK-8法結果顯示各時間點低氧組的細胞數目均高于常氧組，在36，48 h時活力增加顯著(P <0.05)。③流式細胞術結果顯示，與常氧組比較，低氧組處于S期的細胞數量增加，且細胞增殖指數也顯著增加(P<0.05)。④W estern-blot印跡法結果顯示，常氧組細胞缺氧誘導因子1α及血管內皮生長因子蛋白僅有少量的錶達，而低氧組兩蛋白錶達量均呈時間依賴性上調(P<0.05)。以上結果說明低氧可誘導體外培養的大鼠骨髓間充質榦細胞的增殖，又上調瞭缺氧誘導因子1α與血管內皮生長因子蛋白的錶達，隨著低氧時間的延長呈升高趨勢。
배경：작위충자세포래원적골수간충질간세포이식후능부재상대결양적체내배경하생존，시이식성공여부적중요배절。인차연구체외저양배경하골수간충질간세포적생장상태，가이위체내세포이식제공실험의거。목적：관찰체외저양배경대대서골수간충질간세포생장급혈관내피생장인자표체적영향。방법：분리배양대서골수간충질간세포，광경관찰세포적형태；취제3대골수간충질간세포，안양농도분위량조，분별재상양조건하(체적분수위21%양기)화저양조건하(체적분수위3%양기)배양72 h。용CCK-8법화류식세포술검측량조세포적증식정황，Western-blot인적법검측상양조화저양조세포중결양유도인자1α여혈관내피생장인자단백적표체。결과여결론：①성공분리화배양료골수간충질간세포，광경하세포정장사형，형태균일。②CCK-8법결과현시각시간점저양조적세포수목균고우상양조，재36，48 h시활력증가현저(P <0.05)。③류식세포술결과현시，여상양조비교，저양조처우S기적세포수량증가，차세포증식지수야현저증가(P<0.05)。④W estern-blot인적법결과현시，상양조세포결양유도인자1α급혈관내피생장인자단백부유소량적표체，이저양조량단백표체량균정시간의뢰성상조(P<0.05)。이상결과설명저양가유도체외배양적대서골수간충질간세포적증식，우상조료결양유도인자1α여혈관내피생장인자단백적표체，수착저양시간적연장정승고추세。
BACKGROUND:Whether transplanted bone marrow mesenchymal stem cells under hypoxic conditions can survive is crucial for the successful celltransplantation. Therefore, studies on the growth of bone marrow mesenchymal stem cells under hypoxic conditions in vitro can provide experimental evidence for in vivo celltransplantation. OBJECTIVE:To observe the expression of vascular endothelial growth factor in bone marrow mesenchymal stem cells under hypoxia. METHODS:Rat bone marrow mesenchymal stem cells were obtained and cultured, and observed under light microscopy. Passage 3 cells were cultured under normoxia (21%O2) and hypoxia (3%O2 hours. Then cellcounting kit-8 assay and flow cytometry were employed to detect cellproliferation in the two groups. Western blot assay was adopted to detect the expression of hypoxia-inducible factor-1αand vascular endothelial growth factor in the two groups. RESULTS AND CONCLUSION:(1)Rat bone marrow mesenchymal stem cells were obtained and cultured successful y, which were fusiform cells and had uniform shape under the light microscope. (2)The results of cellcounting kit-8 assay showed that the number of cells in the hypoxic group was higher than that in the normoxic group at each time point, and cellviability increased significantly at hours 36 and 48 (P<0.05). (3)The results of flow cytometry demonstrated that the proportion of cells in S phase and cellproliferation index in the hypoxic group were significantly increased, compared with the normoxic group (P<0.05). (4)Western blot results showed ), respectively, for 72 that there was a smal amount of the expression of hypoxia-inducible factor-1αand vascular endothelial growth factor in the normoxic group, but the expression of these two proteins in the hypoxic group was increased in a time-dependent manner (P<0.05). These findings suggest that hypoxia can induce proliferation of rat bone marrow mesenchymal stem cells cultured in vitro, and also raise hypoxia-inducible factor-1αand vascular endothelial growth factor expression in a time-dependent manner.
