【摘要】：目的 關(guān)節(jié)軟骨容易發(fā)生損傷和退變,再生能力差,對損傷軟骨的實(shí)驗(yàn)治療主要集中在細(xì)胞的治療上,包括應(yīng)用成熟軟骨細(xì)胞及其他軟骨祖細(xì)胞。骨髓間充質(zhì)干細(xì)胞(MSCs)作為成熟軟骨細(xì)胞的替代品越來越受到重視。MSCs可以很容易從骨髓中獲得,增殖傳代能力強(qiáng),細(xì)胞均一性好,具多向分化潛能,能分化為各種類型組織,包括軟骨和骨。但轉(zhuǎn)移的MSCs在軟骨缺損部位還沒有產(chǎn)生令人滿意的關(guān)節(jié)軟骨。一個(gè)可能的問題是局部沒有足夠能刺激移植細(xì)胞分化的細(xì)胞因子。體外研究報(bào)告顯示,特異的蛋白因子能夠促進(jìn)成人MSCs向軟骨細(xì)胞分化,提高體內(nèi)軟骨修復(fù)能力。相關(guān)的研究表明,TGF-β1,TGF-β2,TGF-β3,FGF,BMP-2,BMP-6,IGF-1等生長因子具有成軟骨能力,因此可以在局部加入這些因子促進(jìn)MSCs細(xì)胞向軟骨細(xì)胞分化。但外源性細(xì)胞因子易于流失、作用時(shí)間短、價(jià)格昂貴。如果將這些細(xì)胞因子基因?qū)隡SCs中,通過自分泌相應(yīng)細(xì)胞因子誘導(dǎo)MSCs向軟骨細(xì)胞分化,以此基因修飾的MSCs作為種子細(xì)胞來構(gòu)建組織工程軟骨,用于軟骨缺損和退變治療具有重要意義。 目前,應(yīng)用IGF-1基因轉(zhuǎn)染MSCs國內(nèi)外報(bào)道較少。為研究通過IGF-1基因轉(zhuǎn)染來促使MSCs向軟骨細(xì)胞分化,我們采用密度梯度離心法,與貼壁培養(yǎng)法相結(jié)合,對存在于骨髓中的間充質(zhì)干細(xì)胞進(jìn)行分離、純化和體外培養(yǎng)擴(kuò)增,并對其生物學(xué)特性進(jìn)行初步研究。通過構(gòu)建含有GFP和IGF-1基因的共表達(dá)載體,將IGF-1轉(zhuǎn)入MSCs,使MSCs向軟骨細(xì)胞分化,通過Ⅱ型膠原測定來判斷是否穩(wěn)定表達(dá),以期為開展IGF-1基因治療軟骨缺損的研究奠定基礎(chǔ)。 方法 (一)兔骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及其生物學(xué)活性觀察
[Abstract]:Objective the articular cartilage is prone to damage and degeneration, and its regeneration ability is poor. The experimental treatment of damaged cartilage is mainly focused on the cell therapy, including the application of mature chondrocytes and other chondroprogenitor cells. As a substitute for mature chondrocytes, bone marrow mesenchymal stem cells (BMSCs) have attracted more and more attention. MSCs can be easily obtained from bone marrow. Can differentiate into various types of tissue, including cartilage and bone. But the metastatic MSCs did not produce satisfactory articular cartilage at the site of cartilage defect. One possible problem is that there are not enough cytokines locally to stimulate the differentiation of transplanted cells. In vitro studies show that specific protein factors can promote the differentiation of adult MSCs into chondrocytes and improve the ability of cartilage repair in vivo. Related studies have shown that TGF- 尾 1 TGF- 尾 2 and TGF- 尾 3 FGF- 尾 3 FGF- 2 BMP-6 IGF-1 have the ability to form cartilage, so they can be added locally to promote the differentiation of MSCs cells into chondrocytes. But exogenous cytokines are easy to lose, short time and expensive. If these cytokine genes were introduced into MSCs, MSCs was induced to differentiate into chondrocytes by autocrine, and MSCs modified with these genes was used as seed cells to construct tissue engineered cartilage. It is of great significance to treat cartilage defect and degeneration. At present, there are few reports about IGF-1 gene transfection into MSCs at home and abroad. In order to study the differentiation of MSCs into chondrocytes by IGF-1 gene transfection, we used density gradient centrifugation, combined with adherent culture, to isolate, purify and amplify mesenchymal stem cells in bone marrow. And its biological characteristics were preliminarily studied. By constructing a co-expression vector containing GFP and IGF-1 genes, IGF-1 was transferred into MSCs, to differentiate MSCs into chondrocytes, and type 鈪，

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