本文選題：甲狀旁腺激素相關(guān)蛋白 + BM-MSCs��； 參考：《第三軍醫(yī)大學(xué)》2013年博士論文

【摘要】：背景： 近年來，由于軟骨組織退行性改變和缺損而引起的諸如腰椎間盤突出癥、退行性骨關(guān)節(jié)炎等在內(nèi)的退行性骨關(guān)節(jié)疾病越來越多。關(guān)節(jié)軟骨相關(guān)的退化性關(guān)節(jié)疾病嚴(yán)重影響超過三分之一的世界人口的關(guān)節(jié)健康。雖然治療軟骨組織缺損的外科技術(shù)已得到廣泛的研究和發(fā)展，但是現(xiàn)存的治療手段中沒有任何一種能從根本上治療軟骨組織缺損。因此，優(yōu)化關(guān)節(jié)軟骨病變的治療策略具有很高的社會(huì)和經(jīng)濟(jì)重要性。隨著組織工程技術(shù)在骨科領(lǐng)域相關(guān)研究的不斷深入和發(fā)展，為骨科退行性相關(guān)疾病的治療開辟出一條嶄新的治療途徑。 目前采用組織工程學(xué)技術(shù)構(gòu)建的椎間盤替代物研究尚處于起步階段，研究主要集中于支架材料、種子細(xì)胞和生物活性因子三個(gè)方面。骨髓間充質(zhì)干細(xì)胞(Bone marrowmesenchymal stem cells，BM-MSCs)作為一種常用的種子細(xì)胞，已廣泛應(yīng)用于組織工程修復(fù)中。為克服組織工程中種子細(xì)胞不斷衰減，傳代后種子細(xì)胞老化退變，退變的種子細(xì)胞生物學(xué)功能下降等問題，大量的外源性生長因子加入到支架材料中，以維持種子細(xì)胞的生物學(xué)功能。但所添加的生長因子由于不具有靶向結(jié)合支架材料的能力，不能很好滯留在支架材料中發(fā)揮生物學(xué)功能，從而不能對種子細(xì)胞起到持續(xù)性的促進(jìn)作用。膠原蛋白，尤其是I型膠原蛋白是目前最常用的組織工程支架材料，因此I型膠原是組織工程材料靶向性的重要靶點(diǎn)。 甲狀旁腺激素相關(guān)蛋白(Parathyroid hormone related protein，PTHrP)是1987年從惡性高血鈣腫瘤患者的腫瘤中提取純化的。它是由141個(gè)氯基酸組成，其N-端PTHrP(1-34)與N-端甲狀旁腺激素(N-PTH)氨基酸序列具有很高的相同性。PTHrP的生理活性除了類似N-PTH外，還具有其他的生物功能，如促進(jìn)鈣的傳遞，抑制骨的重吸收，促進(jìn)骨髓間充質(zhì)干細(xì)胞成軟骨分化，同時(shí)抑制軟骨細(xì)胞老化。同時(shí)，膠原結(jié)合域(collagen-binding domain, CBD)是由七個(gè)氨基酸構(gòu)成的寡肽序列(TKKTLRT)組成，具有與膠原緊密結(jié)合的功能，被廣泛用于膠原結(jié)合相關(guān)活性分子的重組表達(dá)中。 目的： 在本實(shí)驗(yàn)中，我們利用基因重組技術(shù)構(gòu)建了新型生物活性重組蛋白CBD-PTHrP，該重組蛋白具有雙重的生物學(xué)功能：一方面可以通過其中的PTHrP成分發(fā)揮誘導(dǎo)骨髓間充質(zhì)干細(xì)胞成軟骨分化和抑制軟骨細(xì)胞肥大的生物學(xué)功能，另一方面該融合蛋白又能夠通過CBD序列的偶聯(lián)，將PTHrP錨定在膠原蛋白支架材料上，從而使PTHrP能對種子細(xì)胞起到持續(xù)靶向性的作用。因此，利用基因重組技術(shù)構(gòu)建新型生物活性因子解決種子細(xì)胞在實(shí)驗(yàn)中易老化退變的問題能為構(gòu)建新型軟骨組織工程替代材料奠定基礎(chǔ)。 方法： 1、CBD-PTHrP重組融合蛋白的表達(dá)、純化和膠原靶向性結(jié)合能力鑒定 將CBD的編碼序列構(gòu)建到引物兩端，以cDNA為模板利用PCR的方法獲得編碼CBD-PTHrP和NAT-PTHrP兩種融合蛋白的基因序列，然后連接到pET-32a原核表達(dá)載體中。轉(zhuǎn)化TOP10感受態(tài)細(xì)胞后篩選鑒定出陽性克隆，然后提取質(zhì)粒轉(zhuǎn)化Rosetta(DE3)感受態(tài)細(xì)胞，利用IPTG誘導(dǎo)CBD-PTHrP和NAT-PTHrP兩種融合蛋白表達(dá)，經(jīng)Ni柱純化得到目的蛋白。將濃度為0,0.5,1,2,4,8,12,16μm的融合蛋白添加到鋪有膠原的96孔板中，利用類ELISA的方法檢測兩種融合蛋白對膠原的靶向結(jié)合能力。 2、人源骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)和傳代鑒定 抽取人骨髓，利用密度梯度離心法分離得到骨髓間充質(zhì)干細(xì)胞，然后利用DMEM/F12+10%FBS培養(yǎng)基進(jìn)行培養(yǎng)，在細(xì)胞傳至三代時(shí)，收取細(xì)胞利用流式細(xì)胞術(shù)對細(xì)胞表面抗原：CD34、CD44、CD45、CD73、CD90、CD105進(jìn)行檢測，對所分BM-MSCs的分子標(biāo)記進(jìn)行鑒定。 3、CBD-PTHrP促BM-MSCs向軟骨細(xì)胞分化和抑制軟骨細(xì)胞老化的實(shí)驗(yàn)研究 收集培養(yǎng)的BM-MSCs細(xì)胞，制成細(xì)胞小球后利用成軟骨細(xì)胞誘導(dǎo)培養(yǎng)基進(jìn)行培養(yǎng)，每2-3天換液一次，兩周后用100ng/ml的NAT-PTHrP和CBD-PTHrP蛋白分別處理，并設(shè)空白對照組，再誘導(dǎo)培養(yǎng)兩周后，收集細(xì)胞小球，分別提取RNA和總蛋白，利用Real-time PCR和Western blot檢測COL1A1、COL2A1、COL10A1和Sox-9在mRNA和蛋白水平上的表達(dá)情況，細(xì)胞小球石蠟包埋切片后，利用番紅-O染色檢測軟骨形成情況，免疫組織化學(xué)染色檢測COL1A1、COL2A1、COL10A1和Sox-9在細(xì)胞小球組織中的表達(dá)情況。 結(jié)果： 1、構(gòu)建了pET-32a-NAT-PTHrP和pET-32a-CBD-PTHrP重組質(zhì)粒，，并誘導(dǎo)表達(dá)和純化得到了CBD-PTHrP和NAT-PTHrP兩種融合蛋白。ELISA檢測405nm吸光度結(jié)果提示：相比于NAT-PTHrP蛋白，CBD-PTHrP蛋白在膠原材料上的殘留量更多，具有更強(qiáng)的膠原結(jié)合能力。 2、分離得到了原代人BM-MSCs，免疫標(biāo)記的鑒定結(jié)果為CD34和CD45陰性，排除了所分細(xì)胞為造血細(xì)胞和內(nèi)皮細(xì)胞的可能性；同時(shí)CD44、CD73、CD90和CD105表面抗原為陽性，明確了所分細(xì)胞為BM-MSCs。 3、 Real-time PCR和Western blot的結(jié)果顯示，相比于空白對照組，CBD-PTHrP和NAT-PTHrP蛋白都能顯著的誘導(dǎo)COL2A1和Sox-9基因在mRNA和蛋白水平的表達(dá)，有效的抑制COL1A1和COL10A1基因在mRNA和蛋白水平的表達(dá)。番紅-O染色表明相比于空白對照組，添加CBD-PTHrP和NAT-PTHrP兩種融合蛋白進(jìn)行誘導(dǎo)的細(xì)胞小球中有更多的軟骨細(xì)胞形成。免疫組織化學(xué)的檢測結(jié)果也表明CBD-PTHrP和NAT-PTHrP都能誘導(dǎo)COL2A1和Sox-9蛋白的表達(dá)和抑制COL1A1和COL10A1蛋白的表達(dá)，這與Real-time PCR和Western blot的實(shí)驗(yàn)結(jié)果是一致的。 結(jié)論： 相比于NAT-PTHrP蛋白，在CBD短肽的作用下，CBD-PTHrP蛋白能更好的錨定在I型膠原上，提示CBD-PTHrP蛋白在動(dòng)物和體外水平上都能更好的結(jié)合到膠原支架材料上，對其中的種子細(xì)胞起到持續(xù)的誘導(dǎo)作用。同時(shí)，CBD-PTHrP蛋白具有與NAT-PTHrP蛋白一樣的促進(jìn)BM-MSCs向軟骨細(xì)胞分化的生物學(xué)作用，并能夠有效的抑制軟骨細(xì)胞的肥大，提示在PTHrP的N端加上CBD短肽對其生物學(xué)活性沒有任何影響，使得PTHrP能通過CBD短肽持續(xù)的錨定在膠原支架上，進(jìn)而持續(xù)的誘導(dǎo)膠原支架中的BM-MSCs向軟骨細(xì)胞分化和抑制軟骨細(xì)胞肥大。以上實(shí)驗(yàn)結(jié)果為CBD-PTHrP應(yīng)用到基于BM-MSCs的軟骨組織工程中奠定了堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Background:
Degenerative osteoarthrosis, such as protrusion of lumbar intervertebral disc and degenerative osteoarthritis, is increasing in recent years. Degenerative joint diseases related to articular cartilage seriously affect the joint health of more than 1/3 of the world's population. Although the treatment of cartilage defects is treated Surgical techniques have been widely studied and developed, but none of the existing treatments can fundamentally treat cartilage defects. Therefore, the treatment strategy for the optimization of articular cartilage lesions is of high social and economic importance. The treatment of degenerative diseases in Department of orthopedics opens up a new way of treatment.
Bone marrowmesenchymal stem cells (BM-MSCs), a common seed cell, has been widely used in tissue engineering as a common seed cell, which is mainly focused on three aspects of scaffold materials, seed cells and bioactive factors. In order to overcome the problems of continuous attenuation of seed cells in tissue engineering, aging and degeneration of seed cells after generation, degradation of biological function of degenerative seed cells, a large number of exogenous growth factors are added to the scaffold materials to maintain the biological function of seed cells, but the added growth factors do not have target binding branches. The ability of the scaffold can not be well retained in the scaffold material to play biological functions, which can not promote the continuous promotion of seed cells. Collagen, especially type I collagen, is the most commonly used scaffold material for tissue engineering, so I collagen is an important target for the targeting of tissue engineering materials.
The parathyroid hormone related protein (Parathyroid hormone related protein, PTHrP) is extracted from a malignant tumor of hypercalcemia in 1987. It is composed of 141 chloroacids. The N- terminal PTHrP (1-34) is highly identical to the N- terminal parathyroid hormone (N-PTH) amino acid sequence. The physiological activity of.PTHrP is similar to that of N-P. TH also has other biological functions, such as promoting the transfer of calcium, inhibiting the reabsorption of bone, promoting the differentiation of bone marrow mesenchymal stem cells into chondrocytes and inhibiting the aging of cartilage cells. At the same time, the collagen binding domain (collagen-binding domain, CBD) is a oligopeptide sequence composed of seven amino acids (TKKTLRT), which is closely associated with collagen. Functions are widely used in recombinant expression of collagen binding related active molecules.
Objective:
In this experiment, we constructed a new bioactive recombinant protein CBD-PTHrP using gene recombination technology, which has dual biological functions: on one hand, it can play the biological function of inducing chondrodifferentiation of bone marrow mesenchymal stem cells and inhibiting chondrocyte hypertrophy by its PTHrP components. On the other hand, the biological function of the recombinant protein can be induced. The protein can also be anchored to the collagen scaffold material by the coupling of the CBD sequence, which can enable the PTHrP to be targeted to the seed cells. Therefore, it is possible to construct a new type of cartilage tissue engineering by constructing a new bioactive factor using gene recombination technology to solve the problem that the seed cells can easily degenerate in the experiment. It lays the foundation for alternative materials.
Method錛

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