本文選題：T淋巴細胞疫苗 + BXSB鼠��； 參考：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 研究背景 系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus，SLE)是一種常見的自身免疫性疾病，累及全身多系統(tǒng)和器官，發(fā)病機制復(fù)雜，，臨床變化多端，繼發(fā)感染發(fā)生率高，診治困難，是一種嚴(yán)重危害人類健康的疾病。其廣泛分布于世界各地，全世界的患病率大約是70／10萬-100／10萬。如果以全國13億人口計算，我國的SLE患者已達112-160萬之多，且多見于年輕女性。 SLE的發(fā)病與多種因素有關(guān)，一般認(rèn)為是遺傳素質(zhì)、性激素、感染、藥物和環(huán)境等相互間錯綜復(fù)雜的作用引起免疫調(diào)節(jié)功能紊亂，導(dǎo)致SLE的發(fā)生、持續(xù)和不易緩解。由于病因尚未明確，為其根治帶來困難。近20年來，隨著現(xiàn)代醫(yī)學(xué)的迅速發(fā)展，早期診斷的手段增多和治療水平的提高，SLE的10年生存率已達92％，預(yù)后明顯改善。然而糖皮質(zhì)激素結(jié)合免疫抑制劑藥物仍然是目前治療SLE的首選藥物。一般病例采用大劑量糖皮質(zhì)激素治療后小劑量維持，對急性暴發(fā)性危重SLE患者用激素沖擊療法，效果不理想時聯(lián)合使用細胞毒藥物(主要是環(huán)磷酰胺和硫唑嘌呤)。采用這種常規(guī)治療方法可使大部分患者病情穩(wěn)定，但用藥量大、時間長，藥物的副作用不容忽視，如誘發(fā)或加重感染、糖尿病、消化道潰瘍、穿孔或消化道出血，長期應(yīng)用可引起高血壓和動脈粥樣硬化、骨折及骨缺血性壞死、精神異常、柯興綜合征、痤瘡、多毛等。60％以上患者最后死于激素的副作用和／或并發(fā)癥。嚴(yán)重影響患者的生存期和生活質(zhì)量。 因此尋求SLE新的治療方法一直是國內(nèi)外研究的熱點。大劑量免疫球蛋白沖擊療法、血漿置換療法等，雖能使病情得到暫時緩解，同樣由于未能針對病因治療，且花費高，不能廣泛應(yīng)用。新型免疫抑制劑包括B細胞治療、抗CD20抗單克隆抗體治療、T細胞治療、抗細胞因子治療、補體治療等大部分處于動物實驗階段，其確切治療效果尚須大量臨床對照試驗證實。近年來造血干細胞移植治療取得了令人鼓舞的療效，但存在高風(fēng)險和技術(shù)條件要求高，部分患者在動員階段不能耐受，價格昂貴，較難推廣。 由于SLE是以T和B淋巴細胞功能異常為主要特點的自身免疫性疾病，自身反應(yīng)性T淋巴細胞的活化及由此介導(dǎo)的自身免疫反應(yīng)是本病發(fā)生的中心環(huán)節(jié)。研究發(fā)現(xiàn)SLE發(fā)病過程中自身抗原應(yīng)答性輔助性T細胞(help T cell,Th)增加。從狼瘡鼠克隆的T細胞系能向B細胞提供協(xié)助，使之產(chǎn)生IgG型抗DNA抗體。50％的這些T細胞克隆中能識別核小體中的T細胞抗原表位。在人及鼠SLE的Th細胞克隆中，識別核小體抗原表位的TCRα鏈能結(jié)合不同TCRβ鏈，形成TCRαβ二肽鏈，識別多種T細胞抗原表位，協(xié)助多種B細胞克隆，產(chǎn)生多種自身抗體。在SLE遷延過程中，T細胞反復(fù)受自身抗原激活而增值，從而使突變率增加，其突變的T細胞能促使自身應(yīng)答B(yǎng)細胞產(chǎn)生抗DNA抗體。這些抗體與相應(yīng)自身抗原結(jié)合，形成免疫復(fù)合物，沉積于腎小球基底膜、肝臟、中樞神經(jīng)系統(tǒng)、小血管壁等多種器官及組織，活化補體、多型核白細胞及血小板，致局部炎癥及小血管炎，使血管阻塞，而顯示為多種臨床病癥。如能通過T細胞免疫抑制自身反應(yīng)性T淋巴細胞的活化，控制SLE的發(fā)展，可能是治療SLE的理想方法。 1981年Bennun等對Lewis大鼠實驗性自身免疫性腦脊髓炎(Experimentalautoimmune encephalomyelitis，EAE)的研究時，從發(fā)病鼠淋巴組織分離到一株髓鞘堿性蛋白(Myelin basic protein，MBP)特異性的自身反應(yīng)性T細胞。該細胞輸給正常大鼠可誘發(fā)EAE，但若在轉(zhuǎn)輸前用放射線照射或用絲裂霉素處理的方法滅火該細胞，則受鼠反而獲得對MBP免疫誘導(dǎo)的EAE的抵抗力。此時第一次提出T細胞疫苗(T cell vaccination)的概念，即用滅活或低于致病劑量的自身反應(yīng)性T細胞或其T細胞受體(T cell receptor,TCR)多肽作為疫苗接種，誘導(dǎo)或觸發(fā)調(diào)節(jié)網(wǎng)絡(luò)產(chǎn)生特異性抑制作用，從而清除自身反應(yīng)性T細胞，達到防治自身免疫性疾病的作用。近年來T細胞疫苗與自身免疫性疾病的關(guān)系得到廣泛研究，T細胞疫苗在自身免疫性疾病中的應(yīng)用也得到廣泛關(guān)注。在多發(fā)性硬化、類風(fēng)濕性關(guān)節(jié)炎、器官、組織移植術(shù)后，自身免疫性甲狀腺炎，免疫性心肌炎等疾病的臨床研究中均取得了令人滿意的結(jié)果，為治療難治性自身免疫性疾病的治療提供了新思路。本課題即運用T細胞疫苗免疫的方法對SLE動物模型BXSB鼠進行試驗性治療，分析有關(guān)實驗室指標(biāo)變化，旨在為T細胞疫苗臨床治療系統(tǒng)性紅斑狼瘡提供實驗依據(jù)。 目的 1、用BXSB鼠脾細胞制備T細胞疫苗。 2、觀察T細胞疫苗免疫前后BXSB鼠24小時尿蛋白定量、血清ANA抗體、ds-DNA抗體水平的動態(tài)變化，從而探討T細胞疫苗免疫在一定時間內(nèi)對BXSB鼠病情的影響。 3、觀察T細胞疫苗免疫前后BXSB鼠血清IL-18水平動態(tài)變化，從而探討T細胞疫苗免疫在一定時間內(nèi)對BXSB鼠免疫狀態(tài)影響的有關(guān)機制。 方法 1、實驗動物 雄性BXSB鼠12只。隨機分為2組。A組為2只，用來制備T細胞疫苗。B組10只，實驗組，用來觀察T細胞疫苗對BXSB鼠的影響。 2、BXSB鼠脾臟淋巴細胞分離： 無菌條件下將BXSB鼠脾臟取下，制成脾細胞懸液，加入淋巴細胞分離液，離心，取淋巴細胞，用Hanks液清洗2次，最后用RPMI1640培養(yǎng)液調(diào)整細胞濃度到1×10~8／ml。臺盼蘭染法鑒定活細胞數(shù)95％。 3、T細胞疫苗的制備 用完全培養(yǎng)液調(diào)整細胞至2×10~5／ml，加伴刀豆球蛋白A(ConA)混合后移置5％CO_237℃孵箱中培養(yǎng)48h后收集細胞，然后加入絲裂霉素C(mitomycin C)處理細胞，PBS洗滌后，用RPMI1640培養(yǎng)液調(diào)整細胞至1×10~8／ml備用。 4、T細胞疫苗皮下免疫： 取1×10~7細胞皮下免疫BXSB鼠，每周1次，連續(xù)3次，并于免疫前和首次免疫后第1w、2w、4w觀察有關(guān)指標(biāo)變化。 5、觀察指標(biāo)及檢測方法： 于免疫前和首次免疫后第1w、2w、4w將BXSB鼠斷尾取血，分離血清，ELISA法測血清ANA抗體、ds-DNA抗體和IL-18水平。用代謝籠收集BXSB鼠24小時尿量，考馬斯量藍染色法檢測24小時尿蛋白定量。 結(jié)果 1、T細胞疫苗免疫前后BXSB鼠24小時尿蛋白定量差異有顯著意義(F=22．177，P=0．000)，呈遞減趨勢。免疫后1w、2w、4w均低于免疫前。免疫后1w與免疫前無顯著差異(P=0．519)，而免疫后2w和4w水平顯著低于免疫前和免疫后1w(P0．05)，且免疫后4w水平低于免疫后2w(P=0．037)。 2、T細胞疫苗免疫前后BXSB鼠血清ANA抗體水平差異有顯著意義(F=15．757，P=0．000)，呈下降趨勢。免疫后1w、2w、4w均低于免疫前。免疫后1w與免疫前無顯著差異(P=0．317)，而免疫后2w和4w水平明顯低于免疫前和免疫后1w(P0．05)，但兩者之間無顯著差異(P=0．072)。 3、T細胞疫苗免疫前后BXSB鼠血清ds-DNA抗體水平有顯著差異(F=16．735，P=0．000)，呈下降趨勢。免疫后1w、2w、4w均低于免疫前。免疫后1w與免疫前無顯著差異(P=0．569)，而免疫后2w和4w水平顯著低于免疫前和免疫后1w(P0．05)，但兩者之間無顯著差異(P=0．056)。 4、T細胞疫苗免疫前后BXSB鼠血清IL-18水平差異有顯著性意義(F=34．583，P=0．000)，呈遞減趨勢。免疫后1w、2w、4w均低于免疫前。免疫后1w與免疫前無顯著差異(P=0．130)，而免疫后2w和4w水平顯著低于免疫前和免疫后1w(P0．05)，免疫后4w水平繼續(xù)降低，顯著低于免疫后2w(P=0．006)。 5、首次免疫后4w，其中1只BXSB鼠死亡。 結(jié)論 1、T細胞疫苗免疫BXSB鼠后2周其24小時尿蛋白定量、自身抗體水平即開始下降，免疫后4w仍維持較低水平。從而推測T細胞疫苗在一定時間內(nèi)可抑制BXSB鼠B細胞活化，減少自身抗體的產(chǎn)生，減輕腎臟損傷，延緩病情發(fā)展。 2、T細胞疫苗免疫BXSB鼠后2周其血清IL-18水平開始顯著降低，免疫后4w繼續(xù)降低。提示T細胞疫苗在調(diào)節(jié)免疫細胞及細胞因子的相互聯(lián)系中有重要作用。 3、T細胞疫苗治療BXSB鼠有一定的有效性和安全性。為T細胞疫苗的臨床應(yīng)用提供了實驗依據(jù)。
[Abstract]:Research background
Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is a common autoimmune disease involving multiple systems and organs of the whole body. The pathogenesis is complex, clinical variable, high incidence of secondary infection and difficulty in diagnosis and treatment. It is a disease which is seriously harmful to human health. It is widely distributed all over the world and the prevalence rate in the world. It is about 70 / 100 thousand -100 / 100 thousand. If we calculate 1 billion 300 million people in China, the number of SLE patients in China is up to 112-160, and most of them are in young women.
The incidence of SLE is related to a variety of factors. It is generally believed that the complex effects of sex hormones, infections, drugs and the environment cause the disorder of immune regulation, which lead to the occurrence of SLE, which is not clear and difficult to cure. In the past 20 years, with the rapid development of modern medicine The 10 year survival rate of SLE has reached 92%, and the prognosis is obviously improved. However, glucocorticoid combined with immunosuppressive drugs is still the first choice for the treatment of SLE. The general case adopts a small dose of glucocorticoid after the treatment of large dose of glucocorticoid, and the use of hormone for acute critical SLE patients. Combined use of cytotoxic drugs (mainly cyclophosphamide and azathioprine) when the effect is not satisfactory. The use of this routine treatment can make most patients stable, but with a large amount of medicine and long time, the side effects of the drug can not be ignored, such as induced or aggravated infection, diabetes, peptic ulcer, perforation or gastrointestinal bleeding. Period application can cause hypertension and atherosclerosis, fracture and osteonecrosis of bone, psychosis, Cushing's syndrome, acne, and hairy, and more than.60% in the final death of hormone side effects and / or complications. It seriously affects the life and quality of life of the patients.
Therefore, the search for new treatment methods of SLE has always been a hot spot at home and abroad. Large dose immunoglobulin shock therapy, plasma exchange therapy, etc., can make the disease be temporarily relieved, also because it can not be used for etiological treatment, and can not be widely used. The new immunosuppressive agents include B cell therapy, anti CD20 anti monoclonal antibody treatment. The treatment, T cell therapy, anti cytokine therapy, and complement therapy are mostly in the experimental stage of animal experiment. The exact therapeutic effects of the treatment are still confirmed by a large number of clinical trials. In recent years, the treatment of hematopoietic stem cells has achieved encouraging results, but high risk and technical requirements are high, and some patients are not tolerated at the mobilization stage. It is very expensive and difficult to promote.
Since SLE is an autoimmune disease characterized by T and B lymphocyte dysfunction, the activation of its own reactive T lymphocyte and its mediated autoimmune reaction are the central link of this disease. The study found that the autoantigen responsive T fine cell (help T cell, Th) increased during the pathogenesis of SLE. From the clone of lupus mice The T cell line can provide assistance to B cells to identify the T cell antigen epitopes in the nucleosomes of these T cell clones that produce IgG anti DNA antibody.50%. In the Th cell clone of human and mouse SLE, the TCR alpha chain identifying the nucleosome epitopes can combine with different TCR beta chains to form a TCR alpha beta two peptide chain and identify a variety of antigen epitopes. A variety of B cell clones contribute to the production of a variety of autoantibodies. In the process of SLE migration, T cells are repeatedly activated by their own antigens, which increase the mutation rate, and the mutant T cells can induce their own response to B cells to produce anti DNA antibodies. These antibodies combine with the corresponding autoantigens and form immune complexes, deposited in the glomerular basement membrane, and the liver A variety of organs and tissues such as dirty, central nervous system, small vessel wall, activating complement, polymorphonuclear leukocytes and platelets, causing local inflammation and small vasculitis, causing vascular obstruction, and showing a variety of clinical symptoms. It is possible to control the development of SLE by inhibiting the activation of the T lymphocyte and controlling the development of SLE. It may be ideal for the treatment of SLE. Method.
In the study of the experimental autoimmune encephalomyelitis (Experimentalautoimmune encephalomyelitis, EAE) of Lewis rats in 1981, Bennun isolated from the lymphoid tissue of the infected rat to a specific self reactive T fine cell of the myelin alkaline protein (Myelin basic protein, MBP). This cell could induce EAE, but if it was transferred to the normal rats When the cells were put out by radiation or treated with mitomycin, the mice received the resistance to EAE induced by MBP. At this time, the concept of the T cell vaccine (T cell vaccination) was first proposed, that is, the self reactive T fine cell or its T cell receptor (T cell receptor, TCR) peptides with inactivated or lower than the pathogenic dose. Vaccination, inducing or triggering the regulatory network to produce specific inhibitory effects, thus scavenging the self reactive T cells and achieving the role of preventing autoimmune diseases. In recent years, the relationship between T cell vaccine and autoimmune diseases has been widely studied. The application of T cell vaccine in autoimmune diseases has also been widely concerned. The clinical studies of multiple sclerosis, rheumatoid arthritis, organ and tissue transplantation, autoimmune thyroiditis, immune myocarditis and other diseases have obtained satisfactory results and provide new ideas for the treatment of refractory autoimmune diseases. This topic is the use of T cell vaccine immunization to the SLE animal model. BXSB rats were treated with experimental treatment, and the changes of laboratory indicators were analyzed. The aim was to provide experimental evidence for the treatment of systemic lupus erythematosus with T cell vaccine.
objective
1, T cell vaccine was prepared by BXSB mouse splenocytes.
2, to observe the 24 hour urine protein quantitative, serum ANA antibody and ds-DNA antibody level of BXSB mice before and after immunization of T cell vaccine, and to explore the effect of T cell vaccine immunization on the condition of BXSB mice in a certain time.
3, the dynamic changes of IL-18 level in serum of BXSB mice were observed before and after the immunization of T cell vaccine, and the mechanism of the effect of T cell vaccine immunization on the immune status of BXSB mice in a certain period of time was discussed.
Method
1, experimental animals
A total of 12 male BXSB rats were randomly divided into 2 groups, 2 in group.A, which were used to prepare 10 T cell vaccine group.B, and the experimental group was used to observe the effect of T cell vaccine on BXSB rats.
2, splenic lymphocyte separation in BXSB rats:
Under the aseptic condition, the spleen of BXSB rat was taken down to make the splenic cell suspension, add the lymphocyte separation liquid, centrifuge, take the lymphocyte and clean the cell with Hanks solution for 2 times. Finally, the cell concentration was adjusted by the RPMI1640 culture solution to the 1 x 10~8 / ml. table and the number of living cells was identified by the trypan blue staining method.
3, preparation of T cell vaccine
The cells were adjusted to 2 x 10~5 / ml with complete culture medium and mixed with concanavin A (ConA) and then transferred to 5%CO_237 centigrade incubator to collect the cells. Then the cells were treated with mitomycin C (mitomycin C). After the PBS was washed, the cells were adjusted to 1 * 10~ 8 / reserve by the RPMI1640 culture solution.
4, subcutaneous immunization of T cell vaccine:
1 BXSB 10~7 cells were subcutaneously immunized with BXSB mice 1 times a week for 3 consecutive times, and the changes of the indexes were observed before and after 1W, 2W and 4W after the first immunization.
5, observation index and detection method:
The blood was taken from the tail of BXSB rats before and after the first immunization, 1W, 2W, and 4W. Serum ANA, ds-DNA antibody and IL-18 level were measured by ELISA. The urine volume of BXSB rats was collected for 24 hours by metabolic cage, and the urine protein was measured by Kaumas blue staining method for 24 hours.
Result
1, before and after immunization of T cell vaccine, the quantitative difference of 24 hours urine protein in 24 hours was significant (F=22.177, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between 1W and immune before immunization (P=0.519), while the 2W and 4W levels after immunization were significantly lower than those before and after immunization, and the level of immune 1W was lower than that of immunization. After immunization, 2W (P=0.037).
2, there was a significant difference in the level of ANA antibody in serum of BXSB mice before and after immunization with T cell vaccine (F=15.757, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.317), but the 2W and 4W levels were significantly lower than those before and after immunization, but there was no significant difference between them. Difference (P=0.072).
3, the level of ds-DNA antibody in serum of BXSB mice before and after immunization with T cell vaccine was significantly different (F=16.735, P=0.000), and decreased. 1W, 2W, 4W were lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.569), but 2W and 4W levels were significantly lower than those before and after immunization, but there was no significant difference between the two groups. P=0.056).
4, there was a significant difference in the serum IL-18 level of BXSB mice before and after immunization with T cell vaccine (F=34.583, P=0.000), which decreased. 1W, 2W, 4W were all lower than before immunization. There was no significant difference between immune 1W and pre immunization (P=0.130), while the 2W and 4W levels after immunization were significantly lower than those before and after immunization, and the level of immune after immunization continued to decrease. It was significantly lower than 2W (P=0.006) after immunization.
5, after the first immunization of 4W, 1 of the BXSB mice died.
conclusion
1, T Cell Vaccine Immunized BXSB mice for 24 hours after 2 weeks of urine protein quantitative, the level of autoantibody began to decrease, and the 4W remained low after immunization. Thus, it was suggested that T cell vaccine could inhibit the activation of B cells in BXSB mice, reduce the production of autoantibodies, reduce the injury of kidney and postpone the development of the disease.
2, the serum IL-18 level of BXSB mice was significantly reduced after immunization with T cell vaccine for 2 weeks, and the 4W continued to decrease after immunization. It suggested that the T cell vaccine played an important role in regulating the interaction of immune cells and cytokines.
3, T cell vaccine is effective and safe in the treatment of BXSB mice. It provides experimental evidence for the clinical application of T cell vaccine.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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