本文關(guān)鍵詞：構(gòu)建組織工程軟骨中機(jī)械刺激的作用研究　出處：《浙江大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 軟骨組織工程 機(jī)械刺激 軟骨細(xì)胞 骨髓間充質(zhì)干細(xì)胞 定向分化 TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路 纖維蛋白凝膠——聚氨酯支架復(fù)合物

【摘要】：機(jī)械刺激是軟骨組織工程中非常重要的調(diào)控因子。本文以軟骨細(xì)胞和骨髓間充質(zhì)干細(xì)胞為種子細(xì)胞,研究了機(jī)械刺激在關(guān)節(jié)軟骨組織工程中的作用。 首先研究了機(jī)械刺激對(duì)牛膝關(guān)節(jié)不同軟骨細(xì)胞亞群在PRG4、HAS1、HAS2mRNA表達(dá)和PRG4、透明質(zhì)酸蛋白質(zhì)合成方面的影響。發(fā)現(xiàn)機(jī)械刺激可以誘導(dǎo)軟骨細(xì)胞亞群發(fā)生顯型的改變以適應(yīng)其環(huán)境。軟骨細(xì)胞各個(gè)亞群之間的相互作用對(duì)透明質(zhì)酸的合成有促進(jìn)作用。使用混合亞群的軟骨細(xì)胞,在最佳的機(jī)械刺激條件和生物化學(xué)條件下,可以誘導(dǎo)復(fù)合物表層的細(xì)胞重新分化具有淺表區(qū)細(xì)胞顯型,從而構(gòu)建出較完整的關(guān)節(jié)軟骨層次結(jié)構(gòu)和功能性的軟骨滑液界面。這對(duì)于通常以來(lái)自整個(gè)關(guān)節(jié)的各軟骨細(xì)胞亞群混合細(xì)胞作為種子細(xì)胞的組織工程有重要的意義。 由于軟骨細(xì)胞作為種子細(xì)胞存在來(lái)源不足,獲取手段復(fù)雜且對(duì)患者損傷較大的缺點(diǎn),繼而研究了以hBMSCs為種子細(xì)胞,體外構(gòu)建軟骨組織過(guò)程中機(jī)械刺激的作用。為了建立體外hBMSCs成軟骨細(xì)胞培養(yǎng)體系,研究了纖維蛋白凝膠——聚氨酯支架體系是否支持體外hBMSCs向軟骨細(xì)胞的誘導(dǎo)分化。通過(guò)測(cè)量細(xì)胞的GAG/DNA值,以Real-time PCR測(cè)量細(xì)胞的mRNA水平,和組織學(xué)及免疫生物化學(xué)染色方法,對(duì)支架培養(yǎng)和微團(tuán)培養(yǎng)進(jìn)行了比較。實(shí)驗(yàn)結(jié)果表明與微團(tuán)培養(yǎng)體系相比,在最佳接種密度(5x106個(gè)細(xì)胞／支架)下,細(xì)胞支架復(fù)合物具有相似的軟骨細(xì)胞標(biāo)志性蛋白合成量GAG/DNA,較高的軟骨細(xì)胞標(biāo)志性基因COL2、AGG mRNA水平,和較低的成骨細(xì)胞標(biāo)志性基因Sp7、ALP、BSP2 mRNA水平,同時(shí)還具有能夠?yàn)樾律M織的積聚提供空間的優(yōu)點(diǎn),有利于進(jìn)一步使用生物力學(xué)的手段優(yōu)化體外干細(xì)胞向軟骨細(xì)胞的誘導(dǎo)分化。 在不同的外源TGF-βl濃度下,研究了機(jī)械刺激對(duì)hBMSCs分化過(guò)程的影響,并使用TGF-βⅠ型受體抑制劑LY364947阻斷TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路,研究了TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路在該過(guò)程中的作用。實(shí)驗(yàn)結(jié)果表明機(jī)械剌激可以誘導(dǎo)纖維蛋白凝膠——聚氨酯支架培養(yǎng)的hBMSCs向軟骨細(xì)胞分化,前提條件是外源TGF-β濃度較低且培養(yǎng)液中含有地塞米松。由于TGF-β本身可誘導(dǎo)hBMSCs成軟骨細(xì)胞,過(guò)高的外源TGF-β濃度下機(jī)械刺激的作用會(huì)被覆蓋。機(jī)械刺激通過(guò)TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路誘導(dǎo)hBMSCs向軟骨細(xì)胞分化,TGF-β受體抑制劑可以阻斷TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路從而抑制機(jī)械刺激誘導(dǎo)干細(xì)胞向軟骨細(xì)胞分化的作用。 對(duì)hBMSCs生物支架復(fù)合物施加不同強(qiáng)度和頻率的循環(huán)動(dòng)壓力和表面剪切摩擦力,以觀察不同力學(xué)環(huán)境下hBMSCs的反應(yīng)。發(fā)現(xiàn)機(jī)械刺激的頻率和幅度可以調(diào)控hBMSCs向軟骨細(xì)胞分化的過(guò)程。在本文所采用的機(jī)械刺激范圍內(nèi),較高的機(jī)械刺激頻率和較高的動(dòng)壓力幅度更有利于hBMSCs向軟骨細(xì)胞分化,更有利于抑制軟骨細(xì)胞肥大化。由此說(shuō)明在以BMSCs為種子細(xì)胞的軟骨組織工程研究中,對(duì)細(xì)胞支架復(fù)合物施加適當(dāng)強(qiáng)度的機(jī)械刺激非常重要。 將視黃酸β受體抑制劑LE135與機(jī)械刺激相結(jié)合,研究其對(duì)hBMSCs分化過(guò)程的影響。發(fā)現(xiàn)在微團(tuán)培養(yǎng)和支架培養(yǎng)體系中,視黃酸β受體抑制劑LE135對(duì)外源TGF-β和機(jī)械刺激誘導(dǎo)的hBMSCs向軟骨細(xì)胞分化的過(guò)程有抑制作用。同樣的條件下,LE135對(duì)hBMSCs向成骨細(xì)胞分化的過(guò)程沒(méi)有影響。
[Abstract]:Mechanical stimulation is a very important regulatory factor in cartilage tissue engineering. In this paper, chondrocytes and bone marrow mesenchymal stem cells were used as seed cells, and the role of mechanical stimulation in articular cartilage tissue engineering was studied.
The first study of mechanical stimulation on knee joints of different cartilage cell subsets in PRG4, HAS1, HAS2mRNA and PRG4 expression, affect the synthesis of hyaluronic acid protein. It is found that mechanical stimulation can induce changes of chondrocyte subpopulations phenotype to adapt to its environment. The cartilage cell subpopulations interaction has a promoting effect on the synthesis of hyaluronic acid. The use of mixed cartilage cell subsets, in the best mechanical and chemical conditions, can induce the complex surface of the cell differentiation with superficial cell phenotype, in order to build a more complete articular cartilage structure and function of cartilage synovial fluid interface. This usually takes for the cartilage cell subsets of mixed cells from the entire joint as seed cells in tissue engineering has important significance.
The chondrocytes as seed cell sources exist insufficient means of acquiring complex and larger defects injury patients, then studied with hBMSCs as seed cells, effects of mechanical stimulation of cartilage tissue in vitro. In the process of building a hBMSCs in order to establish in vitro chondrogenic culture system of fibrin gel - polyurethane stent system supports differentiation in vitro hBMSCs to chondrocytes. By measuring cellular GAG/DNA, Real-time PCR mRNA to measure the cell level, staining and histological and immunological biochemistry, to support training and micromass culture were compared. The experimental results show that compared with the micromass culture system, the optimum inoculation density (5x106 cells / scaffold), cell scaffold composite has the sign of similar GAG/DNA protein synthesis of cartilage cells, cartilage cell marker COL2 high, AGG MRNA level and low osteoblast marker genes Sp7, ALP, BSP2 mRNA level, at the same time, it also has the advantages of providing space for the accumulation of new tissue, which is conducive to further optimize the differentiation of stem cells into chondrocytes in vitro.
In different concentrations of exogenous TGF- beta L, studied the mechanical stimulation effect on the differentiation of hBMSCs, and use the TGF- receptor type inhibitor LY364947 beta TGF- signaling pathway, on the role of TGF- beta signaling pathway in this process. The experimental results show that the mechanical stimulating induced by fibrin gel - polyurethane scaffold hBMSCs chondrogenic differentiation, is a prerequisite for exogenous TGF- beta concentration was low and medium containing dexamethasone. Because TGF- beta itself can chondrogenic hBMSCs, effects of mechanical stimulation of high concentration of exogenous TGF- beta would be covered. HBMSCs were induced to chondrocytes by TGF- B signal transduction machinery stimulation of TGF- beta receptor inhibitor can inhibit the TGF- beta signaling pathways to inhibit the mechanical stimulation induced stem cells differentiating into chondrocytes.
Cyclic shear and friction surface pressure applied with different intensity and frequency on hBMSCs biological scaffold, to observe the different mechanical environment hBMSCs reaction. Found that the frequency and amplitude of mechanical stimulation can regulate the differentiation of hBMSCs into chondrocytes. This paper used by the mechanical stimulus range, high frequency and high mechanical stimulation the dynamic pressure amplitude is more conducive to the differentiation of hBMSCs into chondrocytes, more conducive to inhibit chondrocyte hypertrophy. Therefore in the BMSCs as the seed cells in cartilage tissue engineering, applying the appropriate strength on the cell scaffold composite mechanical stimulation is very important.
The retinoic acid receptor inhibitor LE135 and the combination of mechanical stimulation, to study its effect on the differentiation of hBMSCs. Found in micromass culture and culture support system, the process of retinoic acid receptor inhibitor LE135 on induced by exogenous TGF- beta and mechanical hBMSCs chondrogenic differentiation was inhibited. The same condition under the influence of LE135 on hBMSCs differentiation of osteoblasts from there.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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