聚集素蛋白（Clusterin）在心脏移植冷缺血再灌注损伤中的保护作用与机理研究
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摘要
第一部分供体心脏表达clusterin显著降低同基因心脏移植的冷缺血再灌注损伤
[目的]心脏移植中冷缺血保存和随后的热灌注损伤统称为冷缺血再灌注损伤,是引起移植物失功的初始因素,而且还可以导致移植心脏冠状血管病。Clusterin是具有细胞保护功能的伴侣蛋白,几乎在所有哺乳动物组织中表达,具有参与生殖、脂质转运、抑制补体激活和调节凋亡等多种作用。本实验拟探讨供体心脏表达的clusterin在同基因心脏移植冷缺血再灌注损伤中的保护作用及机制。
[方法]RT-PCR和免疫组化染色检测野生型C57BL/6J小鼠(WT B6)和clusterin基因敲除小鼠(CLU-KO,C57BL/6J)心脏组织中的clusterin表达；分别取上述两组小鼠的心脏,在4℃生理盐水中保存8h,然后行同基因腹部异位心脏移植；移植前,分别检测保存8h后两组保存液中心肌细胞释放的肌酸激酶(CK)和乳酸脱氢酶(LDH)含量；移植后15min和24h行心脏移植物评分,评估移植心脏功能；HE染色观察两组心脏移植物病理变化；免疫组化检测两组心脏移植物内髓过氧化物酶(MPO)的表达情况,原位末端脱氧核苷酸转移酶标记法(TUNEL法)检测两组心脏移植物内的细胞凋亡情况。
[结果]RT-PCR证实WT B6小鼠心脏组织组成性的表达clusterin,免疫组化染色发现,clusterin蛋白主要表达在血管内皮组织和心肌细胞表面,而CLU-KO小鼠心脏内未见clusterin表达。与CLU-KO组相比,WT B6组心脏在4℃生理盐水中保存8h后,保存液中CK和LDH的释放量显著降低(CK:784.25±90.71 U/L VS610.96±60.02UL,p=0.0226；LDH:0.744±0.129 VS 0.548±0.076,p= 0.0134)。移植后15mmin和24h,WT B6组心脏移植物评分分别为2.3和3.5,而CLU-KO组心脏移植物评分则分别为1和3,两者相比,差异显著,有统计学意义(p=0.002；p=0.0271)。HE染色证明,WT B6组心脏移植物血管周围炎性反应、淋巴细胞浸润和心肌细胞的坏死均比CLU-KO组减轻。MPO染色和TUNEL检测发现,WT B6组心脏移植物MPO阳性细胞数和凋亡细胞数较CLU-KO组均显著减少(p=0.0002；p=0.0316)。
[结论]供心表达clusterin能显著降低心脏移植过程中冷缺血再灌注损伤。因此,上调供移植心脏中的clusterin表达可能具有保护供心并降低冷缺血再灌注损伤的潜能。
第二部分Clusterin在心肌细胞冷保存损伤中的保护作用及机制
[目的]在第一部分实验中已经证实：供心表达clusterin后能减轻冷缺血再灌注损伤。但是,clusterin表达后通过何种机制降低冷缺血再灌注损伤,特别是冷保存损伤,目前尚不清楚。本实验拟分离clusterin基因敲除(CLU-KO)小鼠心肌细胞,并转入clusterin基因,使心肌细胞表达clusterin,在体外进一步研究clusterin在心肌细胞冷保存损伤中的保护作用及机制。
[方法]分离培养CLU-KO小鼠的心肌细胞,经RT-PCR鉴定后,将永生化SV40基因转入心肌细胞,建立小鼠心肌细胞系(MHC)。分别将pHEX6300或pHEX6300-CLU转染入MHC,获得MHC-Con和MHC-CLU两种细胞,Western Blot检测转染后两组心肌细胞clusterin蛋白的表达；两组心肌细胞在4℃生理盐水中保存6～8h,分别在不同时间点检测两组保存液中心肌细胞的LDH释放量和细胞膜流动性的改变。
[结果]分离培养的小鼠心肌细胞,经RT-PCR鉴定证实,α-心肌肌球蛋白重链(a-MHC)和间隙连接蛋白43 (connexin 43)表达阳性,而β-心肌肌球蛋白重链(β-MHC)和α-骨骼肌肌动蛋白(a-skeletal actin)表达则为阴性。SV40永生化的小鼠心肌细胞转染pHEX6300或pHEX6300-CLU后,Western Blot检测证明,MHC-CLU表达clusterin蛋白,而MHC-Con未见clusterin蛋白表达。两组心肌细胞在4℃生理盐水中保存8h后,MHC-CLU组释放的LDH量从1h的22.66±7.53%升高到8h的28.24±1.78%,而MHC-Con组则从1h的31.39±3.42%升高到8h的92.16±4.07%,两组间比较,差异显著,有统计学差异(p<0.0001)。两组心肌细胞在4℃生理盐水中保存6h后,MHC-CLU膜稳定性与Oh相比增加了30%,而MHC-Con则降低了15%。
[结论]心肌细胞表达clusterin后,通过稳定细胞膜,防止冷保存中心肌细胞膜的破裂,并降低心肌细胞LDH释放,进而抑制心肌细胞坏死,发挥保护作用。
第三部分重组的clusterin蛋白(rCLU)在心脏移植冷缺血再灌注损伤中的保护作用及机制
[目的]前两部分的研究结果证明：(1)供心表达clusterin能显著降低心脏移植过程中冷缺血再灌注损伤；(2)心肌细胞表达clusterin后,通过稳定细胞膜,防止冷保存中心肌细胞膜的破裂,并降低心肌细胞LDH释放,进而抑制心肌细胞坏死,发挥保护作用。本研究拟构建重组的clusterin蛋白(rCLU),并将其加入UW保存液中,探讨rCLU在人内皮细胞系以及小鼠心脏移植中能否发挥抵御冷缺血再灌注损伤的作用及其潜在的作用机制。
[方法]体外合成rCLU并经Western Blot鉴定。(1)体外实验：用含不同浓度rCLU的4℃UW液分别保存人脐静脉内皮细胞(HUVECs)和肾小球内皮细胞(HRGECs);冷保存24h后,分别检测两种细胞的LDH释放量,以确定合适的rCLU浓度。上述两种内皮细胞分别在含lmg/ml rCLU的4℃UW液中保存24h,以相同浓度的牛血清白蛋白(BSA)为阳性对照,FDA-EB法检测两种内皮细胞的死亡率；流式细胞仪检测]-CLU在两种细胞表面的结合率；在不同时间点检测上述保存体系中,两种内皮细胞细胞膜的流动性改变。(2)体内实验：以C57BL/6J小鼠心脏作为供体,在含1mg/ml rCLU的4℃UW液保存24h,以相同浓度的BSA为阳性对照,不同时间点检测保存液中心脏释放的LDH量；供心保存24h后,行同基因腹部异位心脏移植。参照第一部分的实验方法,移植后15min和24h行心脏移植物评分,评估移植心脏功能；移植后24h,行HE染色检测心脏移植物病理学变化；免疫组化染色检测心脏移植物内MPO的表达；TUNEL法检测心肌细胞的凋亡情况。
[结果]Western Blot鉴定结果说明合成的rCLU为异二聚体。体外实验证明,1mg/ml rCLU的4℃UW液与相同浓度的BSA相比,前者能显著降低HUVECs冷保存过程中的LDH释放(29.49±6.07% VS88.27±8.3%, P=0.0039); FDA-EB染色半定量计数亦表明,1mg/ml rCLU的4℃UW液能降低冷保存体系中HUVECs的坏死率；流式细胞术检测证明,rCLU能够结合在HUVECs细胞膜表面,增加细胞膜的流动性；而相同情况下处理的HRGECs,我们也得到了类似的结果。体内实验证实,1mg/ml rCLU的4℃UW液能显著降低小鼠供心在冷保存过程中的LDH释放；心脏移植后,rCLU能有利于心脏移植物功能的恢复并降低血清中CK和LDH值,同时亦能减轻移植物中血管周围炎症、中性粒细胞的浸润以及心肌细胞的坏死和凋亡
[结论]rCLU能保护供移植器官,减轻其冷保存损伤。因此,在UW液中加入rCLU有望作为一个新的策略,优化供移植器官组织或细胞的冷保存方案。
PartⅠReduction of Cold Ischemia-Reperfusion Injury by Graft-Expressing Clusterin in Heart Transplantation
【Objective】Cold ischemia-reperfusion injury (IRI) is one of primary factors for early graft dysfunction and is associated with rejection episodes in heart transplantation. Clusterin (CLU) is a cytoprotective protein with chaperone activity. This study was designed to examine the impact of donor-expressing clusterin on cold IRI.
【Methods】Donor hearts from wild type C57BL/6J (H-2b; B6 WT) versus CLU knockout C57BL/6J (H-2b; CLU-KO) mice were stored at 4"C saline for 8 hours followed by heterotopical transplantation to B6 WT mice. The expression of CLU in the hearts of WT B6 mice or CLU-KO mice was determined by RT-PCR and immunohistochemical analysis. At the end of the preservation, the severity of cardiac injury was determined by release of creatine kinase (CK) and lactate dehydrase (LDH). The function recovery of heart grafts was determined by palpation at 15 minutes and 24 hours after transplantation. At 24 hours after transplantation, tissue injury was tested by HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.
【Results】WT B6 mice hearts constitutively expressed clusterin, and mature clusterin protein was localized mostly in the endothelium as well as on the cell surface of cardiac myocytes. As compared to CLU-KO hearts, WT hearts were more resistant to cold injury during cold preservation. Compared with the CLU-KO hearts, the CK and LDH levels released from WT hearts were significantly lower, (CK:784.25±90.71 U/L VS 610.96±60.02 U/L, p= 0.0226; LDH:0.744±0.129 VS 0.548±0.076, p= 0.0134). WT hearts had a better function recovery from a prolonged cold preservation following transplantation, indicated by the median score 2.3 in WT group compared to 1 in CLU-KO group at 15 minutes (p= 0.002), or 3.5 in WT group compared to 3 in clusterin deficient group at 24 hours (p= 0.0271). The better graft function of clusterin-expressing grafts was significantly correlated with less degree of both neutrophil infiltration and cardiac injury including myocytic apoptosis and necrosis.
【Conclusion】Clusterin expression renders donor hearts resistance to cold IRI in transplantation, suggesting that up-regulation of clusterin expression in donor hearts may have potential for protecting heart grafts from cold IRI.
PartⅡProtection of Cultured Myocytes from Cold-Induced Cell Death by Clusterin Expression
【Objective】We have demonstrated that clusterin expression renders donor hearts resistance to cold IRI in transplantation. But the mechanism by which CLU prevents cell death at cold temperature is not completely understood. This study was designed to investigate the cytoprotective activity of clusterin at cold temperature in cultured myocytes.
【Methods】CLU-KO myocyte line (MHC) established from primary myocytes isolated from the heart of a CLU-KO mouse, followed by immortalization with origin deficient SV40 DNA. The phenotype of cloned MHC was confirmed by RT-PCR. MHC was converted to clusterin-expressing myocytes (MHC-CLU) by stable expression of pHEX6300 vector containing human CLU isoform 1 cDNA, while MHC with stable expression of empty pHEX6300 vector were used as clusterin negative control cells (MHC-Con). Clusterin protein in protein extracts of cultured myocytes was examined by Western blot. Cell death/necrosis at 4℃in saline was measured using the levels of LDH release. The change of membrane fluidity in CLU-expressing MHC-CLU cells versus CLU null MHC-Con cells was investigated following exposure to cold temperature.
【Results】The phenotype of cloned MHC was confirmed by its positive expression of both a-cardiac myosin heavy chain and connexin 43 transcripts, and negative expression of (3-cardiac myosin heavy chain and a-skeletal actin, using RT-PCR. Ectopic expression of clusterin protein in cellular extracts of MHC-CLU versus MHC-Con was determined by Western Blot. Only a slight increase in LDH release was seen in CLU-expressing MHC-CLU cell cultures, from 22.66±7.53% after 1 hr of incubation to 28.24±1.78% after 8 hrs of incubation, while in CLU null MHC-Con cell cultures, LDH release was largely induced following the exposure to 4℃, indicated by the increase of LDH release from 31.39±3.42% after 1 hr to 92.16±4.07% after 8 hrs of incubation (p<0.0001). Further study also shows that the membrane fluidity in MHC-CLU cells increased following cold exposure, evidenced by approximately 30% increase at the end of 6 hrs of incubation, while there was a trend of a slow decrease of membrane fluidity in MHC-Con cells.
【Conclusion】Ectopic expression of CLU in CLU-KO myoytes stabilizes the cell membrane fluidity, resulting in protection of cells from cell membrane disruption, indicated by LDH release, at cold temperature, suggesting that CLU may function as a chaperone and can stabilize the cell membrane by its chaperone activity at cold temperature.
PartⅢReduction of Cold Ischemia/Reperfusion Injury of Donor Organs by Supplement of Recombinant Clusterin Protein in Cold Preservation Solution
【Objective】Donor organ injury during cold preservation prior to transplantation negatively impacts graft survival. Clusterin (CLU) is a chaperonic protein, and its expression confers donor hearts resistance to cold ischemic injury. This study was to evaluate if supplement of recombinant CLU protein (rCLU) protects donor organs from injury during cold storage with University of Wisconsin (UW) solution.
【Methods】Western blot analysis of recombinant cluterin (rCLU) protein, purified from HEK 293 cell cultures. Human endothelial cell cultures were used as an in vitro model. Heart transplantation in mice was used as an in vivo model. Cell death was measured using lactate dehydrogenase (LDH) release or using a fluorescence microscope after stained with fluorescein diacetate-ethidium bromide solution. The intensity of rCLU on cell surface was measured using a flow cytometer. The cell membrane fluidity of endothelial cells was measured at different time points. Donor injury was determined by biochemical evaluation, its functional recovery and HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.
【Results】Western blot analysis showed that rCLU protein, isolated from HEK 193 cell cultures, was a heterodimer. Supplement of rCLU to UW solution remarkably protected cultured human endothelial cells from cold-induced cell necrosis, evidenced by a decrease in LDH release or in the number of ethidium bromide-stained necrotic cells. The protective activity of rCLU was associated with enhanced membrane fluidity at cold temperature. During cold storage of heart organs in UW solution, supplement of rCLU significantly reduced LDH release from heart tissue. In preclinical model of transplantation, heart grafts after cold preservation with rCLU-containing UW solution had a better functional recovery and less severity of perivascular inflammation, neutrophil infiltration and cardio injury including apoptosis and necrosis that correlated with lower levels of serum creatine kinase and LDH in recipients.
【Conclusion】Our data suggest that supplement of CLU protein in a cold preservation solution may have potential in the improvement of cold preservation of donor organs in transplantation.
[目的]心脏移植中冷缺血保存和随后的热灌注损伤统称为冷缺血再灌注损伤,是引起移植物失功的初始因素,而且还可以导致移植心脏冠状血管病。Clusterin是具有细胞保护功能的伴侣蛋白,几乎在所有哺乳动物组织中表达,具有参与生殖、脂质转运、抑制补体激活和调节凋亡等多种作用。本实验拟探讨供体心脏表达的clusterin在同基因心脏移植冷缺血再灌注损伤中的保护作用及机制。
[方法]RT-PCR和免疫组化染色检测野生型C57BL/6J小鼠(WT B6)和clusterin基因敲除小鼠(CLU-KO,C57BL/6J)心脏组织中的clusterin表达；分别取上述两组小鼠的心脏,在4℃生理盐水中保存8h,然后行同基因腹部异位心脏移植；移植前,分别检测保存8h后两组保存液中心肌细胞释放的肌酸激酶(CK)和乳酸脱氢酶(LDH)含量；移植后15min和24h行心脏移植物评分,评估移植心脏功能；HE染色观察两组心脏移植物病理变化；免疫组化检测两组心脏移植物内髓过氧化物酶(MPO)的表达情况,原位末端脱氧核苷酸转移酶标记法(TUNEL法)检测两组心脏移植物内的细胞凋亡情况。
[结果]RT-PCR证实WT B6小鼠心脏组织组成性的表达clusterin,免疫组化染色发现,clusterin蛋白主要表达在血管内皮组织和心肌细胞表面,而CLU-KO小鼠心脏内未见clusterin表达。与CLU-KO组相比,WT B6组心脏在4℃生理盐水中保存8h后,保存液中CK和LDH的释放量显著降低(CK:784.25±90.71 U/L VS610.96±60.02UL,p=0.0226；LDH:0.744±0.129 VS 0.548±0.076,p= 0.0134)。移植后15mmin和24h,WT B6组心脏移植物评分分别为2.3和3.5,而CLU-KO组心脏移植物评分则分别为1和3,两者相比,差异显著,有统计学意义(p=0.002；p=0.0271)。HE染色证明,WT B6组心脏移植物血管周围炎性反应、淋巴细胞浸润和心肌细胞的坏死均比CLU-KO组减轻。MPO染色和TUNEL检测发现,WT B6组心脏移植物MPO阳性细胞数和凋亡细胞数较CLU-KO组均显著减少(p=0.0002；p=0.0316)。
[结论]供心表达clusterin能显著降低心脏移植过程中冷缺血再灌注损伤。因此,上调供移植心脏中的clusterin表达可能具有保护供心并降低冷缺血再灌注损伤的潜能。
第二部分Clusterin在心肌细胞冷保存损伤中的保护作用及机制
[目的]在第一部分实验中已经证实：供心表达clusterin后能减轻冷缺血再灌注损伤。但是,clusterin表达后通过何种机制降低冷缺血再灌注损伤,特别是冷保存损伤,目前尚不清楚。本实验拟分离clusterin基因敲除(CLU-KO)小鼠心肌细胞,并转入clusterin基因,使心肌细胞表达clusterin,在体外进一步研究clusterin在心肌细胞冷保存损伤中的保护作用及机制。
[方法]分离培养CLU-KO小鼠的心肌细胞,经RT-PCR鉴定后,将永生化SV40基因转入心肌细胞,建立小鼠心肌细胞系(MHC)。分别将pHEX6300或pHEX6300-CLU转染入MHC,获得MHC-Con和MHC-CLU两种细胞,Western Blot检测转染后两组心肌细胞clusterin蛋白的表达；两组心肌细胞在4℃生理盐水中保存6～8h,分别在不同时间点检测两组保存液中心肌细胞的LDH释放量和细胞膜流动性的改变。
[结果]分离培养的小鼠心肌细胞,经RT-PCR鉴定证实,α-心肌肌球蛋白重链(a-MHC)和间隙连接蛋白43 (connexin 43)表达阳性,而β-心肌肌球蛋白重链(β-MHC)和α-骨骼肌肌动蛋白(a-skeletal actin)表达则为阴性。SV40永生化的小鼠心肌细胞转染pHEX6300或pHEX6300-CLU后,Western Blot检测证明,MHC-CLU表达clusterin蛋白,而MHC-Con未见clusterin蛋白表达。两组心肌细胞在4℃生理盐水中保存8h后,MHC-CLU组释放的LDH量从1h的22.66±7.53%升高到8h的28.24±1.78%,而MHC-Con组则从1h的31.39±3.42%升高到8h的92.16±4.07%,两组间比较,差异显著,有统计学差异(p<0.0001)。两组心肌细胞在4℃生理盐水中保存6h后,MHC-CLU膜稳定性与Oh相比增加了30%,而MHC-Con则降低了15%。
[结论]心肌细胞表达clusterin后,通过稳定细胞膜,防止冷保存中心肌细胞膜的破裂,并降低心肌细胞LDH释放,进而抑制心肌细胞坏死,发挥保护作用。
第三部分重组的clusterin蛋白(rCLU)在心脏移植冷缺血再灌注损伤中的保护作用及机制
[目的]前两部分的研究结果证明：(1)供心表达clusterin能显著降低心脏移植过程中冷缺血再灌注损伤；(2)心肌细胞表达clusterin后,通过稳定细胞膜,防止冷保存中心肌细胞膜的破裂,并降低心肌细胞LDH释放,进而抑制心肌细胞坏死,发挥保护作用。本研究拟构建重组的clusterin蛋白(rCLU),并将其加入UW保存液中,探讨rCLU在人内皮细胞系以及小鼠心脏移植中能否发挥抵御冷缺血再灌注损伤的作用及其潜在的作用机制。
[方法]体外合成rCLU并经Western Blot鉴定。(1)体外实验：用含不同浓度rCLU的4℃UW液分别保存人脐静脉内皮细胞(HUVECs)和肾小球内皮细胞(HRGECs);冷保存24h后,分别检测两种细胞的LDH释放量,以确定合适的rCLU浓度。上述两种内皮细胞分别在含lmg/ml rCLU的4℃UW液中保存24h,以相同浓度的牛血清白蛋白(BSA)为阳性对照,FDA-EB法检测两种内皮细胞的死亡率；流式细胞仪检测]-CLU在两种细胞表面的结合率；在不同时间点检测上述保存体系中,两种内皮细胞细胞膜的流动性改变。(2)体内实验：以C57BL/6J小鼠心脏作为供体,在含1mg/ml rCLU的4℃UW液保存24h,以相同浓度的BSA为阳性对照,不同时间点检测保存液中心脏释放的LDH量；供心保存24h后,行同基因腹部异位心脏移植。参照第一部分的实验方法,移植后15min和24h行心脏移植物评分,评估移植心脏功能；移植后24h,行HE染色检测心脏移植物病理学变化；免疫组化染色检测心脏移植物内MPO的表达；TUNEL法检测心肌细胞的凋亡情况。
[结果]Western Blot鉴定结果说明合成的rCLU为异二聚体。体外实验证明,1mg/ml rCLU的4℃UW液与相同浓度的BSA相比,前者能显著降低HUVECs冷保存过程中的LDH释放(29.49±6.07% VS88.27±8.3%, P=0.0039); FDA-EB染色半定量计数亦表明,1mg/ml rCLU的4℃UW液能降低冷保存体系中HUVECs的坏死率；流式细胞术检测证明,rCLU能够结合在HUVECs细胞膜表面,增加细胞膜的流动性；而相同情况下处理的HRGECs,我们也得到了类似的结果。体内实验证实,1mg/ml rCLU的4℃UW液能显著降低小鼠供心在冷保存过程中的LDH释放；心脏移植后,rCLU能有利于心脏移植物功能的恢复并降低血清中CK和LDH值,同时亦能减轻移植物中血管周围炎症、中性粒细胞的浸润以及心肌细胞的坏死和凋亡
[结论]rCLU能保护供移植器官,减轻其冷保存损伤。因此,在UW液中加入rCLU有望作为一个新的策略,优化供移植器官组织或细胞的冷保存方案。
PartⅠReduction of Cold Ischemia-Reperfusion Injury by Graft-Expressing Clusterin in Heart Transplantation
【Objective】Cold ischemia-reperfusion injury (IRI) is one of primary factors for early graft dysfunction and is associated with rejection episodes in heart transplantation. Clusterin (CLU) is a cytoprotective protein with chaperone activity. This study was designed to examine the impact of donor-expressing clusterin on cold IRI.
【Methods】Donor hearts from wild type C57BL/6J (H-2b; B6 WT) versus CLU knockout C57BL/6J (H-2b; CLU-KO) mice were stored at 4"C saline for 8 hours followed by heterotopical transplantation to B6 WT mice. The expression of CLU in the hearts of WT B6 mice or CLU-KO mice was determined by RT-PCR and immunohistochemical analysis. At the end of the preservation, the severity of cardiac injury was determined by release of creatine kinase (CK) and lactate dehydrase (LDH). The function recovery of heart grafts was determined by palpation at 15 minutes and 24 hours after transplantation. At 24 hours after transplantation, tissue injury was tested by HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.
【Results】WT B6 mice hearts constitutively expressed clusterin, and mature clusterin protein was localized mostly in the endothelium as well as on the cell surface of cardiac myocytes. As compared to CLU-KO hearts, WT hearts were more resistant to cold injury during cold preservation. Compared with the CLU-KO hearts, the CK and LDH levels released from WT hearts were significantly lower, (CK:784.25±90.71 U/L VS 610.96±60.02 U/L, p= 0.0226; LDH:0.744±0.129 VS 0.548±0.076, p= 0.0134). WT hearts had a better function recovery from a prolonged cold preservation following transplantation, indicated by the median score 2.3 in WT group compared to 1 in CLU-KO group at 15 minutes (p= 0.002), or 3.5 in WT group compared to 3 in clusterin deficient group at 24 hours (p= 0.0271). The better graft function of clusterin-expressing grafts was significantly correlated with less degree of both neutrophil infiltration and cardiac injury including myocytic apoptosis and necrosis.
【Conclusion】Clusterin expression renders donor hearts resistance to cold IRI in transplantation, suggesting that up-regulation of clusterin expression in donor hearts may have potential for protecting heart grafts from cold IRI.
PartⅡProtection of Cultured Myocytes from Cold-Induced Cell Death by Clusterin Expression
【Objective】We have demonstrated that clusterin expression renders donor hearts resistance to cold IRI in transplantation. But the mechanism by which CLU prevents cell death at cold temperature is not completely understood. This study was designed to investigate the cytoprotective activity of clusterin at cold temperature in cultured myocytes.
【Methods】CLU-KO myocyte line (MHC) established from primary myocytes isolated from the heart of a CLU-KO mouse, followed by immortalization with origin deficient SV40 DNA. The phenotype of cloned MHC was confirmed by RT-PCR. MHC was converted to clusterin-expressing myocytes (MHC-CLU) by stable expression of pHEX6300 vector containing human CLU isoform 1 cDNA, while MHC with stable expression of empty pHEX6300 vector were used as clusterin negative control cells (MHC-Con). Clusterin protein in protein extracts of cultured myocytes was examined by Western blot. Cell death/necrosis at 4℃in saline was measured using the levels of LDH release. The change of membrane fluidity in CLU-expressing MHC-CLU cells versus CLU null MHC-Con cells was investigated following exposure to cold temperature.
【Results】The phenotype of cloned MHC was confirmed by its positive expression of both a-cardiac myosin heavy chain and connexin 43 transcripts, and negative expression of (3-cardiac myosin heavy chain and a-skeletal actin, using RT-PCR. Ectopic expression of clusterin protein in cellular extracts of MHC-CLU versus MHC-Con was determined by Western Blot. Only a slight increase in LDH release was seen in CLU-expressing MHC-CLU cell cultures, from 22.66±7.53% after 1 hr of incubation to 28.24±1.78% after 8 hrs of incubation, while in CLU null MHC-Con cell cultures, LDH release was largely induced following the exposure to 4℃, indicated by the increase of LDH release from 31.39±3.42% after 1 hr to 92.16±4.07% after 8 hrs of incubation (p<0.0001). Further study also shows that the membrane fluidity in MHC-CLU cells increased following cold exposure, evidenced by approximately 30% increase at the end of 6 hrs of incubation, while there was a trend of a slow decrease of membrane fluidity in MHC-Con cells.
【Conclusion】Ectopic expression of CLU in CLU-KO myoytes stabilizes the cell membrane fluidity, resulting in protection of cells from cell membrane disruption, indicated by LDH release, at cold temperature, suggesting that CLU may function as a chaperone and can stabilize the cell membrane by its chaperone activity at cold temperature.
PartⅢReduction of Cold Ischemia/Reperfusion Injury of Donor Organs by Supplement of Recombinant Clusterin Protein in Cold Preservation Solution
【Objective】Donor organ injury during cold preservation prior to transplantation negatively impacts graft survival. Clusterin (CLU) is a chaperonic protein, and its expression confers donor hearts resistance to cold ischemic injury. This study was to evaluate if supplement of recombinant CLU protein (rCLU) protects donor organs from injury during cold storage with University of Wisconsin (UW) solution.
【Methods】Western blot analysis of recombinant cluterin (rCLU) protein, purified from HEK 293 cell cultures. Human endothelial cell cultures were used as an in vitro model. Heart transplantation in mice was used as an in vivo model. Cell death was measured using lactate dehydrogenase (LDH) release or using a fluorescence microscope after stained with fluorescein diacetate-ethidium bromide solution. The intensity of rCLU on cell surface was measured using a flow cytometer. The cell membrane fluidity of endothelial cells was measured at different time points. Donor injury was determined by biochemical evaluation, its functional recovery and HE stain, the expression of MPO was detected by immunohistochemistry, and the cell apoptosis was detected by TUNEL assay with cell counted.
【Results】Western blot analysis showed that rCLU protein, isolated from HEK 193 cell cultures, was a heterodimer. Supplement of rCLU to UW solution remarkably protected cultured human endothelial cells from cold-induced cell necrosis, evidenced by a decrease in LDH release or in the number of ethidium bromide-stained necrotic cells. The protective activity of rCLU was associated with enhanced membrane fluidity at cold temperature. During cold storage of heart organs in UW solution, supplement of rCLU significantly reduced LDH release from heart tissue. In preclinical model of transplantation, heart grafts after cold preservation with rCLU-containing UW solution had a better functional recovery and less severity of perivascular inflammation, neutrophil infiltration and cardio injury including apoptosis and necrosis that correlated with lower levels of serum creatine kinase and LDH in recipients.
【Conclusion】Our data suggest that supplement of CLU protein in a cold preservation solution may have potential in the improvement of cold preservation of donor organs in transplantation.
引文
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