【摘要】：目的:觀察四點(diǎn)彎曲應(yīng)力刺激對(duì)人骨髓間充質(zhì)干細(xì)胞的增殖活性以及細(xì)胞周期的影響。 方法:實(shí)驗(yàn)于2010.9—2011.3在大連醫(yī)科大學(xué)附屬第一醫(yī)院中英再生醫(yī)學(xué)中心完成。 1.使用全骨髓培養(yǎng)法分離人骨髓間充質(zhì)干細(xì)胞。進(jìn)行原代培養(yǎng),擴(kuò)增后備用。 2.利用四點(diǎn)彎曲應(yīng)力細(xì)胞加載儀(四川大學(xué)華西醫(yī)學(xué)院與電子科技大學(xué)聯(lián)合研制,專利號(hào):(CN2534576 and CN1425905),取第4~ 6代細(xì)胞接種于加力板(BD No.353135),行爬片處理。分組:空白組,不加力;實(shí)驗(yàn)組,實(shí)驗(yàn)組頻率均為0.5HZ,加力60min,分別受張應(yīng)力1000μstrain、3000μstrain、5000μstrain的機(jī)械信號(hào)刺激,空白組不受力學(xué)信號(hào)刺激,其他培養(yǎng)條件與實(shí)驗(yàn)組一致。 3.細(xì)胞行力學(xué)干預(yù)后,用倒置相差顯微鏡觀察細(xì)胞形態(tài)、排列。分別用四甲基偶氮唑細(xì)胞計(jì)數(shù)法、流式細(xì)胞儀周期分析、共聚焦顯微鏡檢測(cè)鈣離子含量來(lái)測(cè)量細(xì)胞增殖和細(xì)胞周期。所有實(shí)驗(yàn)均重復(fù)三次。 4.統(tǒng)計(jì)學(xué)處理:實(shí)驗(yàn)數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差來(lái)表示,使用SPSS16.0軟件包進(jìn)行單因素方差分析,P0.05具有統(tǒng)計(jì)學(xué)意義,P0.01具有顯著統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1.利用離心法結(jié)合全骨髓培養(yǎng)法培養(yǎng)細(xì)胞增殖迅速,傳代穩(wěn)定。 2.四點(diǎn)彎曲應(yīng)力細(xì)胞加載儀性能穩(wěn)定,準(zhǔn)確,培養(yǎng)板未出行裂痕,變形等情況。細(xì)胞接種加力板后12 ~24h貼壁,貼壁良好,24 h后細(xì)胞覆蓋率約達(dá)80%~90%,無(wú)細(xì)菌污染現(xiàn)象,可適用于應(yīng)力刺激體外細(xì)胞效應(yīng)的實(shí)驗(yàn)研究。 3.通過(guò)四甲基偶氮唑法細(xì)胞計(jì)數(shù)檢測(cè)細(xì)胞增殖、流式細(xì)胞技術(shù)檢測(cè)細(xì)胞周期及PI指數(shù),激光共聚焦顯微鏡鈣離子探針檢測(cè)細(xì)胞內(nèi)鈣離子濃度,發(fā)現(xiàn)適當(dāng)大小的機(jī)械應(yīng)力刺激對(duì)人骨髓間充質(zhì)干細(xì)胞增殖具促進(jìn)作用,其中3000μstrain的應(yīng)力刺激作用最明顯,過(guò)強(qiáng)的機(jī)械應(yīng)力刺激(5000μstrain)對(duì)細(xì)胞不具有促增殖效應(yīng),并可能存在抑制作用。 結(jié)論:適當(dāng)?shù)臋C(jī)械應(yīng)力刺激可以促進(jìn)人骨髓間充質(zhì)干細(xì)胞增殖。
[Abstract]:Aim: to observe the effects of four point bending stress stimulation on proliferation activity and cell cycle of human bone marrow mesenchymal stem cells. Methods: the experiment was carried out in the first affiliated Hospital of Dalian Medical University. Human bone marrow mesenchymal stem cells were isolated by whole bone marrow culture. The primary culture was carried out, and then the amplification was carried out. 2. 2. A four-point bending stress cell loading instrument (Huaxi Medical College of Sichuan University and University of Electronic Science and Technology, patent number: (CN2534576 and CN1425905) was used to inoculate the 4th to 6th generation cells on the BD No.353135. The frequency of the experimental group was 0.5 HZ, and the experimental group was exposed to mechanical signal of 1000 渭 strain,3000 渭 strain,5000 渭 strain for 60 minutes, while the blank group was not stimulated by mechanical signal, and the other culture conditions were the same as that of the experimental group. After mechanical intervention, the morphology and arrangement of cells were observed by inverted phase contrast microscope. The cell proliferation and cell cycle were measured by the method of tetramethylazo cell count, flow cytometry and confocal microscope. All experiments were repeated three times. 4. Statistical processing: the experimental data were expressed as mean 鹵standard deviation, using SPSS16.0 software package for univariate ANOVA (P0.05) had statistical significance (P0.01). Results: 1. The cell proliferation was rapid and the passage was stable by centrifugation and whole bone marrow culture. 2. The four-point bending stress cell loader is stable, accurate, no crack, deformation and so on. The cell coverage rate was about 80% after 24 hours of good adhesion, and there was no bacterial contamination, which could be applied to the experimental study of stress stimulated cell effect in vitro. Cell cycle and PI index were measured by flow cytometry and intracellular calcium concentration was detected by laser confocal microscope calcium probe. It was found that mechanical stress stimulation of appropriate size could promote the proliferation of human bone marrow mesenchymal stem cells, of which 3000 渭 strain stress stimulation was the most obvious, and excessive mechanical stress stimulation (5000 渭 strain) had no proliferative effect on the cells, and might have inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells. Conclusion: proper mechanical stress stimulation can promote the proliferation of human bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：2236163