本文選題：低氧 + 脂肪間充質(zhì)干細(xì)胞　； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討低氧微環(huán)境對脂肪間充質(zhì)干細(xì)胞成肌腱分化影響的作用及可能機(jī)制。方法:1、選取清潔級3周大小的SD大鼠,于兩側(cè)腹股溝處無菌分離脂肪墊,剪取脂肪組織,分離獲取脂肪間充質(zhì)干細(xì)胞(adipose tissue-derived mesenchymal stem cells,ADSCs),24h后首次換液,此后間隔2天更換新的培養(yǎng)液,大約培養(yǎng)7-10天后,細(xì)胞融合約至80%-90%時經(jīng)胰酶消化傳代擴(kuò)增培養(yǎng),第3代用于以后的實(shí)驗(yàn)。2、取P3 ADSCs用于細(xì)胞表面標(biāo)記鑒定,測試CD29、CD31、CD45、CD90表達(dá)水平。3、取P3代ADSCs用于鑒定其多向分化潛能,用成脂或成骨誘導(dǎo)培養(yǎng)基誘導(dǎo)培養(yǎng),并在誘導(dǎo)28天時,用油紅O染色或茜素紅染色。4、ADSCs按照密度為4000個/孔,接種于96孔板中,隨機(jī)分為低氧組及常氧組,放入相應(yīng)孵箱中孵育,分別在孵育1,3,5,7天時運(yùn)用CCK8方法檢測脂肪間充質(zhì)干細(xì)胞在不同氧濃度環(huán)境下的增殖情況,其中常氧環(huán)境下,體外三氣培養(yǎng)箱中氧濃度為21%,低氧環(huán)境下,體外三氣培養(yǎng)箱中氧濃度為5%。5、P3 ADSCs接種于六孔板中,進(jìn)行劃痕試驗(yàn),以檢測脂肪間充質(zhì)干細(xì)胞在不同氧濃度條件下的遷移能力。6、P3 ADSCs置于低氧和常氧微環(huán)境中處理3d,運(yùn)用Q-PCR和細(xì)胞免疫熒光染色檢測缺氧誘導(dǎo)因子-1α(Hypoxia-inducible factor-1α,HIF-1α)及Scleraxis(SCX)的表達(dá)。7、P3ADSCs中加入含有0、5、10、15μM濃度的HIF-1α阻斷劑2-Methoxyestradiol(2-MeOE2)的誘導(dǎo)培養(yǎng)基中,隨機(jī)分為4組,放入低氧孵箱中處理24h,具體分組為: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)),運(yùn)用熒光定量pcr(quatative-pcr,q-pcr)方法檢測hif-1α(hypoxiainducingfactor-1α,hif-1α)基因的表達(dá)情況。8、adscs中各自加入0μm和最適阻斷劑濃度,各自放入低氧及常氧孵箱中處理24h,運(yùn)用q-pcr方法分別檢測scx和tenomodulin(tnmd)基因的表達(dá)情況。結(jié)果:該實(shí)驗(yàn)成功于脂肪組織中提取adscs,并進(jìn)行了體外擴(kuò)增,倒置相差顯微鏡下觀察p3代以后,adscs呈漩渦樣或“魚群樣”排列生長。通過流式細(xì)胞儀鑒定,adscs表達(dá)cd29和cd90呈強(qiáng)陽性,表達(dá)cd31和cd45呈陰性,多向分化鑒定檢測反映出adscs具有成脂、成骨分化潛能。運(yùn)用cck8方法檢測提示低氧微環(huán)境降低adscs增殖能力,第一天時,兩組增殖情況無明顯差異(p0.05),差異無統(tǒng)計學(xué)意義。第3天至第5天,兩組細(xì)胞增殖率明顯提高,且低氧組的增殖率顯著小于常氧組(p0.05)。第7天時,低氧組增值率顯著低于常氧組(p0.05),差異具有統(tǒng)計學(xué)意義。劃痕實(shí)驗(yàn)提示adscs放置于常氧和低氧條件下處理24h后,低氧組劃痕愈合速度較常氧組明顯下降(p0.05)。細(xì)胞免疫熒光和q-pcr檢測提示hif-1α在低氧組的表達(dá)顯著高于常氧組,adscs誘導(dǎo)3d后scx基因表達(dá)均增高,且低氧組顯著高于常氧組(p0.05),低氧組adscs的hif-1α熒光較常氧組強(qiáng)。q-pcr檢測adscs在低氧條件下阻斷劑2-meoe2阻斷hif-1α?xí)r的最適阻斷濃度,當(dāng)抑制劑濃度為10μm,15μm時,hif-1α的表達(dá)顯著降低(p0.01),且兩組之間hif-1α的表達(dá)無明顯差異(p0.05),提示當(dāng)2-MeOE2濃度為10μM時已達(dá)到最適阻斷濃度,繼續(xù)增加2-MeOE2濃度并不會提高阻斷作用。Q-PCR檢測提示,常氧組中添加了10μM阻斷劑與對照組相比,SCX mRNA、TNMD mRNA的表達(dá)無明顯差異(P0.05),而低氧組中添加了10μM阻斷劑的SCX mRNA、TNMD mRNA表達(dá)顯著低于對照組(P0.01),差異具有統(tǒng)計學(xué)意義。低氧組與常氧組相比,低氧組中的SCX mRNA、TNMD mRNA的表達(dá)均顯著高于常氧組(P0.01)。結(jié)論:ADSCs高表達(dá)間充質(zhì)干細(xì)胞標(biāo)志,同時具有多向分化能力。低氧微環(huán)境(5%O2)對ADSCs的體外增殖和遷移均具有抑制作用。低氧微環(huán)境可促進(jìn)ADSCs體外成肌腱分化,其機(jī)制可能是通過HIF-1α信號途徑實(shí)現(xiàn)的。低氧微環(huán)境為脂肪間充質(zhì)干細(xì)胞在肌腱組織工程技術(shù)中提供更加廣闊的應(yīng)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.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R686

【參考文獻(xiàn)】

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本文編號：1805020