本文選題：超分子水凝膠　切入點(diǎn)：組織工程　出處：《南京大學(xué)》2017年碩士論文

【摘要】：背景:椎間盤退變性疾病是臨床的常見疾病,目前的臨床治療方法無法達(dá)到在結(jié)構(gòu)和功能層面上修復(fù)病變椎間盤。髓核組織工程運(yùn)用具備良好功能的髓核種子細(xì)胞來替代已經(jīng)發(fā)生退變的髓核細(xì)胞,由此恢復(fù)正常的髓核生理構(gòu)造和功能,逆轉(zhuǎn)椎間盤退變過程,成為了治療椎間盤退變的一個(gè)研究方向。在髓核組織工程研究中,細(xì)胞支架對種子細(xì)胞的活性和功能表達(dá)至關(guān)重要。目前大多選用天然材料水凝膠或者是人工合成的高分子聚合物水凝膠,這兩類材料均有其自身的缺點(diǎn)。隨著材料化學(xué)的發(fā)展,一類以小分子寡肽為基本單位的超分子水凝膠因?yàn)榫邆鋬?yōu)秀的生物相容性,良好的力學(xué)性能和近似細(xì)胞外基質(zhì)的結(jié)構(gòu)有望成為用于構(gòu)建髓核組織工程細(xì)胞支架的新的選擇。目的:探究以寡肽(Nap-F-F-Y(P))為基本單位復(fù)合硫酸軟骨素的超分子水凝膠用于構(gòu)建髓核組織工程細(xì)胞支架的可行性。方法:在化學(xué)實(shí)驗(yàn)室運(yùn)用固相合成法合成寡肽(Nap-F-F-Y(P)),質(zhì)譜分析鑒定產(chǎn)物。在堿性磷酸酶作用下發(fā)生自組裝并與硫酸軟骨素復(fù)合成膠,探討不同濃度水凝膠的成膠時(shí)間,宏觀形態(tài),微觀結(jié)構(gòu)以及力學(xué)性質(zhì)等材料學(xué)表征。取12周齡大鼠尾椎椎間盤髓核細(xì)胞原代培養(yǎng)作為種子細(xì)胞,超分子水凝膠復(fù)合髓核細(xì)胞共培養(yǎng)兩周,以不加入水凝膠的普通培養(yǎng)為對照,行細(xì)胞活死染色,CKK-8檢測觀察髓核細(xì)胞活性,細(xì)胞免疫熒光染色觀察髓核細(xì)胞特征表型Ⅱ型膠原及聚集蛋白聚糖(Aggrecan)的表達(dá)情況。結(jié)果:合成的寡肽經(jīng)質(zhì)譜分析后證明結(jié)構(gòu)分子量正確,產(chǎn)物純度較高,可進(jìn)一步運(yùn)用于自組裝成膠,其成膠濃度范圍為0.5%(w/v)至1.5%(w/v),肉眼可見凝膠透明度隨濃度增高而降低,寡肽濃度為1%時(shí),成膠速度最快,宏觀形態(tài)結(jié)構(gòu)最穩(wěn)定。透射電鏡觀察水凝膠微觀結(jié)構(gòu)可見納米纖維排列縱橫交錯(cuò),形成納米級別的孔隙,組成了凝膠內(nèi)部疏松多孔結(jié)構(gòu)。水凝膠應(yīng)變及頻率動態(tài)掃描發(fā)現(xiàn)其力學(xué)性質(zhì)良好,具備組織工程水凝膠粘彈性要求。對于1%濃度的寡肽,硫酸軟骨素復(fù)合后能進(jìn)一步提升其力學(xué)性質(zhì)。大鼠髓核細(xì)胞提取經(jīng)原代培養(yǎng)后形態(tài)均一,類似軟骨細(xì)胞,表型鑒定顯示Ⅱ性膠原及Aggrecan表達(dá)完好。經(jīng)水凝膠培養(yǎng)2周后,活死細(xì)胞染色提示髓核細(xì)胞活性與普通培養(yǎng)無差異,CKK-8試驗(yàn)提示細(xì)胞增殖未受到抑制,細(xì)胞免疫熒光發(fā)現(xiàn)髓核細(xì)胞表型完整。結(jié)論:以寡肽(Nap-F-F-Y(P))為基本單位自組裝成膠并復(fù)合硫酸軟骨素的超分子水凝膠在微觀結(jié)構(gòu)上近似細(xì)胞外基質(zhì),具有良好的粘彈性,生物相容度高,與髓核細(xì)胞共培養(yǎng)后能維持其細(xì)胞活性和表型,且成膠簡便,符合生理環(huán)境,用于髓核組織工程細(xì)胞支架的構(gòu)建具有較好的可行性。
[Abstract]:Background: disc degeneration is a common clinical disease. The current clinical treatment methods cannot repair the diseased intervertebral disc at the structural and functional levels. The nucleus pulposus tissue engineering uses the nucleus pulposus seed cells with good function to replace the degenerated nucleus pulposus cells. Thus restoring normal physiological structure and function of nucleus pulposus and reversing the process of disc degeneration have become a research direction in the treatment of disc degeneration. Cell scaffolds are crucial to the activity and functional expression of seed cells. At present, natural hydrogels or synthetic polymer hydrogels are mostly used, both of which have their own disadvantages. With the development of material chemistry, A class of supramolecular hydrogels based on small molecular oligopeptides because of their excellent biocompatibility. Good mechanical properties and similar extracellular matrix structure are expected to be a new choice for the construction of scaffolds for tissue engineering cells of nucleus pulposus. Objective: to explore the supramolecular hydrogel with Nap-F-F-YPU as the basic unit and chondroitin sulfate as the basic unit. Methods: Nap-F-F-YPX oligoseptide was synthesized in a chemical laboratory by solid phase synthesis, and the product was identified by mass spectrometry. Self-assembly occurred under the action of alkaline phosphatase and was combined with chondroitin sulfate to form glue. The gelation time, macroscopic morphology, microstructure and mechanical properties of hydrogels with different concentrations were studied. The primary culture of nucleus pulposus cells of rat tail vertebrae at 12 weeks of age was used as seed cells. Supramolecular hydrogel combined with nucleus pulposus cells were cultured for two weeks. The activity of nucleus pulposus cells was detected by CKK-8 staining without hydrogel. Cell immunofluorescence staining was used to observe the expression of type 鈪，

本文編號：1695283