输入供体凋亡细胞对受体外周血细胞因子的影响

发布时间：2018-06-08 14:11

本文选题：同种异基因 + 凋亡细胞　；参考：《南方医科大学》2009年硕士论文

【摘要】： 目前,原位肝移植(Orthotopic liver transplantation,OLT)是治疗终末期肝病最有效和常规措施。然而,尽管由于外科技术的标准化、免疫抑制剂的不断研发和应用,移植物的长期存活率没有明显提高。器官移植术后的排斥反应仍是威胁患者和移植物长期存活的主要原因。免疫抑制剂的应用,不仅使器官移植成为终末期肾、肝、心脏疾病可接受的治疗方式,而且显著减少了急性排斥的发生率,同时显著提高了器官移植病人的短期、长期生存率。然而,免疫抑制剂的应用带来了许多问题。首先,要终身连续对免疫系统进行非特异性免疫抑制。第二,当前使用的免疫抑制剂中,没有一种能完全预防急性排斥或者慢性排斥。第三,免疫抑制剂的应用带来了肿瘤和感染的风险。第四,免疫抑制剂的应用增加了移植病人高血压的风险以及由此导致的心血管并发症。因此,诱导受者对供者器官特异性免疫耐受是解决排斥反应最理想的措施,相对免疫抑制疗法,诱导免疫耐受的优势是从根本上避免了排斥反应,又不影响机体的正常抗感染和免疫监视功能,同时避免了药物毒性。而且,耐受的诱导对于最终克服异种移植免疫学障碍也许是最佳途径,因为异种移植免疫抑制需长期高水平的、非特异性的免疫抑制剂来维持。因此,诱导移植免疫耐受性,控制移植物排斥反应,保护移植器官的功能,这无论在同种移植和异种移植中都是十分重要的课题。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、PGE2、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。细胞因子是活化细胞产生的细胞间信号多肽。大多数细胞因子都有多种来源,多种作用靶位和多种功效。细胞因子网络是参与免疫识别、应答反应的重要成分,也是决定免疫反应发生方向的主要因素之一。抗原性物质进入机体后引起免疫系统发生的各种反应变化中包括了细胞因子比例水平的改变,由于后者直接参与前者的发生过程与最后结果表现,从而探讨细胞因子的变现表现对分析以及预测免疫反应发生和走向有一定的意义。我们课题组以前的工作证实,一定数量的供体凋亡脾淋巴细胞预输注可以诱导大鼠心、肝脏移植模型产生特异性免疫耐受,延长移植存活的时间。且通过对凋亡细胞进行示踪实验,发现凋亡的脾淋巴细胞主要聚集并驻留于肝脏,且对肝脏组织的细胞因子的微环境产生了影响。但输入的过程中是否也影响外周的细胞因子微环境,这对下部进一步探讨诱导凋亡的机制有重要的影响。所以本实验主要在前期的基础上,对供体凋亡脾淋巴细胞预输注是否影响外周血微环境进行研究。目的已知凋亡细胞发生免疫调节作用时也会同时伴有细胞因子等微环境的变化。且前期工作证明供体凋亡脾淋巴细胞输注对受体肝脏内细胞因子微环境有影响,本部分对供体凋亡细胞输注后对外周血免疫微环境中一系列重要细胞因子的变化进行观察,以了解供体凋亡脾淋巴细胞输注对外周血细胞因子的影响。方法供体C57BL小鼠14只,8-10周,雄性;受体Balb/c小鼠6-8周,50只,雄性。具体如下: (1)机械研磨法分离脾脏细胞,易得分离液分离淋巴细胞。 (2)紫外线照射法诱导凋亡,流式细胞仪检测细胞凋亡率。 (3)采用尾静脉输入法,每处理组每时间点5只小鼠,预输注处理分组:供体脾脏活细胞处理组(Liver cell treatment group,LG),供体脾脏凋亡细胞处理组(Apoptotic cell treatment group,AG); (4)检测时间点:输注后0h、1h、3h、6h、12h; (5)采用体外心脏采血法,收集样本; (6) Luminex技术液态芯片法检测外周血IL-1β、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、GM-CSF(粒细胞-巨细胞激落刺激因子)、IFN-γ、TNF(肿瘤坏死因子); (7)所有实验数据利用SPSS13.0和GraphPad PRISM 3.02等软件进行统计学分析和作图。统计 ①两因素对各因子作用的分析比较,使用SPSS13.0软件之General LinearModel-Univatiate分析,两处理组各个点的比较,使用SPSS13.0软件之One-way ANOVE分析,方差不齐的,选用Independent Samples近似Test; ②各个处理组中时间点于0h之间的比较,使用SPSS13.0软件之One-way ANOVE分析,方差不齐的,选用Independent Samples近似Test; ③采用GraphPad PRISM 3.02软件进行制图。结果 (1)流式细胞术分析,紫外线诱导的凋亡细胞的比例可达到41.07%以上。 (2)活细胞组对外周细胞因子的影响(因IL-1β、IL-2、IL-4、IL-5、TNF在外周血液中的水平低于仪器的检测水平,未测出):外周血中IFN-γ在6h时细胞因子浓度明显升高(P=0.010,6h),IL-10于3h和6h时细胞因子浓度明显升高(P=0.009,3h;P=0.015,6h),IL-12、GM-CSF、IFN-γ在各个时间点无显著性差异。 (3)凋亡细胞组对外周细胞因子的影响:IL-6、IL-10、IL-12、IFN-γ在各个时间点无明显的差异,GM-CSF在3h时明显升高(P=0.049)。 (4)处理组不同对外周细胞因子微环境的影响: ①IL-6:活细胞组和凋亡细胞组相比于1h时存在显著性差异(P=0.035,1h),活细胞组的水平高于凋亡细胞组。 ②IL-10:活细胞组和凋亡细胞组相比于分别于3h、6h、12时存在显著性差异(P=0.001,3h;P=0.001,6h;P=0.043,12h),活细胞组的水平高于凋亡细胞组。 ③IL-12、GM-CSF、IFN-γ在活细胞组和凋亡细胞组相比中无显著性差异。结论本实验证明凋亡细胞组和活细胞组相比,外周IL-6在1h时有显著性差异和IL-10在3h、6h和12h时存在显著性差异,活细胞组高于凋亡细胞组,其他细胞因子都无显著性差异,而且活细胞输注后,3h、6h的IL-10浓度明显高于0h。凋亡细胞输注后对细胞因子基本无改变,从IL-6和IL-10的结果我们可以推测:活细胞输注后,机体产生了急性炎性反应,使IL-6于1h时升高,而3小时后,机体为了避免炎性反应过度而采取了保护措施,增加IL-10的分泌,阻断了单核、巨噬细胞合成IL-6、TNFα等介质,而凋亡细胞对机体外周血无明显的作用,根据凋亡细胞是一种死细胞,具有完整的包膜,在组织中的清理主要为吞噬作用,可能相对于异种的活细胞引起的炎性反应作用较弱。凋亡细胞输入后,对外周血中GM-CFS浓度在3h时明显升高和活细胞输入后,6h时对外周血中IFN-γ浓度的影响有显著性意义,具体机制还需要进一步研究。而根据以往的研究,凋亡细胞对机体有明确的作用,可以引起免疫耐受的,而且本课题组的前期研究通过对凋亡细胞进行示踪实验,发现凋亡的脾淋巴细胞主要聚集并驻留于肝脏,且对肝脏组织的细胞因子的微环境产生了影响,包括IL-1β、IL-4、IL-10、和IFN-γ的特异性分泌水平提高,从而推测凋亡细胞主要在在肝脏起作用,对外周无明显的影响。但具体的机制还需要进一步对肝组织中其他细胞类型和反应功能的改变,如记忆T细胞、B细胞等进行相续的研究。
[Abstract]:At present, Orthotopic liver transplantation (OLT) is the most effective and conventional measure for the treatment of end-stage liver disease. However, despite the standardization of surgical techniques and the continuous development and application of immunosuppressive agents, the long-term survival rate of the graft is not significantly improved. The rejection after organ transplantation is still a threat to patients and transplant. The main cause of long term survival. The application of immunosuppressive agents not only makes organ transplantation an acceptable treatment for end-stage kidney, liver and heart disease, but also significantly reduces the incidence of acute rejection, and significantly improves the short-term and long-term survival rate of organ transplant patients. However, the application of immunosuppressive agents has brought many questions. Second, none of the immunosuppressants currently used can completely prevent acute rejection or chronic rejection. Third, the application of immunosuppressants brings the risk of cancer and infection. Fourth, the application of immunosuppressive agents increases the hypertension of transplant patients. Therefore, it is the most ideal measure to induce the recipient's organ specific immune tolerance to the rejection reaction. Relative immunosuppressive therapy, the advantage of inducing immune tolerance is to avoid the rejection, and do not affect the normal anti infection and immune surveillance function of the body. Moreover, tolerance induction may be the best way to eventually overcome xenotransplantation immunological barriers, because xenograft immunosuppression needs long-term high levels of non specific immunosuppressive agents to maintain. Therefore, it induces transplant immune tolerance, controls graft rejection, and protects the function of transplant organs. It is a very important topic both in homologous transplantation and xenotransplantation.
The mechanism of transplantation immune tolerance is very complex. Although there are many methods to induce immune tolerance, they are not fully reliable and persistent. Apoptosis is a common physiological and pathological phenomenon in the organism. It is normal organ and tissue development, detrimental, excessive and non functional cells, and maintains itself. The necessary link of steady state. The existing research shows that apoptosis is an important mechanism for the balance and stability of the immune state of the body. Apoptotic cells have the active regulation effect on the immune system. Apoptotic cells can secrete chemotactic factors of lipid, change their membrane structure to express "eat-me" signals, induce phagocytes to scavenging them. When the apoptotic cells were phagocyted by antigen presenting cells and secreted by phagocytic cells such as TGF- beta, PGE2, IL-10 and so on, the specific antigen recognition microenvironment was created, which could induce the related lymphocyte to produce immune tolerance against the apoptotic antigen, without causing inflammatory response. Accordingly, sun Erwei and so on proposed the use of donor apoptosis. A theoretical assumption of donor specific immune tolerance induced by cell infusion receptors.
Cytokine is an intercellular signal polypeptide produced by activated cells. Most of the cytokines have a variety of sources, multiple targets and many functions. Cytokine network is an important component of immune recognition, response response and one of the main factors that determine the direction of the immune response. Antigenic substances are induced to escape from the body. The changes in the various responses of the Phytophthora system include changes in the proportion of cytokines, as the latter directly participates in the process of the former and the performance of the final results, thus exploring the expression of cytokine in the analysis and prediction of the occurrence and direction of the immune response.
The previous work of our group confirmed that a certain number of donor apoptotic splenic lymphocytes pretransfused can induce rat heart, the liver transplantation model produces specific immune tolerance and prolongs the survival time of the transplanted cells. And the apoptotic cells are traced by the apoptotic cells, and the apoptotic splenic lymphocytes mainly gather and reside in the liver. The microenvironment of cytokine in the dirty tissue has an impact. But whether the input process also affects the peripheral cytokine microenvironment, which has an important influence on the mechanism of inducing apoptosis in the lower part. Therefore, this experiment is mainly on the basis of the early stage to affect the peripheral blood microenvironment of donor apoptosis of splenic lymphocytic cells. Research.
objective
It is known that the immunoregulation of apoptotic cells may also be accompanied by changes in the microenvironment such as cytokine, and the earlier work has proved that the donor apoptotic splenic lymphocyte infusion has an effect on the cell factor microenvironment in the recipient liver. This part is a series of important cytokines in the peripheral blood immune microenvironment after the donor apoptotic cells are transfused. Changes were observed in order to understand the effects of donor apoptotic splenic lymphocyte infusion on peripheral blood cytokines.
Method
The donor C57BL mice were 14, 8-10 weeks, male, recipient Balb/c mice 6-8 weeks, 50 male, as follows:
(1) the splenic cells were separated by mechanical attrition, and the lymphocytes were separated easily.
(2) apoptosis was induced by ultraviolet irradiation, and apoptosis rate was detected by flow cytometry.
(3) using the tail vein input method, 5 mice per time point per treatment group, pre infusion treatment group: donor spleen living cell treatment group (Liver cell treatment group, LG), donor spleen apoptotic cell processing group (Apoptotic cell treatment group, AG);
(4) detection time points: 0h, 1H, 3h, 6h, 12h after infusion;
(5) in vitro cardiac blood sampling was used to collect samples.
(6) Luminex technique was used to detect peripheral blood IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, GM-CSF (granulocyte - cytomegalocell irritation factor), IFN- gamma, TNF (tumor necrosis factor);
(7) all experimental data were statistically analyzed and plotted using SPSS13.0 and GraphPad PRISM 3.02 software.
Statistics
(1) the analysis and comparison of the two factors on the role of each factor, using the General LinearModel-Univatiate analysis of SPSS13.0 software, the comparison of each point in the two treatment group, the One-way ANOVE analysis of the SPSS13.0 software, the variance of the variance, and the selection of Independent Samples to approximate Test;
(2) the comparison of the time points between 0h in each treatment group, using the One-way ANOVE analysis of SPSS13.0 software, and using the Independent Samples approximation Test as the variance is uneven.
(3) using GraphPad PRISM 3.02 software for drawing.
Result
(1) flow cytometry analysis showed that the proportion of apoptotic cells induced by UV could reach more than 41.07%.
(2) the influence of the peripheral cytokine in the living cell group (IL-1 beta, IL-2, IL-4, IL-5, TNF in the peripheral blood is lower than the instrument detection level, not measured): the concentration of cytokine in the peripheral blood of IFN- gamma in 6h is significantly increased (P=0.010,6h) and IL-10 at 3H and 6h. There is no significant difference between gamma at all time points.
(3) the effect of apoptotic cell group on peripheral cytokines: IL-6, IL-10, IL-12 and IFN- gamma were not significantly different at all time points, and GM-CSF increased significantly at 3H (P=0.049).
(4) the effect of treatment group on peripheral cytokine microenvironment:
There was significant difference between IL-6: living cell group and apoptotic cell group when compared with 1H (P=0.035,1h), and the level of living cell group was higher than that of apoptotic cell group.
2. There were significant differences between the IL-10: living cell group and the apoptotic cell group at 3h, 6h, and 12 (P=0.001,3h; P=0.001,6h; P=0.043,12h). The level of the living cell group was higher than that of the apoptotic cell group.
There was no significant difference in IL-12, GM-CSF and IFN- gamma between the living cell group and the apoptotic cell group.
conclusion
This experiment showed that the apoptotic cell group and the living cell group had significant difference in the peripheral IL-6 at 1H and the significant difference between the IL-10 in 3h, 6h and 12h. The living cell group was higher than the apoptotic cell group, and there was no significant difference in the other cytokines. Moreover, the IL-10 concentration of 3H and 6h was significantly higher than that of the apoptotic cells after the transfusing of the living cells. The factors are basically unchanged. From the results of IL-6 and IL-10, we can speculate that after the infusion of living cells, the body produces an acute inflammatory response, which raises the IL-6 at 1H, and 3 hours later, the body has taken protective measures to avoid excessive inflammatory response, increased the secretion of IL-10, blocked the mononuclear, and macrophages synthesized IL-6, TNF A and other mediators, and apoptosis The cells have no obvious effect on the peripheral blood of the body. According to the apoptotic cells, a dead cell is a dead cell, with a complete capsule. The cleaning in the tissue is mainly phagocytosis, which may be weaker than that of the xenogeneic living cells. After the input of the apoptotic cells, the concentration of GM-CFS in the peripheral blood is obviously elevated at 3H and the input of living cells. The effect of 6h on the concentration of IFN- gamma in peripheral blood is significant, and the specific mechanism needs further study. According to the previous study, the apoptotic cells have a definite effect on the body and can cause immune tolerance, and the preliminary study of the group was traced by the apoptotic cells, and the apoptotic splenic lymphocytes were found. It mainly aggregates and resides in the liver and has an effect on the microenvironment of cytokine in liver tissue, including the specific secretion level of IL-1 beta, IL-4, IL-10, and IFN- gamma, thus speculates that the apoptotic cells mainly play a role in the liver and have no obvious influence on the outside. Changes in type and response function, such as memory T cells, B cells, etc.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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