论文部分内容阅读
目的研究缺氧、缺血诱导培养对骨髓间充质干细胞(BMSC)生长的影响,并通过检测神经导向因子Slit2表达的变化,证实缺氧、缺血诱导可促使其表达增加,为缺血性脑卒中血管再生研究打下基础。方法雄性SD大鼠1只,鼠龄4~6周龄,体质量在120~160 g,清洁级。采用密度梯度离心法,分离大鼠BMSC,用10%胎牛血清达氏修正依氏培养液(DMEM)进行培养,至第3代细胞行流式细胞仪鉴定;取第3代细胞通过缺氧预处理不同时间(12、24、48 h)进行分组,缺氧预处理后进行缺血培养,观察细胞生长状况;采用酶联免疫吸附分析(ELISA)法,检测细胞上清液中Slit2表达水平的变化;应用激光共聚焦显微镜观察缺氧预处理组缺血培养与正常对照组细胞形态及Slit2蛋白表达形态。结果成功分离培养大鼠BMSC,并通过流式细胞仪鉴定成功;缺氧预处理后BMSC对缺血耐受能力增强,缺氧预处理48 h组细胞存活率较高(80%);ELISA结果显示,缺氧预处理24 h组Slit2分泌水平是(422.66±24.42)ng/L,缺氧后缺血培养24 h Slit2分泌水平是(521.10±20.16)ng/L;与相同时间点正常对照组Slit2分泌水平[(279.63±14.91)ng/L,(326.70±14.85)ng/L]相比,差异有统计学意义(P<0.05);激光共聚焦扫描显微镜显示,缺氧预处理组BMSC较正常对照组BMSC细胞形态大,且荧光强度增强。结论当缺氧、缺血诱导后,BMSC分泌Slit2增加,为进一步体内研究打下实验基础。
Objective To investigate the effects of hypoxia and ischemia-induced culture on the growth of bone marrow-derived mesenchymal stem cells (BMSCs). By detecting the expression of Slit2, we found that hypoxia and ischemia induced the expression of Slit2, Stroke revascularization lay the foundation for the study. Methods Male Sprague-Dawley rats, aged 4 to 6 weeks and weighing 120 to 160 g, were clean. BMSCs were isolated by density gradient centrifugation and cultured in 10% fetal bovine serum Dulbecco’s modified Eagle’s medium (DMEM). The passage 3 cells were identified by flow cytometry. The passage 3 cells were exposed to hypoxia The cells were divided into groups at different times (12,24 and 48 h). After hypoxia preconditioning, the cells were cultured in ischemic condition. The cell growth was observed. The expression of Slit2 in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA) The changes of cell morphology and Slit2 protein expression in ischemic preconditioning and hypoxia preconditioning groups were observed by laser scanning confocal microscopy. Results BMSC was successfully isolated and cultured in vitro and identified by flow cytometry. BMSC enhanced the tolerance to ischemia after hypoxic preconditioning, and the cell survival rate was higher at 48 h (80%). The results of ELISA The level of Slit2 secretion was (422.66 ± 24.42) ng / L at 24 h after hypoxic preconditioning and (521.10 ± 20.16) ng / L at 24 h after hypoxic preconditioning, respectively. Compared with the normal control group The level of Slit2 secretion [(279.63 ± 14.91) ng / L, (326.70 ± 14.85) ng / L] was significantly different (P <0.05). Confocal laser scanning microscopy showed that BMSC in hypoxic preconditioning group The normal control group BMSC cell morphology, and enhanced fluorescence intensity. Conclusions When hypoxia and ischemia induce BMSCs to secrete Slit2, it will lay the foundation for further study in vivo.