本文選題：體外發(fā)散式?jīng)_擊波 + BMSC�。� 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的：通過應(yīng)用低能量發(fā)散式?jīng)_擊波(rESW)對(duì)兔骨髓間充質(zhì)干細(xì)胞(BMSC)進(jìn)行直接刺激,觀察發(fā)散式?jīng)_擊波對(duì)BMSC增殖特性的影響。本研究從BMSC沖擊波刺激的反應(yīng)出發(fā),探求發(fā)散式?jīng)_擊波促進(jìn)關(guān)節(jié)軟骨損傷修復(fù)的理論基礎(chǔ)和作用機(jī)制。方法：在細(xì)胞實(shí)驗(yàn)部分,我們采用差速離心法和懸浮貼壁法從2～3月齡的新西蘭白兔的脛骨骨髓中培養(yǎng)原代培養(yǎng)至P4～P6代。分別從形態(tài)學(xué)、表面BMSC,分子和分化能力(成脂、成骨)等方面對(duì)進(jìn)行鑒定。用不同劑量的發(fā)散式BMSC沖擊波對(duì)懸浮的進(jìn)行直接刺激,根據(jù)沖擊波劑量對(duì)實(shí)驗(yàn)進(jìn)行分組,分為0BMSC四組(Bar為沖擊波壓力單位)。對(duì)沖擊波刺激組與對(duì)照組的bar/1 bar/2 bar/3 bar分別進(jìn)行CCK-8檢測(cè)和BMSC檢測(cè)比較其增殖活性,用統(tǒng)計(jì)學(xué)軟件對(duì)實(shí)驗(yàn)CFU-F結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。在動(dòng)物實(shí)驗(yàn)部分,我們使用與之前實(shí)驗(yàn)等月齡的新西蘭白兔制作膝關(guān)節(jié)軟骨缺損模型,將每只兔子的左側(cè)膝關(guān)節(jié)設(shè)為實(shí)驗(yàn)組,右側(cè)膝關(guān)節(jié)設(shè)為對(duì)照組。實(shí)驗(yàn)組采用微骨折加2bar沖擊波進(jìn)行干預(yù),而對(duì)照組只采用微骨折術(shù)。手術(shù)后24小時(shí)將骨髓等量沖出進(jìn)行體外實(shí)驗(yàn),在不同時(shí)間點(diǎn)對(duì)比CFU-F增殖聚集情況。在對(duì)數(shù)據(jù)結(jié)果進(jìn)行分析統(tǒng)計(jì)時(shí),我們使用BMSC軟件并使用SPSS16.0軟件對(duì)圖像進(jìn)行處理分析。ImageJ結(jié)果：通過骨髓分離技術(shù)我們可以獲得原代在倒置相差顯微鏡下觀BMSC,察細(xì)胞形態(tài),類似成纖維細(xì)胞樣聚集分布。在表面分子分析中,缺乏典型BMSC的造血細(xì)胞抗原CD45分子、CD14分子,也不表達(dá)CD79α和CD90,高表達(dá)CD45和CD81。在誘導(dǎo)分化實(shí)驗(yàn)中,可以被成功誘導(dǎo)為骨細(xì)胞、脂肪細(xì)胞,通過BMSC相應(yīng)的染色得到證實(shí),并檢測(cè)相關(guān)轉(zhuǎn)錄因子表達(dá)。經(jīng)不同的沖擊波進(jìn)行刺激后,表現(xiàn)出不同的增殖特性。無論是CCK-8檢測(cè)還是BMSC檢測(cè),沖擊波刺激CFU-F組都明顯優(yōu)于對(duì)照組,并且2Bar時(shí)其效應(yīng)最強(qiáng),其差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。在動(dòng)物體內(nèi)實(shí)驗(yàn)進(jìn)行CFU-F檢測(cè)中,對(duì)術(shù)后24小時(shí)分離的MSc培養(yǎng),并于第8天、11天、15天進(jìn)行CFU-F檢測(cè),結(jié)果顯示實(shí)驗(yàn)組增殖活性均優(yōu)于對(duì)照組,其結(jié)果具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：1.我們發(fā)現(xiàn)兔BMSC在表面分子檢測(cè)中CD90為陰性,這是與以往文獻(xiàn)中的報(bào)道是不同的。2.發(fā)散式?jīng)_擊波體外刺激BMSC具有促進(jìn)其增殖和聚集作用,在沖擊波劑量為2bar時(shí)其促增值作用最強(qiáng)。3.發(fā)散式?jīng)_擊波對(duì)BMSC的作用呈現(xiàn)劑量相關(guān)性,其對(duì)于沖擊波的軟骨修復(fù)劑量具有指導(dǎo)作用。4.在動(dòng)物實(shí)驗(yàn)中,采用微骨折術(shù)配合沖擊波進(jìn)行軟骨缺損修復(fù)研究,為提出一種改良的微骨折術(shù)提供理論依據(jù)。
[Abstract]:Aim: to investigate the effect of low energy divergent shock wave (rESW) on the proliferation of rabbit bone marrow mesenchymal stem cells (BMSC). Based on the response of BMSC to shock wave stimulation, this study explored the theoretical basis and mechanism of divergent shock wave promoting articular cartilage repair. Methods: in the cell experiment, we used differential centrifugation and suspension adherent method from the tibia bone marrow of New Zealand white rabbits of 2 to 3 months old to primary culture to P4 and P6. Morphology, surface BMSCs, molecular and differentiation ability (adipogenic, osteogenic) were identified. The suspension was directly stimulated by different doses of divergent BMSC shock wave. According to the shock wave dose, the experiment was divided into four groups: 0BMSC (Bar was shock wave pressure unit). The proliferative activity of bar/1 bar/2 bar/3 bar in shock wave stimulation group and control group were detected by CCK-8 and BMSC respectively. The results of CFU-F were statistically analyzed by statistical software. In the part of animal experiment, we used New Zealand white rabbits with the same age to make cartilage defect model of knee joint. The left knee joint of each rabbit was set as experimental group and the right knee joint as control group. The experimental group was treated with microfracture plus 2bar shock wave, while the control group was treated with microfracture. Bone marrow was washed out 24 hours after operation in vitro to compare the proliferation and aggregation of CFU-F at different time points. In the analysis and statistics of the data results, we use BMSC software and SPSS 16.0 software to analyze the image processing. ImageJ results: through bone marrow separation technology, we can obtain primary BMSCs under inverted phase contrast microscope, observe cell morphology. Similar to fibroblast-like aggregation distribution. In surface molecular analysis, CD45, CD14, CD79 偽 and CD90, and CD45 and CD81were not expressed in typical BMSC. In the induction and differentiation experiment, osteocytes and adipocytes could be successfully induced, confirmed by the corresponding staining of BMSC, and the expression of related transcription factors was detected. After different shock waves were stimulated, they showed different proliferative characteristics. Whether CCK-8 test or BMSC test, shock wave stimulation CFU-F group was significantly better than the control group, and the effect of 2Bar was the strongest, the difference was statistically significant (P0.05). In vivo CFU-F test, the MSC isolated 24 hours after operation was cultured and CFU-F was detected on the 8th day, 11th day and 15th day. The results showed that the proliferative activity of the experimental group was better than that of the control group, and the results were statistically significant (P0.05). Conclusion 1. We found that the CD90 of rabbit BMSC was negative in surface molecular detection, which was different from previous reports. The proliferation and aggregation of BMSC were stimulated by divergent shock wave in vitro. When the dose of shock wave was 2bar, the effect of proliferation was the strongest. 3. The effect of divergent shock wave on BMSC is dose-dependent, and it has a guiding effect on cartilage repair dose of shock wave. In animal experiments, microfracture combined with shock wave was used to repair cartilage defects, which provided a theoretical basis for improving microfracture.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R687.4

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本文編號(hào)：2064031