【摘要】： 研究背景 韌帶在維持關(guān)節(jié)穩(wěn)定，協(xié)調(diào)關(guān)節(jié)運(yùn)動(dòng)中發(fā)揮著重要作用。外傷、長(zhǎng)期反復(fù)勞累過度等會(huì)造成韌帶損傷，但由于韌帶組織血供少，損傷后很難完全愈合，最終造成關(guān)節(jié)功能嚴(yán)重障礙的病例。韌帶損傷也使得很多優(yōu)秀的運(yùn)動(dòng)員就此葬送了運(yùn)動(dòng)生涯�，F(xiàn)代外科的發(fā)展已使人類替換病損韌帶成為現(xiàn)實(shí)，外科使用過的替換物包括異種韌帶、同種異體韌帶、自體韌帶和人工合成材科等，但這些措施的遠(yuǎn)期效果都不夠理想。組織工程學(xué)的誕生使體外構(gòu)建有活性的生物韌帶的研制成為可能，為這一疾病的治療提供了新途徑 細(xì)胞成分對(duì)提高韌帶的力學(xué)負(fù)載的承受力，基因激活和韌帶自我更新和調(diào)節(jié)的能力非常重要。但如何選擇理想的種子細(xì)胞?種子細(xì)胞需要怎樣的微環(huán)境才能達(dá)到預(yù)期的功能要求?這些問題都有待進(jìn)一步研究。 本試驗(yàn)的目的是：探索在體外條件下如何誘導(dǎo)BMSCs向韌帶成纖維細(xì)胞分化。因此，本實(shí)驗(yàn)中我們采用與韌帶成纖維細(xì)胞間接共培養(yǎng)和力學(xué)刺激兩種方法，研究體外條件下誘導(dǎo)BMSCs向韌帶成纖維細(xì)胞轉(zhuǎn)化，試圖為體外誘導(dǎo)BMSCs向韌帶成纖維細(xì)胞轉(zhuǎn)化提供可行的方法，為韌帶組織工程學(xué)種子細(xì)胞的研究提供思路。 研究方法 1．采用Percoll液密度梯度離心法和傳代貼壁篩選法相結(jié)合分離獲得骨髓間充質(zhì)細(xì)胞，建立大鼠BMSCs(rBMSCs)和人BMSCs(hBMSCs)體外培養(yǎng)的方法；通過形態(tài)學(xué)方法與細(xì)胞表面特異抗原流式細(xì)胞術(shù)檢測(cè)方法，對(duì)在體外所培養(yǎng)的rBMSCs和hBMSCs進(jìn)行鑒定；采用體外誘導(dǎo)rBMSCs和hBMSCs向成骨細(xì)胞和成脂細(xì)胞分化的方法，對(duì)BMSCs的干細(xì)胞生物學(xué)功能進(jìn)行鑒定。 2．實(shí)時(shí)熒光定量PCR檢測(cè)第一代到第六代大鼠BMSCsⅠ型膠原蛋白，Ⅲ型膠原蛋白和韌粘素-C mRNA的表達(dá)。 3．將大鼠BMSCs和大鼠韌帶成纖維細(xì)胞間接共培養(yǎng)3、6、12天，用實(shí)時(shí)熒光定量PCR檢測(cè)，間接共培養(yǎng)后BMSCs三種韌帶特性蛋白(Ⅰ型膠原蛋白，，Ⅲ型膠原蛋白和韌粘素-C)的mRNA表達(dá)；競(jìng)爭(zhēng)放免法和免疫組化的方法，檢測(cè)細(xì)胞這三種蛋白合成。 4．對(duì)大鼠BMSCs施以10％，1赫茲的周期性牽張刺激不同時(shí)段后，分析細(xì)胞的變形和重排；并用實(shí)時(shí)熒光定量PCR檢測(cè)牽張不同時(shí)間段后，其Ⅰ型、Ⅲ型膠原和韌粘素-C mRNA的表達(dá)；用競(jìng)爭(zhēng)放免法和免疫組化，檢測(cè)細(xì)胞這三種蛋白合成；激光共聚焦顯微鏡觀察力學(xué)刺激對(duì)細(xì)胞骨架的影響。 實(shí)驗(yàn)結(jié)果 1．成功獲得了rBMSCs和hBMSCs兩種干細(xì)胞。我們獲得的細(xì)胞具有干細(xì)胞的形態(tài)學(xué)特征(核大漿少)；細(xì)胞表面特異性抗原鑒定(rBMSCs CD44、CD90表達(dá)陽性而CD34、CD45表達(dá)陰性，hBMSCs CD44表達(dá)陽性CD14、CD45表達(dá)陰性)，且這些特征在第一代到第六代的細(xì)胞中穩(wěn)定；rBMSCs和hBMSCs這兩種細(xì)胞具有向成骨細(xì)胞和脂肪細(xì)胞分化的能力。綜合上述三個(gè)特點(diǎn)，我們認(rèn)為實(shí)驗(yàn)中分離培養(yǎng)的細(xì)胞為骨髓間充質(zhì)干細(xì)胞。 2．Ⅰ型膠原、Ⅲ型膠原和韌粘素-C mRNA在第一代到第六代大鼠BMSCs中均穩(wěn)定表達(dá)。 3．間接共培養(yǎng)6天，BMSCsⅠ型膠原和Ⅲ型膠原的mRNA表達(dá)分別是對(duì)照組的2.0和2.2倍，Ⅰ型膠原和Ⅲ型膠原mRNA與內(nèi)參照GAPDH的相對(duì)表達(dá)量在間接共培養(yǎng)6天組分別為：3.9±0.2和1.9±0.2，對(duì)照組分別為：1.9±0.3和0.8±0.1，間接共培養(yǎng)組與對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P＜0.05)而韌粘素-C的表達(dá)無明顯改變。間接共培養(yǎng)12天，Ⅰ型和Ⅲ型膠原的蛋白含量增加，分別從對(duì)照組的12.4±0.8 ng／μg和5.0±0.4 ng／μg增加到間接共培養(yǎng)組的13.6±1.3 ng／μg和5.9±0.5 ng／μg，間接共培養(yǎng)組與對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P＜0.05)；韌粘素-C mRNA的表達(dá)，為對(duì)照組的2.0倍，對(duì)照組和間接共培養(yǎng)組的相對(duì)表達(dá)量分別為0.07±0.02和0.14±0.02。 4．力學(xué)刺激使細(xì)胞胞體較對(duì)照組變細(xì)長(zhǎng)，細(xì)胞重新定向排列在遠(yuǎn)離牽張力方向60～80°和100～130°這兩個(gè)以90°軸對(duì)稱的區(qū)域。力學(xué)刺激12小時(shí)后，BMSCsⅠ型膠原和Ⅲ型膠原mRNA的表達(dá)與對(duì)照組相比增高有顯著差異(P＜0.05)；24小時(shí)后，兩種膠原蛋白的合成有統(tǒng)計(jì)學(xué)差別。韌粘素-C mRNA的表達(dá)在力學(xué)作用24h和36h后，分別增高到對(duì)照組的2.65±0.03和2.89±0.04倍；細(xì)胞骨架在力學(xué)刺激作用下亦發(fā)生了改變，F(xiàn)-actin隨牽張時(shí)間增長(zhǎng)，含量逐漸減少。 結(jié)論 1．我們分離獲得的rBMSCs和hBMSCs細(xì)胞具有向成骨細(xì)胞和脂肪細(xì)胞分化的能力，且它們表面特征分子和膠原蛋白，在第一代到第六代的細(xì)胞中穩(wěn)定表達(dá)。 2．Ⅰ型膠原、Ⅲ型膠原和韌粘素-C mRNA在第一代到第六代BMSCs中穩(wěn)定表達(dá)。 3．與韌帶成纖維細(xì)胞間接共培養(yǎng)，可以促進(jìn)BMSCsⅠ型、Ⅲ型膠原蛋白和韌粘素-C的合成。 4．力學(xué)刺激可以促進(jìn)BMSCs合成Ⅰ型膠原、Ⅲ型膠原和韌粘素-CmRNA的表達(dá)及Ⅰ型和Ⅲ型膠原蛋白的合成，使細(xì)胞重排，細(xì)胞骨架發(fā)生改變。
[Abstract]:Study Background The ligament plays a role in maintaining the stability of the joint and coordinating the joint movement. It is important to play an important role in the injury of the ligament, which can cause the damage of the ligament, but due to the small blood supply of the ligament, it is difficult to completely heal after the injury, and finally, the joint function is serious. The case of the disorder. The injury of the ligament also causes many excellent athletes to be buried in this place The development of modern surgery has made it a reality to replace the disease-damaged ligament, and the surgical-used replacement includes the different ligament, the allogenic ligament, the autograft and the synthetic material, but the long-term effect of these measures It is not ideal. The birth of tissue engineering makes it possible to build an active biological ligament in vitro and to treat the disease. The invention provides a new way cell component, which is used for improving the bearing force of the mechanical load of the ligament, the gene activation and the ligament self more, The ability to new and adjust is very important. How to select the ideal seed cells? What micro-environment is needed for seed cells? In order to meet the expected functional requirements? Some questions need to be further studied. The purpose of this test is to explore how in vitro conditions BMSCs were induced to differentiate into the ligament fibroblasts. Therefore, in this experiment we used two methods of indirect co-culture and mechanical stimulation with the ligament fibroblasts, and the transformation of BMSCs into the ligament fibroblasts under the condition of in vitro was studied, and the BMSCs were induced to induce BMSCs in vitro. to provide a viable method for the transformation of the ligament fibroblasts, band-organized man Methods 1. The method of study was provided for the study of seed cells.1. The bone marrow-derived mesenchymal cells were obtained by the combination of Percoll liquid density gradient centrifugation and passage-attached screening method, and the in vitro culture of BMSCs (rBMSCs) and human BMSCs (hBMSCs) in rats was established. The method comprises the following steps of: carrying out identification on the rBMSCs and hBMSCs cultured in vitro by using a morphological method and a cell surface specific antigen flow cytometry detection method; and inducing the rBMSCs and hBMSCs into the osteoblast and the hBMSCs in vitro. the biological function of the stem cells of the BMSCs is identified by the method of the differentiation of the adipocyte,2. the real-time fluorescence quantitative PCR detection first generation to the second generation 3,6 and 12 days of BMSCs and the rat's ligament fibroblasts were co-cultured for 3,6 and 12 days, and the three kinds of ligament characteristic proteins of BMSCs were detected by real-time fluorescence quantitative PCR (RT-PCR) and indirect co-culture. Type I collagen, type III collagen egg 4. The expression of the mRNA of BMSCs in the rat and the method of immunohistochemistry was used to detect the synthesis of the three proteins.4. After 10% and 1-Hz periodic stretch of BMSCs were applied to the rat BMSCs for different periods, the deformation and rearrangement of the cells were analyzed. The Expression of Type I, Type 鈪

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