本文選題：多孔支架 + 三維細(xì)胞培養(yǎng)��； 參考：《陜西師范大學(xué)》2012年博士論文

【摘要】：藥物發(fā)現(xiàn)已經(jīng)產(chǎn)生了許多成功的藥物處理和治療方法,然而,也遇到了高失敗率、高成本等問題,也受到許多科學(xué)技術(shù)挑戰(zhàn)的限制,例如需要用更快的速度和更準(zhǔn)確的方式分析藥物候選物。利用活細(xì)胞作為生物識(shí)別元素來進(jìn)行化合物功能驗(yàn)證和毒性測(cè)試的基于細(xì)胞的分析是解決這些問題的一個(gè)有效方法。已有文獻(xiàn)報(bào)道熒光蛋白、微管蛋白、G-蛋白偶聯(lián)受體、細(xì)胞膜和活細(xì)胞等與生物活性成分之間的作用。基于活細(xì)胞的分析對(duì)于藥物篩選而言可以產(chǎn)生與生化分析相比更接近體內(nèi)生理的結(jié)果。在藥物發(fā)現(xiàn)的各個(gè)階段,已有微型和納米級(jí)的基于活細(xì)胞的分析技術(shù)。然而,傳統(tǒng)的體外細(xì)胞試驗(yàn)是利用二維培養(yǎng)的細(xì)胞,缺乏細(xì)胞原有組織的三維微環(huán)境,缺乏細(xì)胞生長(zhǎng)的基質(zhì),也缺乏細(xì)胞生長(zhǎng)的支架,細(xì)胞在二維條件下喪失了許多組織學(xué)相關(guān)的功能,不能表達(dá)其在體內(nèi)條件下的許多特性。因此,二維細(xì)胞培養(yǎng)不能模擬細(xì)胞在體內(nèi)生長(zhǎng)的微環(huán)境,在藥物功效預(yù)測(cè)方面準(zhǔn)確度不高。 不同于傳統(tǒng)的二維細(xì)胞培養(yǎng),三維細(xì)胞培養(yǎng)是將具有三維結(jié)構(gòu)的載體與細(xì)胞在體外共培養(yǎng),最大程度地模擬體內(nèi)微環(huán)境,使細(xì)胞能夠在載體的三維空間結(jié)構(gòu)中生長(zhǎng)、遷移、分化,產(chǎn)生一定的三維組織特異性結(jié)構(gòu),具有細(xì)胞培養(yǎng)直觀性和條件可控性的優(yōu)勢(shì),填補(bǔ)了二維細(xì)胞培養(yǎng)和動(dòng)物實(shí)驗(yàn)的鴻溝,有非常大的發(fā)展?jié)摿�。由于傳質(zhì)的局限性,三維培養(yǎng)可得到不同表型的細(xì)胞,如增殖、非增殖和壞死細(xì)胞。在三維培養(yǎng)時(shí)癌細(xì)胞的異質(zhì)性與完整實(shí)體瘤的多重表型相似,而這比二維培養(yǎng)時(shí)癌細(xì)胞的同質(zhì)性更真實(shí)。三維培養(yǎng)的細(xì)胞在形狀和環(huán)境上與體內(nèi)條件更為相似,而細(xì)胞的形狀和環(huán)境決定細(xì)胞的行為和基因表達(dá)。因此,三維細(xì)胞培養(yǎng)可以更真實(shí)地模擬體內(nèi)細(xì)胞生長(zhǎng)的三維微環(huán)境,細(xì)胞在三維條件下的響應(yīng)更能代表其在體內(nèi)條件下的響應(yīng),基于三維細(xì)胞的藥物篩選方法將會(huì)是更有效的篩選方法。 支架具有制備過程簡(jiǎn)單、快速,對(duì)細(xì)胞所需營(yíng)養(yǎng)物和細(xì)胞的代謝物擴(kuò)散的阻力小,可以滿足多細(xì)胞的聚集等優(yōu)點(diǎn)。已經(jīng)有很多支架被用于細(xì)胞的三維培養(yǎng),其中復(fù)合支架由于整合了各單一材料的優(yōu)點(diǎn)、克服了各單一材料的缺點(diǎn)而引起了廣泛的關(guān)注。 細(xì)胞在支架上的接種方法和培養(yǎng)方法影響細(xì)胞的粘附、增殖和分化等活性,影響細(xì)胞與藥物作用。與傳統(tǒng)的靜態(tài)接種和培養(yǎng)方法相比,動(dòng)態(tài)接種和灌流培養(yǎng)中細(xì)胞懸液直接通過支架的孔,對(duì)于不同組成、孔結(jié)構(gòu)和孔隙率的支架都可以更方便的使?fàn)I養(yǎng)物質(zhì)和氣體通過。因此,動(dòng)態(tài)接種和灌流培養(yǎng)在細(xì)胞生長(zhǎng)和存活方面更有優(yōu)勢(shì),利用這種方式三維培養(yǎng)的細(xì)胞與藥物之間的作用更能反映藥物在體內(nèi)作用的真實(shí)情況。 目前,已經(jīng)有許多種類的填充床細(xì)胞反應(yīng)器被用于細(xì)胞的動(dòng)態(tài)接種和灌流培養(yǎng),特別是被用于肝細(xì)胞高密度培養(yǎng)。這些反應(yīng)器與靜態(tài)細(xì)胞接種和培養(yǎng)方法相比,在細(xì)胞粘附、分布、長(zhǎng)時(shí)間存活和各種類型的細(xì)胞功能等方面已經(jīng)顯示出無可比擬的優(yōu)勢(shì)。然而,目前所報(bào)道的大多數(shù)填充床細(xì)胞反應(yīng)器由于不能提供細(xì)胞生長(zhǎng)的必須環(huán)境,而依賴于二氧化碳培養(yǎng)箱,這就限制了它們的使用。并且,目前還沒有利用填充床細(xì)胞反應(yīng)器進(jìn)行藥物篩選的報(bào)道。 因此,本論文旨在制備適合細(xì)胞生長(zhǎng)的多孔支架,利用多孔支架建立不依賴于二氧化碳培養(yǎng)箱的三維細(xì)胞反應(yīng)器,并利用這個(gè)三維細(xì)胞反應(yīng)器篩選分析傳統(tǒng)中藥中能夠與三維培養(yǎng)的癌細(xì)胞特異性結(jié)合的活性成分。 本論文由六章組成。第一章為引言。引言部分介紹了藥物篩選模型、高通量藥物篩選和基于細(xì)胞模型的高通量藥物篩選；指出了三維細(xì)胞培養(yǎng)的優(yōu)點(diǎn)以及常用方法；總結(jié)了三維細(xì)胞反應(yīng)器的設(shè)計(jì)原則與類型；介紹了以多孔支架為填充物的三維細(xì)胞反應(yīng)器,以及幾種常見的用于制備支架的生物材料和方法；簡(jiǎn)要指出了本論文的研究目的意義以及研究?jī)?nèi)容與關(guān)鍵技術(shù)。 第二章為PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架的制備、表征及生物相容性評(píng)價(jià)。研究了超臨界二氧化碳?jí)毫�、溫度、作用時(shí)間對(duì)PLGA/納米羥基磷灰石/膠原蛋白復(fù)合多孔支架孔結(jié)構(gòu)的影響,結(jié)果表明通過調(diào)節(jié)超臨界二氧化碳?jí)毫Α囟�、作用時(shí)間可得到不同孔徑和孔隙率的PLGA/納米羥基磷灰石/膠原蛋白復(fù)合多孔支架。PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架的化學(xué)表征結(jié)果表明,羥基磷灰石、膠原蛋白均勻的分布于PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架中。體外生物相容性實(shí)驗(yàn)表明,PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架適合人骨肉瘤細(xì)胞MG-63粘附和增殖。體內(nèi)生物相容性實(shí)驗(yàn)表明,支架的植入會(huì)引起一些炎癥反應(yīng),術(shù)后7、14和28d后與支架相連的組織中的TNF-α、IL-1β、IL-6的含量逐漸減小,而IL-10的含量逐漸增加,且TNF-α、IL-1β、IL-6和IL-10的含量與空白對(duì)照組在28d時(shí)基本相似,表明由PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架的植入引起的炎癥反應(yīng)在28d后消除。因此,PLGA/羥基磷灰石/膠原蛋白復(fù)合多孔支架具有良好的體內(nèi)、體外生物相容性,在組織工程中有很大的應(yīng)用潛力。 第三章為PLGA-PEG-PLGA/納米羥基磷灰石/膠原蛋白復(fù)合多孔支架的制備、表征及生物相容性評(píng)價(jià)。研究了超臨界二氧化碳?jí)毫�、溫度、作用時(shí)間對(duì)PLGA-PEG-PLGA/納米羥基磷灰石/膠原蛋白復(fù)合多孔支架孔結(jié)構(gòu)的影響,結(jié)果表明通過調(diào)節(jié)超臨界二氧化碳?jí)毫�、溫度、作用時(shí)間可以得到不同孔徑和孔隙率的PLGA-PEG-PLGA/納米羥基磷灰石/膠原蛋白復(fù)合多孔支架。體外、體內(nèi)生物相容性實(shí)驗(yàn)表明,該支架具有良好的體外、體內(nèi)生物相容性。 第四章為絲素蛋白/膠原蛋白/納米羥基磷灰石復(fù)合多孔支架的制備、表征及生物相容性評(píng)價(jià)。研究了氯化鈉粒徑、氯化鈉和納米羥基磷灰石的重量比及絲素蛋白和膠原蛋白的體積比對(duì)絲素蛋白/膠原蛋白/納米羥基磷灰石復(fù)合多孔支架孔結(jié)構(gòu)的影響,結(jié)果表明采用該方法可以制備孔徑和孔隙率可調(diào)的生物相容性好的絲素蛋白/膠原蛋白/納米羥基磷灰石復(fù)合多孔支架,操作簡(jiǎn)單,耗時(shí)短。體外、體內(nèi)生物相容性實(shí)驗(yàn)表明,該支架具有良好的體外、體內(nèi)生物相容性。 第五章為填充床細(xì)胞反應(yīng)器耦合HPLC/MS藥物篩選系統(tǒng)的建立及其在朱砂七活性成分篩選中的應(yīng)用。建立了一個(gè)通過填充床細(xì)胞反應(yīng)器耦合HPLC/MS藥物篩選系統(tǒng),比較藥物與固定的癌細(xì)胞和活的癌細(xì)胞作用之后生物指紋圖譜的峰面積來評(píng)價(jià)藥物與癌細(xì)胞的結(jié)合度。兩種已知抗癌藥物(紫杉醇和白藜蘆醇)和兩種已知非抗癌藥物(酮洛芬和青霉素G)與模型癌細(xì)胞(Lovo細(xì)胞)之間的相互作用證明,該分析系統(tǒng)用于藥物篩選是可行的,并將該分析系統(tǒng)應(yīng)用于朱砂七提取物中活性成分的篩選分析,篩選出兩種生物活性成分,馬兜磷酸A和馬兜磷酸B。 第六章為三維細(xì)胞反應(yīng)器及其在桃兒七活性成分篩選中的應(yīng)用。建立了一個(gè)新型的三維細(xì)胞反應(yīng)器,通過比較藥物與固定的癌細(xì)胞和活的癌細(xì)胞作用之后生物指紋圖譜的峰面積之間有無顯著性差異來判斷該藥物是否能與癌細(xì)胞特異性結(jié)合。兩種已知抗癌藥物(紫杉醇和白藜蘆醇)和兩種已知非抗癌藥物(酮洛芬和青霉素G)與癌細(xì)胞的作用證明,該三維細(xì)胞反應(yīng)器在篩選與細(xì)胞特異性結(jié)合的生物活性成分方面是可行的。利用該三維細(xì)胞反應(yīng)器對(duì)桃兒七提取物中的活性成分進(jìn)行篩選分析。對(duì)桃兒七提取物中已鑒定的10個(gè)成分的篩選表明,槲皮素-3-O-β-D-葡萄糖苷,山奈酚-3-O-β-D-葡萄糖苷和山荷葉素這3種組分與與Lovo細(xì)胞之間沒有特異性結(jié)合。而L-鬼臼毒素-4-0-β-D-葡萄糖苷、槲皮素、二十六烷酸、山奈酚、鬼臼毒素、鬼臼毒酮或異鬼臼苦酮和去氧鬼臼毒素或4’-去甲鬼臼毒酮可以與Lovo細(xì)胞特異性結(jié)合,它們的結(jié)合度分別為：32.6%±3.1%、29.3%±3.0%、15.2%±1.7%、57.1%±4.8%、68.9%±9.2%、63.6%±8.7%、46.3%±4.8%。 本論文采用不同的方法制備了三種多孔支架,并對(duì)各種支架進(jìn)行了表征和生物相容性評(píng)價(jià),為三維細(xì)胞反應(yīng)器的建立奠定了基礎(chǔ)。并對(duì)現(xiàn)有的三維細(xì)胞反應(yīng)器進(jìn)行突破,提出了兩種不依賴于二氧化碳培養(yǎng)箱的三維細(xì)胞反應(yīng)器,分別將這兩種三維細(xì)胞反應(yīng)器用于中藥提取物中與癌細(xì)胞特異性結(jié)合的活性成分的篩選分析,得到了一些能與癌細(xì)胞特異性結(jié)合的活性成分。本論文提供了一個(gè)從多組分混合物中特異性的篩選和分析生物活性成分新方法。同時(shí),也可以為從天然產(chǎn)物和植物藥物中篩選抗癌候選藥物提供一個(gè)新的思路。
[Abstract]:however , conventional in vitro cell assay is an effective method to address these problems .

The three - dimensional cell culture is different from the traditional two - dimensional cell culture , and the three - dimensional cell culture is characterized in that the carrier and the cell with the three - dimensional structure are cocultured in vitro to maximally simulate the in vivo microenvironment , so that the cell can grow , migrate and differentiate in the three - dimensional space structure of the carrier , and the three - dimensional culture can obtain cells of different phenotypes , such as proliferation , non - proliferation and necrotic cells .

The stent has the advantages of simple preparation process , fast speed , small resistance to the diffusion of metabolites of nutrients and cells required by cells , and the like , and can meet the advantages of multi - cell aggregation and the like .

Compared with the traditional static inoculation and culture method , the cell suspension in the dynamic inoculation and perfusion culture can be more convenient for the nutrient substance and the gas to pass through the hole of the stent . Therefore , the dynamic inoculation and perfusion culture are more advantageous in cell growth and survival , and the effect between the three - dimensional cultured cells and the drug can reflect the real situation of the action of the drug in vivo .

Currently , many kinds of packed bed cell reactors have been used for the dynamic inoculation and perfusion culture of cells , in particular for high density culture of hepatocytes . These reactors have shown an unparalleled advantage in terms of cell adhesion , distribution , long - term survival and various types of cellular functions , as compared to static cell inoculation and culture methods . However , most of the packed bed cell reactors currently reported have limited their use because they do not provide the necessary environment for cell growth , and this limits their use .

Therefore , the purpose of this paper is to prepare a porous scaffold suitable for cell growth , to establish a three - dimensional cell reactor independent of the carbon dioxide incubator by using a porous scaffold , and to use the three - dimensional cell reactor to screen and analyze the active ingredients capable of specifically binding to the three - dimensional cultured cancer cells in the traditional Chinese medicine .

The first chapter introduces drug screening model , high flux drug screening and high flux drug screening based on cell model .
The advantages and common methods of three - dimensional cell culture are pointed out .
The design principle and type of three - dimensional cell reactor are summarized .
Three - dimensional cellular reactor with porous scaffold as filler and several common biological materials and methods for preparing stents were introduced .
This paper briefly points out the research objectives and the research contents and key technologies of this paper .

In chapter 2 , the preparation , characterization and biocompatibility evaluation of PLGA / hydroxyapatite / collagen composite porous scaffolds were studied . The results showed that PLGA / hydroxyapatite / collagen composite porous scaffolds were suitable for the adhesion and proliferation of human osteosarcoma cells MG - 63 . The results showed that PLGA / hydroxyapatite / collagen composite porous scaffolds were suitable for human osteosarcoma cell MG - 63 adhesion and proliferation .

In chapter 3 , the preparation , characterization and biocompatibility evaluation of PLGA - PEG - PLGA / nano hydroxyapatite / collagen composite porous scaffold were studied . The effects of supercritical carbon dioxide pressure , temperature and action time on the pore structure of PLGA - PEG - PLGA / nano hydroxyapatite / collagen composite porous scaffold were studied .

In chapter 4 , the preparation , characterization and biocompatibility of silk fibroin / collagen / nano hydroxyapatite composite porous scaffolds were evaluated . The effects of the weight ratio of sodium chloride , sodium chloride and hydroxyapatite and the volume ratio of silk fibroin and collagen on the pore structure of silk fibroin / collagen / nano hydroxyapatite composite porous scaffolds were studied .

In the fifth chapter , an HPLC / MS drug screening system for packed bed cell reactor was established and its application in the selection of active ingredients in Zhucheng 7 was established . An HPLC / MS drug screening system was established by a packed bed cell reactor . The peak area of the fingerprint was compared between the drugs and fixed cancer cells and living cancer cells .

A new type of three - dimensional cell reactor was established . The three - dimensional cellular reactor was used to screen the active components of the cell - specific cell . The results showed that the three components were : 36.6 % 鹵 3.1 % , 29.3 % 鹵 3.0 % , 15.2 % 鹵 1.7 % , 57.1 % 鹵 4.8 % , 68.9 % 鹵 9.2 % , 63.6 % 鹵 8.7 % , 46.3 % 鹵 4.8 % respectively .

Three kinds of porous scaffolds were prepared by different methods , and the characterization and biocompatibility evaluation of various scaffolds were carried out . Two three - dimensional cell reactors which were not dependent on carbon dioxide incubator were established . Two kinds of three - dimensional cell reactors , which were not dependent on the specific binding of the cancer cells , were screened and analyzed in the traditional three - dimensional cell reactor .
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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