【作者】 毛慧娟；

【导师】 王笑云；

【作者基本信息】 南京医科大学 ， 内科学， 2009， 博士

【摘要】 背景和目的肾脏替代治疗已明显改善了肾衰竭患者的预后,使急性肾衰竭(ARF)死亡率降低至30%,但当合并多器官功能障碍综合征(MODS)时,死亡率仍高达60%-100%。面对严峻的挑战,人们设想并已证明这与目前的人工肾治疗仅仅代替了肾小球滤过功能,而忽略了肾小管的替代,不能完成调节免疫、内分泌、代谢、自身调节等多种功能有关。随着细胞治疗和组织工程学的兴起,人们运用组织工程技术构建生物人工肾小管辅助装置(RAD)来完善、补充现有的人工肾之不足。国际上,美国1997年Humes首次用犬肾近端小管细胞株(MDCK)构建RAD,国内本单位于2001年用猪肾近端小管细胞株(LLC-PK1)构建RAD成功。但用猪肾小管细胞构建的RAD,因为是异种生物细胞,不适用于临床治疗。本研究用原代培养的人肾近端小管细胞构建成功RAD,将其应用于治疗MODS猪模型,观察其疗效及对免疫功能的影响,为进一步临床试用RAD提供必备的技术平台和实验数据。材料和方法(1)细胞培养：原代培养人肾近端小管细胞,传代并鉴定；(2)构建RAD：用培养的人肾近端小管细胞构建RAD；(3)制作MODS合并ARF猪模型：采用盲肠结扎和穿孔(CLP),加双侧输尿管结扎术(UL)。证实该模型达到MODS诊断指标；(4)RAD治疗：用人肾近端小管细胞构建的RAD,与血滤器相连,对MODS合并ARF的猪行RAD串联连续性静—静脉血液滤过(CVVH)的治疗。(5)观察指标：血清Cr、BUN,电解质,血气,血清抗炎因子lL-10、致炎因子TNF-α, IL-6,动物存活时间。(6)设立对照组：分为RAD治疗组(A组)；假RAD治疗组(B组)；未血液净化治疗组(C组)三组,作治疗前后自身对照和组间比较。结果1分离纯化人肾近端小管细胞(PTC),培养5-6天后融合成单层细胞,形态呈鹅卵石样,可传代7-9代,鉴定证实为人肾近端小管细胞,重复培养10次,均能稳定获得同样细胞,每克肾皮质可分离培养出(6-12)×106个PTC结果2 RAD对MODS合并ARF猪的治疗作用：(1)改善血压：A组治疗至24h平均动脉血压(MAP)为88.13±7.62)mmHg,而B组(63.50±11.82),C组(53.50±2.52)(P均<0.01)；(2)A、B两组治疗后血BUN、Cr、K及血气PaO2、HCO3值均较治疗前有明显改善,但两组间无明显差异(P>0.05)。(3)血清TNF-α(pg/ml):至24h时A组为(394.42±35.62)较治疗前平均水平(526.67±40.08)明显下降(P<0.05),同时也明显低于同时间点的B组(531.76±30.23)和C组(552.89±27.81)(P<0.05),B组治疗前、后及C组无明显变化(P>0.05)；(4)A组血清IL-10 (pg/ml)的峰值为(272.36±48.89),明显高于B组(129.84±15.43)和C组(102.59±10.21)(P<0.01)；(5)血清IL-6(pg/ml):A组、B组治疗前后血清IL-6水平无明显改变,但C组血清IL-6水平逐渐升高；(6)延长生存时间：A组生存时间(hrs)(118.73±8.48),明显长于B组(81.20±11.76)和C组(74.96±23.00)(P<0.01),分别延长46.20%、58.39%。结论(1)本研究采用的培养方法可在7天内获得人肾近端小管细胞,且重复性好,细胞数量多,可为进行肾小管上皮细胞治疗提供必需的细胞；(2)人肾近端小管细胞构建的RAD治疗MODS猪,能显著改善低血压,降低血清TNF-α,升高血清IL-10水平,延长生存时间。本研究的创新点：(1)在国内首先用原代培养的人肾近端小管细胞构建RAD;(2)在国内率先开展人肾细胞RAD应用于MODS合并ARF的动物实验,并研究其对免疫调节功能的影响。目的：研究新型血液净化方式——配对血浆滤过吸附(coupled plasma filtration adsorption, CPFA)治疗重症感染并MODS患者的临床疗效及其对患者免疫功能的影响。方法：选择重症感染并MODS的患者13例,采用前瞻性,随机,自身交叉对照研究。每例患者均在常规药物治疗基础上加用CPFA (A)和高容量血滤(B) (high volume hemo filtration, HVHF)各治疗10小时,A,B治疗顺序随机,间隔一夜洗脱期(12h),比较两种治疗方式的临床效果及对C反应蛋白(CRP)、七种血清细胞因子：肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1p(1L-1β)、白细胞介素-10(1L-10)、白细胞介素-1受体拮抗剂(IL-1Ra)、可溶性肿瘤坏死因子受体1和受体2 (sTNFR1、sTNFR2)水平的影响,并观察两种血液净化方式对单核细胞HLA-DR表达及对单核细胞分泌功能的影响。结果：1、CPFA治疗后体温明显下降,平均动脉压(MAP)明显升高且升压药剂量减少,心率有下降趋势。APACHEⅡ评分从治疗前21.77±5.83下降至16.95±5.26,P=0.001。HVHF治疗后,MAP明显升高,体温有下降趋势,心率无明显变化,治疗前、后APACHEⅡ评分为18.75±7.92 vs 15.37±7.34,P=0.007。对APACHE评分、MAP的影响两种治疗方式之间未达到显著性差异(P>0.05)。2、与HVHF相比,CPFA同样能很好地维持机体电解质、酸碱的平衡状态,明显降低血尿素氮、肌酐水平,改善血氧分压和氧合指数。对血小板、血红蛋白无不良影响。也未引起血清白蛋白、免疫球蛋白的丢失减少和补体的激活。3、CPFA治疗后,CRP(mg/L)由141.32±21.52下降至89.21±13.97(P=0.009),而HVHF后CRP有下降趋势,无统计学意义。两种方式对CRP的影响有显著性差异(P=0.041)。4、CPFA治疗过程中,无一例发生凝血、出血、过敏、栓塞等不良反应。5、患者的预期死亡率平均是55.65%,实际生存率69.23%(9/13)2例死亡,2例自动出院。6、CPFA能明显降低血清TNF-α,升高血清IL-1Ra及sTNFR2/TNF-α、IL-1Ra/IL-1β的比值。10h与0h相比,均有统计学意义(P<0.05)。HVHF治疗,血清IL-1β在5h时较0h有下降,至10h又恢复到0h水平；血清IL-1Ra、IL-1Ra/IL-1β比值在5h时上升,至10h又明显下降至0h的水平。CPFA、HVHF对血清细胞因子TNF-α、IL-1Ra、sTNFR2/TNF-α、IL-1Ra/IL-1β的影响有显著性差异(P<0.05)。7.CPFA0h,血清TNF-α、IL-1β、IL-6经过整个装置后的下降率分别为38.95%,41.76%,44.39%；5h时下降率分别降至23.55%,16.18%,7.53%。HVHF除在0h,IL-1β经过血滤器的下降率为45.52%外,0h或5h以上细胞因子浓度在动、静脉端均无差异。(P>0.05)。8、CPFA治疗后,单核细胞HLA-DR的阳性率(%)变化(73.65±11.52 vs 84.32±4.63)有显著性意义(P=0.037)。HVHV治疗后,单核细胞HLA-DR阳性率未有明显改变(P>0.05)。9、CPFA治疗后,全血在体外有或无磷酸脂多糖(LPS)刺激下,分泌TNF-α的量均明显高于治疗前(P<0.05)；而HVHF治疗前、后,全血分泌TNF-α的量未有明显变化(P>0.05)。10、随着CPFA的进行,患者血清抑制体外培养的THP-1细胞分泌TNF-α的功能逐渐减弱,至10h时分泌TNF-α的量较0h时有明显升高。而随着HVHF的进行,患者血清抑制THP-1细胞分泌TNF-α的功能有减弱趋势,但未达到统计学意义。结论：与HVHF相比,CPFA治疗MODS同样安全、有效。但在降低CRP和致炎性细胞因子水平,提高抗炎/致炎因子比值,改善单核细胞抗原呈递功能和分泌功能方面,优于HVHF。CPFA具有重建机体免疫内稳态的作用。本研究的特色和创新点：1、本研究针对的是临床工作中的难点、热点问题——多器官功能障碍综合征的救治,尝试应用新型血液净化方式——血浆滤过吸附技术。关于CPFA的原著研究目前Pubmed检索不足10篇论著。国内该项工作刚刚起步,CPFA治疗MODS尚缺乏成熟的经验。2、我们的研究方法采用前瞻性,随机,自身交叉对照研究,对MODS的救治既符合急诊医学和伦理学的要求,又尽可能获得比较两种血液净化方式的有价值资料,客观评价新型血液净化方式CPFA对MODS的安全性和有效性；3、国外仅有一篇CPFA对少数细胞因子和单核细胞分泌功能影响的研究。本研究在此基础上,进一步研究了CPFA对CRP,对炎症因子与抗炎症因子的平衡,对单核细胞呈递功能的影响等。更多还原

【Abstract】 Background and Purpose Though renal replacement therapy plays a very important role in the treatment of acute renal failure (ARF), patients with ARF complicated with multiple organ dysfunction syndrome (MODS) still have mortality rates of 60-100% despite the application of conventional continuous renal replacement therapy (CRRT). Poor outcomes may be due to the fact that these therapies substitute for the glomerular filtration function only, not for replacing the metabolic, reclamation, and endocrine function of renal tubule in vitro. Humes HD et al first reported the establishment of bioartificial renal tubule assistance device in vitro in 1997. We first constructed the RAD using LLC-PK1 cells in China in 2000. This study is to investigate the effect of RAD containing human renal proximal tubule cells (hPTC) on porcine MODS with ARF. It is necessary to get this date for further clinical application of RAD.Methods (1) Primary culture, subculture and identification of human renal proximal tubule cells (PTC). (2) Fabrication of RAD with hPTCs. (3) Establishment of porcine model with MODS and ARF. All pigs were performed by cecal ligation and puncture (CLP) and bilateral ureteral ligation (UL). (4) Pigs with MODS and ARF were placed on CVVH with either a RAD (A group) or a sham RAD without cells (B group) or without blood purification (C group). (5)Serum ALT, BUN, Cr, electrolytes and tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-10 (IL-10), arterial blood gas were tested at 0,8,16,24 hour after the beginning of the treatment. (6)Vital signs and survival time were recorded.Results (1) After 5-6 days, the cultured cells reached confluence, displaying the typical cobblestone appearance and could be subcultured till 7-9 passages. Morphology of the cells and staining of specific marker validated them as human PTC. (2) Effect of RAD constructed with human renal cells on porcine MODS with ARF showed that:①Mean arterial pressure(MAP, mmHg) was significantly higher in group A than group B and C at 24 hours ((88.13±7.62) vs (63.50±11.82) vs (53.50±2.52))(P<0.01);②The values of BUN、Cr、K+、PaO2、HCO3- were similar during the treatment periods between group A and B (P>0.05),which were better than those of group C;③In group A, TNF-α(pg/ml) was 394.42±35.62 at 24 hours significantly lower than that of pre-treat (526.67±40.08) and group B(531.76±30.23) and C (552.89±27.81) at the same time point.(P<0.05). There were no differences between pre-and post-treatment of group B and C.④Peak level of IL-10 (pg/ml) was significantly higher (P<0.01) in group A (272.36±48.89) than that in group B (129.84±15.43) and group C (102.59±10.21).⑤There was no difference of serum IL-6 between pre-treatment and post-treatment in groups A and B, but serum IL-6 gradually increased in group C.⑥The average survival time in group A was 118.73±8.48 hrs significantly longer than in group B (81.20±11.76 hrs) and C (74.96±23.00 hrs) (P<0.01), which prolonged by 46.20% and 58.39%.Conclusions (1) This culture system can amplify a lot of human PTC s in 7 days. These bred cells can supply for cell therapy. (2) Human RAD can ameliorate MAP, drop the level of serum TNF-α, promote the level of serum IL-10 and prolong the survival time of porcine MODS with ARF.Characteristic and innovation of this study (1) First constructing RAD with primary cultured human PTC in China. (2) Taking the lead in the study on the treatment of human RAD in animal model with MODS and ARF in China. Objective:To investigate the effects of coupled plasma filtration adsorption (CPFA) on the immune function of patients with multiple organ dysfunction syndrome (MODS).Methods:This study was a prospective,pilot,before-and-after self-crossover clinical trial. Thirteen patients diagnosed with MODS and severe infection were randomly allocated to both 10 hours of CPFA and 10 hours of high-volume hemo filtration (HVHF) with a 12-hour interval and in random order. The following clinical indicators including changes in mean arterial pressure (MAP), oxygenation index(PaO2/FiO2), biochemical parameters of liver and kidney function, serum C reactive protein (CRP) and acute physiology and chronic health evaluationⅡ(APACHEⅡ) score were determined before and after the treatment. Serum concentrations of seven cytokines including tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), interleukin-1β(IL-1β), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), and soluble tumor necrosis factor receptors 1 and 2 (sTNFR1 and sTNFR2) were measured during each treatment.The HLA-DR expression by the blood monocytes and the TNF-αproduction by the patients’blood (both spontaneous and lipopolysaccharide stimulated) were tested before and after the treatment.TNF-αproduction of normal human monocytes (THP-1 cells) incubated in vitro with the patients’plasma was also measured. Results①Mean arterial pressure (MAP) increased and dopamine requirement decreased equally after 10 hr of CPFAor of HVHF (P>0.05).APACHE score decreased from 21.77±5.83 to 16.95±5.26 with CPFA(P=0.001) and from 18.75±7.92 to 15.37±7.34 with HVHF(P=0.007),but had no statistically difference between the two therapies (P>0.05).②CPFA improved kidney function and oxygenation index, maintained electrolyte balance and acid base balance effectively, which equal to HVHF (P>0.05). There was no decrease of albumin, immunoglobulin, hemoglobin and thrombocyte count during CPFA or HVHF.③CRP(mg/L) decreased from 141.32±21.52 to 89.21±13.97 with CPFA (P=0.009) and only had decrease trend with HVHF(P>0.05). The two treatments had significant difference in the change of CRP(P=0.041).④No therapy related adverse reactions,including severe haemorrhage, shock, hypersensitivity,were noted and patients tolerated well toward CPFA.⑤The total survival rate of patients was 69.23% (9/13 cases) at the end of the treatment, whose average estimated mortality was 55.65%.⑥During CPFA, the fall in serum TNF-αand rise in serum IL-1Ra coincided with the rise in ratios of sTNFR2/TNF-αand IL-1Ra/IL-1β(p<0.05), which were different from those seen within HVHF (p<0.05).⑦The descending rates of TNF-α、IL-1β、IL-6 were 38.95%,41.76%,44.39% respectively at Oh but decreased to 23.55%,16.18%,7.53% at 5h of CPFAdevice.There was no change of those cytokines between pre-and post-of the hemofiltrator at 0h,5h of HVHF, except that the descending rate of IL-1βwas 45.52% at Oh of HVHF device.⑧HLA-DR expression(%) increased after CPFA (84.32±4.63 vs.73.65±11.52, p=0.037), but there was no change after HVHF (p>0.05).⑨Spontaneous and lipopolysaccharide-induced TNF-αproduction increased over time with CPFA (p=0.038,0.034, respectively), but did not change with HVHF (p>0.05).⑩Patient plasma suppressed the production of TNF-αby cultured normal monocytes. This effect decreased over time with CPFA(p=0.041), but there was no effect with HVHF. (p>0.05) Conclusions CPFA was superior to HVHF in increasing the ratios of antiinflammatory to proinflammatory mediators, improving antigen presentation ability, and restoring leukocyte responsiveness. These findings suggest a potential role for CPFA in the treatment of MODS.更多还原