Abstract研究背景： 目前认为缺血性脑血管病（ICVD）有效的治疗方式是在发病急性期采用溶栓或机械取栓等方式，以早期恢复血供。然而，脑组织短时间内便可发生不可逆缺血坏死，其治疗时间窗很短，临床中大多数患者不能及时接受溶栓或血管内机械取栓等治疗。干细胞治疗是通过将具有自我复制能力和多向分化能力的干细胞移植入患者体内，弥补病变区域凋亡和坏死的细胞，促进局部组织结构和功能的恢复，为治疗ICVD等多种疾病带来新的希望。骨髓间充质干细胞（BMSCs）由于来源广泛，取材方便，扩增周期短，免疫原性弱，可自身取材，没有伦理学问题等优势，已成为干细胞治疗最理想的种子细胞之一。 研究目的： 在局部缺血缺氧的微环境下，到达病灶的BMSCs大多数发生凋亡，严重限制其功能的发挥及其推广应用。丁苯酞（NBP）参与抗脑缺血的多个生理病理环节，具有改善微循环，抑制神经细胞凋亡等作用。本研究拟在体外模拟BMSCs缺氧生长环境，观察NBP对缺氧条件下BMSCs凋亡和神经分化的影响。为进一步将BMSCs应用于临床治疗提供理论依据。 研究方法： 1．采用全骨髓贴壁分离法，通过更换培养液得到纯化的BMSCs。使用倒置显微镜观察细胞形态和生长情况，绘制生长曲线。诱导细胞向成脂、成骨分化，以鉴定细胞的多向分化潜能。并采用流式细胞术检测细胞表面抗原。 2．使用厌氧培养袋联合连二亚硫酸钠（Na2S2O4）构建缺氧环境，缺氧培养24h后，采用四甲基偶氮唑盐（MTT）法测定缺氧组和正常对照组干细胞的细胞活性，采用免疫印迹（Western Blot）法检测两组低氧诱导因子-1α（HIF-1α）表达水平，验证此法构建缺氧模型的可行性。 3．设置常氧组，缺氧组，低浓度NBP组（缺氧+ 1 μmol/L NBP），中浓度NBP组（缺氧+ 10 μmol/L NBP）和高浓度NBP组（缺氧+ 100 μmol/L NBP）。缺氧培养24 h后，采用MTT法测定各组细胞的细胞活性，筛选出缺氧条件下NBP作用于BMSCs的最佳实验浓度。 4．设置常氧组，缺氧组和NBP组（缺氧+10 μmol/L NBP），缺氧培养24 h，采用Annexin V/PI双染法检测各组细胞凋亡率，采用荧光分光光度仪检测各组半胱氨酸天冬氨酸特异性蛋白酶-3（caspase-3）表达水平，评估缺氧模型中BMSCs的凋亡情况和NBP的抗凋亡能力。 5．设置对照组，常氧组，缺氧组和NBP组（缺氧+10 μmol/L NBP），后三组细胞先加入10ug/L碱性成纤维细胞生长因子（bFGF）预诱导24 h，再加入5mmol/L β-巯基乙醇进行神经分化诱导。诱导24 h后，采用免疫细胞化学法观察各组巢蛋白（Nestin），神经元特异性烯醇化酶（NSE）及神经胶质纤维酸性蛋白（GFAP）的表达情况，评估缺氧和NBP对BMSCs神经分化的影响。 研究结果： 1．原代培养培养2周后，可见大量梭形的贴壁细胞呈旋涡状生长。成骨诱导后，茜素红染色可见多处形状不规则的钙化结节。成脂诱导后，油红O染色可见细胞中含有大小不一的圆形脂滴。流式细胞术检测结果显示抗CD44和抗CD99的阳性表达率分别为96.14±1.35%和99.27±0.21%，抗CD45的阳性表达率为1.25±1.12%。 2．MTT实验结果显示缺氧组和正常对照组吸光值分别为0.23±0.07和0.30±0.06，缺氧组细胞活性显著降低（P＜0.05）。Western Blot结果显示正常对照组几乎不表达HIF-1α，缺氧组BMSCs表达HIF-1α水平显著增加（P＜0.05）。 3．MTT试验结果显示，中浓度NBP组和高浓度NBP组的吸光值均明显高于缺氧组（均P＜0.05），低浓度NBP组和缺氧组的吸光值差异无统计学意义（P＞0.05），中浓度NBP组和高浓度NBP组间差异无统计学意义（P＞0.05）。 4．流式细胞术检测结果显示常氧组，缺氧组和NBP组凋亡率分别为1.50±0.16%，44.62±8.63%和30.26±6.81%，缺氧组和NBP组凋亡率均显著高于常氧组（均P＜0.01），且NBP组高于缺氧组（P＜0.05）。荧光分光光度仪检测常氧组，缺氧组和NBP组A405值分别为0.50±0.09，4.93±0.26和3.27±0.35，缺氧组和NBP组caspase-3表达量显著高于常氧组（均P＜0.01），NBP组高于缺氧组（P＜0.05）。 5．常氧组抗Nestin、抗NSE阳性率分别为30.28±6.30%和42.81±13.05%。缺氧组细胞数量明显减少，抗Nestin、抗NSE阳性率分别为32.54±20.68%和36.28±16.16%。NBP组细胞数量明显多于缺氧组，抗Nestin、抗NSE阳性率分别为24.15±13.26%和29.31±11.07%。三组抗Nestin、抗NSE阳性率各组间差异均无统计学意义（均P＞0.05）。三组细胞抗GFAP阳性率均较低，各组间差异均无统计学意义（均P＞0.05）。 结论： 1．全骨髓贴壁分离法是体外分离BMSCs的有效方法。 2．厌氧培养袋联合Na2S2O4可用于模拟短时间BMSCs缺氧环境。 3．10 μmol/L NBP作为缺氧条件下作用于BMSCs的最佳实验浓度，可有效抑制缺氧条件下BMSCs凋亡。 4．缺氧条件和NBP对BMSCs向神经分化均无显著影响，但NBP可通过抑制缺氧条件下细胞凋亡，促进增殖的方式，增加BMSCs向神经分化细胞的绝对数量。
Background Nowadays, the effective treatment of ischemic cerebrovascular disease (ICVD) is thrombolysis or mechanical recanalization in the acute phase. These measures could recovery blood supply in early. However, the treatment time window of irreversible ischemic necrosis is very short, most of the patients in the clinical cannot promptly receive thrombolysis or endovascular treatment. By transplanting the stem cells which with replicate and multi-directional differentiation ability into patients, to make up the functional cells loss of the lesions area where lots cells occurred apoptosis and necrosis, to restore local tissue structure and function. Stem cell therapy brings new hope to cure a variety of diseases such as ischemic cerebrovascular disease. Because of their advantages such as extensive sources, easiness in obtaining materials, short amplification cycle, weak immunogenicity and without ethical issues, Bone marrow mesenchymal stem cells (BMSCs) has become the most ideal seed cells of stem cell therapy. Purposes Under the Anoxic-ischemic microenvironment, the most BMSCs which arrived in lesions would occur apoptosis. The function and application were severely limited. Dl-3n-butylphthalide (NBP) play a important role in multiple physiological and pathological link of anti cerebral ischemia. It can inhibit nerve cell apoptosis, and promote microcirculation improvement. This study intends to simulation BMSCs anoxic microenvironment in vitro, and observe the effect of NBP on apoptosis and neural differentiation of BMSCs under anoxic condition. This study also provides the theory basis for the application of BMSCs in clinical diseases. Methods 1. The method of whole bone marrow adherent was used for cell separation in this study. The purified BMSCs were harvested by changing L-DMEM. Inverted microscope was used for observing the cell morphology and growth situation. The growth curve was drawn by counting the number of cells. Through the induction of Adipogenic and osteogenesis differentiation of BMSCs, to identify the multi-directional differentiation potential of BMSCs. And using flow cytometry to detect cell surface antigen. 2. Anaerobicbag and sodium hydrosulfite(Na2S2O4)were combined to simulate the anoxic environment. And BMSCs of control group were cultured in normal oxygen environment. In anoxic environment 24h, the method of methyl thiazolyl tetrazolium (MTT) was used for the detect the cell activity of anoxia group and normoxic group BMSCs. And the level of HIF-1 alpha was detected by Western Blot to validate the feasibility of the anoxic model. 3. BMSCs were divided into five groups, normoxic group, anoxia group, low concentration group (anoxia + 1 μmol/L NBP), moddle concentration group (anoxia + 10 μmol/L NBP) and high concentration group (anoxia + 100 μmol/L NBP). In anoxia environment 24h, the method of MTT was used for the detect the cell activity. According to the results of MTT, to Screening the best experimental concentrations of NBP for BMSCs in anoxic conditions. 4. BMSCs were divided into three groups, normoxic group, anoxia group, NBP group (anoxia + 10μmol/L NBP). In anoxic environment 24h, the cell apoptosis rate was detected by flow cytometry to detect Annexin V-FITC/PI. Fluorescence spectrophotometer meter was used for detecting cysteinyl aspartate-specific protease-3(caspase - 3). To evaluate the apoptosis of BMSCs in anoxia model, and the anti-apoptotic ability of NBP. 5. BMSCs were divided into four groups, the control group, the normoxic group, the anoxia group and the NBP group (anoxia + 10 μmol/L NBP). Among them, the last three groups were induced neuronal differentiation by same method. After induction, the positive expression rates of Nest protein (Nestin), neuron specific enolization enzyme (NSE) and glial fibers acidic protein (GFAP) of each group were detected by immunocytochemistry. To assess the effect of anoxia and NBP on neural differentiation of BMSCs. Results 1. After 2 weeks of the original generation, a large number of spindle adherent cells grown as spiral shaped. Under the influence of inducing osteogenesis culture solution, a lot of calcified nodules of different sizes could be observed after alizarin red staining. After induction of adipogenic differentiation, intracellular lipid droplets could be observed more apparently oil red O staining. The results of flow cytometry showed that the positive expression rate of anti-CD44 and anti-CD99 were 96.14±1.35% and 99.27±0.21%, anti-CD45 positive expression rate of 1.25±1.12%. 2. The results of MTT showed that absorbance value of the anoxic group and the control group were 0.23±0.07 and 0.30±0.06 respectively. Cell activity of the anoxic group decreased significantly (P < 0.05). Western Blot results showed that the control group hardly expressed HIF - 1 alpha. However, the level of HIF - 1 alpha of the anoxic group increased significantly (P < 0.05). 3. The results of MTT showed that the absorbance value of the middle and the high concentration group were both obviously higher than that of the anoxic group (both P < 0.05). However, there was no statistically significant difference of the absorbance values between the low concentration group and the anoxic group (P > 0.05). The difference of the absorbance values between the middle concentration group and the high concentration group had no statistical significance (P > 0.05). 4. The results of flow cytometry showed that the apoptosis rates of the normoxic group, the anoxic group and the NBP group were 0.50±0.09,4.93±0.26 and 3.27±0.35 respectively. Compared with the normoxic group, the apoptosis rates of the anoxic group and the NBP group were significantly increased (P < 0.01). And the apoptosis rate of the NBP group was significantly lower than that of the anoxic group (P < 0.05). The results of fluorescence spectrophotometer instrument testing showed that the A405 values of the normoxic group, the anoxic group and the NBP group were 0.50±0.09,4.93±0.26 and 3.27±0.35 respectively. Compared with the normoxic group, the expression of caspase 3 of the anoxic group and the NBP group were significantly increased (both P < 0.01). And compared with the anoxic group, the expression of caspase 3 of the NBP group was significantly decreased (P < 0.05). 5. The results of immunocytochemistry showed that the positive rates of anti-Nestin and anti-NSE of the normoxic group were 30.28±6.30% and 42.81±13.05% respectively. The cell population of the anoxic group cells was decreased significantly. The positive rates of anti-Nestin and anti-NSE of the anoxic group were 32.54±20.68% and 36.28 ±16.16% respectively. The cell population of the NBP group was significantly more than the anoxic group. The positive rates of anti-Nestin and anti-NSE of the NBP group were 24.15±13.26% and 29.31±11.07% respectively. There was no difference between the three groups about the positive rates of anti-Nestin and anti-NSE (P > 0.05). The positive rate of anti-GFAP in the later three groups were all low, difference between each group had no statistical significance (P > 0.05). Conclusion 1. The whole bone marrow adherent separation method is an effective method for separation of BMSCs in vitro. 2. Combination of anaerobicbag and Na2S2O4 can be used for simulating BMSCs anoxic environment in a short time. 3. 10 umol/L NBP as the best experimental concentrations for BMSCs in anoxic condition, could inhibit BMSCs apoptosis under anoxic environment effectively. 4. Anoxia condition and NBP both had no significant effect for neural differentiation of BMSCs. But NBP can inhibit apoptosis, promote proliferation, increase the absolute number of neural cell differentiation under anoxic conditions. NBP combine with BMSCs could play an important role in nerve repair.