利用胚胎干细胞及肾脏脱细胞支架进行肾脏再生的研究

发布时间：2018-03-12 12:26

  本文选题：脱细胞支架　切入点：生物材料　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：随着慢性肾功能不全发病率的逐年增高,终末期肾病(ESRD)已经成为严重困扰公众的社会问题。目前对于ESRD的治疗方式主要有两种：血液透析及肾脏移植。血液透析容易引起较多的并发症且效率较低,长期治疗效果较差,容易给患者带来较大的痛苦以及经济负担。而肾脏移植能够完整替代肾脏的功能,但是存在肾源短缺严重以及严重的免疫排斥反应问题。干细胞与肾脏再生医学的发展为这一难题带来了新的希望。其中细胞-脱细胞支架技术的发展成为近年来肾脏再生领域研究的热点。所谓细胞-脱细胞支架技术是指将动物天然肾脏进行去细胞化,获得无细胞的肾脏细胞外基质(ECM)—肾脏脱细胞支架,然后应用干细胞或者成体细胞对脱细胞支架进行再细胞化,进行体外或体内培养后,获得有功能的再生肾脏。肾脏细胞种类复杂,因此,选用合适的干细胞在支架内进行分化,是选择种子细胞的依据。胚胎干细胞是一种多能干细胞,拥有强大的增殖能力与分化能力,且在体外已能够向肾脏细胞系分化,是一种良好的种子细胞。本研究拟采用胚胎干细胞,对肾脏脱细胞支架进行再细胞化,获得有功能、可移植的肾脏。第一部分 肾脏脱细胞支架的建立与生物学特性的检测目的建立快速有效的脱细胞支架流程,获得适合细胞种植的肾脏脱细胞支架。方法：1.肾脏的获取：实验动物选择雄性Wista大鼠,体重在200-300g之间。应用10%水合氯醛麻醉后,取腹正中切口,暴露腹主动脉,应用24G套管针插入,将肾脏血液冲出,然后取出肾脏。2.脱细胞支架的制备：应用0.5%的十二烷基磺酸钠(SDS)溶液以及蒸馏水对肾脏进行持续灌注,速度为0.5ml/min,肾脏变为白色半透明时换PBS溶液冲洗,将残留的SDS溶液冲洗干净。3.脱细胞支架鉴定：(1)形态学及免疫学检测：应用HE以及Masson染色检测的方法鉴定脱细胞支架中细胞去除情况。应用免疫组化检测肾脏脱细胞前后Ⅳ型胶原及纤连蛋白的表达情况。扫描电镜检测显微结构保留情况。(2) 分子生物学检测：应用DNA及胶原检测试剂盒检测肾脏脱细胞支架中DNA及胶原的含量；应用ELISA的方法检测脱细胞支架中HGF、TGF及VEGF的含量。(3)血管系统检测：应用血管造影的方式在体外检测血管系统的完整性；将肾脏脱细胞支架原位移植到大鼠体内,进一步检测其通畅性及严密性。结果：1.大体观：肾脏经过SDS及双蒸水灌注后,逐渐变为白色半透明状。2.HE及Masson染色：显示细胞成分去除彻底,仅剩胶原成分。3.免疫组化：显示脱细胞支架中Ⅳ型胶原以及纤连蛋白保留较好。4.扫描电镜显示肾小球的基底膜等膜性结构保留完整。5.分子生物学检测：DNA含量在脱细胞之后低于正常的5%以下；胶原含量在脱细胞前后变化不明显；HGF、TGF以及VEGF等细胞因子的含量变化不明显。6.血管系统检测：血管造影显示肾脏血管系统保留完整,无造影剂漏出,血管分支结构显示清楚。脱细胞支架原位移植血管夹放开后,血液运行通畅,无明显漏出。HE显示血液充盈到肾脏内部,2月后形成血栓及炎性细胞浸润。结论：应用SDS等脱细胞剂及自制设备灌注大鼠肾脏,能够获得完整的肾脏脱细胞支架,建立了有效的肾脏脱细胞支架制备流程。第二部分 肾脏脱细胞支架的再细胞化及再生肾脏的功能鉴定目的：应用胚胎干细胞对肾脏脱细胞支架进行再细胞化,获得有一定功能、可移植的肾脏。方法：1.种子细胞选用小鼠胚胎干细胞,在体外进行培养,应用免疫组化检测干细胞标志物,如OCT4.NANOG等干细胞标志物,以确保其在细胞种植之前保持干细胞特性。2.将胚胎干细胞消化、收集,调整其细胞浓度至1×107/ml。经肾动脉灌注3ml,在-50cmH2O负压下经输尿管灌注3ml。静置过夜,连续灌注2天。3.再生肾的培养：肾脏脱细胞种植再细胞化后,通过自制灌注设备,应用DMEM/F12培养基进行循环灌注培养,温度37℃,连续培养7天。4.再生肾的鉴定：再生肾脏经过体外培养后,进行HE染色鉴定；体外制备改良的Hank‘s液,进行功能鉴定；将再生肾脏移植到大鼠体内,收集静脉血及尿液,进行肾脏功能的体内测定。结果：1.胚胎干细胞经过鉴定,OCT4.NANOG以及SOX2等干细胞标志物表达良好,表明其干细胞特性保持较好。2.经过细胞种植以及再生肾脏体外培养,肾脏脱细胞支架的肾小球部位以及部分肾小管部位有较多细胞贴附。3.经过功能测定,再生肾脏较脱细胞支架产生尿液(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)较脱细胞支架更多。结论：胚胎干细胞为良好的肾脏组织工程种子细胞,经过再细胞化,获得了有一定形态结构及功能、可以移植的再生肾脏。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692;R318.08

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