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P>0.05,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(P>0.05); RAPA组与PBS组比较有显著性统计学差异(P<0.01);RAPA+凋亡细胞联用与单纯使用RAPA组比较无统计学差异(P>0.05);
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(P>0.05),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(P>0.05)。
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,P<0.05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。
Backgroud:
Organ transplantation is the most effective treatment to end-stage organ disease, but graft rejection is still an obstacle against long-term patient and graft survival,the application of conventional immunosuppressive agents can effectively inhibit acute rejection, but can not solve the fundamental cause of the chronic graft rejection, and long-term application in patients will increase the chance of infection, poisoning, and the incidence of cancer and other diseases, besides,it is still expensive. Thus, the solution of rejection after organ transplantation is to induce the recipient to the donor graft immune tolerance, immune tolerance means the body's immune system is led to non-antigen-specific immune response after exposure to certain antigens, once the formation of such immune unresponsiveness succeeds, it is not necessary to maintain any immunosuppressive agent, so it will solve post-transplant rejection, granting graft and host with a long-term coexistence. However, at present in transplantation immunology area,there is a challenge that how to induce immune tolerance of transplant recipient? Accordingly, Professor Sun erwei proposed his hypothesis of using donor's apoptotic cells to induce receptor's immunological tolerance. Apoptosis is a common physiological and pathological phenomenon in the body, the hypothesis indicates that the apoptotic cells can initiatively regulate the immune function, apoptotic cells send to APC initiative through swallowing the signal transmission of "eat me signal",it makes the phagocytic uptake of apoptotic cells were able to quickly and carried by donor antigens which is specifically concentrated within and recipient APC, thereby providing a large number of donor MHC antigens by recipient APC to effectively presented to T cells in a short time, while uptake of apoptotic cells by APC, the APC to promote secretion of immunosuppressive factors, and to promote regulatory T cells'(Treg) proliferation to induce immune tolerance. Fsdok and others also found that it can inhibit the secretion of several inflammatory factors, and promoted TGF-β, IL-10 and some other immune-regulating factors' production, when a large number of apoptotic cells were phagocyted by macrophage. Voll, who found that in response of LPS stimulated peripheral blood lymphocytes, then by adding apoptotic cells, it not only inhibited the secretion of IL-2, IL-1B, TNF-αand other inflammatory factors,but also boosted the immune inhibitory factor IL-10 generation. The research group has successfully established a pre-infusion of donor apoptotic cells induced small animal's heart, liver transplant tolerance model, but it is not an obvious effect of induction immune tolerance when the simple infusion of apoptotic cells on large animal transplantation model, similarly, many scholars in China and abroad-induced immune tolerance of adult large animal transplantation conducted a number of attempts, but they did not get the ideal outcome, however, the apoptotic cells combined with immunosuppressive drugs induce large animal transplantation immune tolerance has not been reported.
Currently how to build an adult large animal model and clinical induction of immune tolerance has become the main direction and objective of transplantation immunology academic. Because the immune system of the big animals is relatively complex,and they have very strong rejection, and it is too expensive, so in this discussion, we established the skin graft model in mice,the mouse transplantation rejection is strong, and the cost of mice is lower, to induce immune tolerance of the recipient mice by pre-infusion of apoptotic cells and combined with low doses of immunosuppressive drugs to explore its mechanisms and principles by detecting cytokines and changes of Treg cells at different time points after transplantation to do an exploratory study for further research on large animals and clinical induction of immune tolerance.
The topic is the National Natural Science Foundation of China (those mechanisms for apoptosis inducing transplantation immune tolerance) funded projects. In this study, using the same allogeneic transplantation model as mouse skin transplantation,as we know, a skin graft is the strongest of a viable organ transplant rejection, it has been widely used as transplantation model,because observation period is short, easy to operate, a high success rate,easy to control, and so on; Preparation of apoptotic cells have various methods, at present it is commonly recommended that ultraviolet radiation, antibody induction, chemotherapy drug-induced, radiation-induced, hormone-induced and other methods, this experiment adopts dexamethasone drug-induced method. Dexamethasone as a glucocorticoid drugs,it is capable of regulating cell growth, development and inducing death to achieve the maintenance of body tissue stability. That Dexamethasone-induced lymphocytes and other immune cell apoptosis is more stable and easier to operate have been more reported in the literature than the other method.
Part 1 A preliminary study of the dexamethasone induced thymocytes apoptosis of donor
Purpose:To explore the effects of dexamethasone induced apoptosis of mouse thymocytes, in order to find a approach of high stability, easy to control, and a high induce rate of apoptosis for further experiments.
Method:Mulling the healthy adult mouse thymus cell to be suspension, cell count and then adjusting the cell concentration reach 1×106/ml with RPMI-1640 complete medium (containing 10% fetal calf serum), the cells were divided into two groups:one group with dexamethasone mg incubate in 5%CO237℃incubator for 6 hours; the other exposed to UV lamp which placed 20cm Department direct for 15 minutes. Then stain the two groups of post-thymic cell suspension with Annexin V-PI double staining, flow cytometry analysis of thymocytes apoptosis and necrosis.
Result:1、The apoptosis rate of mouse thymocytes which was treated with dexamethasone have a little difference.The thymocytes is treatmented by dexamethasone in the concentration of 1×10-7M, then detect the apoptosis rate by flow cytometry,it is 53.72%, when it is treatmented with dexamethasone in 2×10-5M concentration,the apoptosis rate get to 58.46%,and when the dexamethasone concentration change to 2×10-3M, the apoptosis rate get to 55.72%.
2、Mouse thymocytes was treated with UV irradiation(keep 20cm distance for 15 minutes)and then incubate in 5% CO237℃incubator for 4 hours,and the apoptosis rate is 50.29%.
Conclusions:The dexamethasone-induced apoptosis in mouse thymocytes is complex, time-consuming, when ultraviolet radiation method is simple and concise. However, dexamethasone-induced apoptosis rate in mouse thymocytes average should be higher than the ultraviolet irradiation, and have a low necrosis rate, the results are stable, easy to control,because we can get the highest thymocytes apoptosis rate by dexamethasone in 2×10-5M concentration,, so the following to the recipient mice will use this method.
Part 2 The study of immunosuppressive combine apoptosis inducing immunological tolerance of mouse skin transplantation
Purpose Explore mechanism of donor apoptotic cells with immunosuppressive induceing skin graft immune tolerance
Method 1 Preparation of donor apoptotic cells in the thymus:C57 mice obtained by grinding the thymus lymphocytes were also incubated into the incubator for 6 hours after adding dexamethasone,then centrifugate to acquire mouse thymus apoptosis cells, flow cytometry analysis the apoptosis ratio of thymocytes
2 The recipient mice infusion donor thymus apoptotic cells:respectively 7,3,1 days before operative infusion of donor apoptotic cells in the recipient mice through penile dorsal vein.
3 Establishment of mouse back to back skin transplantation model:the reconciliation of donor mouse back skin with 4-8 suture needle fixed in the recipient mice back, covered with sterile dressing then bandage bandaged.
4 Using flow cytometry to detect the Treg cells in blood and the use of liquid chip technique detect whole blood cytokines at different time points.
Result 1、Skin survival time of 45 cases autologous skin grafts (surgical control group) is more than 1 month, and survival rate was 95%(43/45)
2、Pre-infusion apoptotic cells one,two,three times respectively, the skin survival times are not statistically significant(P> 0.05), but the infusion of apoptotic cells three times,skin grafts survive in mice was significantly extended.
3、82 cases of back-back of c57-> Babl/C mice skin graft surgery,18 cases for PBS group, the skin survival time is average 6.8 days,in which there are 20 cases apoptosis group, the skin survival time is average 7.4 days,the other 22 cases is rapamycin combine to apoptosis group,the time is average 10.68 days. Apoptotic cell group compared to the PBS control group by Kaplan-Meier is statistically significant, is not significant difference(P> 0.05),compared to rapamycin group, the PBS group have a statistically significant,apoptosis joint rapamycin group compare to the rapamycin group,the result is not statistically significant(P> 0.05).
4、78 cases of Babl/C-> C57 mice back-back skin graft surgery,18 cases for PBS group, the skin survival time is average 6.5 days, in which there are 20 cases apoptosis group, the skin survival time is average 7.3 days, the other 19 cases is rapamycin group,the time is average 10:42 days, the other 21 cases is rapamycin combine to apoptosis group,the time is average 10.52 days. Apoptotic cell group compared to the PBS control group by Kaplan-Meier statistically significant, is not significant difference(P> 0.05),but the apoptosis group has an extended tendency. Apoptosis joint rapamycin group was compared to the rapamycin group,the result is not statistically significant(P> 0.05).
Conclusion
1 Infused apoptotic cells for 1 or 2 times, there was not significantly longer to mouse skin graft survival time, but the survival time by infusion of apoptotic cells 3 times had an obvious extend tendency;
2 There is a little effect of apoptosis induce immunosuppressive synergy in mouse skin graft model;
3 Low doses of immunosuppressive drugs can prolong allogeneic skin graft survival time of mice;
4 There is inapparent effect of apoptotic cells combined immunosuppressive synergy in mouse skin graft model.
Part 3 The mechanism study of different immunosuppressive drugs effecting the secretion of whole blood cytokine.
Objective:To study the different immunosuppressive drugs are able to affect different cytokines secretion in blood.
Methods:1、Sampling:Whole blood from healthy volunteers which were effected by different immunosuppressive drugs,incubate for 6 hours,then stimulated by lOul 0.15μg/ml phorbol ester (PMA) combined 10μl 2.5μg/ml ionomycin (IONO) for 6 hours, centrifuge (300 G,5 min),assemble supernatant for detect.
2、Detect the chang of cytokines with Bio-Plex system,compare to the level of cytokines in different immunosuppressive groups.
3、Statistics:statistically anlysis by SPSS10.0,compare the different of experiment and control groups by One Way Anova and LSD,it has statistically significant(P< 0.05).
Results:High concentration, the middle concentration of dexamethasone (DEX) and cyclosporin A (CsA) can inhibit the secretion of MCP-1, but tacrolimus (FK506) and mycophenolic acid (MPA) can not effectively inhibition of cytokine secretion.
Conclusion:DEX and CsA can inhibit the secretion of MCP-1, while FK506 and MPA has no effect on the secretion of MCP-1.
引文
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