血管组织工程种子细胞的建立及意义

目的：利用人Nanog基因转染血管组织工程的种子细胞（血管内皮细胞ECV304）,提高其增殖能力,以在较短时间内获得大量种子细胞,从而为克服血管组织工程种子细胞来源的匮乏及组织工程血管的快速构建提供新途径。方法：制备含有人Nanog基因的复制缺陷型重组腺相关病毒血清2型病毒颗粒rAAV2-hNanog,用rAAV2-hNanog转染ECV304细胞,然后采用RT-PCR检测hNanog基因在转染的ECV304细胞中的表达,再用MTT、免疫荧光及激光共聚焦技术检测hNanog基因对血管内皮细胞ECV304生长的影响。结果：（1）rAAV2-hNanog转染ECV304细胞后,RT-PCR检测表明hNanog基因能在血管内皮细胞ECV304中稳定表达;同时,血管内皮细胞在转染后的增殖能力也显著增强（P〈0.05）。光镜下观察发现,转染的血管内皮细胞的形态无明显改变。结论：转染hNanog基因后,血管内皮细胞的形态无显著变化,但其增殖能力明显增强。利用hNanog基因提高血管内皮细胞的增殖能力,能够克服血管组织工程种子细胞来源不足的瓶颈,为组织工程血管的快速构建及应用提供一种新的方法。     

重组2型腺相关病毒-人骨形成蛋白7转染犬髓核细胞及其对髓核细胞表型的影响

目的:观察重组2型腺相关病毒-人骨形成蛋白7(recombinant adeno-associated virus-2expressinghuman bone morphogenetic protein-7,rAAV2-hBMP7)转染犬髓核细胞情况及其对细胞表型的影响。方法:用1岁龄Beagle犬L4/5椎间盘髓核进行髓核细胞体外培养,取第2代髓核细胞进行实验,实验组以rAAV2-hBMP7(1×105v.g/细胞)转染髓核细胞,对照组以重组2型腺相关病毒-增强型绿色荧光蛋白(recombinantadeno-associated virus-2expressing enhanced green fluorescent protein,rAAV2-EGFP)转染髓核细胞。在转染后4d、7d和14d以PCR检测髓核细胞中hBMP7的mRNA表达,在转染后7d和14d以Western blot法检测髓核细胞中hBMP7蛋白表达。在转染后4d、7d和14d采用RT-PCR法检测蛋白多糖、Ⅰ型胶原和Ⅱ型胶原的mRNA含量,采用二甲基蓝及ELISA法分别检测蛋白多糖、Ⅰ型胶原和Ⅱ型胶原的蛋白含量。结果:实验组髓核细胞表达hBMP7 mRNA及蛋白,并于转染后7d时表达量最高,而对照组无表达。实验组髓核细胞蛋白多糖mRNA及蛋白含量在转染后7d和14d时较4d时明显增高,14d时较7d时亦明显升高(P0.05),对照组各时间点无统计学差异(P0.05);4d时实验组与对照组比较无明显差异,7d和14d时较对照组明显增加(P0.05)。同组各时间点Ⅰ型胶原mRNA及蛋白含量均无统计学差异,两组各时间点间比较无统计学差异(P0.05)。实验组髓核细胞Ⅱ型胶原mRNA及蛋白含量在转染后7d和14d时较4d时明显增高,14d时较7d亦明显升高(P0.05),对照组各时间点无统计学差异(P0.05);实验组4d时与对照组比较无明显差异,7d和14d时较对照组明显增加(P0.05)。结论:rAAV2-hBMP7转染犬髓核细胞后能表达hBMP7蛋白,并能提高其蛋白多糖及Ⅱ型胶原含量。     

腺相关病毒载体转染供肝方法学的实验研究

目的 探讨腺相关病毒载体基因转染的有效途径和方法.方法 我们设计肝动脉、门静脉、双重灌注3种途径和传统、循环、夹闭3种转染方法.通过观察肝脏灌注后颜色、检测肝脏功能和肝细胞转染率,确定灌注途径和转染方法,并探讨其安全性.结果 肝脏灌注后颜色无明显差别,无肿胀或花斑出现,血流开放以后肝脏颜色迅速恢复正常,3种灌注途径ALT比较差异无统计学意义(F=0.343,1.265,0.055,P＞0.05);1周后肝脏组织均有免疫荧光染色,并且携带增强型绿色荧光蛋白基因的腺相关病毒载体,转染率比较差异无统计学意义(F=0.080,0.091,0.045,P＞0.05).传统法的转染率略低于循环法,而夹闭法的转染率在各时间段均高于前两种方法,差异有统计学意义(F=3.880,2.976,5.129,P＜0.05).3种方法转染率随时间逐渐升高,6周左右达到高峰,以后缓慢下降.结论 经肝动脉灌注是基因转染的有效途径,夹闭法可以提高目的基因的转染率,两者均无肝脏功能损害,腺相关病毒载体的转染呈缓慢、持续的过程.     

人降钙素基因相关肽重组腺相关病毒在原代培养大鼠阴茎海绵体平滑肌细胞中的表达及其作用

目的:观察腺相关病毒载体介导的人降钙素基因相关肽(hCGRP)基因在原代培养的大鼠阴茎海绵体平滑肌(CCSM)细胞的表达及其作用,探讨其应用于勃起功能障碍(ED)基因治疗的可行性。方法:原代培养的SD大鼠CCSM细胞随机分为4组:实验组、空病毒组、示踪剂组和对照组,分别以可分泌表达hCGRP重组腺相关病毒VssH-GCMV-hCGRP、空病毒VssHGCMV和表达绿色荧光蛋白(GFP)重组腺相关病毒VssCMV-GFP进行体外转染或不予任何处理。采用斑点印迹试验检测培养液中的hCGRP和放射免疫法检测转染细胞中的环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)水平,观察hCGRP和示踪剂GFP的表达及其对CCSM细胞的影响。结果:重组腺相关病毒载体能有效地将外源基因hCGRP导入大鼠CCSM细胞并稳定表达,与空病毒组和对照组相比,该病毒明显刺激大鼠CCSM细胞中cAMP水平升高[(48.7±1.1)nmol/L对(12.3±1.2)nmol/L和(7.8±1.4)nmol/L,P均<0.01],培养液中可检测到免疫反应性hCGRP。结论:可分泌表达生物活性肽重组腺相关病毒基因转移系统可有效进行多肽类在CCSM细胞过表达,并影响该细胞的cAMP水平,可被应用于ED的基因治疗。     

腺相关病毒载体介导p27Kip1基因转染骨肉瘤MG-63细胞的研究

目的 观察p27Kip1基因转染后对骨肉瘤MG-63细胞增殖和生存能力的影响.方法 重组质粒在内的三质粒共转染包装、收集腺相关病毒颗粒,检测病毒滴度并感染MG-63细胞,细胞免疫组织化学检测p27Kip1基因的表达,并行细胞增殖实验、细胞周期分析,探讨p27Kip1基因对骨肉瘤MG-63细胞的体外作用.结果 三质粒成功共转染293细胞,X-gal染色观察转染率为60%;采用CPE法(微量全细胞病变法)检测病毒滴度1.6×108 PFU/ml;细胞免疫细胞化学鉴定p27Kip1基因高表达;细胞生长曲线显示转染组细胞增殖明显减慢;流式细胞仪观察转染AAV-p27Kip1组细胞周期见S、G2期细胞比例明显降低,G0/G1期细胞比例增高.结论 p27Kip1基因重组腺相关病毒感染骨肉瘤MG-63细胞后能高表达目的 基因,并阻滞细胞于G1/S期,从而显著抑制骨肉瘤MG-63细胞增殖.     

2型重组腺相关病毒/增强型绿色荧光蛋白转染胰腺癌细胞PANC-1的体外研究

基因治疗的关键是目的基因的高效转染和表达。我们采用2型重组腺相关病毒（rAAV2／EGFP）对人胰腺癌细胞PANC-1进行体外基因转移，观察EGFP基因能否在PANC-1中有效表达及其细胞毒性，探讨rAAV2介导目的基因转导胰腺癌细胞的效率。[第一段]     

生物人工肾小管10年回顾与展望

多脏器功能障碍综合征的高病死率与急性肾小管上皮细胞坏死密切相关.血液透析疗法可以替代肾小球的滤过功能,不能替代肾小管的功能,生物人工肾小管辅助装置具备肾小管的功能,但是这一装置还有许多的缺陷.本文从生物人工.肾小管辅助装置的设计、肾小管的重吸收功能、分泌功能、代谢功能、免疫功能等几个方面对已经取得的研究进展做综述,并对其发展趋势做出展望.     

生物人工肾小管治疗肾功能衰竭的研究进展

利用生物学与工程学原理制造出一种能模拟人体器官功能的装置 ,在这种装置中种植有成活的肾小管细胞并使其持续发挥相应的生物效应 ,从而替代肾功能衰竭者丧失的肾小管功能。     

可分泌表达人降钙素基因相关肽重组腺相关病毒体外转染培养的血管内皮细胞及其表达

目的观察可分泌表达人降钙素基因相关肽(humancalcitoningene-relatedpeptide,hCGRP)重组腺相关病毒(rAAV)体外转染培养的血管内皮细胞(endothelialcellsofvessel,ECV)及其作用,探讨其应用于血管性勃起功能障碍基因治疗的可行性。方法分别以可分泌表达hCGRP的重组腺相关病毒VssHGCMV-hCGRP、表达报告基因GFP中的重组腺相关病毒VssCMV-GFP和空病毒pssHGCMV体外转染培养的ECV细胞,采用斑点印迹试验检测培养基中的hCGRP,以放射免疫法检测转染细胞中的cAMP和cGMP水平,观察GFP和hCGRP的表达及其对ECV细胞的影响。结果腺相关病毒载体能有效地将外源基因hCGRP导入血管内皮细胞并稳定表达,VssHGCMV-hCGRP病毒组细胞中cAMP水平(45.74±3.45)nmol/L较pssHGCMV空病毒组(14.84±0.65)nmol/L和对照组(12.74±0.65)nmol/L明显高(P<0.01),并可在实验组24h细胞培养液中检测到免疫反应性hCGRP。结论可分泌表达生物活性肽重组腺相关病毒基因转移系统可有效进行多肽类在血管内皮细胞过表达,并可选择性刺激该细胞中cAMP水平升高,提示hCGRP可作为血管性勃起功能障碍基因治疗的有效候选基因。     

人p27kip1基因腺相关病毒载体的构建

目的 克隆人肿瘤转移抑制基因p27kip1,利用基因重组技术,构建其腺相关病毒载体,为进一步研究骨肉瘤的基因治疗建立一个平台.方法 从人正常胎盘组织中提取总RNA,RT-PCR获取p27kip1基因(ORF)cDNA序列,并将其克隆至腺相关病毒载体pAAV-MCS,构建人p27kip1基因腺相关病毒载体.结果 人胎盘组织抽提总RNA,并通过RT-PCR获得目的 基因ORF,经TA克隆后,将目的 基因连人pAAV-MCS中,送测序结果 无误.结论 成功构建的重组质粒pAAV-MCS-p27kip1,将为进一步研究p27kip1蛋白的生物学效应提供基础,为p27kip1基因应用于骨肉瘤的治疗提供实验依据.     

Research into the development of a wearable bioartificial kidney with a continuous hemofilter and a bioartificial tubule device using tubular epithelial cells

Current hemodialysis treatment is insufficient because of intermittent treatment and loss of tubular function. In order to overcome the loss of tubular function, a bioartificial kidney has been developed consisting of continuous hemofiltration (CHF) with 10 L/day of filtrate and a bioartificial tubule device using proximal tubular epithelial cells and hollow fiber membranes. Ten L/day of CHF enabled plasma levels of urea, creatinine, uric acid and, beta2-microglobulin in eight renal failure patients to be maintained at remarkably low levels. The concept was tested with 6 L (4 mL/min) of 10 L/day (7 mL/min) filtrate regenerated by a bioartificial tubule device and 4 L/day (3 mL/min) replaced by food and drinks. Lewis lung cancer-porcine kidney 1 (LLC-PK1) cells with a cell density of 107 cells/mL were seeded inside polysulfone hollow fiber modules four times at 1 h intervals while rotating the module 90 degrees each time, and were cultured for 48 h to form confluent monolayers. The leak rates of urea and creatinine across LLC-PK1 cell-attached polysulfone membrane modules (membrane areas: 56 cm2 and 4000 cm2) were investigated. Via conversion from 56 m2 to 1 m2 hollow fiber modules with LLC-PK1 cells for 24 h, the transport rates of H2O, glucose and Na+ were, respectively, 40, 65 and 35% of the target transported amounts from 6 L/day of filtrate. The rates are expected to approach 100% when 4-5 g/dL of albumin is added to the basal portion of the medium since the results were obtained without the addition of albumin for colloidal osmotic pressure.     

生物人工肾小管体外构建及对氨基酸、钠离子、肌酐转运功能的研究

目的探讨体外构建生物人工肾小管(RAD)的方法及5h内RAD在体外对肌酐(Cr)、葡萄糖(Glu)、钠离子(Na )和天冬氨酸、苏氨酸、丝氨酸、谷氨酸的转运功能。方法采用猪肾近曲小管上皮细胞株(LLC-PK1)体外培养,然后植入FH66血液滤过器内腔继续培养两周构建RAD。测定RAD对Na 、Cr、葡萄糖及4种氨基酸的转运功能,并与未植入LLC-PK1的滤过系统做对照,并观察哇巴因、根皮苷对转运的特异性抑制作用。结果RAD组肌酐滤过率明显低于对照组(P<0.01)。RAD组Na 、葡萄糖、氨基酸的滤过率在哇巴因及根皮苷作用后明显低于对照组(P<0.01);去除药物抑制因素后,滤过率又能恢复,与对照组比较差异有显著性意义。结论体外构建的RAD具有对氨基酸、葡萄糖、钠离子、肌酐的选择性转运功能,且能在5h内稳定维持。     

Culture and characterization of human hepatocytes obtained after graft reduction for liver transplantation: a reliable source of cells for a bioartificial liver

This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 x 10(6) and 38 x 10(6) cells/g of tissue (median value 14.73 x 10(6) cells/g), and the viability was 61.17 +/- 27.43%. The total number of cells ranged between 57.6 x 10(6) and 12 150 x 10(6) cells (median value: 740 x 10(6) cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 +/- 1.77 micro g/mL). Urea production was detected during the first week (peak value at day 6: 17.12 +/- 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.     

Evaluation of water and electrolyte transport of tubular epithelial cells under osmotic and hydraulic pressure for development of bioartificial tubules

Our aim was to develop bioartificial tubules using tubular epithelial cells and artificial membranes and evaluate the function of water and electrolyte transport by various tubular epithelial cells. The cells were cultivated onto extracellular matrix (ProNectin F) coating polycarbonate membrane. Water transport from the apical to the basolateral site of cells was examined using a modified Ussing chamber module. Water transport under colloidal osmotic pressure on the apical site and hydraulic pressure on the basolateral site were higher in JTC-12, LLC-PK1 cells than in MDCK cells. Water transport under osmotic plus hydraulic pressure was highest in LLC-PK1 cells. We made bioartificial tubules using LLC-PK1 cells and polysulfone hollow fiber cartridges. Water and Na ion transport function was high, and BUN and creatinine passage was recognized in these bioartificial tubules. BUN and creatinine concentrations of reabsorption fluid in these bioartificial tubules were significantly lower than those concentrations of control media and of noncell attached polysulfone hollow fiber cartridges. Though LLC-PK1 cells were more preferable cells for the use of bioartificial tubules in terms of water and electrolyte transport, the passage of BUN and creatinine was not appropriate for clinical use. To select more preferable cells for bioartificial tubules which transport water and electrolytes and do not induce passage of uremic toxins is necessary.     

Isolation, propagation and characterization of primary tubule cell culture from human kidney

Proximal tubule cells (PTC) are the major cell type in the cortical tubulointerstitium. Because PTC play a central role in tubulointerstitial pathophysiology, it is essential to prepare pure PTC from kidney tissue to explore the mechanisms of tubulointerstitial pathology. The authors have successfully refined and characterized primary cultures of human PTC using Percoll density gradient centrifugation as a key PTC enrichment step. The cells obtained by this method retain morphological and functional properties of PTC and are minimally contaminated by other renal cells. In particular, the primary isolates have characteristics of epithelial cells with uniform polarized morphology, tight junction and well-formed apical microvilli. Cytokeratin is uniformly and strongly expressed in the isolates. Brush border enzyme activities and PTC transport properties are retained in the isolates. This method therefore provides an excellent in vitro model for the physiologic study of the human proximal tubule.     

Transport characteristics of human proximal tubule cells in primary culture

Summary: In order to establish an in vitro model for studying human proximal tubule transport, primary culture of human proximal tubule cells (PTC) was carried out using an improved technique and the properties of these cells were characterised in detail. Using a combination of collagenase treatment, mechanical sieving and isopycnic ultracentrifugation, large numbers of highly purified populations of PTC were isolated and propagated from histologically normal regions of human nephrectomy specimens. Cultured human PTC demonstrated typical histologic and ultrastructural morphologies, well-preserved brush border enzyme activities, and cyclic adenosine monophosphate (cAMP) production which was stimulated by parathyroid hormone (PTH) but not by vasopressin. Tight confluence, as evidenced by relative impermeability to the paracellular diffusion of inulin, was achieved on porous membrane inserts within 6–8 days. Confluent monolayers generated Na⁺, K⁺, Cl^?, HCO₃^? and PO₄^3- concentration gradients between apical and basolateral medium compartments, which correlated well with the reabsorption processes known to occur in human PTC in vivo. A number of polarised transport systems were demonstrated, including phlorizin-inhibitable apical Na⁺-glucose transport, PTH-inhibitable apical Na⁺-phosphate transport, probenecid-inhibitable organic anion transport and quinine-inhibitable organic cation transport. Using microspectrofluorimetric and ²²Na⁺ uptake measurements, pharmacologically distinct apical and basolateral sodium-hydrogen exchangers (NHE) were identified. Apical NHE was significantly inhibited by micromolar concentrations of phorbol esters, ethylisopropylamiloride (EIPA) and 3-methylsulphonyl-4-piperidino-benzoylguanidine methanesulphonate (HOE694). the mean resting intracellular pH of human PTC was 7.23 ± 0.04 and the mean intrinsic buffering capacity following a 20 mmol/L NH₄Cl prepulse was 28.45 ± 0.96 mmol/L/unit pH. the results suggest that human PTC, prepared for culture as described herein, maintain morphological and physiological properties characteristic of the segment in vivo. the method therefore provides a useful model for the study of highly polarised transport processes in the human proximal tubule.     

Metabolic outcomes and renal function after simultaneous kidney/pancreas transplantation compared with kidney transplantation alone for type 2 diabetes mellitus patients

In this study, we aimed to compare the metabolic outcomes, renal function, and survival outcomes of simultaneous pancreas and kidney transplantation (SPK) and kidney transplantation alone (KTA) among end-stage kidney disease (ESKD) patients with type II diabetes mellitus (T2DM). Patients with ESKD and T2DM who underwent KTA (n = 85) or SPK (n = 71) in a transplant center were retrospectively reviewed. Metabolic profiles, renal function, and survival outcomes were assessed repeatedly at different follow-up time points. Propensity score procedures were applied to enhance between-group comparability. The levels of renal and metabolic outcomes between SPK and KTA over time were examined and analyzed using mixed-model repeated-measures approaches. The median follow-up period was 1.8 years. Compared with KTA, SPK resulted in superior metabolic outcomes and renal function, with lower levels of glycated hemoglobin (HbA1c; P = 0.0055), fasting blood glucose (P < 0.001), triglyceride (P = 0.015), cholesterol (P = 0.0134), low-density lipoprotein (P = 0.0161), and higher estimated glomerular filtration rate (eGFR; P < 0.001). SPK provided better metabolic outcomes and renal function. The survival outcomes of the recipients and grafts were comparable between the two groups.     

Cytokines, T cells and proliferative glomerulonephritis

SUMMARY: The glomerulus, by virtue of its functional role as a filter, is vulnerable to injury in the context of inflammatory responses, with the potential involvement of a number of different inflammatory processes. Recent work has provided insights into the role of T cells in proliferative glomerulonephritis, particularly in determining patterns of injury and outcomes in cresentic forms of glomerulonephritis. Experimental models have shown that in proliferative glomerulonephritis, cytokines play important roles both in determining T helper cell phenotype (particularly in the context of T helper cell 1 responses) and (from T cell themselves) in activating effectors of injury. Conversely, some cytokines regulate T cell responses to limit injury. There is an emerging role in other areas of the inflammatory response for cytokines traditionally thought to be involved predominantly in the injurious T cell response. These include regulatory T cells, the interaction between resident renal cells and leukocytes and the development of renal fibrosis. Cytokine-based therapies are entering clinical practice in other diseases. However, a number of challenges and questions remain to be answered before translating basic understanding into clinical practice in immune glomerular injury.     

Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria

The primary hyperoxalurias, PH1 and PH2, are inherited disorders caused by deficiencies of alanine:glyoxylate aminotransferase and glyoxylate reductase, respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end-stage renal failure and systemic oxalosis. To provide a non-invasive means of accessing kidney cells from individuals with primary hyperoxaluria, we have derived primary cultures of renal proximal tubule cells from the urine of these patients. The cells stain positively for the epithelial markers pan-cytokeratin and zonula occludens 1 and the proximal tubule marker γ-glutamyl transpeptidase. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed glyoxylate reductase at the mRNA level, illustrating their potential value as a source of renal material from these individuals.