Experimental Study on the Cryopreservation of LLC-PK1 Epithelial Cells with Hypoxic UW Solution

The effects of oxygen partial pressure on cryopreservation of the cells with organ preservation solution were explored. Hypoxic UW solution was made by purging the UW solution with argon. The pig proximal tubule epithelial cells （LLC-PK1 cells） were cryopreserved in hypoxic UW solution （Ar-UW group） or standard UW solution （UW group） at 4℃ for 48 h. Trypan blue staining and LDH detection were performed to evaluate the injury of the cells. The results showed that the oxygen partial pressure in Ar-UW group was significantly declined from 242±6 mmHg to 83±10 mmHg. After cryopreservation at 4℃ for 48 h, LDH leakage rate and Trypan blue-stained rate in Ar-UW group were （11.3±3.4）% and （10.5±4.7）%, respectively, which were significantly lower than in UW group [（49.5±6.9）% and （47.6±9.3）% respectively, both P〈0.01]. It was concluded that lower oxygen partial pressure of UW solution was more beneficial to the cryopreservation of LLC.     

A modified CZ-1 preserving solution for organ transplantation: comparative study with UW preserving solution

Background The University of Wisconsin colloid based preserving solution （UW solution） is the most efficient preserving solution for multiorgan transplantation. Unfortunately, unavailability of delayed organ preserving solutions hindered further progression of cardinal organ transplantation in China. In this study, we validated an organ preserving Changzheng Organ Preserving Solution （CZ-1 solution） and compared it with UW solution.
Methods A series of studies were conducted on how and how long CZ-1 solution could preserve the kidneys, livers, hearts, lungs and pancreas of New Zealand rabbits and SD rats. Morphology of transplanted organs was studied by visible microscopy and electron microscopy; biochemical and physiological functions and the survival rate of the organs during prolonged cold storage were studied.
Results There was no significant difference between CZ-1 and UW solutions in preserving the kidneys, livers, hearts or lungs of rabbits; kidneys, livers, intestinal mucosa or pancreases of SD rats or five deceased donors＇ testicles. In some aspects, such as preserving rabbits＇ hearts, rats＇ intestinal mucosa and pancreases, the effect of CZ-1 solution was superior to UW solution. CZ-1 could safely preserve kidneys for 72 hours, livers for 24 hours, hearts for 18 hours and lungs for 8 hours for SD rats. Twelve kidneys preserved in cold CZ-1 solution for 22-31 hours were transplanted successfully and the mean renal function recovery time was （3.83±1.68） days.
Conclusions CZ-1 solution is as effective as UW solution for organ preservation. The development of CZ-1 solution not only reduces costs and improves preservation of organs, but also promotes future development of organ transplantation in China.     

Studies on the development of the multi-organ preservation solution

At PreSent, UW solution is the ~ efficient multiogn PreSent solution for ~. lhause ourcountly is laCk of Prolonged multi~Ogu PreSerVation soluhon laal UW ~on, the baber developnd of caldhaloaf haplantation is ~ctedll]. Our h~ be toexplore the multi~ogu PreSerVation solution in lop. After5 yealS effort, we succ~ developed a ~ed [J'WSOlution: NO. I Ch~ Mulh-Ogu ~ sation (CZ-l ~on) and has accoedished ~ acments and some clinical aPPlication re~.~ ~ ~ P~ ~ (l) caf ~:Inade in our hafta…     

SMO保存液对犬肾低温保存期间皮质细胞凋亡的影响

目的:探讨自制上海多器官保存液(Shanghai multi-organ preservation solution,SMO液)对低温保存期间犬肾皮质细胞凋亡的影响,以探讨SMO液的作用机制.方法:犬肾分别以0～4℃的SMO液、高渗枸橼酸盐腺嘌呤液(HC-A)或UW液(University of Wisconsin solution)灌洗保存,在低温保存各时间点取肾皮质标本作形态学观察;采用TUNEL法检测皮质细胞凋亡发生情况;以硫代巴比妥酸法测定各保存液组肾皮质丙二醛(MDA)含量的改变,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性变化.结果:低温保存第1天光镜观察细胞水肿、胞质空泡化、肾小管坏死等改变,三组相似,但保存2 d后SMO组和UW组形态学改变明显轻于HC-A组.随低温保存时间延长,各保存液组肾皮质细胞凋亡的发生增加,而SMO组凋亡指数显著低于同时点HC-A液组 (P＜0.05).随着低温保存时间延长,各保存液组肾皮质MDA含量增加,保存1～3 d SMO液组MDA含量显著低于HC-A液组 (P＜0.05);低温保存中SMO液组、HC-A液组、UW液组肾皮质SOD酶活性无明显差异.结论:SMO液可以提高肾皮质保存效果,可能与其减少低温保存期间肾皮质细胞凋亡和抑制脂质过氧化损伤有关.     

多器官保存液的实验研究和临床应用进展

UW液是目前器官移植中常用的多器官保存液,HTK液、Celsior液在肾脏、肝脏、胰腺移植中的应用也日益成熟,逐渐成为UW液可靠的替代品。最新研究发现向现有器官保存液中加入保护性因子可以减轻低温缺血损伤,改善器官保存效果。本文对近期多器官保存液实验研究和临床应用进展作一综述。     

低温灌洗UW液和Euro-Collins液保存离体鼠肺效果的组织形态学比较研究

目的　比较UW液和Euro Collins液的肺保存效果。方法　选用 2 1只Wistar大鼠 ,应用 10℃的UW液和Euro Collins液同时分别进行左、右肺动脉内灌洗 ,灌洗前主肺动脉内快速推注PGE1。灌洗结束后于肺充气状态下切取左、右肺 ,并迅速分别置于 10℃的UW液及Euro Collins液中保存。分别于 0 ,2 ,4,6h取材 ,在光镜及电镜下观察肺组织形态学变化 ,并互相比较 ,以判定组织活力 ,确定肺保存效果。结果　低温灌洗 10℃的UW液肺保存效果优于低温灌洗Euro Collins液。 6h后 ,组织损伤表现为不可逆性。结论　在肺保存不超过 6h时 ,UW液比Euro Collins液更有效     

上海多器官保存液保存离体大鼠肝脏的实验研究

目的:观察上海多器官保存液(Shanghai-mutil-organ solution,SMO液)对离体大鼠肝脏的保存效果,探讨应用SMO液保存离体供肝的可行性.方法:SD大鼠随机分为SMO液、UW液和HTK液保存组,建立离体肝脏单纯低温保存模型,保存液保存8、16、24、36 h分析肝脏组织能量代谢情况,观察肝组织形态学改变和肝脏细胞凋亡情况.结果:保存16、24、36 h,SMO液组肝组织三磷酸腺苷(ATP)、磷酸腺苷总量(TAN)及Atkinson能荷(AEC)均明显高于同时点HTK液组 (P＜0.05),与同时点UW液组无显著差异;形态学检查见SMO液组组织损伤较同时点HTK液组轻,除细胞肿胀较同时间点UW液组明显外,其余表现基本一致.保存24、36 h,SMO液组凋亡指数明显低于HTK液组(P＜0.05),而与UW液组无明显差异.结论:SMO液对大鼠离体肝脏的保存效果总体上与UW液相当,优于HTK液,仅在防止细胞水肿方面较UW液稍差.     

肝移植3种器官保存液的比较研究

目的 比较UW液、Celsior液和HTK液时移植供肝灌注保存的效果.方法 回顾性分析2002年1月～2004年3月施行的成人首次肝脏移植且术前诊断为良性肝病者263例,根据所采用的灌注保存液分为lAW液组(161例)、Celsior组(45例)和HTK组(57例),3组患者年龄、性别及原发病分布,术前肝功能指标及分级,肝移植术式及供肝冷,热缺血时间差异均无显著性,比较3组患者术后早期移植肝功能指标、血管及胆道并发症发生率和移植物存活率.结果 3组患者肝移植术后早期血清丙氨酸氨基转移酶、门冬氨酸氨基转移酶、碱性磷酸酶和γ-谷氨酰转肽酶差异均无显著性;UW液组术后第7天血清总胆红素(117.4±96.3)μmol/L高于Celsior组[(85.1±77.9)μmol/L,P=0.047]和HTK组[(86.5±82.6)μmol/L,P=0.033];HTK组术后即刻凝血酶原时间(21.9±5.8)s低于UW液组[(24.8±6.2)s,P=0.004]和Celsior组[(26.4±7.0)s,P=0.0011;HTK组凝血酶原时问峰值(24.0±5.2)s低于Celsior组[(27.5±6.1)s,P=0.002].UW液组发生移植肝原发无功能2例(1.2%),但组间差异无显著性(P=0.528).3组患者术后血管并发症、胆道并发症及排斥反应发生率和再次移植率差异均无显著性.移植物1年及3年存活率分别为:UW液组80.7%和73.9%,Celsior组91.1%和82.2%,HTK组94.7%和80.7%,存活时间差异无显著性(P=0.099).结论 UW液、Celsior液和HTK液对移植供肝灌注保存的效果差异无显著性.     

UW液、Celsior液和HTK液低温保存生物人工肝用L-02细胞的效果比较

目的 比较UW液、Celsior液和HTK液4℃常规低温保存生物人工肝用L-02细胞的效果。方法 制备好的L-02细胞悬液分以下3组：UW液保存组（UW液组）;Celsior液保存组（CS液组）;HTK液保存组（HTK液组）。各组细胞于4℃低温保存72h后,分别测定细胞存活率及死亡率（流式细胞术）,ALT及LDH释放,尿素合成功能及白蛋白分泌功能。结果 UW液较Celsior液和HTK液显著提高了4℃常规低温保存保存72h的L-02细胞的存活率[(60.05 ± 4.23)% vs (50.12 ± 3.99)%、(44.20 ±4.67)%],均P<0.05;降低了细胞死亡率[(39.95±4.23)%vs (49.88 ±3.99)%、(55.80%±4.67)%], 均P<0.05;抑制了ALT、LDH释放(均P <0.05);更好地维持了L-02细胞尿素合成功能[ (1.03 ± 0.23)mmol/L vs (0.80± 0.14)mmol/L、(0.48± 0.05)mmol/L]和白蛋白分泌功能[(8.36 ±1.38 )mg/L vs (6.41±1.25)mg/L、(5.19±0.41)mg/L), 均P<0.05。结论 同Celsior液和HTK液相比,使用UW液4℃常规低温保存L-02肝细胞可以明显的提高复温后细胞存活率,降低低温损伤引起的ALT、LDH释放,有效的保护肝细胞尿素合成功能和白蛋白分泌功能,但保存时间不应超过72h。     

Addition of ulinastatin to preservation solution promotes protection against ischemia-reperfusion injury in rabbit lung

Background  The composition of the lung preservation solution used in lung graft procurement has been considered the key to minimize lung injury during the period of ischemia. Low-potassium dextran glucose (LPDG), an extracellular-type solution, has been adopted by most lung transplantation centers, due to the experimental and clinical evidences that LPDG is superior to intracellular-type solutions. Ulinastatin has been shown to attenuate ischemia-reperfusion (I/R) injury in various organs in animals. We supposed that the addition of ulinastatin to LPDG as a flushing solution, would further ameliorate I/R lung injury than LPDG solution alone.

Methods  Twelve male New Zealand white rabbits were randomly divided into 2 groups. Using an alternative in situ lung I/R model, the left lung in the control group was supplied and preserved with LPDG solution for 120 minutes. In the study group 50 000 U/kg of ulinastatin was added to the LPDG solution for lung preservation. Then re-ventilation and reperfusion of the left lung were performed for 90 minutes. Blood gas analysis (PaO₂, PaCO₂), mean pulmonary artery pressure (MPAP) and serum TNF-α level were measured intermittently. The pulmonary water index (D/W), tissue myeloperoxidase (MPO) activity, tissue malondialdehyde (MDA) content and morphologic changes were analyzed.

Results  The study group showed significantly higher PaO₂ and lower MPAP at the end of reperfusion. Serum TNF-α level, left lung tissue MPO and MDA in the study group were significantly lower than those in the control group. D/W and pathologic evaluation were also remarkably different between the two groups.

Conclusions  This study indicated that better lung preservation could be achieved with the use of an ulinastatin modified LPDG solution. Ulinastatin further attenuated lung I/R injury, at least partly by reducing oxidative reactions, inhibiting the release of inflammatory factors and neutrophils immigration.

     

自制多器官保存液对兔肾低温延时保存的形态学观察

目的：观察长征１号（ＣＺ－１）多器官保存液对兔肾低温保存后的形态学变化。方法：将新西兰雄性兔肾脏分别用２～４℃ＣＺ－１液、ＵＷ液及ＨＣ－Ａ液５０～１００ｍｌ灌注，待肾脏变白后，切断所有血管，将肾脏取下放入装有ＣＺ－１液、ＵＷ液和ＨＣ－Ａ液保存罐中２～４℃保存，供取材作形态学观察。结果：７２ｈ以内ＣＺ－１液保存组肾脏组织光镜和电镜下形态学无明显改变，而且ＣＺ－１液保存组低温保存肾脏组织的变化以及细胞和细胞器的变性均缓于ＵＷ液与ＨＣ－Ａ液保存组。结论：ＣＺ－１液能低温保存肾脏在７２ｈ之内。     

尼可地尔改善心脏停搏液对心脏的保护作用

目的　观察在HTK液和UW液中加入尼可地尔 (Nicorandil)对心肌保存液保存效果的影响。方法　实验分为 4组 :UW液 (U)组 ;HTK液 (H)组 ;UW液组 +Nicorandil(U +N)组 ;HTK液 +Nicorandil(H +N)组 ;每组 8只大白鼠。麻醉和抗凝后 ,快速取下鼠心并悬挂在Langendorff灌注模型上灌注 ,测定血流动力学基础值。分别用四组保存液灌停离体鼠心并低温 (4℃ )浸泡保存 6h。复温、复灌后再次测定血流动力学值 ,留取标本分别测定心肌水含量、心肌酶漏出量、心肌细胞ATP和CP含量和心肌细胞超微结构变化。结果　心肌血流动力学恢复率 :H +N组>U +N组 >H组 >U组。心肌水含量 :4组间差异无显著意义。灌脉流出液中心肌酶漏出量 :H +N组 U +N组 >H组 >U组。心肌细胞超微结构变化 :H +N组心肌损害最轻 ,H组轻于U +N组 ,U组心肌损害最严重。结论　HTK液和UW液都是比较合适的心肌保存液 ,HTK液对心肌的保护作用优于UW液 ,钾离子通道开放剂Nicorandil能够善器官保存液HTK液和UW液对心肌的保存效果。     

各种心肌保存液对鼠心低温保存效果的形态学观察

目的 研究在冷缺血期3种液体对鼠心保存的效果。方法 将离体鼠心分别用HTK液，UW液及St.ThomasⅡ液灌注并4℃冷冻保存6h。利用离体鼠心非循环式Lan-gendorff灌流功能测定模型，复灌1h后，取心肌组织做心镜电镜观察。结果 经过HTK液保存后的鼠心细胞损伤较轻，UW液鼠心细胞损伤较重，但冠脉血管内膜剥脱较重。St.ThomasⅡ液鼠心细胞损伤最生。结论 HTK液心肌保存效果最好，高钾对血管内皮有损害。     

Extended preservation of human liver grafts with UW solution

The fate of 185 cadaveric liver homografts preserved for four to 24 hours with University of Wisconsin (UW) solution was compared with that of 180 grafts preserved for three to 9 1/2 hours with conventional Euro-Collins solution. Although the average preservation time of the UW-preserved livers was almost twice as long as that of the Euro-Collins-preserved livers, the UW-preserved grafts survived at a higher rate; permitted equal patient survival; and had a lower rate of primary nonfunction, a reduced need for retransplantation, and a lower rate of hepatic artery thrombosis. There was no correlation between the time of preservation of UW-preserved grafts up to 24 hours and liver function abnormalities in the first postoperative week. In contrast, livers preserved with Euro-Collins solution for more than five hours had significantly increased perturbations of hepatic function tests. The potential revolutionary effect of the UW solution on liver transplantation is described herein.     

含血低钾右旋糖酐液肺保存效果的实验研究

目的:比较含与不含20%自体静脉血的低钾右旋糖酐(LPD)液的肺保存效果。方法:将12只犬分为2组,分别用含与不含20%自体静脉血的LPD液灌注,10℃保存12h后,分别在离体状态下,连续通气和温静脉血再灌注10min。比较供肺流入、流出血的氧分压、二氧化碳分压、呼吸道峰压及再灌注后离体肺的湿/干重量比,并行病理学检查。结果:两组间氧分压犤(21.55±3.94)kPa与(20.90±1.16)kPa犦、二氧化碳分压犤(4.25±0.15)kPa与(4.32±0.11)kPa犦、呼吸道峰压犤(19.00±1.41)kPa与(18.50±1.87)kPa犦及再灌注后离体肺的湿/干重量比(6.47±0.41与6.09±0.28)之间差异无统计学意义(P>0.05),病理切片均未见肺泡内出血。结论:含与不含20%自体静脉血的LPD液的肺保存效果差异无统计学意义,肺保存效果并未因灌注液中加血而明显改善。     

低氧有助于小鼠骨髓源间充质干细胞的定向分化

目的本研究在测量小鼠体内股骨骨髓正常氧分压的基础上设定相应的体外低氧浓度(3%),研究低氧环境对小鼠骨髓源间充质干细胞向内皮细胞系定向分化功能的影响。方法用微电极测量正常小鼠股骨骨髓的氧分压,并以此为依据设定体外培养的低氧浓度。将小鼠骨髓源单个核细胞置于3%(低氧)和20%(常氧)氧浓度下用内皮细胞专用培养基(EGM-2)进行定向诱导分化培养,培养至第2代进行Dil-Ac-LDL摄取实验和FITC-UEA结合实验双染色鉴定。对3%和20%氧浓度下培养的内皮样细胞用MTT法进行增生能力的鉴定;用荧光染色法对其进行黏附能力的比较。结果小鼠股骨骨髓生理性氧分压浓度为(21.55±3.40)mm Hg(1 mm Hg=0.133 k Pa),相当于(2.8±0.45)%氧体积分数(约为3%)。在不同氧浓度下培养的细胞,DilAc-LDL摄取实验和FITC-UEA结合实验均为阳性。。MTT细胞增生实验显示低氧(3%)培养的细胞吸光度为0.31±0.05,常氧(20%)培养的细胞吸光度为0.16±0.01(P<0.01,n=5)。低氧培养的细胞,其黏附率为(8.72±2.95)%,常氧培养的内皮样细胞,其黏附率为(3.91±0.72)%(P<0.05,n=5)。结论 3%的氧浓度是骨髓生理状态下的氧浓度。在此浓度下,骨髓源间充质干细胞可被诱导分化为内皮样细胞。与常氧相比,低氧培养的内皮样细胞具有较高的增生和黏附能力。     

静脉输注高氧液治疗家兔单肺通气期间低氧血症

目的 探索高氧液对兔单肺通气期间低氧血症的治疗作用. 方法 家兔30只,随机分为对照组(C组)和高氧液组(H组)各15只. 单肺通气(OLV) 30 min后,对照组静脉输入9 g/L生理盐水100 mL,高氧液组输入高氧液100 mL. 输注完毕后,记录OLV前及OLV后30 min(治疗前)及静脉输液后10 min(治疗后) pH,PaO2,PaCO2, PvO2,SaO2及SvO2等指标. 结果与OLV前相比,两组单肺通气30 min后,SaO2 [(91.0±3.5)%, (91.0±2.9)%], PaO2[(10.7±1.2) kPa, (10.9±1.2)kPa], PvO2[(8.3±1.4) kPa, (8.3±1.5)kPa], SvO2[(57.0±2.3)%, (57.0±3.8)%]均明显下降,而PaCO2[(5.9±0.6) kPa, (5.6±0.6)kPa]显著升高(P<0.01);对照组在输液后10 min各指标仍呈下降趋势,高氧液组在治疗后SaO2(95.0±3.2)%, PaO2(11.9±1.5)kPa, PvO2(9.2±1.4)kPa, SvO2(63.0±3.1)%明显升高,与对照组相应各指标[(89.0±2.7)%, (10.3±1.1)kPa, (8.1±1.2)kPa, (55.0±3.1)%]相比有显著性差异(P<0.05). 结论 静脉输注高氧液对OLV期间低氧血症有明显的治疗作用.     

含氢保存液降低大鼠供心保存过程中氧化应激和炎性损伤

目的 观察含氢保存液对大鼠供心保存过程中氧化应激和炎性损伤的影响。方法 32只SD大鼠随机分4组(n=8):对照组(保存液为HTK液),H1组(保存液为含氢浓度约0.2 mmol/L的HTK液),H2组(保存液为含氢浓度约0.4 mmol/L的HTK液),H3组(保存液为含氢浓度约0.8 mmol/L的HTK液)。采用Langendorff离体大鼠心脏灌注法,心脏分别在各组保存液中冷存(4℃) 6 h,复灌后硫代巴比妥酸法检测心肌组织中MDA含量,黄嘌呤氧化酶法检测心肌组织中SOD活力,ELISA法检测心肌组织中8-羟基脱氧鸟苷、TNF-α、IL-6含量。结果 供心冷保存(4℃) 6 h后,H1、H2、 H3组MDA含量低于对照组(P<0.01,P<0.05)。H2、H3组SOD活力高于对照组(P<0.01,P<0.05);对照组8-羟基脱氧鸟苷水平高于H1、H2、H3组(P<0.01),对照组TNF-α、IL-6含量也高于H1、H2、H3组(P<0.01,P<0.05)。结论 含氢保存液能降低供心保存过程中的心肌氧化损伤,减少炎性细胞因子的产生。     

自制长征-1号多器官保存液的动物实验和部分临床应用研究

目的：研制我国自制的多器官保存液,以满足日益发展的国内器官移植手术的需要。方法;在全面分析国外最先进的多器官保存液ＵＷ液配方的基础上,研制成长征－１号多器官保存液。改进有：（１）用低分子右旋糖酐－４０替代羟乙基淀粉’（２）用蔗糖替代木棉粮;（３）去掉ＵＷ液中添加成分;（４）添加钙离子拮抗剂维拉帕米;（５）使ＰＨ值进一步偏碱性。     

器官保存研究进展及展望

维持器官保存期间移植物的活力,是器官移植成功的前提条件和根本保障。目前,单纯低温保存(cold storage,CS)仍是当前各大器官移植中心首选、最常用的保存方法。随着供体需求量的增加,越来越多的老年供体和边缘供体被用于临床,这对传统的器官保存方法提出了更高的要求。文中着重对器官保存的理论、病理生理改变以及器官保存技术等方面的最新进展进行综述。