本文選題：脫細胞支架　切入點：生物材料　出處：《山東大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：隨著慢性腎功能不全發(fā)病率的逐年增高,終末期腎病(ESRD)已經(jīng)成為嚴重困擾公眾的社會問題。目前對于ESRD的治療方式主要有兩種：血液透析及腎臟移植。血液透析容易引起較多的并發(fā)癥且效率較低,長期治療效果較差,容易給患者帶來較大的痛苦以及經(jīng)濟負擔(dān)。而腎臟移植能夠完整替代腎臟的功能,但是存在腎源短缺嚴重以及嚴重的免疫排斥反應(yīng)問題。干細胞與腎臟再生醫(yī)學(xué)的發(fā)展為這一難題帶來了新的希望。其中細胞-脫細胞支架技術(shù)的發(fā)展成為近年來腎臟再生領(lǐng)域研究的熱點。所謂細胞-脫細胞支架技術(shù)是指將動物天然腎臟進行去細胞化,獲得無細胞的腎臟細胞外基質(zhì)(ECM)—腎臟脫細胞支架,然后應(yīng)用干細胞或者成體細胞對脫細胞支架進行再細胞化,進行體外或體內(nèi)培養(yǎng)后,獲得有功能的再生腎臟。腎臟細胞種類復(fù)雜,因此,選用合適的干細胞在支架內(nèi)進行分化,是選擇種子細胞的依據(jù)。胚胎干細胞是一種多能干細胞,擁有強大的增殖能力與分化能力,且在體外已能夠向腎臟細胞系分化,是一種良好的種子細胞。本研究擬采用胚胎干細胞,對腎臟脫細胞支架進行再細胞化,獲得有功能、可移植的腎臟。第一部分 腎臟脫細胞支架的建立與生物學(xué)特性的檢測目的建立快速有效的脫細胞支架流程,獲得適合細胞種植的腎臟脫細胞支架。方法：1.腎臟的獲�。簩嶒瀯游镞x擇雄性Wista大鼠,體重在200-300g之間。應(yīng)用10%水合氯醛麻醉后,取腹正中切口,暴露腹主動脈,應(yīng)用24G套管針插入,將腎臟血液沖出,然后取出腎臟。2.脫細胞支架的制備：應(yīng)用0.5%的十二烷基磺酸鈉(SDS)溶液以及蒸餾水對腎臟進行持續(xù)灌注,速度為0.5ml/min,腎臟變?yōu)榘咨胪该鲿r換PBS溶液沖洗,將殘留的SDS溶液沖洗干凈。3.脫細胞支架鑒定：(1)形態(tài)學(xué)及免疫學(xué)檢測：應(yīng)用HE以及Masson染色檢測的方法鑒定脫細胞支架中細胞去除情況。應(yīng)用免疫組化檢測腎臟脫細胞前后Ⅳ型膠原及纖連蛋白的表達情況。掃描電鏡檢測顯微結(jié)構(gòu)保留情況。(2) 分子生物學(xué)檢測：應(yīng)用DNA及膠原檢測試劑盒檢測腎臟脫細胞支架中DNA及膠原的含量；應(yīng)用ELISA的方法檢測脫細胞支架中HGF、TGF及VEGF的含量。(3)血管系統(tǒng)檢測：應(yīng)用血管造影的方式在體外檢測血管系統(tǒng)的完整性；將腎臟脫細胞支架原位移植到大鼠體內(nèi),進一步檢測其通暢性及嚴密性。結(jié)果：1.大體觀：腎臟經(jīng)過SDS及雙蒸水灌注后,逐漸變?yōu)榘咨胪该鳡睢?.HE及Masson染色：顯示細胞成分去除徹底,僅剩膠原成分。3.免疫組化：顯示脫細胞支架中Ⅳ型膠原以及纖連蛋白保留較好。4.掃描電鏡顯示腎小球的基底膜等膜性結(jié)構(gòu)保留完整。5.分子生物學(xué)檢測：DNA含量在脫細胞之后低于正常的5%以下；膠原含量在脫細胞前后變化不明顯；HGF、TGF以及VEGF等細胞因子的含量變化不明顯。6.血管系統(tǒng)檢測：血管造影顯示腎臟血管系統(tǒng)保留完整,無造影劑漏出,血管分支結(jié)構(gòu)顯示清楚。脫細胞支架原位移植血管夾放開后,血液運行通暢,無明顯漏出。HE顯示血液充盈到腎臟內(nèi)部,2月后形成血栓及炎性細胞浸潤。結(jié)論：應(yīng)用SDS等脫細胞劑及自制設(shè)備灌注大鼠腎臟,能夠獲得完整的腎臟脫細胞支架,建立了有效的腎臟脫細胞支架制備流程。第二部分 腎臟脫細胞支架的再細胞化及再生腎臟的功能鑒定目的：應(yīng)用胚胎干細胞對腎臟脫細胞支架進行再細胞化,獲得有一定功能、可移植的腎臟。方法：1.種子細胞選用小鼠胚胎干細胞,在體外進行培養(yǎng),應(yīng)用免疫組化檢測干細胞標志物,如OCT4.NANOG等干細胞標志物,以確保其在細胞種植之前保持干細胞特性。2.將胚胎干細胞消化、收集,調(diào)整其細胞濃度至1×107/ml。經(jīng)腎動脈灌注3ml,在-50cmH2O負壓下經(jīng)輸尿管灌注3ml。靜置過夜,連續(xù)灌注2天。3.再生腎的培養(yǎng)：腎臟脫細胞種植再細胞化后,通過自制灌注設(shè)備,應(yīng)用DMEM/F12培養(yǎng)基進行循環(huán)灌注培養(yǎng),溫度37℃,連續(xù)培養(yǎng)7天。4.再生腎的鑒定：再生腎臟經(jīng)過體外培養(yǎng)后,進行HE染色鑒定；體外制備改良的Hank‘s液,進行功能鑒定；將再生腎臟移植到大鼠體內(nèi),收集靜脈血及尿液,進行腎臟功能的體內(nèi)測定。結(jié)果：1.胚胎干細胞經(jīng)過鑒定,OCT4.NANOG以及SOX2等干細胞標志物表達良好,表明其干細胞特性保持較好。2.經(jīng)過細胞種植以及再生腎臟體外培養(yǎng),腎臟脫細胞支架的腎小球部位以及部分腎小管部位有較多細胞貼附。3.經(jīng)過功能測定,再生腎臟較脫細胞支架產(chǎn)生尿液(1.8±0.7ul/min vs 4.9±1.4ul/min)更少,但其中排出肌酐(1.4±1.4mg/dl)以及尿素氮(26.5±1.6mg/dl)較脫細胞支架更多。結(jié)論：胚胎干細胞為良好的腎臟組織工程種子細胞,經(jīng)過再細胞化,獲得了有一定形態(tài)結(jié)構(gòu)及功能、可以移植的再生腎臟。
[Abstract]:Along with the increasing incidence of chronic renal failure, end-stage renal disease (ESRD) has become a serious social problem plagued the public for the treatment of ESRD. At present there are two main ways: hemodialysis and renal transplantation. Hemodialysis can cause many complications and low efficiency, long-term treatment effect is poor, easy to bring greater the pain and the economic burden for patients. Kidney transplantation can completely replace kidney function, but there are serious and serious shortage of kidney immune rejection problems. The development of stem cell and regenerative medicine kidney brings new hope for this problem. The development of technology - the cell scaffold of acellular kidney regeneration has become a hotspot in recent years the field of research. The cell scaffold technology refers to natural animal kidney cells to obtain kidneys, extracellular matrix without cells (ECM)- The kidney scaffold, then the application of stem cells or somatic cells on the acellular scaffold recellularization in vitro or in vivo, after culture, regenerated kidney function. Renal cell complex types, therefore, choose the appropriate stem cell differentiation on the scaffolds, is the choice of seed cells of embryonic stem cell basis. Is a kind of pluripotent stem cell proliferation and differentiation, have strong ability, and can differentiate into renal cells in vitro, is a kind of good seed cells. This study intends to use embryonic stem cells on kidney acellular support to cells, obtain a function, can be transplanted kidney detection. Objective to establish the first part of the kidney acellular scaffold and the biological characteristics of the establishment of the rapid and effective process of cell scaffold, for cells grown kidney acellular scaffold. Methods: We obtained 1. of the kidney: experimental Animal male Wista rats weighing between 200-300g. Using 10% chloral hydrate anesthesia, abdominal incision, abdominal aorta was exposed, the application of 24G trocar insertion, the renal blood rushed out, then remove the kidney.2. acellular scaffold preparation: Twelve sodium dodecyl sulfate 0.5% (SDS) and distilled solution water continuous perfusion of kidney, speed of 0.5ml/min, the kidney into white translucent in PBS solution, SDS solution rinse residue.3. acellular scaffold identification: (1) the morphological and immunological detection: using HE and Masson staining detection and cell removal of cell scaffold. Immunohistochemistry detection of renal cells before and after removal of type IV collagen and fibronectin expression. Scanning electron microscopy, the microstructure of retention situation. (2) molecular biology detection: application of DNA and collagen detection test kit for detecting The content of DNA and collagen in kidney acellular scaffold; application of ELISA detection method of acellular scaffold of HGF, content of TGF and VEGF. (3) vascular system detection: application of angiography in detection of vascular integrity in vitro system; kidney transplantation in situ removal will scaffold into rats, further testing its patency and strictness. Results: 1. general observation: after kidney SDS and double distilled water after reperfusion, gradually become white and translucent.2.HE and Masson staining showed: cell component removal completely, only collagen.3. immunohistochemistry: show off type IV collagen scaffold and fibronectin with well preserved.4. scanning electron microscopy showed that glomerular the basement membrane membrane structure of intact.5. molecular biology detection: the content of DNA in acellular after lower than normal below 5%; in the acellular collagen content did not change significantly before and after HGF, T; The content changes of GF and VEGF cytokines was.6. vascular system detection: angiography showed renal vascular system intact, no contrast agent leakage, vascular branch structure showed clearly. Decellularized vascular clamp release after orthotopic transplantation, blood running smooth, no obvious leakage of.HE showed blood filling to the kidney, after February thrombosis and inflammatory cell infiltration. Conclusion: the application of SDS acellular agent and self-made kidney equipment in rats, and can obtain a complete kidney acellular scaffold, establish effective kidney acellular scaffold preparation process. The second part kidney acellular scaffold recellularization and regeneration of kidney function and identification Objective: application of embryo stem cells on kidney acellular stent recellularization, have certain functions, can be transplanted kidney. Methods: 1. seed cells of mouse embryonic stem cells in vitro. Culture, immunohistochemical detection of stem cell markers, such as OCT4.NANOG stem cell markers, to ensure that the.2. will maintain stem cells of embryonic stem cells in the digestive cells collected before planting, and adjust the cell concentration to 1 * 107/ml. by renal artery perfusion of 3ml, in -50cmH2O under negative pressure by static ureteral perfusion 3ml. overnight, 2 days of continuous perfusion culture.3. regeneration kidney: kidney acellular plant cells after reperfusion through the self-made equipment, application of DMEM/F12 medium circulating perfusion culture, the temperature of 37 DEG C, continuous cultivation and identification of.4. 7 days: the regeneration of renal kidney regeneration after in vitro culture, HE staining; Hank s liquid preparation modified in vitro system, identify the function; the regeneration of kidney transplantation into rats, collecting venous blood and urine, determination of renal function in vivo. Results: 1. embryonic stem cells after identification, OCT4.NANOG And SOX2 expression of stem cell markers, showed the characteristics of stem cells to maintain good.2. after planting and regeneration of in vitro cultured kidney cells, renal glomerular acellular scaffold site and part of the renal tubular parts have more cells attached after.3. function test, regeneration of kidney compared with acellular scaffold produce urine (1.8 + 0.7ul/min 4.9 + vs 1.4ul/min) less, but the discharge of creatinine (1.4 + 1.4mg/dl) and urea nitrogen (26.5 + 1.6mg/dl) with acellular scaffold more. Conclusion: embryonic stem cells for good kidney tissue engineering seed cells, after recellularization, obtained the structure and function of a certain shape, can be transplanted kidney regeneration.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R692;R318.08

【相似文獻】

中國期刊全文數(shù)據(jù)庫 前10條

1 王偉;馬利林;陸麗萍;陳瑞新;于秀;;未經(jīng)預(yù)處理同種活性生物脫細胞支架的免疫原性及支架張力(英文)[J];中國組織工程研究與臨床康復(fù);2008年41期

2 王輝;陳雄生;周盛源;黃俊俊;蔡_"藝;;凍干韌帶脫細胞支架材料的生物相容性及優(yōu)勢[J];中國組織工程研究與臨床康復(fù);2011年29期

3 周敏;劉長建;衛(wèi)志慶;劉昭;蔣雪峰;喬彤;冉峰;;bFGF預(yù)載脫細胞支架構(gòu)建小口徑人工血管的研究[J];中國修復(fù)重建外科雜志;2008年03期

4 周敏;劉長建;衛(wèi)志慶;喬威;喬彤;冉峰;張明;;以肝素固化脫細胞支架構(gòu)建的小口徑抗凝人工血管[J];中國組織工程研究與臨床康復(fù);2008年36期

5 張炎,劉太華,姜宗來,張秀花,劉波,李玉泉;全生物化組織工程血管構(gòu)建的初步研究[J];生物醫(yī)學(xué)工程學(xué)雜志;2005年03期

6 冉峰;劉長建;周敏;衛(wèi)志慶;喬威;劉昭;喬彤;;脫細胞支架復(fù)合犬骨髓源內(nèi)皮祖細胞構(gòu)建組織工程血管[J];中國組織工程研究與臨床康復(fù);2008年32期

7 武欣;谷涌泉;段紅永;陳兵;李建新;吳英鋒;張淑文;汪忠鎬;張建;;改良酶學(xué)方法獲得異種血管脫細胞支架[J];中國組織工程研究與臨床康復(fù);2011年47期

8 鹿亮;郭全義;楊啟友;袁玫;張莉;黃靖香;趙斌;彭江;許文靜;盧世璧;;豬來源關(guān)節(jié)軟骨脫細胞支架的生物安全性研究[J];中國醫(yī)藥生物技術(shù);2010年01期

9 龐超見;佟曉杰;劉貴波;李奇;賈樺;王瑩;高海;;VEGF在小劑量超短波促進種植骨髓間充質(zhì)干細胞脫細胞支架修復(fù)大鼠坐骨神經(jīng)損傷中的表達[J];中國醫(yī)科大學(xué)學(xué)報;2010年04期

10 ;[J];;年期

中國重要會議論文全文數(shù)據(jù)庫 前3條

1 張永珍;王棟;熊紹虎;黨瑞山;張傳森;;促進靜脈脫細胞支架材料內(nèi)皮化的實驗研究[A];中國解剖學(xué)會2011年年會論文文摘匯編[C];2011年

2 任昊楨;施曉雷;陶亮;肖江強;肖江強;馬虎成;丁義濤;;兩種不同脫細胞技術(shù)構(gòu)建大鼠肝臟脫細胞支架的比較研究[A];2013中國器官移植大會論文匯編[C];2013年

3 陳華勇;柏樹令;樸成哲;;組織工程小血管脫細胞支架的結(jié)構(gòu)研究[A];中國解剖學(xué)會2011年年會論文文摘匯編[C];2011年

中國博士學(xué)位論文全文數(shù)據(jù)庫 前2條

1 劉雙德;利用豬腎臟脫細胞支架進行腎臟再生的研究[D];山東大學(xué);2015年

2 管勇;利用胚胎干細胞及腎臟脫細胞支架進行腎臟再生的研究[D];山東大學(xué);2016年

中國碩士學(xué)位論文全文數(shù)據(jù)庫 前2條

1 周盛源;韌帶脫細胞支架構(gòu)建的實驗研究[D];第二軍醫(yī)大學(xué);2010年

2 姜楠;不同方法制備大鼠肝臟脫細胞支架及其免疫原性和細胞相容性研究[D];第四軍醫(yī)大學(xué);2013年

，

本文編號：1601557