同种异基因骨髓间充质干细胞诱导大鼠肾移植免疫低反应性的实验研究
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摘要
第一部分大鼠骨髓间充质干细胞的体外分离、培养与鉴定
目的：建立稳定的大鼠间充质干细胞(Mesenchymal stem cell，MSC)体外分离、培养、鉴定方法，为进一步开展细胞移植提供细胞来源。
方法：选用雄性清洁级Wistar大鼠，月龄1月，处死消毒后取其长骨，用含15％胎牛血清L-DMEM冲洗骨髓腔，收集冲洗液，采用密度梯度离心法分离获取细胞，培养后取贴壁细胞进行传代培养。取第3代生长状态良好的细胞，胰蛋白酶消化制成细胞悬液计数并记录生长曲线。细胞悬液加入二甲亚砜和胎牛血清培养基以冻存。在骨向诱导条件下：加入地塞米松、β-甘油磷酸钠、抗坏血酸后，进行培养15天后碱性磷酸酶(ALP)染色。在成脂诱导条件下：加入青霉素、链霉素、转铁蛋白、胰岛素、亚油酸、地塞米松、3-异丁基-1甲基黄嘌呤，培养5天后，改用加入快速胰岛素而无胎牛血清培养基培养5天，予油红O染色。
结果：MSC分离后在培养瓶中24h后就有细胞贴壁，全量换液去除未贴壁细胞，第3-4d细胞生长明显，传代后细胞生长迅速约5-6d即可长满培养瓶底，达融合状态。三代后即可得到较为纯化的MSC成纤维样细胞，并至少可传至10代以上，冻存对细胞生长无明显影响。在脂肪细胞诱导分化过程中可以观察到诱导后第二天即有含脂质的空泡在细胞内累积，至4-5天脂质含量更为丰富。在骨细胞分化中，MScs形成聚集物或小结，并增加碱性磷酸酶的表达，3天后出现明显的钙沉积。经染色证实诱导均获成功。
结论：1)MSC是一种能够自我更新，至少能够体外传代达到10代，有很强增殖能力的干细胞，并在一定条件下，可向多种细胞分化，具有多向分化特性；2)以约200／cm~2密度接种，采用0.25％胰酶消化5 min对于Wistar大鼠MSCs的生长及消化传代效果较好；3)通过密度梯度离心及贴壁筛选培养法获取大鼠MSC的方法简便易行，可提供稳定的大鼠MSC。
意义：研究证实了MSC具有很强增殖能力、多向分化潜能，获取方法简便易行，为进一步开展细胞移植治疗提供了细胞来源。
第二部分大鼠肾移植模型的改良
目的：以改良方法建立成功率较高的大鼠肾移植模型。
方法：Wistar大鼠作为肾移植供体，Lewis大鼠作为受体，采用重力低温原位灌注对供体的左肾灌注，受体左肾切除后行供受体腹主动脉-腹主动脉两定点端侧吻合，肾静脉-肾静脉间断端端吻合，输尿管膀胱瓣与受体膀胱吻合。术后第2天使用术中预留线结扎右肾动脉，替代右肾切除以减轻受体手术打击，提高手术成功率。
结果：预试验中共施行大鼠肾移植30次60X(30对)，成活21只，供体、受体手术时间分别为50±14、75±22min，热缺血时间1min左右，冷缺血时间为60±11min，手术时间共为140±30 min，并发症依次有吻合口出血、排斥反应、肾衰、感染、血栓形成、输尿管梗阻等，成功率70％。正式试验时，作者施行大鼠肾移植36次72只(36对)，成活34只，供体、受体手术时间分别为35±12、65±10min，热缺血时间1min左右，冷缺血时间为40±8min，手术时间共为110±20 min，成功率约95％，其中一只是由于肾静脉吻合口出血而导致失败，另一只为净动脉血栓形成。
结论：本模型建立的改良为重力原位恒压灌注；原位修整供肾，取肾、移植肾均在左侧；动、静脉均采取直接吻合法；采用输尿管带膀胱瓣吻合法；延迟肾切除。整个手术均在显微镜下进行，手术难度较大，但效果较好。
意义：通过手术方面的改进，成功地改良了大鼠肾移植模型，并使手术所带来的对采用该模型进一步实验的影响减至最小。
第三部分MSCs移植免疫调节作用及与CsA协同作用的研究
目的：观察大鼠MSCs对大鼠移植肾的影响，了解MSCs的免疫调节，探讨使用MSCs减少免疫抑制剂环孢素A(Cyclosporine A，CsA)剂量甚至诱导免疫耐受的可能性。
方法：将实验动物随机分为4组进行肾移植，分别接受不同的预处理和术后治疗。(1)MSC诱导组(第1组)：受者于肾移植前1周、开放血流前、术后1周、2周分别输注来源于Wistar大鼠的MSC1×10~7，术后无特殊药物治疗；(2)CsA治疗组(第2组)：受者不接受预处理，术后每日腹腔内注射CsA0.5mg／kg；(3)MSC诱导+常规剂量CsA组(第3组)：受者接受MSC诱导方法同单纯MSC诱导组，并术后每日给予CsA0.5mg／kg；(4)阴性对照组(第4组)：受者不接受任何预处理，肾移植术后腹腔内注射PBS作为对照。术后于第3、5、10、15、25、40天取血测定各组血肌酐值，于术后第4天各组取2只大鼠肾脏病理，RT-PcR分析大鼠肾脏IL-1β，TNF-α，TGF-β1等相关炎性因子产生情况，观察其余大鼠生存时间。
结果：CsA治疗组(第2组)或联合疗法组(第3组)，与阴性对照组(第4组)相比，显著延长动物存活期(P=0.002，0.003)。与阴性对照组相比，MSC诱导组(第1组)存活期确实有所延长(P=0.001)，但比第2组(P=0.036)及第3组(P=0.045)要短。阴性对照组的SCr与第1、2、3组相比，从第5天开始显著升高(P=0.000，0.011，0.000)，第2组与第1以及第3组相比第5天出现SCr的升高(P＜0.05)，但三组间在其余几天检测上没有显著差异。病理研究发现第4组移植肾出现了典型的严重急性排斥，第1组表现出明显减轻的急性排斥，第2及第3组与第1组相比，又有明显改善。包括IL-1β，TNF-α及TGF-β1在内的所有炎症介质的mRNA产物，第2组结果与其余各组相比明显要少(P＜0.01)，第1组与第3组在某些炎症介质的产物上也有轻度减少。
小结：MSCs能够对免疫反应产生下调作用，减少部分炎症介质的产生，在移植后初期保护移植器官的功能并延长动物的存活期。但这种作用弱于CsA疗法。此外，MSCs联合小剂量CsA疗法，能保护移植器官的功能，但与CsA单一疗法相比并不延长动物的存活期。
意义：本研究中，MSCs在体内免疫下调作用有限，且可能与CsA在体内存在相互影响，影响免疫抑制效果。因此，主张通过应用MSCs以减少CsA剂量的观点需要进一步研究。
Ⅰ. Isolation, Culturing and Identify of Rat MSCsfrom the Bone Marrow in vitro
Objective: To establish immortalized rat MSC lines in vitro, to providesource for cellular transplant.
Methods: One month old male Wistar rats was killed and degermed to getlong bones. Bone marrow cells were collected by flushing the longbones with DMEM medium (supplemented with 15% fetal bovineserum) and purified by density centrifuge,after Cultured the adherentcells were chosen for serial subcultivation. The third generation cellsin good growth condition was harvested and trypsinized to form cellsuspension,counted and noted the growth curve. The cell suspensionwas reserved in frozen condition supplemented with DMSO andfetal bovine serum medium. In osteocyte differentiationcondition: cells were cultured with dexamethasone,β-glycerolphosphate and ascorbate for 15 days,then stained with alkalinephosphatase(ALP). In adipocyte differentiation condition: cells werecultured with penicillin,streptomycin, transferrin,insulin;linoleic acid,dexamethasone and 3-isobutyl-1-methylxanthin for 5days,subsequently, cells were cultured for 5 days with MSC culturemedium supplemented with rapid insulin without FBS, thenconfirmed by Oil Red O staining.
Results: Adherent cells were observed in cell culture flasks after theIsolated MSC cultured for 24h,nonadherent cells were removed bychanging the culture medium.
The cell growth was obvious on the 3-4d,and after passage the cellsgrown rapidly to fill the floor of cell culture flasks and achievedconfluency. Purified MSC fibroblast-like cells could be harvestedafter 3 generations,and could passage for at least 10 generations.There was no obvious influence to cell growth when reserved infrozen condition. In the adipocyte differentiation, the accumulation oflipid-rich vacuoles within cells was observed on day 2 afterinduction, and the lipid was richer on the 4-5 day. In the osteocytedifferentiation, the MSCs formed aggregates or nodules andincreased their expression of alkaline phosphatase, and calciumaccumulation was evident after 3 days. The inductions wereconfirmed with staining.
Conclusion: 1)MSCs are a type of stem cells with strong proliferativeactivity and sustain self-renewal at least for 10 passage. They have afeature of pluripotent and could differentiate to a variety of cell types in some condition; 2)Inoculated by a density of 200/cm~2 andtrypsinized with 0.25% trypsin for 5 min are effective for the growthand passage of Wistar rat MSCs; 3)The methods of obtaining ratMSC with density gradient centrifugation and adherence cultivationwas convenient,and could provid immortalized rat MSC lines.
Significance: Our research demonstrates extensive proliferation andpluripotent of MSC, and provids a convenient harvest method forcellular transplant therapy.
ⅡEstablishment of a Modified Model of Rat Renal Transplantation
Objective: To establish a modified model of rat renal transplantation forhigh achievement ratio.
Methods: Wistar and Lewis mice were used as the donors and therecipients. Left donor's kidney was perfused with ice-cold solution.End-to-side anatomosis of recipient's abdominal aorta to the donor'swas performed between two points. End-to-end anastomosis of bothrenal veins was performed interrupted. Urinary tract reconstructionwas accomplished by suturing the donor ureter with a bladder patchto the recipient bladder. A silk thread was remained in the operation and this silk thread was tigated later in the second day after theoperation instead of nephrectomy to reduce the strike of the extraoperation for increasing the operative achievement ratio.
Result: In advance test of 30 operations on 60 rats, 21 rats were survived,the operative time of the donor's was 50±14min, and the recipient'swas 75±22min., including the warm ischemia time (about 1min), thecold ischemia time (60±11min), the whole operative time was140±30 min. Complications including anastomosis hemorrhage atarkery, rejection, renal failure, infection, thrombosis and uretalobstruction were observed. Achievement ratio is about 70%. Informal test of 36 operations on 72 rats, 34 rats were survived, theoperative time of the donor's was 35±12min, and the recipient's was65±10min. including the warm ischemia time (about 1 min), the coldischemia time (40±8min). Achievement ratio is about 95%. Onereason of the abortive cases was anastomosis of both renal veinshemorrhage, and another was thrombosis of renal artery.
Conclusion: The modifiability of this model of rat renal transplantation isthe infusion of left kidney with gravity at constant pressure andprepared at prime position, and the grafted kidney was put at the leftside. Direct anastomosis is used in artery-to-artery and vein-to-vein.Urinary tract reconstruction was accomplished by suturing the donorureter with a bladder patch. Delay the nephrectomy. The whole operation is processed under the microscope. Although it is difficultin operation, the effect is satisfying.
Significances: The modifiability in operation is successful to establish amodel of rat renal transplantation, and reduce the effect on the modelwith the operation.
ⅢMSCs Modulate Immune Responses Combined withCyclosporine A in Transplantation
Objective: To observe the effect of rat MSCs in rat renaltransplantation, study the immunological regulation and reparationof MSCs,and investigate the consideration that reduce dose ofCyclos-porine A (CsA) and even induce immune tolerance byapplication of MSCs.
Methods: Rats bearing renal allografts were divided randomly into fourgroups, which was given different fore treatmen and postoperationtherapy respectively.(1)MSC monotherapy group (group 1): therecipients were engrafted respectively one week beforetransplantation, before reperfusion,one week after operation and twoweeks after operation, with MSC(1×10~7, purified from Wistar rat bone marrow cells) by intravenous injection,and without specialdrug treatment; (2)CsA therapy (group 2): CsA was injected to therecipients intraperitoneally at a dosage of 0.5 mg/kg/d from day 2after transplantation without fore treatment; (3)MSC and commondose CsA combination therapy (group 3): the recipients were treatwith MSC the same as group 1,and CsA was injected at a dosage of0.5 mg/kg/d after transplantation;(4)No therapy group (group 4): nofore treatment was given, and PBS was injected intraperitoneallyafter transplantation as control. We tested the values of S-Cr of eachanimal in four groups on day 3,5,10,15,25 and 40 aftertransplantation. Graft histology of 2 recipients from each group washarvested on day 4, the production of inflammatory mediators,including IL-1β, TNF-αand TGF-β1, was analyzed by RT-PCR.Observed survival days of other animals.
Results: Group treated with CsA monotherapy (group 2) orcombination therapy (group 3) prolonged animal survivalsignificantly compared with no therapy group (group 4) (P=0.002,0.003).Indeed, animal survival of MSC monotherapy group (group 1)was prolonged compared with group 4 (P=0.001), but shortened thangroup 2 (P=0.036) and group 3(P=0.045).The recipients without anytreatment(Group 4)exhibited significantly elevated values of S-Crfrom day5 compared with group 1,2,3 (P=0.000,0.011,0.000), and the recipients in group 2 showed elevated values of S-Cr on day 5compared with group1 or group3 (P＜0.05), but there is no differenceamong group 1, group2 or group 3 on other testing days. Grafthistology shown that allografts of animals in group4 exhibitedfindings typical of severe acute rejection. Allografts of recipientswith MSC monotherapy(group1) showed findings of acute rejectionwith considerably milder severity. But allografts of recipients ingroup 2 and group 3 were markedly well preserved compared withgroup 1. The production of mRNA of all inflammatory mediators weanalyzed, including IL-1β, TNF-αand TGF-β1, was significantlydecreased in the allografts treated with a therapy of CsA (group 2) ascompared with all other groups (P＜0.01). MSC monotherapy (group1) or combination therapy (group 3) decreased production of someinflammatory mediators mildly;
Conclusion: MSCs can downregulate the immune response, reduce theproduction of some inflammatory mediators, preserve the graftfunction in the initial stage after transplantation and prolong theanimal survival. But this effect was weaker than CsA therapy.Moreover, MSCs therapy combined with low-dose CsA, couldprotect the graft function, but can not prolong the animal survivaltime compared with CsA monotherapy.
Significance: Our results suggest that down the downregulation of MSCs in immune response in vivo is limited, and indicate a potentialinteraction between MSC and CsA activities,which impact theimmune suppression. Therefore, previously considered reducing doseof CsA by application of MSCs need to be carefully investigated.
目的：建立稳定的大鼠间充质干细胞(Mesenchymal stem cell，MSC)体外分离、培养、鉴定方法，为进一步开展细胞移植提供细胞来源。
方法：选用雄性清洁级Wistar大鼠，月龄1月，处死消毒后取其长骨，用含15％胎牛血清L-DMEM冲洗骨髓腔，收集冲洗液，采用密度梯度离心法分离获取细胞，培养后取贴壁细胞进行传代培养。取第3代生长状态良好的细胞，胰蛋白酶消化制成细胞悬液计数并记录生长曲线。细胞悬液加入二甲亚砜和胎牛血清培养基以冻存。在骨向诱导条件下：加入地塞米松、β-甘油磷酸钠、抗坏血酸后，进行培养15天后碱性磷酸酶(ALP)染色。在成脂诱导条件下：加入青霉素、链霉素、转铁蛋白、胰岛素、亚油酸、地塞米松、3-异丁基-1甲基黄嘌呤，培养5天后，改用加入快速胰岛素而无胎牛血清培养基培养5天，予油红O染色。
结果：MSC分离后在培养瓶中24h后就有细胞贴壁，全量换液去除未贴壁细胞，第3-4d细胞生长明显，传代后细胞生长迅速约5-6d即可长满培养瓶底，达融合状态。三代后即可得到较为纯化的MSC成纤维样细胞，并至少可传至10代以上，冻存对细胞生长无明显影响。在脂肪细胞诱导分化过程中可以观察到诱导后第二天即有含脂质的空泡在细胞内累积，至4-5天脂质含量更为丰富。在骨细胞分化中，MScs形成聚集物或小结，并增加碱性磷酸酶的表达，3天后出现明显的钙沉积。经染色证实诱导均获成功。
结论：1)MSC是一种能够自我更新，至少能够体外传代达到10代，有很强增殖能力的干细胞，并在一定条件下，可向多种细胞分化，具有多向分化特性；2)以约200／cm~2密度接种，采用0.25％胰酶消化5 min对于Wistar大鼠MSCs的生长及消化传代效果较好；3)通过密度梯度离心及贴壁筛选培养法获取大鼠MSC的方法简便易行，可提供稳定的大鼠MSC。
意义：研究证实了MSC具有很强增殖能力、多向分化潜能，获取方法简便易行，为进一步开展细胞移植治疗提供了细胞来源。
第二部分大鼠肾移植模型的改良
目的：以改良方法建立成功率较高的大鼠肾移植模型。
方法：Wistar大鼠作为肾移植供体，Lewis大鼠作为受体，采用重力低温原位灌注对供体的左肾灌注，受体左肾切除后行供受体腹主动脉-腹主动脉两定点端侧吻合，肾静脉-肾静脉间断端端吻合，输尿管膀胱瓣与受体膀胱吻合。术后第2天使用术中预留线结扎右肾动脉，替代右肾切除以减轻受体手术打击，提高手术成功率。
结果：预试验中共施行大鼠肾移植30次60X(30对)，成活21只，供体、受体手术时间分别为50±14、75±22min，热缺血时间1min左右，冷缺血时间为60±11min，手术时间共为140±30 min，并发症依次有吻合口出血、排斥反应、肾衰、感染、血栓形成、输尿管梗阻等，成功率70％。正式试验时，作者施行大鼠肾移植36次72只(36对)，成活34只，供体、受体手术时间分别为35±12、65±10min，热缺血时间1min左右，冷缺血时间为40±8min，手术时间共为110±20 min，成功率约95％，其中一只是由于肾静脉吻合口出血而导致失败，另一只为净动脉血栓形成。
结论：本模型建立的改良为重力原位恒压灌注；原位修整供肾，取肾、移植肾均在左侧；动、静脉均采取直接吻合法；采用输尿管带膀胱瓣吻合法；延迟肾切除。整个手术均在显微镜下进行，手术难度较大，但效果较好。
意义：通过手术方面的改进，成功地改良了大鼠肾移植模型，并使手术所带来的对采用该模型进一步实验的影响减至最小。
第三部分MSCs移植免疫调节作用及与CsA协同作用的研究
目的：观察大鼠MSCs对大鼠移植肾的影响，了解MSCs的免疫调节，探讨使用MSCs减少免疫抑制剂环孢素A(Cyclosporine A，CsA)剂量甚至诱导免疫耐受的可能性。
方法：将实验动物随机分为4组进行肾移植，分别接受不同的预处理和术后治疗。(1)MSC诱导组(第1组)：受者于肾移植前1周、开放血流前、术后1周、2周分别输注来源于Wistar大鼠的MSC1×10~7，术后无特殊药物治疗；(2)CsA治疗组(第2组)：受者不接受预处理，术后每日腹腔内注射CsA0.5mg／kg；(3)MSC诱导+常规剂量CsA组(第3组)：受者接受MSC诱导方法同单纯MSC诱导组，并术后每日给予CsA0.5mg／kg；(4)阴性对照组(第4组)：受者不接受任何预处理，肾移植术后腹腔内注射PBS作为对照。术后于第3、5、10、15、25、40天取血测定各组血肌酐值，于术后第4天各组取2只大鼠肾脏病理，RT-PcR分析大鼠肾脏IL-1β，TNF-α，TGF-β1等相关炎性因子产生情况，观察其余大鼠生存时间。
结果：CsA治疗组(第2组)或联合疗法组(第3组)，与阴性对照组(第4组)相比，显著延长动物存活期(P=0.002，0.003)。与阴性对照组相比，MSC诱导组(第1组)存活期确实有所延长(P=0.001)，但比第2组(P=0.036)及第3组(P=0.045)要短。阴性对照组的SCr与第1、2、3组相比，从第5天开始显著升高(P=0.000，0.011，0.000)，第2组与第1以及第3组相比第5天出现SCr的升高(P＜0.05)，但三组间在其余几天检测上没有显著差异。病理研究发现第4组移植肾出现了典型的严重急性排斥，第1组表现出明显减轻的急性排斥，第2及第3组与第1组相比，又有明显改善。包括IL-1β，TNF-α及TGF-β1在内的所有炎症介质的mRNA产物，第2组结果与其余各组相比明显要少(P＜0.01)，第1组与第3组在某些炎症介质的产物上也有轻度减少。
小结：MSCs能够对免疫反应产生下调作用，减少部分炎症介质的产生，在移植后初期保护移植器官的功能并延长动物的存活期。但这种作用弱于CsA疗法。此外，MSCs联合小剂量CsA疗法，能保护移植器官的功能，但与CsA单一疗法相比并不延长动物的存活期。
意义：本研究中，MSCs在体内免疫下调作用有限，且可能与CsA在体内存在相互影响，影响免疫抑制效果。因此，主张通过应用MSCs以减少CsA剂量的观点需要进一步研究。
Ⅰ. Isolation, Culturing and Identify of Rat MSCsfrom the Bone Marrow in vitro
Objective: To establish immortalized rat MSC lines in vitro, to providesource for cellular transplant.
Methods: One month old male Wistar rats was killed and degermed to getlong bones. Bone marrow cells were collected by flushing the longbones with DMEM medium (supplemented with 15% fetal bovineserum) and purified by density centrifuge,after Cultured the adherentcells were chosen for serial subcultivation. The third generation cellsin good growth condition was harvested and trypsinized to form cellsuspension,counted and noted the growth curve. The cell suspensionwas reserved in frozen condition supplemented with DMSO andfetal bovine serum medium. In osteocyte differentiationcondition: cells were cultured with dexamethasone,β-glycerolphosphate and ascorbate for 15 days,then stained with alkalinephosphatase(ALP). In adipocyte differentiation condition: cells werecultured with penicillin,streptomycin, transferrin,insulin;linoleic acid,dexamethasone and 3-isobutyl-1-methylxanthin for 5days,subsequently, cells were cultured for 5 days with MSC culturemedium supplemented with rapid insulin without FBS, thenconfirmed by Oil Red O staining.
Results: Adherent cells were observed in cell culture flasks after theIsolated MSC cultured for 24h,nonadherent cells were removed bychanging the culture medium.
The cell growth was obvious on the 3-4d,and after passage the cellsgrown rapidly to fill the floor of cell culture flasks and achievedconfluency. Purified MSC fibroblast-like cells could be harvestedafter 3 generations,and could passage for at least 10 generations.There was no obvious influence to cell growth when reserved infrozen condition. In the adipocyte differentiation, the accumulation oflipid-rich vacuoles within cells was observed on day 2 afterinduction, and the lipid was richer on the 4-5 day. In the osteocytedifferentiation, the MSCs formed aggregates or nodules andincreased their expression of alkaline phosphatase, and calciumaccumulation was evident after 3 days. The inductions wereconfirmed with staining.
Conclusion: 1)MSCs are a type of stem cells with strong proliferativeactivity and sustain self-renewal at least for 10 passage. They have afeature of pluripotent and could differentiate to a variety of cell types in some condition; 2)Inoculated by a density of 200/cm~2 andtrypsinized with 0.25% trypsin for 5 min are effective for the growthand passage of Wistar rat MSCs; 3)The methods of obtaining ratMSC with density gradient centrifugation and adherence cultivationwas convenient,and could provid immortalized rat MSC lines.
Significance: Our research demonstrates extensive proliferation andpluripotent of MSC, and provids a convenient harvest method forcellular transplant therapy.
ⅡEstablishment of a Modified Model of Rat Renal Transplantation
Objective: To establish a modified model of rat renal transplantation forhigh achievement ratio.
Methods: Wistar and Lewis mice were used as the donors and therecipients. Left donor's kidney was perfused with ice-cold solution.End-to-side anatomosis of recipient's abdominal aorta to the donor'swas performed between two points. End-to-end anastomosis of bothrenal veins was performed interrupted. Urinary tract reconstructionwas accomplished by suturing the donor ureter with a bladder patchto the recipient bladder. A silk thread was remained in the operation and this silk thread was tigated later in the second day after theoperation instead of nephrectomy to reduce the strike of the extraoperation for increasing the operative achievement ratio.
Result: In advance test of 30 operations on 60 rats, 21 rats were survived,the operative time of the donor's was 50±14min, and the recipient'swas 75±22min., including the warm ischemia time (about 1min), thecold ischemia time (60±11min), the whole operative time was140±30 min. Complications including anastomosis hemorrhage atarkery, rejection, renal failure, infection, thrombosis and uretalobstruction were observed. Achievement ratio is about 70%. Informal test of 36 operations on 72 rats, 34 rats were survived, theoperative time of the donor's was 35±12min, and the recipient's was65±10min. including the warm ischemia time (about 1 min), the coldischemia time (40±8min). Achievement ratio is about 95%. Onereason of the abortive cases was anastomosis of both renal veinshemorrhage, and another was thrombosis of renal artery.
Conclusion: The modifiability of this model of rat renal transplantation isthe infusion of left kidney with gravity at constant pressure andprepared at prime position, and the grafted kidney was put at the leftside. Direct anastomosis is used in artery-to-artery and vein-to-vein.Urinary tract reconstruction was accomplished by suturing the donorureter with a bladder patch. Delay the nephrectomy. The whole operation is processed under the microscope. Although it is difficultin operation, the effect is satisfying.
Significances: The modifiability in operation is successful to establish amodel of rat renal transplantation, and reduce the effect on the modelwith the operation.
ⅢMSCs Modulate Immune Responses Combined withCyclosporine A in Transplantation
Objective: To observe the effect of rat MSCs in rat renaltransplantation, study the immunological regulation and reparationof MSCs,and investigate the consideration that reduce dose ofCyclos-porine A (CsA) and even induce immune tolerance byapplication of MSCs.
Methods: Rats bearing renal allografts were divided randomly into fourgroups, which was given different fore treatmen and postoperationtherapy respectively.(1)MSC monotherapy group (group 1): therecipients were engrafted respectively one week beforetransplantation, before reperfusion,one week after operation and twoweeks after operation, with MSC(1×10~7, purified from Wistar rat bone marrow cells) by intravenous injection,and without specialdrug treatment; (2)CsA therapy (group 2): CsA was injected to therecipients intraperitoneally at a dosage of 0.5 mg/kg/d from day 2after transplantation without fore treatment; (3)MSC and commondose CsA combination therapy (group 3): the recipients were treatwith MSC the same as group 1,and CsA was injected at a dosage of0.5 mg/kg/d after transplantation;(4)No therapy group (group 4): nofore treatment was given, and PBS was injected intraperitoneallyafter transplantation as control. We tested the values of S-Cr of eachanimal in four groups on day 3,5,10,15,25 and 40 aftertransplantation. Graft histology of 2 recipients from each group washarvested on day 4, the production of inflammatory mediators,including IL-1β, TNF-αand TGF-β1, was analyzed by RT-PCR.Observed survival days of other animals.
Results: Group treated with CsA monotherapy (group 2) orcombination therapy (group 3) prolonged animal survivalsignificantly compared with no therapy group (group 4) (P=0.002,0.003).Indeed, animal survival of MSC monotherapy group (group 1)was prolonged compared with group 4 (P=0.001), but shortened thangroup 2 (P=0.036) and group 3(P=0.045).The recipients without anytreatment(Group 4)exhibited significantly elevated values of S-Crfrom day5 compared with group 1,2,3 (P=0.000,0.011,0.000), and the recipients in group 2 showed elevated values of S-Cr on day 5compared with group1 or group3 (P＜0.05), but there is no differenceamong group 1, group2 or group 3 on other testing days. Grafthistology shown that allografts of animals in group4 exhibitedfindings typical of severe acute rejection. Allografts of recipientswith MSC monotherapy(group1) showed findings of acute rejectionwith considerably milder severity. But allografts of recipients ingroup 2 and group 3 were markedly well preserved compared withgroup 1. The production of mRNA of all inflammatory mediators weanalyzed, including IL-1β, TNF-αand TGF-β1, was significantlydecreased in the allografts treated with a therapy of CsA (group 2) ascompared with all other groups (P＜0.01). MSC monotherapy (group1) or combination therapy (group 3) decreased production of someinflammatory mediators mildly;
Conclusion: MSCs can downregulate the immune response, reduce theproduction of some inflammatory mediators, preserve the graftfunction in the initial stage after transplantation and prolong theanimal survival. But this effect was weaker than CsA therapy.Moreover, MSCs therapy combined with low-dose CsA, couldprotect the graft function, but can not prolong the animal survivaltime compared with CsA monotherapy.
Significance: Our results suggest that down the downregulation of MSCs in immune response in vivo is limited, and indicate a potentialinteraction between MSC and CsA activities,which impact theimmune suppression. Therefore, previously considered reducing doseof CsA by application of MSCs need to be carefully investigated.
引文
1 Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells : implications in transplantation. Transplantation, 2003, 75 :389 - 397.
2 Deans RJ. Mesenchymal stem cells: cell and gene therapy applications. Eur Cytokine Networks,2000,l 1:323-324.
3 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
4 Bartholomew A ,Sturgeon C ,Siatskas M ,et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol ,2002 ,30 :42-48.
5 Peled A,Petit I,Kollet O,et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science,1999,283:845-848.
6 Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellar or nonspecific mitogenic stimuli.Blood,2002,99:3838-3843.
7 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigenspecific T cells to their cognate peptide. Blood, 2003, 101: 3722 -3729.
8 Han Q, Deng W, Zhao RC, et al. Allogeneic adult stem cells establish long-term residence in recipient tissues and facilitate skin transplantation. Exp Hematol, 2003 ; 31 : 158
9 Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect J Bone Joint Surg Am,2003,85-A(10):1927-1935.
10 Wu GD, Nolta JA, Jin YS,et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75(5):679-685.
1 Herzog EL ,Chai L ,Krause DS. Plasticity of marrow-derived stem cells[J] .Blood ,2003 ,102(10) :3483 - 3493.
2 Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model [J]. J Cell Sci ,2000 ,113 (Pt7):1161-1166.
3 Encina NR ,Billotte WG, Hofmann MC. Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma[J]. Lab Invest, 1999 ,79(4) :449-457.
4 Mets T ,Verdonk G. In vitro aging of human bone marrow derived stromal cells[J]. Mech Ageing Dev, 1981, 16(1) :81 - 89.
5 Colter DC ,Class R ,DiGirolamo CM ,et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow [J]. Proc Natl Acad Sci USA, 2000 ,97 (7) :3213 -3218.
6 Waller EK,Huang S , Terstappen L. Changes in the growth properties of CD34 + ,CD382bone marrow progenitors during human fetal development [J]. Blood, 1995 , 86 : 710 - 718.
7 Simmons PJ , Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody ,STRO21[J] .Blood ,1991 ,78 :55 - 62.
8 Healy L ,May G,Gale K,et al. The stem cell antigen CD34 functions as a regulator of hematopoietic cell adhesion [J].Proc Natl Acad Sci USA ,1995 ,92(26) :12240 - 12244.
9 Simmons PJ, Torok-Storb B. CD34 expression by stromal precursors in normal human adult bone marrow[J]. Blood, 1991,78 : 2848-2853.
10 Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics ,self-renewal ,and the osteogenic potential of purified human mesenchymanl stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997; 64: 278-294.
11 Pittenger MF, Mackay AM, Beck S, et al. Multilineage potential of adult human mesenchymal stem cells.Science 1999,284:143-147
12 Uta K, Song R, Djuric Zivka, et al. Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis. J Am Soc Nephrol 2006; 17: 2202-2212
1 Lee S , Jolla LA. A improved of renal transplantation in the rat. 1967, 61: 771-773
2 Silber SJ ,Crudop J . Kidney transplantation in inbred rat. Am J Surg, 1973, 125: 551-553
3 Fisher B ,Lee S. Microvasculary surgeral techniques in research ,with special reference to renal tranplantation in the rat. Surg, 1965, 58: 904-905
4 Fabre J ,Lin SH ,Morris P. Renal transplantation in the rat. Detail of the techniques. Aust N Z J Surg ,1971 ,41: 69-75
5 Kamada N. A description of cuff techniques for renal transplantation in therat. Use in studies of tolerance induction during combine liver grafting. Transplantation ,1985 ,39: 93-95
6 Ernst W. The rat experimental model for organ transplantation : Technique of rat kidney transplantation. Contr Nephrol, 1980, 19: 167-169
7 Buamis TP. Microsurgical renal transplantation techniques in small animals. Invest Urol, 1977, 15: 143-145
1 H Azuma, S Takahara, K Matsumoto, et al. Hepatocyte growth factor prevents development of chronic allograft nephropathy in rats. J Am SocNephrol 12(2001) 1280.
2 A Bartholomew, C Sturgeon, M Siatskas, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30(2002) 42-48.
3 I Seiichiro, CP Felix, EK Gudrun, et al.immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model. Transplantation, 81 (2006) 1589-1595.
4 GD Wu, JA Nolta, YS Jin,et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75(5):679-685.
5 AM De Mattos, AJ Olyaei, WM Bennett. Nephrotoxicity of immunosuppressive drugs: Long-term consequences and challenges for the future. Am J Kidney Dis 35(2002) 333.
6 W Deng, Q Han, L Liao, et al. Allogeneic bone marrow-derived flk-1+Sca-1- mesenchymal stem cells leads to stable mixed chimerism and donor-specific tolerance. Exp Hematol 32(2004) 861.
7 K Le Blanc, I Rasmusson, B Sundberg, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363(2004) 1439.
8 WT Tse, JD Pendleton, WM Beyer, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75(2003) 389.
9 S Aggarwal, MF Pittenger. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(2005) 1815.
10 S Beyth, Z Borovsky, D Mevorach, et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 105(2005) 2214.
11 K Le Blanc, I Rasmusson, C Gotherstrom, et al. Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes. Scand J Immunol 60:(2004) 307.
12 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
1 Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T2cell proliferation by human marrow stromal cells : implications in transplantation. Transplantation, 2003, 75 :389 - 397.
2 Deans RJ. Mesenchymal stem cells: cell and gene therapy applications. Eur Cytokine Networks,2000,l 1:323-324.
3 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
4 Bartholomew A ,Sturgeon C ,Siatskas M ,et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol ,2002 ,30 :42-48.
5 Peled A,Petit I,Kollet O,et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science, 1999,283:845-848.
6 Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellar or nonspecific mitogenic stimuli.Blood,2002,99:3838-3843.
7 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigenspecific T cells to their cognate peptide. Blood, 2003,101: 3722 -3729.
8 McIntosh K, Bartholomew A. Stromal cell modulation of the immune system. Graft, 2000,3:324-328.
9 Sudeepta A, Pitterger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood,2005,105:1815-1822.
10 Rasmusson I,Ringden O,Sundberg B,et al.Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms.Exp Cell Res,2005,305:33-41.
11 Meisel R,Zibert A,Laryea M,et al.Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase mediated tryptophan degradstion.Blood,2004,103:4619-4621.
12 Hariharan S, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 2000;342:605- 12.
13 Tanphaichitr NT, Brennan DC. Infectious complications in renal transplant recipients. Adv Ren Replace Ther 2000;7:131 -46.
14 Han Q , Deng W, Zhao RC , et al. Allogeneic adult stem cells establish long-term residence in recipient tissues and facilitate skin transplantation. Exp Hematol, 2003 ; 31 : 158
15 Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect. J Bone Joint Surg Am,2003,85-A(10):1927-1935.
16 Wu GD, Nolta JA, Jin YS,et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75(5):679-685.
17 Lazarus H, Curtion P, Devine S,et al. Role of mesenchymal stem cells in allogeneic transplantation. Early phase I clinical results.Blood,2000,96:392.
18 Le Blanc K, Tammik L , Sundberg B , et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol, 2003; 57: 11-20.
19 Frassoni FLM, Bacigalupo A, Frasson FLM, et al. Expanded mesenchymal stem cells(MSC), coinfused with HLA identical hemopoietic stem cell transplants, reduce acute and chronic graft versus host disease: a matched pair analysis. Bone Marrow Transplant,2002,29:75.
2 Deans RJ. Mesenchymal stem cells: cell and gene therapy applications. Eur Cytokine Networks,2000,l 1:323-324.
3 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
4 Bartholomew A ,Sturgeon C ,Siatskas M ,et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol ,2002 ,30 :42-48.
5 Peled A,Petit I,Kollet O,et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science,1999,283:845-848.
6 Di Nicola M,Carlo-Stella C,Magni M,et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellar or nonspecific mitogenic stimuli.Blood,2002,99:3838-3843.
7 Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigenspecific T cells to their cognate peptide. Blood, 2003, 101: 3722 -3729.
8 Han Q, Deng W, Zhao RC, et al. Allogeneic adult stem cells establish long-term residence in recipient tissues and facilitate skin transplantation. Exp Hematol, 2003 ; 31 : 158
9 Arinzeh TL, Peter SJ, Archambault MP, et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect J Bone Joint Surg Am,2003,85-A(10):1927-1935.
10 Wu GD, Nolta JA, Jin YS,et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75(5):679-685.
1 Herzog EL ,Chai L ,Krause DS. Plasticity of marrow-derived stem cells[J] .Blood ,2003 ,102(10) :3483 - 3493.
2 Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model [J]. J Cell Sci ,2000 ,113 (Pt7):1161-1166.
3 Encina NR ,Billotte WG, Hofmann MC. Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma[J]. Lab Invest, 1999 ,79(4) :449-457.
4 Mets T ,Verdonk G. In vitro aging of human bone marrow derived stromal cells[J]. Mech Ageing Dev, 1981, 16(1) :81 - 89.
5 Colter DC ,Class R ,DiGirolamo CM ,et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow [J]. Proc Natl Acad Sci USA, 2000 ,97 (7) :3213 -3218.
6 Waller EK,Huang S , Terstappen L. Changes in the growth properties of CD34 + ,CD382bone marrow progenitors during human fetal development [J]. Blood, 1995 , 86 : 710 - 718.
7 Simmons PJ , Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody ,STRO21[J] .Blood ,1991 ,78 :55 - 62.
8 Healy L ,May G,Gale K,et al. The stem cell antigen CD34 functions as a regulator of hematopoietic cell adhesion [J].Proc Natl Acad Sci USA ,1995 ,92(26) :12240 - 12244.
9 Simmons PJ, Torok-Storb B. CD34 expression by stromal precursors in normal human adult bone marrow[J]. Blood, 1991,78 : 2848-2853.
10 Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics ,self-renewal ,and the osteogenic potential of purified human mesenchymanl stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997; 64: 278-294.
11 Pittenger MF, Mackay AM, Beck S, et al. Multilineage potential of adult human mesenchymal stem cells.Science 1999,284:143-147
12 Uta K, Song R, Djuric Zivka, et al. Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis. J Am Soc Nephrol 2006; 17: 2202-2212
1 Lee S , Jolla LA. A improved of renal transplantation in the rat. 1967, 61: 771-773
2 Silber SJ ,Crudop J . Kidney transplantation in inbred rat. Am J Surg, 1973, 125: 551-553
3 Fisher B ,Lee S. Microvasculary surgeral techniques in research ,with special reference to renal tranplantation in the rat. Surg, 1965, 58: 904-905
4 Fabre J ,Lin SH ,Morris P. Renal transplantation in the rat. Detail of the techniques. Aust N Z J Surg ,1971 ,41: 69-75
5 Kamada N. A description of cuff techniques for renal transplantation in therat. Use in studies of tolerance induction during combine liver grafting. Transplantation ,1985 ,39: 93-95
6 Ernst W. The rat experimental model for organ transplantation : Technique of rat kidney transplantation. Contr Nephrol, 1980, 19: 167-169
7 Buamis TP. Microsurgical renal transplantation techniques in small animals. Invest Urol, 1977, 15: 143-145
1 H Azuma, S Takahara, K Matsumoto, et al. Hepatocyte growth factor prevents development of chronic allograft nephropathy in rats. J Am SocNephrol 12(2001) 1280.
2 A Bartholomew, C Sturgeon, M Siatskas, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30(2002) 42-48.
3 I Seiichiro, CP Felix, EK Gudrun, et al.immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model. Transplantation, 81 (2006) 1589-1595.
4 GD Wu, JA Nolta, YS Jin,et al. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 2003,75(5):679-685.
5 AM De Mattos, AJ Olyaei, WM Bennett. Nephrotoxicity of immunosuppressive drugs: Long-term consequences and challenges for the future. Am J Kidney Dis 35(2002) 333.
6 W Deng, Q Han, L Liao, et al. Allogeneic bone marrow-derived flk-1+Sca-1- mesenchymal stem cells leads to stable mixed chimerism and donor-specific tolerance. Exp Hematol 32(2004) 861.
7 K Le Blanc, I Rasmusson, B Sundberg, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363(2004) 1439.
8 WT Tse, JD Pendleton, WM Beyer, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75(2003) 389.
9 S Aggarwal, MF Pittenger. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(2005) 1815.
10 S Beyth, Z Borovsky, D Mevorach, et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 105(2005) 2214.
11 K Le Blanc, I Rasmusson, C Gotherstrom, et al. Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes. Scand J Immunol 60:(2004) 307.
12 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
1 Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T2cell proliferation by human marrow stromal cells : implications in transplantation. Transplantation, 2003, 75 :389 - 397.
2 Deans RJ. Mesenchymal stem cells: cell and gene therapy applications. Eur Cytokine Networks,2000,l 1:323-324.
3 Djouad F , Plence P ,Bony C ,et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood ,2003 ,102:3837-3844.
4 Bartholomew A ,Sturgeon C ,Siatskas M ,et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol ,2002 ,30 :42-48.
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