免疫抑制剂及凋亡细胞诱导移植免疫耐受的实验研究

发布时间：2018-01-10 06:05

本文关键词：免疫抑制剂及凋亡细胞诱导移植免疫耐受的实验研究　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究背景：器官移植是目前治疗器官终末期疾病的最有效的治疗措施,但是移植术后的排斥反应仍是阻碍患者和移植物长期存活的主要原因,传统免疫抑制剂的应用虽然能够有效地抑制急性排斥反应,但是在移植物的慢性排斥方面仍是不能解决其根本原因,并且长期服用免疫抑制剂会增加患者感染、中毒、肿瘤等疾病的发生率,且费用昂贵。因此,解决器官移植术后排斥反应的关键在于诱导受者对供者移植物的免疫耐受,免疫耐受是机体免疫系统在接触某种抗原后所产生的对该抗原的特异性免疫无应答状态,一旦此种免疫无应答状态形成后便无需任何免疫抑制剂维持,从而从更本上解决移植后的排斥反应,达到移植物与宿主能够长期共存的目的。但是,目前摆在移植免疫学界的难题就是如何才能够诱导受者的移植免疫耐受?据此孙尔维等人提出了利用供体凋亡细胞诱导受者免疫耐受的假说。该假说认为凋亡细胞能主动性地调节机体的免疫功能,凋亡细胞通过给其APC主动性地传递吞噬信号"eat me signal"使得吞噬细胞能够快速摄取凋亡细胞并将其所携带的供者抗原被特异性地浓集与受者APC内,从而短时间内提供大量供者MHC抗原,有效地被受者APC提呈给T细胞,最终促进调节性T细胞(Treg)的产生,抑制辅助性T细胞的产生来诱导机体的免疫耐受。国外Fsdok等人也发现巨噬细胞大量吞噬凋亡细胞时,可以抑制一些炎性因子的分泌,促进了TGF-β、IL-10等一些免疫调节因子的产生。Voll等人发现在细菌脂多糖刺激外周血淋巴细胞的反应中加入凋亡细胞不仅能够抑制IL-2、IL-1B、TNF-α等炎性因子的分泌,而且能够促进免疫抑制因子IL-10的产生。本课题组已经成功建立了供者凋亡细胞预输注诱导小动物心脏、肝脏移植免疫耐受模型,但是单纯输注凋亡细胞对大动物的移植模型诱导免疫耐受的作用并不明显,同样,国内外众多学者对诱导成年大动物移植免疫耐受进行了多次尝试,均没有理想的结果,但是凋亡细胞联合免疫抑制剂对大动物移植免疫耐受的诱导实验尚未见报道。目前就如何建立成年大动物和临床诱导免疫耐受已经成为了移植免疫学界的主攻方向和目标。因为大动物免疫系统比较复杂,排斥反应强烈,且购买及饲养价格都比较昂贵,故本课题欲先采用小鼠建立皮肤移植模型,小鼠皮肤移植模型排斥反应较为强烈,并且小鼠免疫系统简单,易操作,费用较为低廉,给受者小鼠预输注凋亡细胞并联合低剂量的免疫抑制剂诱导其免疫耐受,并通过检测其在不同免疫抑制剂作用下的全血中细胞因子的变化来探讨其机制和原理,为下一步大动物和临床诱导免疫耐受做一个探索性研究。本课题为国家自然科学基金(供者凋亡细胞诱导移植免疫耐受的机制)资助项目。本实验利用同种异基因小鼠皮肤移植作为移植模型,皮肤移植是目前可行的器官移植中排斥反应最为强烈的模型,因观察周期短,操作简便,手术成功率高,可控性强等诸多优点而被广泛应用于移植模型的建立；凋亡细胞的制备有多种方法,目前一般采用紫外线照射、抗体诱导、化疗药物诱导、射线诱导、激素诱导等方法,本实验采取地塞米松药物诱导法。地塞米松作为糖皮质激素类药物具有调节细胞生长、发育、死亡以达到维持机体组织细胞平衡稳定的作用。地塞米松诱导淋巴细胞及其它免疫细胞的凋亡较其它方法稳定、易操作已有较多文献报道。第一部分地塞米松诱导供体胸腺细胞的凋亡的初步研究目的：摸索使用地塞米松作为诱导剂对小鼠胸腺细胞凋亡的诱导作用,为下一步实验找到一种稳定性高、可控性强、凋亡率高的诱导方法。方法：将健康成年小鼠胸腺研磨制成细胞悬液,用血球计数板计数后用RPMI-1640完全培养基(含10%胎牛血清)调整细胞浓度为1×106／ml,将所获细胞分装进4个培养皿中进行不同处理：一组置于紫外线灯下20cm处直接照射15分钟后5%C02孵箱37℃恒温培养4小时。另外三组分别加入浓度为1×10-7M、2×10-5M、2×10-3M的地塞米松后置于5%CO2孵箱37℃恒温培养6小时；将处理后的四组胸腺细胞悬液用Annexin V-PI试剂双染后,用流式细胞仪检测胸腺细胞的凋亡和坏死。结果：1、不同浓度地塞米松处理后的小鼠胸腺细胞凋亡率略有不同,经1×10-7M浓度的DEX处理BALB/C小鼠胸腺细胞后,用流式细胞仪检测其凋亡率为53.72%；经2×10-SM浓度的DEX处理BALB/C小鼠胸腺细胞后,检测其凋亡率为58.46%；经2×10-3M浓度的DEX处理BALB/C小鼠胸腺细胞后,测得其凋亡率为55.72%。 2、在紫外线灯下20cm处直接照射15分钟后用5%CO2孵箱37℃恒温培养4小时后的小鼠胸腺细胞,测得其凋亡率为50.29%,；结论：用地塞米松诱导小鼠胸腺细胞凋亡过程复杂、耗时较长,而紫外线照射法操作简便、耗时短。但是用地塞米松诱导的小鼠胸腺细胞凋亡率要高于紫外线照射法,且坏死细胞较少,结果稳定,容易控制,因2×10-5M浓度的DEX处理BALB/C小鼠胸腺细胞凋亡率最高,故下一步实验给受者小鼠注射凋亡细胞采取2×10-5M浓度的地塞米松诱导。第二部分免疫抑制剂及凋亡细胞对诱导小鼠皮肤移植免疫耐受的试验研究目的：探索供体凋亡细胞与免疫抑制剂对小鼠皮肤移植免疫耐受的作用方法：1、制备供体小鼠的胸腺凋亡细胞：采用研磨法获得供者小鼠的胸腺淋巴细胞悬液,经2×10-5M DEX处理后放入5%C02孵箱37℃恒温培养6小时后,获取供体小鼠的胸腺凋亡细胞用Annexin V-PI试剂双染后,流式细胞仪检测胸腺细胞的凋亡率； 2、实验分组：将受者小鼠随机分为四组：PBS对照组、凋亡细胞组、雷帕霉素(RAPA)组、RAPA+凋亡细胞联合组。输注供体胸腺凋亡细胞组别的受者小鼠分别在术前7、3、1天经阴茎背静脉输注供体小鼠的凋亡细胞悬液；接受药物处理的受者小鼠在术前3天开始灌胃给予RAPA药物,1次／d,直到所有移植皮片完全坏死时停药； 3、建立小鼠背对背皮肤移植模型：将修好的供者小鼠背部皮片采用6-8针间断垂直褥式外翻缝合固定在受者小鼠背部,盖上无菌敷料后创可贴加压包扎； 4、术后观察：在行皮肤移植术后第5天打开包扎观察移植皮片存活情况,若植皮与宿主的背底部愈合,色泽一致,无炎症和充血,有毛的皮肤毛发生长良好,则认为未发生排斥反应.50%以上皮片结痂、变硬、坏死、脱落作为排斥标准。结果：1、自体皮肤移植(手术控制组)45例,皮片存活时间1月的存活率为95%(43／45)； 2、受者小鼠术前分别预输注一次、两次、三次凋亡细胞比较移植皮片存活时间没有统计学差异P0.05,但是输注三次凋亡细胞的受者小鼠移植皮片存活时间呈明显延长趋势； 3、C57—Babl/C小鼠背—背皮肤移植手术82例,其中PBS组18例,平均存活时间6.8天；凋亡组共20例平均存活时间7.4天；RAPA组共行手术22例,平均存活时间10.36天；RAPA联合凋亡细胞组共行22例,平均存活时间10.68天。用Kaplan-Meier统计方法分析后PBS对照组与凋亡细胞组移植皮片存活时间比较无统计学差异(P0.05); RAPA组与PBS组比较有显著性统计学差异(P0.01);RAPA+凋亡细胞联用与单纯使用RAPA组比较无统计学差异(P0.05); 4、Babl/C— C57小鼠小鼠背—背皮肤移植手术78例,其中PBS组共18例,平均存活时间6.5天；凋亡组共20例,平均存活时间7.3天；RAPA组共行手术19例,平均存活时间10.42天；RAPA联合凋亡细胞组共行21例,平均存活时间10.52天。用Kaplan-Meier统计方法分析后PBS组与凋亡细胞组移植皮片存活时间比较无统计学差异(P0.05),但是凋亡组较PBS组移植皮片呈延长趋势；RAPA+凋亡细胞联用与单纯使用RAPA组比较无统计学差异(P0.05)。结论：1、分别输注1次、2次、凋亡细胞均对小鼠皮肤移植存活时间无明显延长,但是输注3次凋亡细胞对移植物存活时间有着明显的延长趋势； 2、凋亡细胞对诱导同种异基因小鼠皮肤移植免疫耐受作用不明显。 3、低剂量免疫抑制剂能够延长同种异基因小鼠的皮肤移植存活时间； 4、免疫抑制剂联合凋亡细胞的协同作用在小鼠皮肤移植模型上效果不明显。第三部分：不同免疫抑制剂对全血中细胞因子分泌的机制研究目的：研究不同免疫抑制剂对全血细胞中不同细胞因子分泌的影响。方法：1.取样：取健康成人外周血加入不同浓度的免疫抑制剂培养6 h后加入10μl浓度为0.15μg／ml的佛波酯(PMA)和10μl浓度为2.5 gg／ml的离子霉素(IONO),再培养6 h(37℃,5% CO2)后离心(300 G,5 min),收集上清待测; 2.利用用Bio-Plex悬浮蛋白芯片系统检测细胞因子的变化,比较不同免疫抑制剂组细胞因子的水平。 3.统计：采用SPSS10.0统计分析软件对数据进行统计分析。采用单向方差分析和LSD检验比较实验组与对照组之间的差异,P0.05有统计学意义结果：高浓度,中浓度的地塞米松(DEX)及环孢素A (CsA)都能有效抑制MCP-1的分泌,而他克莫司(FK506)和麦考酚酸(MPA)不能有效抑制细胞因子的分泌。结论：DEX和CsA可有效抑制MCP-1的分泌,而FK506和MPA对MCP-1的分泌没有影响。
[Abstract]:Research background:
Organ transplantation is the treatment of end-stage organ disease the most effective treatment measures, but the main reason for graft rejection is still hinder the long-term graft survival, the application of traditional immunosuppressants can effectively inhibit acute rejection, but in terms of the chronic graft rejection is still not solve the fundamental reason and long-term use of immunosuppressive patients will increase the incidence of infection, poisoning, cancer and other diseases, and expensive. Therefore, the key to solve the rejection after organ transplantation is induced by graft immune tolerance to donor immune tolerance, immune system is generated in contact with certain antigens to the antigen the specific immune unresponsiveness, once the formation of immunological unresponsiveness after without any immunosuppressive maintenance, so as to solve the shift from the Rejection after transplantation, graft and host can achieve the purpose of the long-term coexistence. However, the current problem placed in transplantation immunology circles that how to induce immune tolerance of transplant recipient? The sun Erwei proposed by immune tolerance induced by donor apoptotic cells of the hypothesis. The hypothesis that immune cells apoptosis can take the initiative to regulate the body's cell apoptosis through the APC initiative to transfer signal "eat me signal" phagocytosis of phagocytes can make rapid uptake of apoptotic cells and the donor antigen was carried by specific concentration by APC, so a short period of time to provide a large number of donor MHC antigen. Effectively by recipient APC presenting to T cells and eventually promote regulatory T cells (Treg) generation, inhibition of T helper cells to induce immune tolerance. Fsdok et al also found macrophages A large number of cell phagocytosis of apoptotic cells, can inhibit the secretion of inflammatory cytokines, promote TGF- beta, IL-10 and some other immunomodulatory cytokines produced.Voll et al found that adding apoptosis in lipopolysaccharide stimulated peripheral blood lymphocyte responses can not only inhibit IL-2 secretion of TNF-, IL-1B, and other inflammatory factors, but also can promote the immunosuppressive factor IL-10. The research group has successfully established donor apoptotic cells induced by pre infusion of small animal heart, liver transplantation immune tolerance model, but simply note the apoptotic cells transplantation model on large animal immune tolerance induced by the effect is not obvious, also, many domestic and foreign scholars have made many attempts to the adult animal induced immune tolerance, are not ideal results, but apoptotic cells combined with immunosuppressive agents on immune tolerance induced by transplantation of animal experiments have not yet See the report.
At present, how to set up the adult animal and clinical induction of immune tolerance has become the main direction and goal of transplant immunology circles. Because the immune system of large animal more complex, strong rejection, and the purchase price is relatively expensive and feeding, so this research will first established by skin transplantation model of small rat model of skin allograft rejection in mice more strongly, and the immune system is simple, easy operation, relatively low cost, to recipients immunosuppressive mice pre infusion of apoptotic cells and combined with low doses of induced immune tolerance, and through the inspection of the cells in the effects of different immunosuppressive agents of the blood factor to explore the mechanism and principle for a large animal and clinical immune tolerance induced by doing an exploratory study.
This subject is the National Natural Science Fund (the mechanism of donor apoptotic cells induce immune tolerance) projects. This experiment using murine skin allografts as transplantation model, skin transplantation is the organ transplant rejection is the most feasible in the strong model, because of the short observation period, simple operation, high success rate the establishment of the advantages of strong controllability, and is widely used in transplantation model; apoptotic cell preparation of a variety of methods, the current commonly used ultraviolet irradiation induced antibody induced by chemotherapeutic drugs, radiation, induction, hormone induction and other methods, this experiment take dexamethasone induced by dexamethasone. As glucocorticoids regulate cell growth, development, death in order to achieve the maintenance of body tissue stability. The apoptosis of lymphocytes and other immune cells induced by dexamethasone It is more stable and easier to operate than other methods.
Primary study on the apoptosis of donor thymus cells induced by dexamethasone in the first part
Objective: To explore the induction effect of dexamethasone as an inducer on thymocyte apoptosis in mice, and find a high stability, high controllability and high apoptosis inducing method for next experiment.
Methods: healthy adult mouse thymus and grinded into cell suspension by blood cell counting plate count with RPMI-1640 complete medium (containing 10% fetal bovine serum) the cell concentration was adjusted to 1 * 106 / ml, will be divided into 4 different cell culture processing dish: culture 5%C02 incubator a constant temperature of 37 DEG C group under ultraviolet lamp 20cm under direct irradiation 15 minutes after 4 hours. The other three groups were added at the concentration of 1 * 10-7M, 2 * 10-5M, 2 * 10-3M of dexamethasone after 5%CO2 incubation box at 37 C for 6 hours; the four groups after the treatment of thymus cell suspension with double Annexin V-PI kit after staining, flow cytometry was used to detect the thymocyte apoptosis and necrosis.
Results: 1, thymus cell apoptosis rate after different concentrations of dexamethasone treatment is slightly different, with 1 * 10-7M concentration of DEX BALB/C in mouse thymocytes, detected their apoptotic rate by flow cytometry was 53.72%; with 2 * 10-SM concentration of DEX BALB/C in mouse thymocytes, detected the apoptosis rate was 58.46% with 2 * 10-3M; the concentration of DEX BALB/C in mouse thymocytes, measured the apoptosis rate of 55.72%.
2, after 15 minutes of direct irradiation at 20cm under ultraviolet light, the murine thymocytes were incubated at 37 C at 37 C in 5%CO2 incubator for 4 hours, and the rate of apoptosis was 50.29%.
Conclusion: Dexamethasone induced mouse thymocyte apoptosis is complex, time-consuming, and ultraviolet irradiation method is simple, time is short. But the use of dexamethasone induced mouse thymocyte apoptosis rate is higher than that of ultraviolet irradiation, and the necrotic cells less, stable, easy to control, because the DEX BALB/C in mouse thymocytes apoptosis rate up to 2 * 10-5M concentration, so the next step of the experiment to the recipient mice with apoptotic cells induced by taking 2 * 10-5M concentration of dexamethasone.
Experimental study on immune tolerance of second parts of immunosuppressant and apoptotic cells in inducing skin transplantation in mice
Objective: To explore the effect of donor apoptotic cells and immunosuppressive agents on the immune tolerance of skin transplantation in mice
Methods: 1 cell apoptosis of thymus, preparation of donor mice: donor mice by the method of grinding the thymus lymphocyte suspension, with 2 * 10-5M after DEX treatment in the 5%C02 incubator at 37 C after 6 hours of incubation, cell apoptosis of donor mice thymus acquisition by Annexin V-PI double staining reagent, thymocyte apoptosis the rate of flow cytometry;
2, experiment group: recipient mice were randomly divided into four groups: PBS control group, apoptotic cells group, rapamycin (RAPA) group, RAPA+ group. Apoptotic cells combined with infusion of donor apoptotic cells in thymus groups of recipient mice suspension of apoptotic cells in the preoperative 7,3,1: dorsal penile vein infusion of donor mice respectively.; the treatment operation in recipient mice 3 days before intragastric administration of RAPA drugs, 1 / D, until all the skin graft necrosis completely discontinued when;
3, establish a mouse model of skin transplantation: fixed back-to-back donor mice back skin using 6-8 needle interrupted vertical mattress suture fixation in recipient mice back, covered with sterile dressing after compression bandage bandage;
4, postoperative observation: at fifth days after skin transplantation open dressing grafts survival situation, if the back bottom of skin grafting and host healing, consistent color, no inflammation and hyperemia, hairy skin and hair growth is good, is that no rejection of more than.50% skin knot scab, harden, necrosis and off as the rejection criteria.
Results: 1, 45 cases of autologous skin graft (operation control group), the survival time of skin graft survival time in January was 95% (43 / 45).
2, the recipient mice were pre injected once, two times, and three times before the operation. Compared with the three apoptotic cells, the survival time of grafted skin was not statistically different. P0.05, however, the survival time of the mice who had been transfused for three times was significantly prolonged.
3, C57 - Babl/C - mouse dorsal dorsal skin transplantation in 82 cases, including 18 cases of PBS group, the average survival time of 6.8 days; the apoptosis of 20 cases of the average survival time of 7.4 days; group RAPA underwent surgery in 22 cases, the average survival time of 10.36 days; RAPA group apoptosis were performed in 22 cases, the average survival time for 10.68 days. Using Kaplan-Meier statistical analysis method of PBS control group and group apoptosis skin graft survival time had no significant difference (P0.05); RAPA group and PBS group, there was significant difference (P0.01); and the simple use of RAPA group compared with no significant difference in apoptotic cells in combination with RAPA+ (P0.05);
4, Babl/C - C57 mouse dorsal dorsal skin graft in 78 cases, PBS group 18 cases, the average survival time of 6.5 days; apoptosis were 20 cases, the average survival time of 7.3 days; group RAPA underwent surgery in 19 cases, the average survival time of 10.42 days; RAPA group included 21 cases of apoptosis and the average survival time of 10.52 days. Using Kaplan-Meier statistical analysis method after the PBS group and the group apoptosis skin graft survival time had no significant difference (P0.05), but the apoptosis group than in PBS group of skin grafts increased; the apoptosis of RAPA+ cells associated with the use of RAPA compared with the control group showed no significant difference (P0.05).
Conclusion: 1, 1 times and 2 times of infusion 1 times, the survival time of the skin cells was not prolonged, but the survival time of the apoptotic cells was prolonged obviously after 3 times of infusion.
2, the immunological tolerance of the apoptotic cells to the allogeneic mouse skin transplantation was not obvious.
3, low dose immunosuppressant can prolong the survival time of skin allograft in allogeneic mice.
4, the synergistic effect of immunosuppressive agents combined with apoptotic cells was not effective in the mouse skin transplantation model.
The third part: the study of the mechanism of different immunosuppressive agents on the secretion of cytokines in the whole blood
Objective: To study the effect of different immunosuppressants on the secretion of different cytokines in the whole blood cells.
Methods: 1. samples: healthy adult peripheral blood with different concentrations of immunosuppressant culture phorbol ester 6 h after adding 10 L concentration of 0.15 g / ml (PMA) and 10 L concentration was 2.5 GG / ionomycin ml hormone (IONO), and then cultured for 6 h (37 DEG C, 5% CO2) after centrifugation (300 G, 5 min), the supernatant was collected to be measured;
2. the level of cytokines in different immunosuppressant groups was compared by using Bio-Plex suspension protein chip system to detect the changes of cytokines.
3. statistics: SPSS10.0 statistical analysis software was used to analyze the data. Unidirectional variance analysis and LSD test were used to compare the difference between the experimental group and the control group, P0.05 was statistically significant.
Results: high concentration, moderate concentration of dexamethasone (DEX) and cyclosporine A (CsA) can effectively inhibit MCP-1 secretion, while tacrolimus (FK506) and mycophenolic acid (MPA) can not effectively inhibit cytokine secretion.
Conclusion: DEX and CsA can effectively inhibit the secretion of MCP-1, while FK506 and MPA have no effect on the secretion of MCP-1.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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