PD-L1免疫负调节作用与延长移植物存活及其机制

发布时间：2018-01-06 10:08

本文关键词：PD-L1免疫负调节作用与延长移植物存活及其机制　出处：《华中科技大学》2009年博士论文　论文类型：学位论文

【摘要】： 【目的】 I型糖尿病是一种主要由T细胞介导的，CD4~+、CD8~+T细胞和巨噬细胞浸润胰岛导致分泌胰岛素的β细胞受损，使胰岛素分泌减少，导致胰岛素绝对缺乏。目前已证实该疾病和自身免疫耐受缺失有关，，自身反应性T细胞在I型糖尿病的免疫发病机理中起主导作用。PD-L1已被证实为免疫负性调节受体PD-1的配体，文献证明PD-L1在免疫负性调节及外周耐受中发挥重要作用。本实验构建PD-L1表达载体，转染NIT细胞，通过体内外实验探讨PD-L1免疫调节作用，研究其在链脲佐菌素(STZ)诱导的糖尿病小鼠模型中延长胰岛移植存活时间及其作用机制。【方法】 1．稳定表达pPD-L1的NIT-1细胞株的建立用Lipofectamine~(TM)2000将pPD-L1-EGFP n1和pEGFPn1两个质粒分别转染NIT-1细胞系获得稳定转染细胞株，分别命名为NIT-PD-L1和NIT-EGFP。 2．致敏淋巴细胞的制备分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离Balb／c小鼠脾脏单个核细胞作为致敏淋巴细胞备用。 3．混合细胞培养将经过丝裂霉素C处理的NIT-1、NIT-PD-L1和NIT-EGFP三组细胞作为刺激细胞分别与新鲜分离的脾脏淋巴细胞和(或)上述致敏淋巴细胞进行混合培养7天。 4．细胞增殖实验用CFSE预染上述小鼠淋巴细胞，待细胞混合培养7天后，FCM检测淋巴细胞的增殖。 5．细胞凋亡检测收集上述细胞混合培养7天后的淋巴细胞，Annexin V-cy5和PI染色，FCM检测淋巴细胞的凋亡。 6．细胞毒实验分别用三组刺激细胞NIT-1、NIT-PD-L1和NIT-EGFP体内腹腔注射Balb／c小鼠，间隔1周再腹腔注射一次，细胞免疫后第14天分离小鼠脾淋巴细胞，将刺激细胞与上述获得的脾淋巴细胞以1：10的比例混合培养，3天后收集淋巴细胞作为效应细胞。将NIT-1细胞用CFSE染色，作为靶细胞，并以1：20的比例与效应细胞混合培养，4小时后用PI染色，FCM检测坏死细胞数。 7．细胞因子检测收集上述混合细胞培养上清，ELISA检测细胞因子的分泌水平；收集上述混合培养后的淋巴细胞，FCM检测淋巴细胞内细胞因子的表达。 8．PD-L1诱导同种胰岛移植耐受实验 (1)STZ诱导糖尿病模型用小剂量多次腹腔注射STZ的方法诱导糖尿病模型。 (2)细胞腹腔移植将NIT-1、NIT-EGFP或NIT-PD-L1细胞(1.5×10~7)注射到Balb／c糖尿病模型小鼠腹腔。 (3)血糖、体重、胰岛素释放实验以及生存时间观察移植后每隔一天监测一次血糖、体重及生存时间；在移植后第7天和24天，用放免法检测葡萄糖刺激的胰岛素释放量实验。 (4)细胞增殖、细胞凋亡、培养上清细胞因子及胞内细胞因子的测定方法同上 (5)小鼠血清细胞因子测定移植后第7天，收集小鼠血清，ELISA检测细胞因子的表达。 (6)腹腔淋巴细胞凋亡以及腹腔冲洗细胞免疫荧光染色移植后第7天，收集小鼠腹腔冲洗细胞，FCM检测腹腔淋巴细胞凋亡；免疫荧光染色检测移植细胞的排斥情况。【结果】 1．PD-L1对同种淋巴细胞的调节作用 (1)PD-L1对同种淋巴细胞的增殖与凋亡的影响：新鲜分离的小鼠脾脏淋巴细胞作为反应细胞，与未修饰的NIT-1或PD-L1修饰的NIT-1(NIT-PD-L1)作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率；未修饰的NIT-1致敏淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，FCM检测结果表明未修饰的NIT-1刺激的淋巴细胞增殖率显著高于NIT-PD-L1刺激的增殖率，而NIT-1诱导的淋巴细胞凋亡率低于NIT-PD-L1诱导的淋巴细胞凋亡率； NIT-PD-L1致敏的淋巴细胞作为反应细胞，与未修饰的NIT-1或NIT-PD-L1作为刺激细胞混合培养，NIT-PD-L1刺激的淋巴细胞增殖率显著低于未修饰的NIT刺激的淋巴细胞增殖，且可诱导反应细胞凋亡。结果表明PD-L1可抑制新鲜分离的初次反应性淋巴细胞和致敏淋巴细胞的增殖，并促进其凋亡。 (2)PD-L1对细胞因子的表达与分泌的影响：FCM检测淋巴细胞内细胞因子以及ELISA检测分泌性细胞因子结果表明，与NIT-1或NIT-EGFP诱导的淋巴细胞相比，NIT-PD-L1细胞诱导的淋巴细胞表达与分泌IL-4、IL-10水平显著增高，而IFN-γ水平明显降低。 (3)PD-L1对CTL活性的影响：通过NIT-1、NIT-PD-L1或NIT-EGFP三组细胞分别体内外联合刺激获得的脾淋巴细胞，分别与NIT-1细胞体外共培养后，CFSE与PI双染色，FCM检测结果表明，NIT-1或NIT-EGFP细胞诱导的脾淋巴细胞介导NIT-1靶细胞中度坏死，而NIT-PD-L1细胞诱导的脾淋巴细胞介导NIT-1靶细胞轻度坏死，结果表明PD-L1可体内外抑制CTL的激活与效应。 2．PD-L1延长STZ诱导的糖尿病小鼠胰岛移植物的存活将NIT-1、NIT-PD-L1和NIT-EGFP三组细胞分别移植入STZ诱导的Balb／c糖尿病小鼠腹腔，观察移植前后的血糖、体重、糖刺激的胰岛素释放及生存时间，以及脾脏细胞的增殖与凋亡、细胞因子的表达与分泌，实验结果表明： (1)PD-L1对糖尿病小鼠体重、血糖及存活时间的影响：移植NIT-1、NIT-PD-L1或NIT-EGFP细胞后第3天，各组糖尿病小鼠血糖均降至正常范围，其中移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠血糖，在第7天左右升高并超过正常血糖水平；而移植NIT-PD-L1细胞的糖尿病小鼠可维持正常血糖水平约21天，随后逐渐升高，于移植后第24天血糖升高且超过正常上限(＞11.1mmol／L)；在移植后第7天，移植NIT-1或NIT-EGFP细胞的对照组糖尿病小鼠体重逐渐下降，而移植NIT-PD-L1的糖尿病小鼠体重逐渐增加；移植NIT-PD-L1细胞糖尿病小鼠的生存时间比移植NIT-1或NIT-EGFP细胞的糖尿病小鼠明显延长。 (2)葡萄糖刺激的胰岛素释放：进一步检测葡萄糖刺激的胰岛素释放，由于移植NIT-1或NIT-EGFP细胞的糖尿病小鼠在移植后24天内均死亡，所以24天组仅包括移植NIT-PD-L1细胞的小鼠。检测结果表明，在移植后第7天，移植NIT-1或NIT-EGFP细胞的小鼠胰岛素释放量明显低于正常小鼠，而移植NIT-PD-L1细胞的小鼠胰岛素释放量与正常小鼠对照组相比无显著性差异，表明NIT-PD-L1细胞维持正常功能；但在移植后24天，移植NIT-PD-L1细胞小鼠的胰岛素释放量低于正常对照组小鼠，提示可能发生排斥反应。 (3)PD-L1延长移植物存活的免疫学机制：为了研究PD-L1延长移植物存活的机制，在细胞移植后第7天，分别检测了脾淋巴细胞和腹腔淋巴细胞的增殖与凋亡。FCM检测结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞增殖反应显著高于NIT-PD-L1细胞移植小鼠，而细胞凋亡率低于NIT-PD-L1细胞移植的小鼠；在移植后第7天，FCM检测脾淋巴细胞内细胞因子以及ELISA检测细胞培养上清和血清细胞因子结果表明，移植NIT-1或NIT-EGFP细胞的小鼠脾淋巴细胞内IFN-γ表达明显高于NIT-PD-L1细胞移植小鼠，而IL-4和IL-10显著低于移植NIT-PD-L1细胞的小鼠；脾淋巴细胞培养上清与小鼠血清中细胞因子检测结果与胞内细胞因子的表达类似。上述结果表明PD-L1抑制淋巴细胞增殖同时促进其凋亡；胞内细胞因子和ELISA检测结果显示，PD-L1抑制IFN-γ的表达，而增加IL-4和IL-10的产生，从而促进Th1细胞向Th2细胞漂移。 (4)腹腔冲洗细胞活性：腹腔冲洗细胞免疫荧光染色结果显示，移植NIT-PD-L1细胞的小鼠腹腔中胰岛素阳性细胞数量明显高于移植NIT-1或NIT-EGFP细胞的小鼠，而移植NIT-PD-L1细胞小鼠腹腔中淋巴细胞凋亡数增高，且浸润程度显著低于移植NIT-1或NIT-EGFP细胞的小鼠组。【结论】PD-L1基因修饰的NIT可明显抑制同种淋巴细胞的增殖反应，同时诱导其凋亡；PD-L1可抑制CTL的活性，且可诱导Th2细胞因子的分泌，抑制Th1细胞因子的分泌；从而移植延长糖尿病小鼠移植物存活时间，并维持较长的正常血糖水平，研究结果为进一步通过诱导免疫耐受重建IDDM胰岛细胞功能研究奠定了基础。
[Abstract]:[Objective]
Type I diabetes is a mainly mediated by T cells, CD4~+, leading to insulin secreting beta cell damage in CD8~+T cells and macrophage infiltration in pancreatic islets, the decrease of insulin secretion, leading to absolute insulin deficiency. It has been confirmed that the disease and the deficiency of the immune tolerance, autoreactive T cells play a dominant role of.PD-L1 has been confirmed as a negative regulator of immune ligand receptor PD-1 in the pathogenesis of type I diabetes in the literature that PD-L1 play an important role in regulating the immune negative and peripheral tolerance. The constructed PD-L1 expression vector and transfected into NIT cells in vitro and in vivo study of PD-L1 immunomodulatory effects on the STZ prime (STZ) to extend the survival time of islet transplantation and its mechanism of diabetic mice induced.
[method]
The establishment of 1. stable NIT-1 cell line expressing pPD-L1.
Lipofectamine~ (TM) 2000 pPD-L1-EGFP N1 and two pEGFPn1 plasmids were transfected into NIT-1 cell lines stably transfected cell line, named NIT-PD-L1 and NIT-EGFP.
2. preparation of sensitized lymphocytes
With three group cells stimulated NIT-1, NIT-PD-L1 and NIT-EGFP in vivo by intraperitoneal injection of Balb / c mice, 1 weeks apart with a single intraperitoneal injection of immune cells, fourteenth days after the separation of Balb / c mice spleen mononuclear cells of sensitized lymphocytes as spare.
3. mixed cell culture
Pretreated with mitomycin C for NIT-1, NIT-PD-L1 and NIT-EGFP three cells as spleen and lymphocyte stimulating cells were freshly isolated and (or) the sensitized lymphocytes were co cultured for 7 days.
4. cell proliferation experiment
With the CFSE pre stained mouse lymphocytes, cells to be mixed after 7 days of culture, FCM lymphocyte proliferation.
5. detection of apoptosis
Collecting the cells after 7 days of culture mixed lymphocyte, Annexin V-cy5 and PI staining, detection of apoptosis of FCM lymphocytes.
6. cytotoxicity test
With three group cells stimulated NIT-1, NIT-PD-L1 and NIT-EGFP in vivo by intraperitoneal injection of Balb / c mice, 1 weeks apart with a single intraperitoneal injection of mouse spleen lymphocyte separation, fourteenth days after the stimulation of cellular immunity, cell and the spleen lymphocyte mixed by 1:10 culture, collected 3 days after NIT-1 lymphocytes were used as effector cells. Cells with CFSE staining, as target cells, and to the ratio of 1:20 cells and the effect of mixed culture, 4 hours after PI staining, cell necrosis detection FCM number.
Detection of 7. cytokines
Collect the mixed cell culture supernatant, the secretion level of ELISA cytokine detection; the collection of lymphocytes after mixed culture, the expression of cytokines in FCM lymphocytes in the detection.
Islet allograft tolerance test induced by 8.PD-L1
(1) diabetes model induced by STZ
Induction of diabetic model by low dose repeated intraperitoneal injection of STZ.
(2) cells in peritoneal transplantation
NIT-1, NIT-EGFP or NIT-PD-L1 cells (1.5 * 10~7) was injected into Balb / C diabetic mouse model of peritoneal cavity.
(3) the blood glucose, body weight, insulin release test and survival time were observed
Every day after transplantation to monitor a blood glucose, body weight and survival time; in seventh days and 24 days after transplantation, detected by radioimmunoassay of glucose stimulated insulin release test.
(4) cell proliferation, apoptosis, cytokine secretion culture assay and intracellular cytokine.
(5) determination of mouse serum cell factor
Seventh days after transplantation, serum were collected, to detect the expression of ELISA cytokines.
(6) apoptosis of peritoneal lymphocytes and peritoneal lavage cells by immunofluorescence staining
Seventh days after transplantation, mice peritoneal lavage cells, peritoneal lymphocyte apoptosis detection FCM; rejection of the transplanted cells by immunofluorescence staining.
[results]
The effect of 1.PD-L1 on regulation of allogeneic lymphocytes
(1) effect of PD-L1 on proliferation and apoptosis of allogeneic lymphocytes: freshly isolated mouse spleen lymphocytes as reaction cells with NIT-1 or PD-L1 modified NIT-1 (NIT-PD-L1) as stimulator cells in mixed culture, FCM test results show that the unmodified NIT-1 stimulated lymphocyte proliferation rate was significantly higher than that of NIT-PD-L1 stimulated proliferation rate;
Unmodified NIT-1 sensitized lymphocytes as the reaction cell, and NIT-1 or NIT-PD-L1 as stimulator cells in mixed culture, FCM test results show that the unmodified NIT-1 stimulated lymphocyte proliferation rate was significantly higher than that of NIT-PD-L1 stimulated proliferation rate and lymph cell apoptosis induced by NIT-1 was lower than the rate of lymphocyte apoptosis induced by NIT-PD-L1;
NIT-PD-L1 sensitized lymphocytes as reaction cells, and NIT-1 or NIT-PD-L1 as stimulator cells mixed culture was significantly lower than that of unmodified NIT stimulated lymphocyte proliferation rate NIT-PD-L1 stimulated lymphocyte proliferation and induce apoptosis of lymphocyte reaction. The results show that the initial reaction of PD-L1 can inhibit the freshly isolated and sensitized lymphocytes proliferation, and promote their apoptosis.
(2) effect on the expression of PD-L1 and secretion of cytokines: cytokines of FCM lymphocytes in the detection and ELISA detection of cytokines secretion results show that compared with NIT-1 or NIT-EGFP induced lymphocyte NIT-PD-L1 cell induced lymphocyte expression and secretion of IL-4, IL-10 levels were significantly higher, while IFN- levels decreased significantly.
(3) the effect of PD-L1 on the activity of CTL by NIT-1, NIT-PD-L1 or NIT-EGFP three group cells were obtained in vitro and in vivo combined stimulation of spleen lymphocytes were co cultured with NIT-1 cells in vitro, CFSE and PI double staining, FCM assay showed that the lymphocyte mediated NIT-1 or NIT-EGFP cells induced by moderate necrosis of NIT-1 cells however, spleen lymphocyte mediated NIT-PD-L1 cells induced by mild necrosis of NIT-1 cells, and the results show that the activation effect of PD-L1 and cortisol suppression of CTL.
The survival of 2.PD-L1 prolonged islet graft induced by STZ
NIT-1, Balb / C diabetic mice NIT-PD-L1 and NIT-EGFP three groups of cells were transplanted into STZ induced observation before and after transplantation, blood glucose, body weight, insulin release and survival time of glucose stimulation, and the proliferation and apoptosis of spleen cells, expression and secretion of cytokines, the experimental results show that:
(1) PD-L1 on diabetic mice weight, influence and survival time of blood glucose: the transplantation of NIT-1, NIT-PD-L1 or NIT-EGFP cells after third days, the blood glucose of diabetic mice were decreased to normal range, the transplantation of NIT-1 or NIT-EGFP cells in control group blood glucose in diabetic mice, in about seventh days and increased more than the normal blood glucose level and maintain normal blood glucose; the level of about 21 days can be transplanted NIT-PD-L1 cells in diabetic mice, then gradually increased. In twenty-fourth days after transplantation and increased blood glucose than the upper limit of normal (11.1mmol / L);
On the seventh day after transplantation, transplantation of NIT-1 or NIT-EGFP cells in the control group of diabetic mice weight decreased gradually, and the diabetic mice transplanted NIT-PD-L1 weight increased gradually; the transplanted NIT-PD-L1 cells in diabetic mice survival time was longer than NIT-1 or NIT-EGFP cells transplantation in diabetic mice.
(2) glucose stimulated insulin release: further detection of glucose stimulated insulin release, the transplantation of NIT-1 or NIT-EGFP cells in diabetic mice were killed in 24 days after transplantation, so the 24 day group includes only the transplantation of NIT-PD-L1 cells in mice. The detection results show that in the seventh days after transplantation, transplantation of insulin was significantly lower than that in normal mice NIT-1 or NIT-EGFP cells and mouse insulin release, transplantation of NIT-PD-L1 cells release and normal mice had no significant difference compared to control group, indicating that NIT-PD-L1 cells maintain normal function; but on the 24 day after transplantation, mice transplanted NIT-PD-L1 cells insulin release is lower than that of normal control mice, suggesting that the occurrence of rejection.
(3) PD-L1 extended shift immunological mechanism of plant survival: in order to study the extension of the PD-L1 shift mechanism of plant survival, in cells seventh days after transplantation, respectively to detect.FCM proliferation and apoptosis detection results of splenic lymphocytes and peritoneal lymphocytes showed that the proliferation of splenic lymphocytes of mice transplanted with NIT-1 or NIT-EGFP cells was significantly higher than that of mouse NIT-PD-L1 cells. The apoptosis rate is lower than the NIT-PD-L1 cell transplantation in mice;
On the seventh day after transplantation, cytokine FCM detection and ELISA detection in spleen lymphocytes in cell culture supernatants and serum cytokine results showed that the expression of mouse NIT-PD-L1 cells was significantly higher than that of IFN- gamma spleen lymphocytes of mice transplanted with NIT-1 or NIT-EGFP cells, while IL-4 and IL-10 were significantly lower than the transplantation of NIT-PD-L1 cells of mice spleen lymphocyte culture; cytokine expression the detection results of cytokines in serum and supernatant and intracellular similar. These results indicated that PD-L1 inhibited lymphocyte proliferation and promote apoptosis; results of cytokines and ELISA cells showed that PD-L1 inhibits expression of IFN- gamma, increased IL-4 and IL-10 production, thus promoting Th1 cell to Th2 cell drift.
(4) peritoneal cell activity: peritoneal cell immunofluorescence staining showed that the number of insulin positive cells in mice by intraperitoneal transplantation of NIT-PD-L1 cells was significantly higher than that in the transplantation of NIT-1 or NIT-EGFP cells in mice, and transplanted NIT-PD-L1 cells in mouse peritoneal lymphocyte apoptosis increased, and the degree of infiltration was significantly lower than that of mice transplanted with NIT-1 or NIT-EGFP cells.
[Conclusion] the proliferative response of PD-L1 gene modified NIT can inhibit allogeneic lymphocytes, and induce apoptosis; PD-L1 can inhibit CTL activity and induce Th2 secretion, cytokine secretion, inhibit Th1 cell factor; and diabetic mice transplantation prolongs the survival time of the grafts to normal blood glucose levels and long, research the results for further laid the foundation for the study of IDDM function of islet cell immune tolerance induced by reconstruction.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R587.1;R392

【引证文献】