PD-L1与Kupffer细胞在大鼠肝移植免疫耐受诱导中的作用
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摘要
目的:(1)建立稳定的大鼠肝移植动物模型,观察从BrownNorway(BN)→BN和Lewis(LEW)→BN大鼠肝移植术后移植肝脏的免疫反应、程序性死亡配体1(programmed death ligand 1,PD-L1)以及辅助性T细胞1类(T helper cells-1,Th1)和辅助性T细胞2类(T helper cells-2,Th2)细胞因子在移植肝脏中的表达情况。(2)分离术后7天移植肝脏中的枯否细胞(Kupffer cells,KCs),观察其表达PD-L1的情况以及对淋巴细胞(lympholeukocytes,LCs)的增殖、凋亡以及功能的影响。(3)利用基因克隆技术,通过设计简并性引物,以大鼠肝脏cDNA为模板,采用pUC19质粒载体,克隆大鼠PD-L1基因。(4)构建针对大鼠PD-L1基因编码区的短发夹型RNA(short hairpinribonucleic acid,shRNA)表达质粒PD-L1-shRNA,观察PD-L1-shRNA对KCs PD-L1基因和蛋白表达的抑制效果及其对LCs功能的影响,试图阐明PD-L1与KCs在大鼠肝移植免疫耐受诱导中的作用及机制。
方法:(1)采用Kamada“二袖套法”建立BN→BN和LEW→BN肝移植动物模型。术后观察大鼠存活率和生存质量,并于1、3、5及7天分别活杀6只取外周血及肝脏组织标本。光镜观察肝脏组织形态学改变;全自动生化分析仪检测外周血浆丙氨酸氨基转移酶(alanineaminotransferase,ALT)、天冬氨酸转移酶(aspartate aminotransferase,AST)和总胆红素(total bilirubin,TBIL);ELISA、real-time PCR和免疫组织化学分别检测Th1类细胞因子白介素2(interleukin-2,IL-2)、干扰素γ(interferon-γ,IFN-γ)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和Th2类细胞因子白介素10(interleukin-10,IL-10)在血浆和移植肝脏中的表达情况,免疫组织化学检测PD-L1在两种动物模型移植肝脏中的表达情况。(2)分离术后7天移植肝脏中KCs,real-timePCR和Western-blot分别检测KCs PD-L1的基因和蛋白的表达情况。将KCs与LCs共同培养24h,~3H标记的脱氧核糖核苷(hydrogen3-thymine deoxyriboside,~3H-TdR)掺入法和流式细胞计数分别检测LCs的增殖和凋亡情况,ELISA检测培养上清液中IL-2、IFN-γ、TNF-α和IL-10的含量。(3)利用PCR技术,以大鼠肝脏组织cDNA为模板扩增PD-L1基因片段,PCR产物胶回收后与载体pUC19连接,并转化DH5α感受态细菌,随机挑取白色菌落通过PCR和测序鉴定阳性重组载体的正确性。(4)根据RNA干扰(ribonucleic acid interfere,RNAi)作用原理设计针对大鼠PD-L1的shRNA,利用pRNAT-U6.2质粒,构建重组干扰质粒pPD-L1-A、pPD-L1-B及其阴性对照质粒pPD-L1-C。经PCR及测序鉴定重组成功后,用脂质体转染大鼠KC,转染24小时以后与活化的T细胞共同培养。Real-time PCR和Western bolt检测干扰质粒对KCs PD-L1基因和蛋白表达的抑制作用,ELISA检测上清液中IL-2、INF-γ、TNF-α和IL-10的含量。
结果:(1)采用改良的Kamada“二袖套法”成功建立稳定的大鼠原位肝移植动物模型。发现BN→BN大鼠肝移植后一直未见明显的急性排斥反应表现,可长期存活。而LEW→BN大鼠肝移植术后3天开始出现急性排斥反应病理表现(Banff标准),术后第5至7天比较典型,7只受鼠于16天内全部死亡。LEW→BN组大鼠术后血浆ALT、AST和TBIL呈现进行性升高,而BN→BN组则逐渐降至正常范围,两组差异明显(P＜0.05)。ELISA结果显示:BN→BN组术后7天,血浆中IL-2、INF-γ和TNF-α(分别为210.68±16.82、170.97±14.64和126.4±15.57,pg/ml)的浓度明显低于LEW→BN(分别为753.50±12.68、509.83±17.91和427.97±13.49,pg/ml)组,而IL-10(371.13±17.63,pg/ml)的浓度则明显高于LEW→BN组(187.48±12.89,pg/ml)(P＜0.05)。Real-time PCR和免疫组织化学表明上述细胞因子在两种动物模型移植肝脏中的表达情况与其血浆中的表达趋势一致。免疫组织化学结果显示:术后第1天PD-L1在两组移植肝脏中几乎没有表达。术后第3天PD-L1在BN→BN组PD-L1表达明显增强,第5、7天PD-L1继续呈强阳性表达,并且表达范围逐渐扩大;在LEW→BN组PD-L1表达始终较弱,两组比较有明显差异(P＜0.05)。(2)免疫耐受组中KCs PD-L1的表达明显高于排斥组(P＜0.05)。KCs+LCs共培养组LCs的增殖(13258.34±951.26 cpm)明显低于LCs单独培养组(3047.98±101.42 cpm,P＜0.05),而其凋亡率(8.83±0.37%)却显著高于后者(1.34±0.16%,P＜0.05)。共培养组上清液的IL-2、TNF-α和INF-γ的含量(分别为186.50±12.68、142.83±17.91和129.97±13.49,pg/ml)明显低于LCs单独培养组(分别为481.76±34.53、357.16±19.88和326.4±15.57,pg/ml),但IL-10在共培养组(108.8±12.89 pg/ml)明显高于LCs单独培养组(49.13±17.63 pg/ml,P＜0.05)。(3)采用PCR方法从大鼠肝脏组织中扩增出约873bp的DNA片段,连接到pUC19载体成功构建重组质粒,其扩增的目的片段经PCR和测序验证为PD-L1基因片段,克隆的PD-L1与GenBank中小鼠PD-L1(NM_008798)序列同源性达到88%,其翻译蛋白与小鼠PD-L1理论蛋白同源性为85%。(4)经PCR及测序证实重组干扰质粒pPD-L1-A、pPD-L1-B及其阴性对照质粒pPD-L1-C构建成功,并在脂质体介导下可以高效转染KCs。转染24小时后,RNA干扰组PD-L1表达水平均明显低于未转染组和RNAi对照组。其中pPD-L1-B对KCs PD-L1的表达有较好的抑制效果,抑制率约为79%。pPD-L1-B组上清液中IL-2、IFN-γ和TNF-α的含量(分别为448.70±37.61、327.33±24.35和301.62±17.72,pg/ml)明显高于未转染组(分别为179.60±20.47、153.46±10.70和118.90±15.65,pg/ml),而IL-10的表达则正好相反(分别为40.03±7.81和103.2±17.46,P＜0.05)。
结论:(1)PD-L1在免疫耐受移植肝脏中的表达明显高于急性排斥组,PD-L1可能通过调节LCs的功能,影响Th1和Th2细胞因子的平衡从而参与了移植后免疫反应过程的调控。(2)免疫耐受组移植肝脏中KCs PD-L1的表达水平明显高于急性排斥组。高表达PD-L1的KCs可以显著性抑制LCs的增殖、活化及功能,可能在诱导免疫耐受方面具有重要作用。(3)成功克隆了PD-L1基因,首次构建了pUC19-PD-L1克隆质粒。克隆基因经测序证明与小鼠PD-L1具有较好的同源性。(4)利用克隆的PD-L1可以成功构建出PD-L1-shRNA干扰质粒,其在体外可以有效抑制KCs PD-L1的表达,消除KCs对LCs功能的抑制作用,证实KCs通过高表达PD-L1可以有效抑制LCs功能。表明PD-L1与KCs在肝移植免疫耐受的诱导和维持过程中发挥重要作用。
Objective:(1) To build stable and longtime survival experimental model of rat orthotopic liver transplantation(LT) to investigate the difference of immune reaction between Brown Norway(BN) to BN and Lewis(LEW) to BN LT,expression of programmed death ligand 1(PD-L1) and T help type 1(Th1) and T help type 2(Th2) cytokines in the liver grafts, respectively.(2) To observe the PD-L1 expression of KCs isolated from the two groups on day 7 after LT and its effect on the proliferation,apoptosis and function of LCs.(3) To clone the rat PD-L1 gene using gene cloning technique and degenerated primers with the template of rat liver tissue cDNA and plasmid vector of pUC19.(4) To construct the short hairpin ribonucleic acid(shRNA) targeted PD-L1 and investigate the effect of PD-L1-shRNA on the PD-L1 expression of KCs and cytokines production of LCs,and explore the role of PD-L1 and KCs in the immune tolerance in rat liver transplantation.
Methods:(1) BN to BN and LEW to BN liver transplantation were performed using modified Kamada two-cuff method.Survival rate and general condition of recipients were observed after LT.Every six survival rats were sacrificed at days 1,3,5 and 7 post-transplantation.The pathohistological change of grafts was observed by light microscope and plasma levels of alanine aminotransferase(ALT),aspartase mainotransferase(AST) and total bilirubin(TBIL) were measured with an automatic biochemical analyser.Expression of Th1,including interleukin-2 (IL-2),interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α),and Th2 cytokines,including interleukin-10(IL-10),in plasma and grafts were determined by ELISA,real-time PCR and immunohistochemistry staining, respectively.PD-L1 expression in the allograft was evaluated by immunohistochemistry staining.(2) KCs were isolated from the graft on day 7 after LT,expression of PD-L1 mRNA and protein in KCs was determined by real-time PCR and Western-blot,respectively.KCs and LCs were co-cultured for 24 hours,proliferation and apoptosis of LCs was analyzed by ~3H incorporation and flow cytometry.Supernatant levels of IL-2,IFN-γ, TNF-αand IL-10 were determined by ELISA.(3) PD-L1 gene was amplified from RNA isolated from the liver tissue by PCR and cloned into plasmid pUC19,and then the recombinate plasmid was transformed into DH5αcompetence bacteria.White colonies were randomly selected to identify the amplified PD-L1 gene by PCR and sequencing.(4) Two different recombinant plasmids(pPD-L1-A,pPD-L1-B) targeting rat PD-L1 gene sequences and their unrelated control plasmid pPD-L1-C were constructed based on pRNAT vector,and identified with digestion and sequencing.These recombinant plasmids were transfected into freshly isolated rat KCs with cationic liposome.At 24h post transfection,KCs was co-cultured with rat TCs.Expression changes of PD-L1 mRNA and protein were determined by real-time PCR and Western bolt analysis,respectively. Levels of IL-2,INF-γ,TNF-αand Interlukin-10 in the supernatant were measured by ELISA.
Results:(1) BN to BN and BN to LEW LT model were successfully established.Without using any immunosuppressive therapy,recipients in BN-BN group survived a long time without any obviously acute rejection, but it was observed in LEW-BN group on day 3 after LT and typical characteristic appeared at day 5-7.Similarly,plasma levels of ALT,AST and TBIL were sharply increased in LEW-BN group,while they were gradually decreased to normal in BN-BN group(P＜0.05).ELISA results showed that plasma levels of IL-2,IFN-γand TNF-αin BN-BN group(210.68±16.82, 170.97±14.64 and 126.4±15.57 pg/ml,respectively) were significant lower and that of IL-10(371.13±17.63 pg/ml) were obviously higher than that in LEW-BN(753.50±12.68,509.83±17.91,427.97±13.49 and 187.48±12.89 pg/ml) group on day 7 after LT(P＜0.05).Rea-time PCR and immunohistochemistry showed the change of these cytokines in the grafts was same to that in the plasma.After LT,PD-L1 had no obvious expression in the two groups on day 1.But it elevated on day 3,and kept strong positive on day 5-7 in the BN-BN group,while it kept week-positive in LEW-BN group after LT(P＜0.05).(2) PD-L1 mRNA and protein expression of KCs in BN-BN group was significantly higher than that in the LEW-BN group (P＜0.05).Proliferation of LCs was obviously inhinited in KCs+LCs group (13258.34±951.26 cmp) in comparison with that in LCs group (3047.98±101.42 cpm,P＜0.05).But its apoptosis rate was significantly higher than that in the LCs group(8.83±0.37%and 1.34±0.16%, respectively;P＜0.05).IL-2,TNF-αand INF-γlevels were remarkably high and IL-10 levels were lower in the KCs+LCs group(186.50±12.68, 142.83±17.91,129.97±13.49 and 108.8±12.89 pg/ml,respectively) than that in the LCs group(481.76±34.53,357.16±19.88,326.4±15.57 and 49.13±17.63 pg/ml,respectively;P＜0.05).(3) 873 bp of DNA fragment was amplified from liver tissue by PCR and identified by sequencing.As compared with mouse gene PD-L1(NM_008798) in Genebank,cloned rat PD-L1 gene sequence homology was about 88%,and the homology of cloned rat PD-L1 gene translated protein with the standard protein sequence of mouse PD-L1 was about 85%.(4) Interference recombinant plasmid (pPD-L1-A,pPD-L1-B) and the unrelated control plasmid pPD-L1-C were successfully constructed and identified with PCR and sequencing.At 24h post-transfection,the results of real-time PCR and Western blot assay indicated that these recombinant plasmids carrying different shRNA showed different inhibition on the mRNA and protein expression of PD-L1.The pPD-L1-B was the most efficient plasmid in comparsion with the control. The levels of IL-2、IFN-γand TNF-αin pPD-L1 group were significantly higher and that of IL-10 was obviously lower than that in the untransfection group(448.70±37.61,327.33±24.35,301.62±17.72 and 40.03±7.81 pg/ml in pPD-L1-B group;179.60±20.47,153.46±10.70,118.90±15.65 and 103.2±17.46 pg/ml in no transfection group,respectively;P＜0.05).
Conclusion:(1) Expression of PD-L1 in graft of BN-BN group was significantly higher that that in the LEW-BN group,it may be involved in the regulation of immune reaction by regulation the function of lymphocytes and the balance of Th1 and Th2 cytokines.(2) PD-L1 expression in KCs of immune tolerance grafts was obviously higher than that in the rejection grafts.KCs with high expression of PD-L1 could significantly suppress the proliferation and function of LCs,which may indicate it play an important role in the immune tolerance induction.(3) PD-L1 gene was successfully cloned and ligated into pUC19 vector.The sequence of the cloned gene showed a good homology to mouse PD-L1 gene.(4) The interference plasmid PD-L1-shRNA was successfully constructed according to the cloned PD-L1 gene.It could effectively silence the expression PD-L1 in KCs in vivo and restored the function of LCs.These results indicated that PD-L1 and KCs play a key role in the immune tolerance in liver transplantation.
方法:(1)采用Kamada“二袖套法”建立BN→BN和LEW→BN肝移植动物模型。术后观察大鼠存活率和生存质量,并于1、3、5及7天分别活杀6只取外周血及肝脏组织标本。光镜观察肝脏组织形态学改变;全自动生化分析仪检测外周血浆丙氨酸氨基转移酶(alanineaminotransferase,ALT)、天冬氨酸转移酶(aspartate aminotransferase,AST)和总胆红素(total bilirubin,TBIL);ELISA、real-time PCR和免疫组织化学分别检测Th1类细胞因子白介素2(interleukin-2,IL-2)、干扰素γ(interferon-γ,IFN-γ)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和Th2类细胞因子白介素10(interleukin-10,IL-10)在血浆和移植肝脏中的表达情况,免疫组织化学检测PD-L1在两种动物模型移植肝脏中的表达情况。(2)分离术后7天移植肝脏中KCs,real-timePCR和Western-blot分别检测KCs PD-L1的基因和蛋白的表达情况。将KCs与LCs共同培养24h,~3H标记的脱氧核糖核苷(hydrogen3-thymine deoxyriboside,~3H-TdR)掺入法和流式细胞计数分别检测LCs的增殖和凋亡情况,ELISA检测培养上清液中IL-2、IFN-γ、TNF-α和IL-10的含量。(3)利用PCR技术,以大鼠肝脏组织cDNA为模板扩增PD-L1基因片段,PCR产物胶回收后与载体pUC19连接,并转化DH5α感受态细菌,随机挑取白色菌落通过PCR和测序鉴定阳性重组载体的正确性。(4)根据RNA干扰(ribonucleic acid interfere,RNAi)作用原理设计针对大鼠PD-L1的shRNA,利用pRNAT-U6.2质粒,构建重组干扰质粒pPD-L1-A、pPD-L1-B及其阴性对照质粒pPD-L1-C。经PCR及测序鉴定重组成功后,用脂质体转染大鼠KC,转染24小时以后与活化的T细胞共同培养。Real-time PCR和Western bolt检测干扰质粒对KCs PD-L1基因和蛋白表达的抑制作用,ELISA检测上清液中IL-2、INF-γ、TNF-α和IL-10的含量。
结果:(1)采用改良的Kamada“二袖套法”成功建立稳定的大鼠原位肝移植动物模型。发现BN→BN大鼠肝移植后一直未见明显的急性排斥反应表现,可长期存活。而LEW→BN大鼠肝移植术后3天开始出现急性排斥反应病理表现(Banff标准),术后第5至7天比较典型,7只受鼠于16天内全部死亡。LEW→BN组大鼠术后血浆ALT、AST和TBIL呈现进行性升高,而BN→BN组则逐渐降至正常范围,两组差异明显(P＜0.05)。ELISA结果显示:BN→BN组术后7天,血浆中IL-2、INF-γ和TNF-α(分别为210.68±16.82、170.97±14.64和126.4±15.57,pg/ml)的浓度明显低于LEW→BN(分别为753.50±12.68、509.83±17.91和427.97±13.49,pg/ml)组,而IL-10(371.13±17.63,pg/ml)的浓度则明显高于LEW→BN组(187.48±12.89,pg/ml)(P＜0.05)。Real-time PCR和免疫组织化学表明上述细胞因子在两种动物模型移植肝脏中的表达情况与其血浆中的表达趋势一致。免疫组织化学结果显示:术后第1天PD-L1在两组移植肝脏中几乎没有表达。术后第3天PD-L1在BN→BN组PD-L1表达明显增强,第5、7天PD-L1继续呈强阳性表达,并且表达范围逐渐扩大;在LEW→BN组PD-L1表达始终较弱,两组比较有明显差异(P＜0.05)。(2)免疫耐受组中KCs PD-L1的表达明显高于排斥组(P＜0.05)。KCs+LCs共培养组LCs的增殖(13258.34±951.26 cpm)明显低于LCs单独培养组(3047.98±101.42 cpm,P＜0.05),而其凋亡率(8.83±0.37%)却显著高于后者(1.34±0.16%,P＜0.05)。共培养组上清液的IL-2、TNF-α和INF-γ的含量(分别为186.50±12.68、142.83±17.91和129.97±13.49,pg/ml)明显低于LCs单独培养组(分别为481.76±34.53、357.16±19.88和326.4±15.57,pg/ml),但IL-10在共培养组(108.8±12.89 pg/ml)明显高于LCs单独培养组(49.13±17.63 pg/ml,P＜0.05)。(3)采用PCR方法从大鼠肝脏组织中扩增出约873bp的DNA片段,连接到pUC19载体成功构建重组质粒,其扩增的目的片段经PCR和测序验证为PD-L1基因片段,克隆的PD-L1与GenBank中小鼠PD-L1(NM_008798)序列同源性达到88%,其翻译蛋白与小鼠PD-L1理论蛋白同源性为85%。(4)经PCR及测序证实重组干扰质粒pPD-L1-A、pPD-L1-B及其阴性对照质粒pPD-L1-C构建成功,并在脂质体介导下可以高效转染KCs。转染24小时后,RNA干扰组PD-L1表达水平均明显低于未转染组和RNAi对照组。其中pPD-L1-B对KCs PD-L1的表达有较好的抑制效果,抑制率约为79%。pPD-L1-B组上清液中IL-2、IFN-γ和TNF-α的含量(分别为448.70±37.61、327.33±24.35和301.62±17.72,pg/ml)明显高于未转染组(分别为179.60±20.47、153.46±10.70和118.90±15.65,pg/ml),而IL-10的表达则正好相反(分别为40.03±7.81和103.2±17.46,P＜0.05)。
结论:(1)PD-L1在免疫耐受移植肝脏中的表达明显高于急性排斥组,PD-L1可能通过调节LCs的功能,影响Th1和Th2细胞因子的平衡从而参与了移植后免疫反应过程的调控。(2)免疫耐受组移植肝脏中KCs PD-L1的表达水平明显高于急性排斥组。高表达PD-L1的KCs可以显著性抑制LCs的增殖、活化及功能,可能在诱导免疫耐受方面具有重要作用。(3)成功克隆了PD-L1基因,首次构建了pUC19-PD-L1克隆质粒。克隆基因经测序证明与小鼠PD-L1具有较好的同源性。(4)利用克隆的PD-L1可以成功构建出PD-L1-shRNA干扰质粒,其在体外可以有效抑制KCs PD-L1的表达,消除KCs对LCs功能的抑制作用,证实KCs通过高表达PD-L1可以有效抑制LCs功能。表明PD-L1与KCs在肝移植免疫耐受的诱导和维持过程中发挥重要作用。
Objective:(1) To build stable and longtime survival experimental model of rat orthotopic liver transplantation(LT) to investigate the difference of immune reaction between Brown Norway(BN) to BN and Lewis(LEW) to BN LT,expression of programmed death ligand 1(PD-L1) and T help type 1(Th1) and T help type 2(Th2) cytokines in the liver grafts, respectively.(2) To observe the PD-L1 expression of KCs isolated from the two groups on day 7 after LT and its effect on the proliferation,apoptosis and function of LCs.(3) To clone the rat PD-L1 gene using gene cloning technique and degenerated primers with the template of rat liver tissue cDNA and plasmid vector of pUC19.(4) To construct the short hairpin ribonucleic acid(shRNA) targeted PD-L1 and investigate the effect of PD-L1-shRNA on the PD-L1 expression of KCs and cytokines production of LCs,and explore the role of PD-L1 and KCs in the immune tolerance in rat liver transplantation.
Methods:(1) BN to BN and LEW to BN liver transplantation were performed using modified Kamada two-cuff method.Survival rate and general condition of recipients were observed after LT.Every six survival rats were sacrificed at days 1,3,5 and 7 post-transplantation.The pathohistological change of grafts was observed by light microscope and plasma levels of alanine aminotransferase(ALT),aspartase mainotransferase(AST) and total bilirubin(TBIL) were measured with an automatic biochemical analyser.Expression of Th1,including interleukin-2 (IL-2),interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α),and Th2 cytokines,including interleukin-10(IL-10),in plasma and grafts were determined by ELISA,real-time PCR and immunohistochemistry staining, respectively.PD-L1 expression in the allograft was evaluated by immunohistochemistry staining.(2) KCs were isolated from the graft on day 7 after LT,expression of PD-L1 mRNA and protein in KCs was determined by real-time PCR and Western-blot,respectively.KCs and LCs were co-cultured for 24 hours,proliferation and apoptosis of LCs was analyzed by ~3H incorporation and flow cytometry.Supernatant levels of IL-2,IFN-γ, TNF-αand IL-10 were determined by ELISA.(3) PD-L1 gene was amplified from RNA isolated from the liver tissue by PCR and cloned into plasmid pUC19,and then the recombinate plasmid was transformed into DH5αcompetence bacteria.White colonies were randomly selected to identify the amplified PD-L1 gene by PCR and sequencing.(4) Two different recombinant plasmids(pPD-L1-A,pPD-L1-B) targeting rat PD-L1 gene sequences and their unrelated control plasmid pPD-L1-C were constructed based on pRNAT vector,and identified with digestion and sequencing.These recombinant plasmids were transfected into freshly isolated rat KCs with cationic liposome.At 24h post transfection,KCs was co-cultured with rat TCs.Expression changes of PD-L1 mRNA and protein were determined by real-time PCR and Western bolt analysis,respectively. Levels of IL-2,INF-γ,TNF-αand Interlukin-10 in the supernatant were measured by ELISA.
Results:(1) BN to BN and BN to LEW LT model were successfully established.Without using any immunosuppressive therapy,recipients in BN-BN group survived a long time without any obviously acute rejection, but it was observed in LEW-BN group on day 3 after LT and typical characteristic appeared at day 5-7.Similarly,plasma levels of ALT,AST and TBIL were sharply increased in LEW-BN group,while they were gradually decreased to normal in BN-BN group(P＜0.05).ELISA results showed that plasma levels of IL-2,IFN-γand TNF-αin BN-BN group(210.68±16.82, 170.97±14.64 and 126.4±15.57 pg/ml,respectively) were significant lower and that of IL-10(371.13±17.63 pg/ml) were obviously higher than that in LEW-BN(753.50±12.68,509.83±17.91,427.97±13.49 and 187.48±12.89 pg/ml) group on day 7 after LT(P＜0.05).Rea-time PCR and immunohistochemistry showed the change of these cytokines in the grafts was same to that in the plasma.After LT,PD-L1 had no obvious expression in the two groups on day 1.But it elevated on day 3,and kept strong positive on day 5-7 in the BN-BN group,while it kept week-positive in LEW-BN group after LT(P＜0.05).(2) PD-L1 mRNA and protein expression of KCs in BN-BN group was significantly higher than that in the LEW-BN group (P＜0.05).Proliferation of LCs was obviously inhinited in KCs+LCs group (13258.34±951.26 cmp) in comparison with that in LCs group (3047.98±101.42 cpm,P＜0.05).But its apoptosis rate was significantly higher than that in the LCs group(8.83±0.37%and 1.34±0.16%, respectively;P＜0.05).IL-2,TNF-αand INF-γlevels were remarkably high and IL-10 levels were lower in the KCs+LCs group(186.50±12.68, 142.83±17.91,129.97±13.49 and 108.8±12.89 pg/ml,respectively) than that in the LCs group(481.76±34.53,357.16±19.88,326.4±15.57 and 49.13±17.63 pg/ml,respectively;P＜0.05).(3) 873 bp of DNA fragment was amplified from liver tissue by PCR and identified by sequencing.As compared with mouse gene PD-L1(NM_008798) in Genebank,cloned rat PD-L1 gene sequence homology was about 88%,and the homology of cloned rat PD-L1 gene translated protein with the standard protein sequence of mouse PD-L1 was about 85%.(4) Interference recombinant plasmid (pPD-L1-A,pPD-L1-B) and the unrelated control plasmid pPD-L1-C were successfully constructed and identified with PCR and sequencing.At 24h post-transfection,the results of real-time PCR and Western blot assay indicated that these recombinant plasmids carrying different shRNA showed different inhibition on the mRNA and protein expression of PD-L1.The pPD-L1-B was the most efficient plasmid in comparsion with the control. The levels of IL-2、IFN-γand TNF-αin pPD-L1 group were significantly higher and that of IL-10 was obviously lower than that in the untransfection group(448.70±37.61,327.33±24.35,301.62±17.72 and 40.03±7.81 pg/ml in pPD-L1-B group;179.60±20.47,153.46±10.70,118.90±15.65 and 103.2±17.46 pg/ml in no transfection group,respectively;P＜0.05).
Conclusion:(1) Expression of PD-L1 in graft of BN-BN group was significantly higher that that in the LEW-BN group,it may be involved in the regulation of immune reaction by regulation the function of lymphocytes and the balance of Th1 and Th2 cytokines.(2) PD-L1 expression in KCs of immune tolerance grafts was obviously higher than that in the rejection grafts.KCs with high expression of PD-L1 could significantly suppress the proliferation and function of LCs,which may indicate it play an important role in the immune tolerance induction.(3) PD-L1 gene was successfully cloned and ligated into pUC19 vector.The sequence of the cloned gene showed a good homology to mouse PD-L1 gene.(4) The interference plasmid PD-L1-shRNA was successfully constructed according to the cloned PD-L1 gene.It could effectively silence the expression PD-L1 in KCs in vivo and restored the function of LCs.These results indicated that PD-L1 and KCs play a key role in the immune tolerance in liver transplantation.
引文
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