层流剪切力对骨髓间充质干细胞增殖和分化的影响及血管来源的初步探讨

发布时间：2018-01-24 15:15

  本文关键词： CD146~+血管周围细胞 间充质干细胞 层流剪切力 细胞周期抑制 凋亡 微重力 软骨诱导分化　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：目的：1.分离、纯化CD146+血管周围细胞,并进行软骨诱导,探讨其成为软骨修复组织工程的种子细胞可行性；进一步揭示间充质干细胞(MSCs)的血管周围来源。2.研究层流剪切力(laminar shear stress; LSS)对体外培养的大鼠MSCs增殖和凋亡的影响；并初步探讨其抗凋亡机制。3.在体外微重力条件下三维诱导大鼠MSCs向类髓核细胞分化,为退变椎间盘的修复提供种子细胞。方法：1.从大鼠脂肪组织中分离、纯化CD146+血管周围细胞；进行软骨诱导；通过RT-PCR和Western blot检测软骨分化；进一步检测CD146+血管周围细胞的迁移能力。2.大鼠MSCs分组予以生理范围内的层流剪切力力学干预。通过流式细胞学检测细胞周期和凋亡；RT-PCR检测各组细胞的Bcl-2、Bax凋亡相关基因的mRNA表达情况。3.在微重力条件下采用颗粒培养法(pellet culture)诱导骨髓MSCs向髓核细胞分化,建立三个细胞培养组：实验组(0ng/ml TGF-β1和微重力),阳性对照组(10ng/ml TGF-β1和微重力),空白对照组(0ng/ml TGF-β1)。WST-8检测细胞增殖；组织化学和RT-PCR检测细胞分化。结果：1.CD146+血管周围细胞成功从S-D大鼠脂肪组织中分离、纯化培养；流式细胞检测显示：CD146阳性,CD45、CD56、CD34阴性；Sox9和Aggrecan在1mRNA和蛋白水平上均高于对照组；细胞迁移实验显示实验组较高。2.在加载生理力度的LSS (15-dyne/cm2)后,MSCs的DNA合成明显受到抑制；细胞周期检测发现：在细胞加载LSS (15 dyne/cm2)后的不同时间点,处于S和G2/M期的细胞百分含量随着时间变化有明显的下降；细胞凋亡检测显示：,实验组的活细胞比率由59.39%上升到82.37%,24小时保持在81.55%,实验组的凋亡细胞比率由40.61%下降到17.49%,24小时保持在18.83%；生理范围内的力学刺激促进抗凋亡基因的表达,即Bcl-2表达增加,Bcl-2/Bax比率增高。3.诱导培养第三天,TGF-βl对MSCs的增殖有轻度的促进作用；诱导培养第七天,组织染色显示微重力下两组都有胶原和蛋白聚糖存在；蛋白聚糖含量检测实验组显著增高；RT-PCR结果显示微重力条件下软骨细胞标志基因(Sox-9,蛋白聚糖,Ⅱ型胶原)表达量增高；实验组蛋白多糖与胶原的比率(proteoglycans/collagen)是阳性对照组的3.4倍,进一步提示MSC向髓核细胞方向分化。结论：1.成功分离、纯化CD146+血管周围细胞；诱导后CD146+血管周围细胞向软骨细胞方向分化；CD146+血管周围细胞有较好的细胞迁移能力。2.生理范围内的LSS引起MSCs细胞周期抑制和抗凋亡作用,介导MSCs的休眠,这有利于MSCs的干细胞特性保持。3.MSCs在微重力条件下自发向髓核细胞方向分化,证明三维立体培养是诱导MSCs向髓核细胞分化的条件之一,可为椎间盘退变性疾病的干细胞移植治疗提供种子细胞。
[Abstract]:Objective to isolate and purify the perivascular cells of CD146 and to investigate the feasibility of becoming seed cells for cartilage repair tissue engineering. To further reveal the sources of mesenchymal stem cells (MSCs) around blood vessels. 2. To study laminar shear stress in laminar flow. Effects of LSS on proliferation and apoptosis of rat MSCs in vitro; The mechanism of anti-apoptosis was also discussed. 3. Three-dimensional differentiation of rat MSCs into nucleus pulposus cells was induced by microgravity in vitro. To provide seed cells for the repair of degenerated intervertebral disc. Methods: 1. Isolated and purified CD146 perivascular cells from adipose tissue of rats. Cartilage induction; Cartilage differentiation was detected by RT-PCR and Western blot. The migration ability of perivascular cells of CD146 was further measured. Rat MSCs was divided into two groups and subjected to laminar shear stress intervention in physiological range. Cell cycle and apoptosis were detected by flow cytology. Bcl-2 was detected by RT-PCR. Expression of Bax apoptosis-related gene mRNA. 3. Bone marrow MSCs was induced to differentiate into nucleus pulposus cells by granular culture under microgravity. Three cell culture groups were established: experimental group (0 ng / ml TGF- 尾 1 and microgravity), positive control group (10 ng / ml TGF- 尾 1 and microgravity). The cell proliferation was detected by TGF- 尾 _ (1) and WST-8 in the blank control group (0 ng / ml TGF- 尾 _ (1)). Results the perivascular cells were isolated and cultured successfully from the adipose tissue of S-D rats. Flow cytometry showed that CD146 was positive and CD45, CD56 and CD34 were negative. Sox9 and Aggrecan were significantly higher than those of the control group at 1 mRNA and protein levels. The cell migration assay showed that the DNA synthesis of LSS was significantly inhibited after the physiological stress of 15-dyne / cm ~ (2). Cell cycle analysis showed that the percentage content of cells in S and G _ 2 / M phase decreased significantly at different time points after cell loading with LSS 15 dyne / cm ~ (2). Cell apoptosis test showed that the percentage of living cells in the experimental group increased from 59.39% to 82.37 and remained at 81.55% for 24 hours. The percentage of apoptotic cells in the experimental group decreased from 40.61% to 17.49 and remained at 18.83 for 24 hours. Mechanical stimulation in physiological range promoted the expression of anti-apoptotic genes, that is, the expression of Bcl-2 increased, the ratio of Bcl-2 / Bax increased .3.The third day of induction culture. TGF- 尾 l slightly promoted the proliferation of MSCs. After 7th days of induction and culture, tissue staining showed the presence of collagen and proteoglycan in both groups under microgravity. The content of proteoglycan was significantly higher in the experimental group. RT-PCR results showed that the expression of chondrocyte marker gene (Sox-9, proteoglycan, type 鈪，

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