本文選題：纖維支架 + 生物反應(yīng)器　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：前言:急性肝衰竭(acute liver failure,ALF)是指無(wú)基礎(chǔ)肝臟疾病患者,在肝臟受到各種創(chuàng)傷后(物理、化學(xué)、生物性等),發(fā)生的肝功能損害或衰竭的急癥,其發(fā)病急,病程進(jìn)展迅速,未加治療自然病程死亡率高達(dá)80%[1,2]。目前尚無(wú)特效治療方法,內(nèi)科保守治療,患者死亡率仍高達(dá)50%以上,臨床最有效的治療方法是肝移植,但由于供肝的短缺,限制了肝移植的廣泛應(yīng)用[3]。大部分急性肝衰竭的患者未能等到可移植肝源就已死亡,因此急切需要一種暫時(shí)可以替代肝臟功能的機(jī)器,給予急性肝功能衰竭患者充足的時(shí)間過(guò)渡到肝移植狀態(tài)或肝細(xì)胞再生狀態(tài),人工肝是治療肝衰竭患者的有效途徑,可在短時(shí)間內(nèi)達(dá)到部分或全部替代肝臟功能的目的,為急性肝衰竭患者贏得寶貴時(shí)間[4]。近十年來(lái),人工肝研究取得了長(zhǎng)足進(jìn)展,從早期簡(jiǎn)單的機(jī)械型人工肝進(jìn)步至生物型人工肝,再到更為合理的混合型(機(jī)械+生物型)生物人工肝。生物反應(yīng)器作為生物人工肝的核心部分[4-7],主要包括:中空纖維型,結(jié)合微載體培養(yǎng)型,細(xì)胞球懸浮培養(yǎng)型等多種,但對(duì)于臨床治療所要求的1010數(shù)量級(jí)細(xì)胞量,極少有生物反應(yīng)器能達(dá)到上述要求[8,9]。本課題組經(jīng)過(guò)多年的努力,研發(fā)了基于NASA微重力培養(yǎng)反應(yīng)器[6,10]的微載體貼附懸浮式生物生物反應(yīng)器的人工肝支持系統(tǒng)原理機(jī)和商品化機(jī)器,并在大動(dòng)物(西藏小型豬及食蟹猴)上進(jìn)行驗(yàn)證[11,12]。為了進(jìn)一步向臨床需求靠攏,我們?cè)谝陨系幕A(chǔ)上研制纖維支架循環(huán)灌注式生物反應(yīng)器,以期達(dá)到臨床治療要求數(shù)量級(jí)的肝細(xì)胞,并在后續(xù)實(shí)驗(yàn)中嘗試對(duì)108細(xì)胞進(jìn)行培養(yǎng),擴(kuò)增至109-1010的肝細(xì)胞進(jìn)行長(zhǎng)達(dá)15d的培養(yǎng),并初步取得成功,其主要由纖維支架反應(yīng)罐,外置灌流系統(tǒng),氧氣檢測(cè)系統(tǒng),二氧化碳檢測(cè)系統(tǒng),溫度控制系統(tǒng),懸浮攪拌系統(tǒng)等構(gòu)成;其具有獨(dú)特的特點(diǎn):①.逆重力式灌流,降低流體剪切力;②.外置循環(huán)灌注培養(yǎng)模式,增加液體交換效率;③.全自動(dòng)氧氣、二氧化碳、酸堿度檢測(cè)調(diào)整系統(tǒng);④.體型變小,重量變輕,易于運(yùn)輸。第一章纖維支架載體肝細(xì)胞生物相容性評(píng)價(jià)目的:對(duì)纖維支架材料的構(gòu)成,理化性質(zhì)加以分析,完成對(duì)纖維支架循環(huán)灌注式生物反應(yīng)器的設(shè)計(jì)及組裝,檢測(cè)纖維支架材料與肝細(xì)胞之間的生物相容性。方法:采用電鏡,X射線光電子能譜分析(X-ray photoelectron spectroscopy,XPS)等技術(shù)分析纖維支架材料特征,元素構(gòu)成,表面電荷及氨基酸分析纖維支架材料表面粘附氨基酸分子;通過(guò)高溫高壓(121℃,45min),強(qiáng)酸(10%鹽酸,浸泡24h)強(qiáng)堿(10%碳酸氫鈉,浸泡24h)處理后通過(guò)電鏡觀察其表面性質(zhì)及破損情況分析其穩(wěn)定性。在纖維支架載體上接種C3A細(xì)胞,并隨后采用Live/dead試劑盒細(xì)胞生長(zhǎng)狀況進(jìn)行評(píng)價(jià)。結(jié)果:纖維支架載體,此載體長(zhǎng)10cm,寬1.13cm,為細(xì)胞提供接近11.3cm2貼附面積,電鏡下觀察纖維直徑均一,多層交叉,形成多種空隙,面積體積比大,可以提供較大細(xì)胞生長(zhǎng)表面積。此支架主要成分為C,N,O分別74.32%,2.84%,22.84%,密度為p=0.2254mmol/g。支架表面攜帶賴氨酸,攜帶正電荷,增強(qiáng)細(xì)胞粘附。在高溫,高壓,強(qiáng)酸,強(qiáng)堿處理后,纖維支架材料均呈現(xiàn)出穩(wěn)定狀態(tài),電鏡下未觀察到纖維破裂、變形、融化等不良現(xiàn)象。細(xì)胞對(duì)纖維支架載體貼附良好,5d內(nèi)細(xì)胞活力維持大于90%,與對(duì)照組比較無(wú)顯著差異(P0.05)。結(jié)論:纖維支架材料面積體積比大,高溫高壓強(qiáng)酸強(qiáng)堿處理后理化性質(zhì)穩(wěn)定,細(xì)胞帖附緊密生長(zhǎng)旺盛,肝細(xì)胞與支架細(xì)胞相容性良好。第二章纖維支架循環(huán)灌注式生物反應(yīng)器大規(guī)模培養(yǎng)肝細(xì)胞目的:使用纖維支架循環(huán)灌注式生物反應(yīng)器大規(guī)模培養(yǎng)肝細(xì)胞,達(dá)到1010臨床治療要求水平,為新型生物人工肝系統(tǒng)提供可靠的生物反應(yīng)器部件。方法:按照說(shuō)明調(diào)試機(jī)器,校準(zhǔn)PH、溫度溶氧探頭,并提前預(yù)濕載體(600-700ml全培,37℃C,2cm/s空轉(zhuǎn)24h;將108的C3A細(xì)胞量,并消化離心成50ml懸濁液,接種入反器,4H后取樣檢測(cè)細(xì)胞懸浮情況,隨后每日取樣進(jìn)行臺(tái)盼藍(lán)染色、結(jié)晶紫染色、細(xì)胞計(jì)數(shù),Q-PCR、電鏡觀察;每日取培養(yǎng)基上清送檢葡萄糖、白蛋白、ALT、AST、乳酸。結(jié)果:細(xì)胞生長(zhǎng)良好,在15d的培養(yǎng)中,細(xì)胞從1.07×108增長(zhǎng)至約90-100×108個(gè),結(jié)晶紫染色,染色面積逐漸增加,至15d載體面積貼附約為70%-80%,細(xì)胞活力維持在75%以上,ALT、AST漏出水平低,細(xì)胞損傷小,細(xì)胞基因表達(dá)水平較平板對(duì)照組顯著增高,電鏡觀察發(fā)現(xiàn)細(xì)胞-細(xì)胞間連接增多。結(jié)論:纖維支架循環(huán)灌注式生物反應(yīng)器可大規(guī)模培養(yǎng)肝細(xì)胞至109數(shù)量級(jí),細(xì)胞活力維持良好,細(xì)胞功能進(jìn)一步增強(qiáng)是優(yōu)良的肝細(xì)胞大規(guī)模培養(yǎng)反應(yīng)器。
[Abstract]:Acute liver failure ( ALF ) refers to acute liver failure ( ALF ) , which refers to a patient with no basic liver disease . It is urgent for liver function damage or failure after various trauma ( physical , chemical , biological , etc . ) in the liver . There is no special effect treatment method . The mortality rate of the patients is still up to over 50 % . The most effective treatment method is liver transplantation , but due to the shortage of donor liver , the widespread use of liver transplantation is limited . Most of the patients with acute liver failure can not wait until the transplant liver source has died . Therefore , there is a need for a machine which can replace the liver function temporarily , so that the patients with acute liver failure can be given sufficient time to transition to the state of liver transplantation or liver cell regeneration , and the artificial liver is an effective way for treating liver failure patients , and the purpose of partial or total replacement of liver function can be achieved in a short time , and the precious time for patients with acute liver failure can be achieved . In recent ten years , we have made great progress in artificial liver research , from simple mechanical artificial liver to biological artificial liver , and then to a more reasonable hybrid ( mechanical + biological ) bioartificial liver . The biological reactor is composed of fiber support reaction tank , external perfusion system , oxygen detection system , carbon dioxide detection system , temperature control system , suspension stirring system , etc . The biological reactor is mainly composed of fiber support reaction tank , external perfusion system , oxygen detection system , carbon dioxide detection system , temperature control system , suspension stirring system and so on . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was increased from 1.07 脳 108 to about 90 - 100 脳 108 , the cell viability was maintained at over 75 % , the level of ALT and AST was lower , the cell damage was small , the expression level of the cells was higher than that of the control group . Conclusion : The fibrous scaffold circulating perfusion bioreactor can be used for large - scale culture of hepatocytes to 109 orders of magnitude , the viability of the cells is maintained well , and the cell function is further enhanced to be an excellent large - scale culture reactor for hepatocytes .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.3

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