【摘要】： 研究背景 韌帶在維持關節(jié)穩(wěn)定，協(xié)調關節(jié)運動中發(fā)揮著重要作用。外傷、長期反復勞累過度等會造成韌帶損傷，但由于韌帶組織血供少，損傷后很難完全愈合，最終造成關節(jié)功能嚴重障礙的病例。韌帶損傷也使得很多優(yōu)秀的運動員就此葬送了運動生涯。現(xiàn)代外科的發(fā)展已使人類替換病損韌帶成為現(xiàn)實，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，但這些措施的遠期效果都不夠理想。組織工程學的誕生使體外構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 細胞成分對提高韌帶的力學負載的承受力，基因激活和韌帶自我更新和調節(jié)的能力非常重要。但如何選擇理想的種子細胞?種子細胞需要怎樣的微環(huán)境才能達到預期的功能要求?這些問題都有待進一步研究。 本試驗的目的是：探索在體外條件下如何誘導BMSCs向韌帶成纖維細胞分化。因此，本實驗中我們采用與韌帶成纖維細胞間接共培養(yǎng)和力學刺激兩種方法，研究體外條件下誘導BMSCs向韌帶成纖維細胞轉化，試圖為體外誘導BMSCs向韌帶成纖維細胞轉化提供可行的方法，為韌帶組織工程學種子細胞的研究提供思路。 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合分離獲得骨髓間充質細胞，建立大鼠BMSCs(rBMSCs)和人BMSCs(hBMSCs)體外培養(yǎng)的方法；通過形態(tài)學方法與細胞表面特異抗原流式細胞術檢測方法，對在體外所培養(yǎng)的rBMSCs和hBMSCs進行鑒定；采用體外誘導rBMSCs和hBMSCs向成骨細胞和成脂細胞分化的方法，對BMSCs的干細胞生物學功能進行鑒定。 2．實時熒光定量PCR檢測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。 3．將大鼠BMSCs和大鼠韌帶成纖維細胞間接共培養(yǎng)3、6、12天，用實時熒光定量PCR檢測，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；競爭放免法和免疫組化的方法，檢測細胞這三種蛋白合成。 4．對大鼠BMSCs施以10％，1赫茲的周期性牽張刺激不同時段后，分析細胞的變形和重排；并用實時熒光定量PCR檢測牽張不同時間段后，其Ⅰ型、Ⅲ型膠原和韌粘素-C mRNA的表達；用競爭放免法和免疫組化，檢測細胞這三種蛋白合成；激光共聚焦顯微鏡觀察力學刺激對細胞骨架的影響。 實驗結果 1．成功獲得了rBMSCs和hBMSCs兩種干細胞。我們獲得的細胞具有干細胞的形態(tài)學特征(核大漿少)；細胞表面特異性抗原鑒定(rBMSCs CD44、CD90表達陽性而CD34、CD45表達陰性，hBMSCs CD44表達陽性CD14、CD45表達陰性)，且這些特征在第一代到第六代的細胞中穩(wěn)定；rBMSCs和hBMSCs這兩種細胞具有向成骨細胞和脂肪細胞分化的能力。綜合上述三個特點，我們認為實驗中分離培養(yǎng)的細胞為骨髓間充質干細胞。 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。 3．間接共培養(yǎng)6天，BMSCsⅠ型膠原和Ⅲ型膠原的mRNA表達分別是對照組的2.0和2.2倍，Ⅰ型膠原和Ⅲ型膠原mRNA與內參照GAPDH的相對表達量在間接共培養(yǎng)6天組分別為：3.9±0.2和1.9±0.2，對照組分別為：1.9±0.3和0.8±0.1，間接共培養(yǎng)組與對照組相比有統(tǒng)計學差異(P＜0.05)而韌粘素-C的表達無明顯改變。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)組與對照組相比有統(tǒng)計學差異(P＜0.05)；韌粘素-C mRNA的表達，為對照組的2.0倍，對照組和間接共培養(yǎng)組的相對表達量分別為0.07±0.02和0.14±0.02。 4．力學刺激使細胞胞體較對照組變細長，細胞重新定向排列在遠離牽張力方向60～80°和100～130°這兩個以90°軸對稱的區(qū)域。力學刺激12小時后，BMSCsⅠ型膠原和Ⅲ型膠原mRNA的表達與對照組相比增高有顯著差異(P＜0.05)；24小時后，兩種膠原蛋白的合成有統(tǒng)計學差別。韌粘素-C mRNA的表達在力學作用24h和36h后，分別增高到對照組的2.65±0.03和2.89±0.04倍；細胞骨架在力學刺激作用下亦發(fā)生了改變，F(xiàn)-actin隨牽張時間增長，含量逐漸減少。 結論 1．我們分離獲得的rBMSCs和hBMSCs細胞具有向成骨細胞和脂肪細胞分化的能力，且它們表面特征分子和膠原蛋白，在第一代到第六代的細胞中穩(wěn)定表達。 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。 3．與韌帶成纖維細胞間接共培養(yǎng)，可以促進BMSCsⅠ型、Ⅲ型膠原蛋白和韌粘素-C的合成。 4．力學刺激可以促進BMSCs合成Ⅰ型膠原、Ⅲ型膠原和韌粘素-CmRNA的表達及Ⅰ型和Ⅲ型膠原蛋白的合成，使細胞重排，細胞骨架發(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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