低氧微环境对脂肪间充质干细胞成肌腱分化影响的研究

发布时间：2018-04-26 07:14

本文选题：低氧 + 脂肪间充质干细胞　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:探讨低氧微环境对脂肪间充质干细胞成肌腱分化影响的作用及可能机制。方法:1、选取清洁级3周大小的SD大鼠,于两侧腹股沟处无菌分离脂肪垫,剪取脂肪组织,分离获取脂肪间充质干细胞(adipose tissue-derived mesenchymal stem cells,ADSCs),24h后首次换液,此后间隔2天更换新的培养液,大约培养7-10天后,细胞融合约至80%-90%时经胰酶消化传代扩增培养,第3代用于以后的实验。2、取P3 ADSCs用于细胞表面标记鉴定,测试CD29、CD31、CD45、CD90表达水平。3、取P3代ADSCs用于鉴定其多向分化潜能,用成脂或成骨诱导培养基诱导培养,并在诱导28天时,用油红O染色或茜素红染色。4、ADSCs按照密度为4000个/孔,接种于96孔板中,随机分为低氧组及常氧组,放入相应孵箱中孵育,分别在孵育1,3,5,7天时运用CCK8方法检测脂肪间充质干细胞在不同氧浓度环境下的增殖情况,其中常氧环境下,体外三气培养箱中氧浓度为21%,低氧环境下,体外三气培养箱中氧浓度为5%。5、P3 ADSCs接种于六孔板中,进行划痕试验,以检测脂肪间充质干细胞在不同氧浓度条件下的迁移能力。6、P3 ADSCs置于低氧和常氧微环境中处理3d,运用Q-PCR和细胞免疫荧光染色检测缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)及Scleraxis(SCX)的表达。7、P3ADSCs中加入含有0、5、10、15μM浓度的HIF-1α阻断剂2-Methoxyestradiol(2-MeOE2)的诱导培养基中,随机分为4组,放入低氧孵箱中处理24h,具体分组为:ADSCs低氧0μM 2-MeOE2(hypoxia,0μM 2-MeOE2,HM0))、adscs低氧5μm2-meoe2(hypoxia,5μm2-meoe2,hm5))、adscs低氧10μm2-meoe2(hypoxia,10μm2-meoe2,hm10))、adscs低氧15μm2-meoe2(hypoxia,15μm2-meoe2,hm15)),运用荧光定量pcr(quatative-pcr,q-pcr)方法检测hif-1α(hypoxiainducingfactor-1α,hif-1α)基因的表达情况。8、adscs中各自加入0μm和最适阻断剂浓度,各自放入低氧及常氧孵箱中处理24h,运用q-pcr方法分别检测scx和tenomodulin(tnmd)基因的表达情况。结果:该实验成功于脂肪组织中提取adscs,并进行了体外扩增,倒置相差显微镜下观察p3代以后,adscs呈漩涡样或“鱼群样”排列生长。通过流式细胞仪鉴定,adscs表达cd29和cd90呈强阳性,表达cd31和cd45呈阴性,多向分化鉴定检测反映出adscs具有成脂、成骨分化潜能。运用cck8方法检测提示低氧微环境降低adscs增殖能力,第一天时,两组增殖情况无明显差异(p0.05),差异无统计学意义。第3天至第5天,两组细胞增殖率明显提高,且低氧组的增殖率显著小于常氧组(p0.05)。第7天时,低氧组增值率显著低于常氧组(p0.05),差异具有统计学意义。划痕实验提示adscs放置于常氧和低氧条件下处理24h后,低氧组划痕愈合速度较常氧组明显下降(p0.05)。细胞免疫荧光和q-pcr检测提示hif-1α在低氧组的表达显著高于常氧组,adscs诱导3d后scx基因表达均增高,且低氧组显著高于常氧组(p0.05),低氧组adscs的hif-1α荧光较常氧组强。q-pcr检测adscs在低氧条件下阻断剂2-meoe2阻断hif-1α时的最适阻断浓度,当抑制剂浓度为10μm,15μm时,hif-1α的表达显著降低(p0.01),且两组之间hif-1α的表达无明显差异(p0.05),提示当2-MeOE2浓度为10μM时已达到最适阻断浓度,继续增加2-MeOE2浓度并不会提高阻断作用。Q-PCR检测提示,常氧组中添加了10μM阻断剂与对照组相比,SCX mRNA、TNMD mRNA的表达无明显差异(P0.05),而低氧组中添加了10μM阻断剂的SCX mRNA、TNMD mRNA表达显著低于对照组(P0.01),差异具有统计学意义。低氧组与常氧组相比,低氧组中的SCX mRNA、TNMD mRNA的表达均显著高于常氧组(P0.01)。结论:ADSCs高表达间充质干细胞标志,同时具有多向分化能力。低氧微环境(5%O2)对ADSCs的体外增殖和迁移均具有抑制作用。低氧微环境可促进ADSCs体外成肌腱分化,其机制可能是通过HIF-1α信号途径实现的。低氧微环境为脂肪间充质干细胞在肌腱组织工程技术中提供更加广阔的应用前景。
[Abstract]:Objective: To investigate the effect and possible mechanism of hypoxia microenvironment on the differentiation of adipose mesenchymal stem cells (MSCs) into tendon. Methods: 1, SD rats with 3 weeks of cleaning grade were selected to separate fat pads from both sides of groin, and the adipose tissue was cut, and the adipose tissue-derived mesenchymal stem cells (ADSCs) was isolated and obtained. After 24h for the first time, the new medium was replaced for 2 days. After 7-10 days, the cells were cultured for about 7-10 days. When the cell fusion was about 80%-90%, the third generation was used for the later experimental.2. P3 ADSCs was used to identify the cell surface markers and test the CD29, CD31, CD45, CD90 expression level.3, and take P3 ADSCs to identify its multidirectional differentiation potential. Ability to induce culture with lipid or osteogenic induction medium, and when induced 28 days,.4 with oil red O or alizarin red, ADSCs according to density of 4000 / holes, 96 Kong Banzhong, randomly divided into low oxygen group and normal oxygen group, and incubated in the incubator to incubate 1,3,5,7 days with CCK8 method to detect fat mesenchymal stem cells respectively. In the environment of different oxygen concentration, under the environment of atmospheric oxygen, the oxygen concentration in the three gas incubator was 21%. Under the environment of low oxygen, the oxygen concentration in the three gas incubator was 5%.5, and the P3 ADSCs was inoculated in the six hole plate, and the scratch test was carried out to detect the migration ability of the fat mesenchymal stem cells under different oxygen concentration conditions,.6, P3 ADSCs The expression of hypoxia inducible factor -1 alpha (Hypoxia-inducible factor-1 alpha, HIF-1 a) and Scleraxis (SCX) was detected by Q-PCR and cell immunofluorescence staining in the low oxygen and atmospheric microenvironment, and 4 groups were randomly divided into 4 groups in the inducible medium containing 0,5,10,15 micron M concentration. In the incubator of hypoxia, 24h is treated, and the specific group is: ADSCs hypoxia 0 mu M 2-MeOE2 (hypoxia, 0 mu M 2-MeOE2, HM0), ADSCs hypoxia 5 mu m2-meoe2 (hypoxia, 5 mu m2-meoe2). The expression of HIF-1 alpha (hypoxiainducingfactor-1 alpha, HIF-1 alpha) gene was detected by the method.8. ADSCs was added to the ADSCs and the concentration of 0 micron m and the optimum blocker. The 24h was treated in the hypoxia and atmospheric incubator respectively. The expression of SCX and tenomodulin (tnmd) gene was detected by Q-PCR method. In vitro amplification, after the inverted phase microscope observation, after P3 generation, ADSCs was arranged in a whirlpool or "fish sample". Through the flow cytometry, the expression of CD29 and CD90 was strongly positive, the expression of CD31 and CD45 was negative, and the identification and detection of multidirectional differentiation showed that ADSCs had lipid formation and osteogenic differentiation potential. CCK8 method was used to detect and extract the ADSCs. The proliferation of ADSCs was reduced by hypoxia microenvironment. There was no significant difference in proliferation between the two groups at the first day (P0.05). There was no significant difference in the proliferation rate between third days and fifth days. The proliferation rate of the two groups was significantly higher than that of the normal oxygen group (P0.05). At seventh days, the rate of value added in the hypoxia group was significantly lower than that of the normal oxygen group (P0.05). The scratch test suggested that the healing speed of scratch in the hypoxia group was significantly lower than that of the normal oxygen group (P0.05). The expression of HIF-1 alpha in the hypoxia group was significantly higher than that of the normal oxygen group (P0.05). The expression of HIF-1 in the hypoxia group was significantly higher than that in the normal oxygen group. The expression of SCX gene in the hypoxia group was higher than that in the ADSCs induced 3D, and the hypoxia group was significantly higher than that in the hypoxia group. Oxygen group (P0.05), the HIF-1 alpha fluorescence of ADSCs in hypoxic group was stronger than that of the normal oxygen group, and the optimal concentration of ADSCs was detected by ADSCs when the blocker 2-meoe2 blocked HIF-1 alpha under the hypoxic condition. When the inhibitor concentration was 10 u m and 15 micron, the expression of HIF-1 a decreased significantly (P0.01), and there was no significant difference in the expression of HIF-1 alpha between the two groups. 10 mu M had reached the optimum blocking concentration, and continued to increase the concentration of 2-MeOE2 did not improve the.Q-PCR detection of blocking action. The expression of SCX mRNA, TNMD mRNA was not significantly different (P0.05), and the expression of SCX mRNA was not significantly different from the control group in the normal oxygen group, and the SCX mRNA was added with 10 micron M blockers in the hypoxia group. 0.01) the difference was statistically significant. Compared with the normal oxygen group, the expression of SCX mRNA and TNMD mRNA in the hypoxia group was significantly higher than that of the normal oxygen group (P0.01). Conclusion: ADSCs expressed high expression of mesenchymal stem cells and had the ability to differentiate at the same time. Low oxygen microenvironment (5%O2) had inhibitory effect on the proliferation and migration of ADSCs in vitro. The environment can promote the differentiation of ADSCs tendon in vitro, and its mechanism may be realized through the HIF-1 alpha signal pathway. Low oxygen microenvironment provides a broader prospect for the application of adipose mesenchymal stem cells in tendon tissue engineering.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R686

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