供体凋亡细胞输注诱导大鼠胰岛移植免疫耐受的研究

发布时间：2018-05-25 01:37

本文选题：凋亡细胞 + 免疫耐受　；参考：《第一军医大学》2007年博士论文

【摘要】： 研究背景胰岛移植重建胰岛素分泌系统，是一种有望彻底根治糖尿病的治疗方法。2000年，Edmonton方案的成功，标志着胰岛移植取得了突破性的进展，从而带动了胰岛移植临床试验性治疗在世界范围内广泛展开。但是免疫排斥以及免疫抑制剂所带来的副作用和潜在危险性仍是难以克服的障碍。目前普遍认为，解决异体移植排斥反应的关键在于诱导受体对供体的细胞、组织或器官产生免疫耐受。由于移植免疫耐受的机制十分复杂，尽管诱导免疫耐受的方法众多，但均未能达到完全可靠持久的特异性耐受。细胞凋亡是生物体内普遍存在的一种生理和病理现象，是正常器官和组织发育、清除有害的、过量的和无功能细胞、维持自身稳态的必要环节。现有研究表明：凋亡是机体免疫状态保持平衡和稳定的重要作用机制，凋亡细胞对免疫系统存在着主动的调节作用。凋亡细胞能分泌脂类趋化因子，改变自身胞膜结构表达“eat-me”信号，诱导吞噬细胞对其进行清除；同时凋亡细胞被抗原提呈细胞吞噬后，通过吞噬细胞分泌抑制性免疫因子如TGF-β、PGE_2、IL-10等，造成了特殊的抗原识别微环境，可以促使相关淋巴细胞针对凋亡抗原产生免疫耐受，而不会引起炎性免疫应答。据此，孙尔维等提出利用供体凋亡细胞输注受体诱导供体特异性免疫耐受的理论设想。我们课题组以前的工作证实，凋亡细胞在体外对T淋巴细胞活化增殖有直接抑制作用；凋亡细胞预输注可以显著延长大鼠心脏移植模型、肝移植模型存活时间。因此，我们拟应用糖尿病大鼠模型进行同种异体胰岛移植，以供体凋亡脾淋巴细胞静脉输注的方法，观察胰岛移植物的存活时间，了解凋亡细胞调节免疫反应过程参与的淋巴细胞类型、细胞因子及分子基础，探求延长胰岛移植物存活的方法。目的初步探讨凋亡细胞静脉输注诱导胰岛移植免疫耐受的方法及作用机制，为寻找诱导胰岛移植免疫耐受的方法开辟新的途径，提供新的依据。方法 1、直线加速器照射对体外培养大鼠脾细胞凋亡的影响：采用研磨法获得Wistar大鼠脾淋巴细胞悬液。将脾细胞悬液加入细胞培养瓶，分四组，A组为对照组，B、C、D组应用Varian医用直线加速器照射，各组吸收剂量分别为：1.5Gy、2.0Gy、3.0Gy。各组细胞处理后置于37℃，5％CO_2恒温培养箱培养。分别在培养后4h、8h、12h应用流式细胞仪检测细胞凋亡率； 2、大鼠胰岛细胞的分离纯化：采用胶原酶P胰管灌注消化，短期低温培养后Ficoll-400不连续梯度纯化Wistar大鼠胰岛； 3、建立糖尿病动物模型：以STZ(56mg／kg·体重)经腹腔一次性注射，制备SD大鼠实验性糖尿病模型(连续2次血糖＞16.7mmol／L)： 4、供体凋亡细胞输注对糖尿病胰岛移植物的影响：以雄性Wistar大鼠为供体，雄性SD大鼠为受体。将糖尿病SD大鼠随机分为Hank's液注射(A组)、正常供体脾细胞预输注(B组)、供体凋亡脾细胞预输注(C组)及供体坏死脾细胞预输注(D组)四组。各组在预处理7天后行胰岛移植，将1000IEQ的胰岛细胞移植入各组糖尿病大鼠的肾包囊内，比较各组移植物生存时间上的差异。同时各实验组分别在预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后应用放射免疫法测定大鼠血清胰岛素水平。供体凋亡脾细胞预输注糖尿病SD大鼠则在不同的移植时期(包含血糖正常期、排斥期两个时期)手术将移植物载体肾取出；其余各实验组在糖尿病SD大鼠移植物排斥后2天，取出其移植物载体肾，做胰岛素的免疫组化检测。 5、供体凋亡细胞输注对胰岛移植糖尿病大鼠混合淋巴细胞反应的影响：将糖尿病SD大鼠按上述方法分组，各组分别于预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后取大鼠脾脏，分离淋巴细胞，与丝裂霉素C处理的供体Wistar大鼠和无关第三品系F344／N大鼠脾淋巴细胞进行单向混合淋巴细胞培养，MTT法观察受体淋巴细胞增殖反应； 6、供体凋亡细胞输注对胰岛移植糖尿病大鼠T淋巴细胞亚群及CD4~+CD25~+T细胞的影响：将糖尿病SD大鼠按上述方法分组，各组分别于预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后取外周血及大鼠脾脏制备的淋巴细胞悬液，应用流式细胞仪检测CD8~+、CD4~+、CD4~+CD25~+／CD4~+T淋巴细胞比率。 7、供体凋亡细胞输注对胰岛移植糖尿病大鼠细胞因子微环境的影响：将糖尿病SD大鼠按上述方法分组，各组分别于预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后取外周血清及大鼠脾组织标本，应用Luminex多功能流式点阵仪液相芯片法检测IL-2、IFN-γ、IL-4、IL-10含量；同时应用酶联免疫吸附法检测外周血清TGF-β1含量。结果 1、直线加速器高能X线照射Wistar大鼠脾细胞，吸收剂量1.5Gy组于4h后开始有明显细胞凋亡发生，而后随时间推移凋亡率增加。2.0Gy组于4h后即有显著凋亡发生，至8h达到凋亡高峰(61.17±3.70)％(P＜0.050)，但于12h凋亡率略有下降。3.0Gy组凋亡率无显著升高，而坏死率明显增加。 2、每条Wistar大鼠胰腺经分离、纯化后平均可获取的胰岛细胞团数量为(1063.91±84.74)IEQ，DTZ染色显示纯度为(70.51±6.20)％。高糖刺激时胰岛B细胞胰岛素的释放量为低糖刺激时的2.72倍； 3、以STZ(120mg／kg·体重)一次性经腹腔注射的方法，可成功诱发SD大鼠的试验性糖尿病，糖尿病模型建模成功率为94％； 4、供体凋亡脾细胞预输注组糖尿病SD大鼠胰岛移植物生存时间与其它三组比较具有显著性差异(P＜0.050)，中位生存时间(MST)为(31.00±6.41)天，最长达42天。正常脾细胞输注组移植物生存时间也有所延长，MST为(12.00±2.97)天(P＜0.050)。预输注Hank's液及坏死细胞组则无此效应，MST分别为(6.00±1.67)、(6.00±1.10)天； 5、供体预输注凋亡脾细胞组移植后7天、14天糖尿病大鼠血清胰岛素水平较移植前显著增高，分别达(19.26±4.50)mIU／ml、(20.70±6.33)mIU／ml(P＜0.050)，排斥后胰岛素水平回落。正常细胞组移植后7天胰岛素水平亦明显增高(P＜0.050)，14天时则明显下降，而预输注Hank's液及坏死细胞组则无明显变化。 6、供体凋亡细胞预输注组胰岛移植物在受体血糖正常时取出做组织切片，经Insulin免疫组织化学染色证实有表达胰岛素阳性的胰岛细胞团存在，呈棕褐色。而在被排斥后取出的移植物进行免疫组化检测，则未发现胰岛素阳性的胰岛细胞团存在。输注Hank's液组、输注供体正常细胞或坏死细胞组的移植物在被排后取出行Insulin免疫组化染色检测，亦无胰岛素阳性细胞存在。 7、供体凋亡细胞预输注后7天SD大鼠的脾细胞对丝裂霉素C处理的Wistar大鼠脾细胞的增殖反应较接受Hank's液输注组、供体正常脾细胞组及供体坏死脾细胞组明显降低(P＜0.050)，直至移植后7天、14天抑制效应仍为显著，排斥后则明显升高(P＜0.050)；且与无关第三品系F344／N大鼠脾细胞的增殖反应比较，两者差异明显，移植前、移植后7、14天各时间点相互比较均有显著性差异(P＜0.050) 8、各处理组糖尿病大鼠接受胰岛移植后，无论外周血还是脾脏组织CD4~+T细胞和CDS~+T细胞比例均显著升高(P＜0.050)，各组间无明显差异(P＞0.050)。 9、供体凋亡细胞预输注后7天，糖尿病大鼠外周血和脾脏CD4~+CD25~+T细胞比例均明显高于预输注Hank's液组、供体正常细胞组及供体坏死细胞组，分别占CD4~+T细胞的(11.49±1.29)％、(12.44±1.63)％(P均＜0.050)；移植后7天、14天仍保持较高比例，排斥后则有所下降。 10、胰岛移植前各实验组糖尿病大鼠外周血和脾脏组织IL-2水平均无显著性差异(P＞0.050)。移植后各组均明显升高，但供体凋亡细胞输注组在移植后7天、14天的IL-2水平明显低于Hank's液输注组、供体正常细胞组及供体坏死细胞组，有显著性差异(P＜0.050)；排斥后明显升高。 11、胰岛移植前各实验组糖尿病大鼠外周血和脾脏组织IFN-γ水平亦无显著性差异(P＞0.050)。移植后各组均明显升高，但移植后7天、14天，供体凋亡细胞输注组与Hank's液输注组、供体正常细胞组及供体坏死细胞组相比水平较低，有显著性差异(P＜0.050)。排斥后则升高显著。 12、胰岛移植前各实验组糖尿病大鼠外周血和脾脏组织IL-4水平无显著性差异(P＞0.050)；移植后7天、14天直至排斥后预输注Hank's液组、供体正常细胞组、供体凋亡细胞组及供体坏死细胞组IL-4水平均无明显改变。 13、输注供体凋亡细胞后7天，糖尿病大鼠外周血和脾脏组织IL-10水平显著升高；直至移植后7天、14天仍明显高于其它各组，有显著性差异(P＜0.050)；移植物排斥后则降低。同时，接受Hank's液预输注组、供体正常细胞组及供体坏死细胞组无明显变化。 14、输注供体凋亡细胞后7天，，糖尿病大鼠血清TGF-β1水平明显升高，并在胰岛移植后7，14天仍可维持较高水平(P＜0.050)；移植物排斥后TGF-β1水平下降。而其余Hank's液预输注组、供体正常细胞组及供体坏死细胞组无明显变化。结论 1、直线加速器高能X线照射能在体外简便安全有效诱导大鼠脾细胞凋亡。 2、采用胶原酶P胰管灌注消化，短期低温培养后Ficoll-400不连续梯度纯化能分离、纯化出较大数量且功能良好的大鼠胰岛细胞。 3、预输注供体凋亡细胞能显著延长同种异体大鼠胰岛移植物的存活时间。应用供体凋亡细胞诱导胰岛移植免疫耐受是一条可行的途径。 4、以单向混合淋巴细胞培养实验证实，预输注供体凋亡细胞的受体鼠淋巴细胞对供体淋巴细胞刺激的增殖反应具有明显抑制作用。 5、供体凋亡细胞预输注能显著升高胰岛移植糖尿病大鼠外周血和脾脏CD4~+CD25~+T细胞比例。 6、供体凋亡细胞预输注可使胰岛移植糖尿病大鼠血清和脾脏IL-2、IFN-γ呈现较低水平，而显著升高IL-10、TGF-β水平，IL-4则无明显改变，提示供体凋亡细胞输注诱导胰岛移植免疫耐受可能包含调节Th1／Th2、Th3平衡，纠正免疫偏离的作用；并且更为重要的机制可能是，Tr、Tr1等尤其是CD4~+CD25~+T调节性淋巴细胞的参与。
[Abstract]:Research background
Islet transplantation to reconstruct the insulin secretion system is a promising cure for diabetes in.2000 years. The success of the Edmonton scheme marks a breakthrough in islet transplantation, leading to extensive clinical trials of islet transplantation in the world. However, immune rejection and immunosuppressive agents have been carried out. The side effects and potential risks are still difficult to overcome. It is widely believed that the key to the solution of allograft rejection is to induce the receptor to produce immune tolerance to the cells, tissues or organs of the donor. The mechanism of the transplantation immune tolerance is very complex, although there are many methods to induce immune tolerance, but they have not been achieved. Cell apoptosis is a common physiological and pathological phenomenon in living organisms. It is a necessary link in normal organs and tissue development, eliminating harmful, excessive and non functional cells and maintaining homeostasis. The existing study shows that apoptosis is an important role in maintaining balance and stability of the immune state of the body. The apoptotic cells can regulate the immune system actively. The apoptotic cells can secrete the chemotactic factor of lipid, change the cell membrane structure to express "eat-me" signal and induce the phagocyte to scavenging it. At the same time, the apoptotic cells are phagocyted by the antigen presenting cells and through the phagocytic cells secreting the inhibitory immune factors such as TGF- beta, PG E_2, IL-10 and so on, cause a special antigen recognition microenvironment, which can induce the related lymphocyte to produce immune tolerance against apoptotic antigen, without causing inflammatory response. Accordingly, sun Erwei put forward the theory of using donor apoptotic cell infusion receptor to induce donor specific immune tolerance. In fact, the apoptotic cells have a direct inhibitory effect on the proliferation of T lymphocytes in vitro, and the preinfusion of apoptotic cells can significantly prolong the rat model of heart transplantation and the survival time of the liver transplantation model. Therefore, we should use the diabetic rat model for allogenic islet transplantation with the method of intravenous infusion of donor apoptotic splenic lymphocytes. To investigate the survival time of the islet graft, understand the lymphocyte types, cytokines and molecular basis of the apoptotic cells regulating the immune response process, and explore the methods of prolonging the survival of the islet grafts.
objective
In order to find a new way to induce the immune tolerance of islet transplantation, this paper provides a new basis for finding the immune tolerance of islet transplantation induced by apoptotic cell vein infusion.
Method
1, the effect of linear accelerator irradiation on the apoptosis of splenocytes of rats in vitro: the splenic lymphocyte suspension of Wistar rats was obtained by grinding method. Splenic cell suspension was added into cell culture bottle, four groups were added, group A was used as control group, B, C, and D group were irradiated with Varian medical linear accelerator. The absorption doses of each group were 1.5Gy, 2.0Gy and 3.0Gy. in each group, respectively. The cells were incubate at 37 C and incubate in 5%CO_2 incubator. The apoptosis rate was detected by flow cytometry after 4h, 8h and 12h respectively.
2, isolation and purification of rat islet cells: using collagenase P pancreatic duct perfusion and digestion, after short-term hypothermia culture, Ficoll-400 was purified from Wistar rat islets in discontinuous gradient.
3, establish an animal model of diabetes: an experimental diabetic model of SD rats (2 consecutive blood glucose > 16.7mmol / L) was prepared by intraperitoneal injection of STZ (56mg / kg / weight):
4, the effect of donor apoptotic cell infusion on diabetic islet graft: male Wistar rats as donors and male SD rats as receptors. The diabetic SD rats were randomly divided into Hank's liquid injection (A group), normal donor splenocytes preinfusion (group B), donor apoptotic splenocytes preinfusion (C group) and donor necrotic splenocytes pre infusion (D group) in each group (group D). Each group was divided into four groups. The islet transplantation was performed 7 days after pretreatment, and the islet cells of 1000IEQ were transplanted into the renal cysts of each group of diabetic rats. The differences in the survival time of each group were compared. At the same time, the experimental groups were seventh days after pretreatment (before the islet transplantation), 7 days after the islet transplantation, 14 days after the islet transplantation, and the radioimmunoassay was used to determine the serum of rats after rejection. Insulin level. SD rats with donor apoptotic splenocytes pretransfused to diabetic rats during the different period of transplantation (including the normal period of blood glucose, two periods of rejection) to remove the graft carrier kidney; the rest of the experimental groups were taken out of the transplant carrier kidney for 2 days after the graft rejection of diabetic rats, and made the immunohistochemical detection of insulin.
5, the effect of donor apoptotic cells infusion on the mixed lymphocyte reaction in diabetic rats: diabetic SD rats were grouped according to the methods mentioned above, seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, spleenes were isolated from rats after rejection, and the donor Wis treated with mitomycin C Unidirectional mixed lymphocyte culture was carried out on tar rats and spleen lymphocytes from F344 / N rats without the third strain, and the proliferation of recipient lymphocytes was observed by MTT.
6, the effect of donor apoptotic cells infusion on T lymphocyte subsets and CD4~+CD25~+T cells in diabetic rats: diabetic SD rats were grouped according to the methods mentioned above, seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, and lymphocytic suspension prepared from peripheral blood and rat spleen. The ratio of CD8~+, CD4~+, CD4~+CD25~+ / CD4~+T lymphocyte was detected by flow cytometry.
7, the effect of donor apoptotic cells infusion on the microenvironment of cytokines in diabetic rats: groups of diabetic SD rats were grouped according to the above methods, each group was seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, after rejection, the peripheral blood and rat spleen tissue specimens were taken, and Luminex multifunction flow point was applied. The contents of IL-2, IFN-, IL-4 and IL-10 were detected by liquid phase microarray, and the content of TGF- beta 1 in peripheral serum was detected by enzyme-linked immunosorbent assay.
Result
1, the splenic cells of Wistar rats were irradiated by high energy X-ray in the linear accelerator. After the absorption of 1.5Gy group, there was obvious apoptosis in the group of 4h, and then the apoptosis rate increased in the.2.0Gy group after 4h, and reached the peak (61.17 + 3.70)% (P < 0.050) to the peak of apoptosis (P < 0.050), but the apoptosis rate of 12h decreased slightly in the.3.0Gy group. The rate of necrosis was significantly increased and the necrosis rate increased significantly.
2, the pancreas of each Wistar rat was separated, and the average number of islet cell masses was (1063.91 + 84.74) IEQ after purification, and the purity of DTZ staining showed (70.51 + 6.20)%. The release of insulin in islet B cells was 2.72 times as high as that of low glucose stimulation.
3, STZ (120mg / kg / body weight) by intraperitoneal injection can successfully induce experimental diabetes in SD rats, and the success rate of modeling is 94%.
4, the survival time of pancreatic islet graft in SD rats was significantly different from that of the other three groups (P < 0.050), the median survival time (MST) was (31 + 6.41) days and the longest was 42 days. The survival time of the allograft in the normal splenocytes infusion group was also prolonged, and MST was (12 + 2.97) days (P < 0.050). Pre infusion Hank There was no such effect in's solution and necrotic cell group, MST was (6 + 1.67), (6 + 1.10) days.
5, on the 7 day after donor pre infusion, the serum insulin level in the 14 day diabetic rats increased significantly (19.26 + 4.50) mIU / ml, (20.70 + 6.33) mIU / ml (P < 0.050) and the level of insulin after rejection. The insulin level in the normal cell group was also significantly higher (P < 0.050) on the 7 day after the transplantation (P < 0.050), and the level of insulin was obvious on the day of the transplantation. However, there was no significant change in pre infusion of Hank's and necrotic cells.
6, the pancreatic islet graft in the donor cell preinfusion group was taken out of the tissue section at the normal blood glucose level. The insulin positive islet cell group was found to be brown in the presence of Insulin immunohistochemical staining, and the insulin positive islet cells were not found in the graft after the rejection. In the Hank's solution group, the graft of the donor donor normal cell or the necrotic cell group was detected by Insulin immunohistochemical staining after being discharged, and no insulin positive cells existed.
7, the proliferation response of splenocytes in the spleen cells treated with mitomycin C on the spleen cells treated by mitomycin C was significantly lower than that of the Hank's liquid infusion group, the donor normal splenocytes group and the donor necrotic splenocytes group (P < 0.050) in the SD rats treated with mitomycin C (P < 0.050), and the inhibitory effect was still significant in the 14 day after the transplantation (P < < < < < < 0. 0.050) and compared with the proliferation reaction of spleen cells of F344 / N rats, the difference was obvious. Before transplantation, there were significant differences in each time point of 7,14 days after transplantation (P < 0.050).
8, the proportion of CD4~+T cells and CDS~+T cells in both peripheral and spleen tissues increased significantly (P < 0.050), and there was no significant difference between each group (P > 0.050).
9, the proportion of peripheral blood and spleen CD4~+CD25~+T cells in the diabetic rats was significantly higher than that of the pre infusion group 7 days after the apoptosis of the donor cells. The donor normal cell group and the donor necrotic cell group accounted for (11.49 + 1.29)% of the CD4~+T cells and (12.44 + 1.63)% (P < 0.050), respectively, and still kept a high proportion on the 14 day after the transplantation, and on the 14 day after the transplantation. Then it fell down.
10, there was no significant difference in the IL-2 level between the peripheral blood and the spleen in the experimental groups before the islet transplantation (P > 0.050). All groups were significantly higher after transplantation, but the IL-2 level of the donor apoptotic cell infusion group was significantly lower than that of the Hank's infusion group at 7 days after the transplantation, and the donor normal cell group and the donor necrotic cell group were significant. The difference was (P < 0.050), and the difference was significantly increased after rejection.
11, there was no significant difference in the level of IFN- gamma in the peripheral blood and spleen tissues of the experimental groups before the islet transplantation (P > 0.050). All groups were significantly higher after transplantation, but the donor group was compared with the Hank's liquid infusion group at 7 days and 14 days after transplantation, and there was a significant difference in the level of the donor normal cell group and the donor necrotic cell group. (P < 0.050). After rejection, the increase was significant.
12, there was no significant difference in the IL-4 level between the peripheral blood and the spleen tissues of the experimental groups before the islet transplantation (P > 0.050), and 7 days after the transplantation, 14 days after the transplantation and the pre transfusion group Hank's solution, the donor normal cell group, the donor apoptotic cell group and the donor necrotic cell group had no significant changes in the IL-4 water.
13, on the 7 day after infusion of donor apoptotic cells, the levels of IL-10 in peripheral blood and spleen tissues of diabetic rats were significantly higher than those of other groups until 7 days after transplantation. There was a significant difference (P < 0.050), while the graft rejection was reduced. At the same time, the group of donor normal cells and donor necrotic cells group were not clear. Change.
14, the level of serum TGF- beta 1 in diabetic rats was significantly increased at 7 days after infusion of donor cells, and still maintained a high level in 7,14 days after islet transplantation (P < 0.050), and the level of TGF- beta 1 decreased after graft rejection, but there was no significant change in the normal cell group and donor necrotic cell group in the rest of the Hank's solution group.
conclusion
1, high energy X-ray irradiation of linear accelerator can be used in vitro to induce apoptosis of rat spleen cells in a simple, safe and effective way.
2, the collagenase P pancreatic duct was perfused and digested. After short term low temperature culture, Ficoll-400 discontinuous gradient purification could be separated and the large number and well functioning rat islet cells were purified.
3, pre infusion of donor apoptotic cells can significantly prolong the survival time of allogeneic rat islet grafts.
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R657.5;R392.4

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