本文選題：凋亡細(xì)胞 + 免疫耐受�。� 參考：《第一軍醫(yī)大學(xué)》2007年博士論文

【摘要】： 研究背景 胰島移植重建胰島素分泌系統(tǒng)，是一種有望徹底根治糖尿病的治療方法。2000年，Edmonton方案的成功，標(biāo)志著胰島移植取得了突破性的進(jìn)展，從而帶動了胰島移植臨床試驗性治療在世界范圍內(nèi)廣泛展開。但是免疫排斥以及免疫抑制劑所帶來的副作用和潛在危險性仍是難以克服的障礙。目前普遍認(rèn)為，解決異體移植排斥反應(yīng)的關(guān)鍵在于誘導(dǎo)受體對供體的細(xì)胞、組織或器官產(chǎn)生免疫耐受。由于移植免疫耐受的機(jī)制十分復(fù)雜，盡管誘導(dǎo)免疫耐受的方法眾多，但均未能達(dá)到完全可靠持久的特異性耐受。細(xì)胞凋亡是生物體內(nèi)普遍存在的一種生理和病理現(xiàn)象，是正常器官和組織發(fā)育、清除有害的、過量的和無功能細(xì)胞、維持自身穩(wěn)態(tài)的必要環(huán)節(jié)。現(xiàn)有研究表明：凋亡是機(jī)體免疫狀態(tài)保持平衡和穩(wěn)定的重要作用機(jī)制，凋亡細(xì)胞對免疫系統(tǒng)存在著主動的調(diào)節(jié)作用。凋亡細(xì)胞能分泌脂類趨化因子，改變自身胞膜結(jié)構(gòu)表達(dá)“eat-me”信號，誘導(dǎo)吞噬細(xì)胞對其進(jìn)行清除；同時凋亡細(xì)胞被抗原提呈細(xì)胞吞噬后，通過吞噬細(xì)胞分泌抑制性免疫因子如TGF-β、PGE_2、IL-10等，造成了特殊的抗原識別微環(huán)境，可以促使相關(guān)淋巴細(xì)胞針對凋亡抗原產(chǎn)生免疫耐受，而不會引起炎性免疫應(yīng)答。據(jù)此，孫爾維等提出利用供體凋亡細(xì)胞輸注受體誘導(dǎo)供體特異性免疫耐受的理論設(shè)想。我們課題組以前的工作證實，凋亡細(xì)胞在體外對T淋巴細(xì)胞活化增殖有直接抑制作用；凋亡細(xì)胞預(yù)輸注可以顯著延長大鼠心臟移植模型、肝移植模型存活時間。因此，我們擬應(yīng)用糖尿病大鼠模型進(jìn)行同種異體胰島移植，以供體凋亡脾淋巴細(xì)胞靜脈輸注的方法，觀察胰島移植物的存活時間，了解凋亡細(xì)胞調(diào)節(jié)免疫反應(yīng)過程參與的淋巴細(xì)胞類型、細(xì)胞因子及分子基礎(chǔ)，探求延長胰島移植物存活的方法。 目的 初步探討凋亡細(xì)胞靜脈輸注誘導(dǎo)胰島移植免疫耐受的方法及作用機(jī)制，為尋找誘導(dǎo)胰島移植免疫耐受的方法開辟新的途徑，提供新的依據(jù)。 方法 1、直線加速器照射對體外培養(yǎng)大鼠脾細(xì)胞凋亡的影響：采用研磨法獲得Wistar大鼠脾淋巴細(xì)胞懸液。將脾細(xì)胞懸液加入細(xì)胞培養(yǎng)瓶，分四組，A組為對照組，B、C、D組應(yīng)用Varian醫(yī)用直線加速器照射，各組吸收劑量分別為：1.5Gy、2.0Gy、3.0Gy。各組細(xì)胞處理后置于37℃，5％CO_2恒溫培養(yǎng)箱培養(yǎng)。分別在培養(yǎng)后4h、8h、12h應(yīng)用流式細(xì)胞儀檢測細(xì)胞凋亡率； 2、大鼠胰島細(xì)胞的分離純化：采用膠原酶P胰管灌注消化，短期低溫培養(yǎng)后Ficoll-400不連續(xù)梯度純化Wistar大鼠胰島； 3、建立糖尿病動物模型：以STZ(56mg／kg·體重)經(jīng)腹腔一次性注射，制備SD大鼠實驗性糖尿病模型(連續(xù)2次血糖＞16.7mmol／L)： 4、供體凋亡細(xì)胞輸注對糖尿病胰島移植物的影響：以雄性Wistar大鼠為供體，雄性SD大鼠為受體。將糖尿病SD大鼠隨機(jī)分為Hank's液注射(A組)、正常供體脾細(xì)胞預(yù)輸注(B組)、供體凋亡脾細(xì)胞預(yù)輸注(C組)及供體壞死脾細(xì)胞預(yù)輸注(D組)四組。各組在預(yù)處理7天后行胰島移植，將1000IEQ的胰島細(xì)胞移植入各組糖尿病大鼠的腎包囊內(nèi)，比較各組移植物生存時間上的差異。同時各實驗組分別在預(yù)處理后第7天(即胰島移植術(shù)前)、胰島移植術(shù)后7天、14天、排斥后應(yīng)用放射免疫法測定大鼠血清胰島素水平。供體凋亡脾細(xì)胞預(yù)輸注糖尿病SD大鼠則在不同的移植時期(包含血糖正常期、排斥期兩個時期)手術(shù)將移植物載體腎取出；其余各實驗組在糖尿病SD大鼠移植物排斥后2天，取出其移植物載體腎，做胰島素的免疫組化檢測。 5、供體凋亡細(xì)胞輸注對胰島移植糖尿病大鼠混合淋巴細(xì)胞反應(yīng)的影響：將糖尿病SD大鼠按上述方法分組，各組分別于預(yù)處理后第7天(即胰島移植術(shù)前)、胰島移植術(shù)后7天、14天、排斥后取大鼠脾臟，分離淋巴細(xì)胞，與絲裂霉素C處理的供體Wistar大鼠和無關(guān)第三品系F344／N大鼠脾淋巴細(xì)胞進(jìn)行單向混合淋巴細(xì)胞培養(yǎng)，MTT法觀察受體淋巴細(xì)胞增殖反應(yīng)； 6、供體凋亡細(xì)胞輸注對胰島移植糖尿病大鼠T淋巴細(xì)胞亞群及CD4~+CD25~+T細(xì)胞的影響：將糖尿病SD大鼠按上述方法分組，各組分別于預(yù)處理后第7天(即胰島移植術(shù)前)、胰島移植術(shù)后7天、14天、排斥后取外周血及大鼠脾臟制備的淋巴細(xì)胞懸液，應(yīng)用流式細(xì)胞儀檢測CD8~+、CD4~+、CD4~+CD25~+／CD4~+T淋巴細(xì)胞比率。 7、供體凋亡細(xì)胞輸注對胰島移植糖尿病大鼠細(xì)胞因子微環(huán)境的影響：將糖尿病SD大鼠按上述方法分組，各組分別于預(yù)處理后第7天(即胰島移植術(shù)前)、胰島移植術(shù)后7天、14天、排斥后取外周血清及大鼠脾組織標(biāo)本，應(yīng)用Luminex多功能流式點(diǎn)陣儀液相芯片法檢測IL-2、IFN-γ、IL-4、IL-10含量；同時應(yīng)用酶聯(lián)免疫吸附法檢測外周血清TGF-β1含量。 結(jié)果 1、直線加速器高能X線照射Wistar大鼠脾細(xì)胞，吸收劑量1.5Gy組于4h后開始有明顯細(xì)胞凋亡發(fā)生，而后隨時間推移凋亡率增加。2.0Gy組于4h后即有顯著凋亡發(fā)生，至8h達(dá)到凋亡高峰(61.17±3.70)％(P＜0.050)，但于12h凋亡率略有下降。3.0Gy組凋亡率無顯著升高，而壞死率明顯增加。 2、每條Wistar大鼠胰腺經(jīng)分離、純化后平均可獲取的胰島細(xì)胞團(tuán)數(shù)量為(1063.91±84.74)IEQ，DTZ染色顯示純度為(70.51±6.20)％。高糖刺激時胰島B細(xì)胞胰島素的釋放量為低糖刺激時的2.72倍； 3、以STZ(120mg／kg·體重)一次性經(jīng)腹腔注射的方法，可成功誘發(fā)SD大鼠的試驗性糖尿病，糖尿病模型建模成功率為94％； 4、供體凋亡脾細(xì)胞預(yù)輸注組糖尿病SD大鼠胰島移植物生存時間與其它三組比較具有顯著性差異(P＜0.050)，中位生存時間(MST)為(31.00±6.41)天，最長達(dá)42天。正常脾細(xì)胞輸注組移植物生存時間也有所延長，MST為(12.00±2.97)天(P＜0.050)。預(yù)輸注Hank's液及壞死細(xì)胞組則無此效應(yīng)，MST分別為(6.00±1.67)、(6.00±1.10)天； 5、供體預(yù)輸注凋亡脾細(xì)胞組移植后7天、14天糖尿病大鼠血清胰島素水平較移植前顯著增高，分別達(dá)(19.26±4.50)mIU／ml、(20.70±6.33)mIU／ml(P＜0.050)，排斥后胰島素水平回落。正常細(xì)胞組移植后7天胰島素水平亦明顯增高(P＜0.050)，14天時則明顯下降，而預(yù)輸注Hank's液及壞死細(xì)胞組則無明顯變化。 6、供體凋亡細(xì)胞預(yù)輸注組胰島移植物在受體血糖正常時取出做組織切片，經(jīng)Insulin免疫組織化學(xué)染色證實有表達(dá)胰島素陽性的胰島細(xì)胞團(tuán)存在，呈棕褐色。而在被排斥后取出的移植物進(jìn)行免疫組化檢測，則未發(fā)現(xiàn)胰島素陽性的胰島細(xì)胞團(tuán)存在。輸注Hank's液組、輸注供體正常細(xì)胞或壞死細(xì)胞組的移植物在被排后取出行Insulin免疫組化染色檢測，亦無胰島素陽性細(xì)胞存在。 7、供體凋亡細(xì)胞預(yù)輸注后7天SD大鼠的脾細(xì)胞對絲裂霉素C處理的Wistar大鼠脾細(xì)胞的增殖反應(yīng)較接受Hank's液輸注組、供體正常脾細(xì)胞組及供體壞死脾細(xì)胞組明顯降低(P＜0.050)，直至移植后7天、14天抑制效應(yīng)仍為顯著，排斥后則明顯升高(P＜0.050)；且與無關(guān)第三品系F344／N大鼠脾細(xì)胞的增殖反應(yīng)比較，兩者差異明顯，移植前、移植后7、14天各時間點(diǎn)相互比較均有顯著性差異(P＜0.050) 8、各處理組糖尿病大鼠接受胰島移植后，無論外周血還是脾臟組織CD4~+T細(xì)胞和CDS~+T細(xì)胞比例均顯著升高(P＜0.050)，各組間無明顯差異(P＞0.050)。 9、供體凋亡細(xì)胞預(yù)輸注后7天，糖尿病大鼠外周血和脾臟CD4~+CD25~+T細(xì)胞比例均明顯高于預(yù)輸注Hank's液組、供體正常細(xì)胞組及供體壞死細(xì)胞組，分別占CD4~+T細(xì)胞的(11.49±1.29)％、(12.44±1.63)％(P均＜0.050)；移植后7天、14天仍保持較高比例，排斥后則有所下降。 10、胰島移植前各實驗組糖尿病大鼠外周血和脾臟組織IL-2水平均無顯著性差異(P＞0.050)。移植后各組均明顯升高，但供體凋亡細(xì)胞輸注組在移植后7天、14天的IL-2水平明顯低于Hank's液輸注組、供體正常細(xì)胞組及供體壞死細(xì)胞組，有顯著性差異(P＜0.050)；排斥后明顯升高。 11、胰島移植前各實驗組糖尿病大鼠外周血和脾臟組織IFN-γ水平亦無顯著性差異(P＞0.050)。移植后各組均明顯升高，但移植后7天、14天，供體凋亡細(xì)胞輸注組與Hank's液輸注組、供體正常細(xì)胞組及供體壞死細(xì)胞組相比水平較低，有顯著性差異(P＜0.050)。排斥后則升高顯著。 12、胰島移植前各實驗組糖尿病大鼠外周血和脾臟組織IL-4水平無顯著性差異(P＞0.050)；移植后7天、14天直至排斥后預(yù)輸注Hank's液組、供體正常細(xì)胞組、供體凋亡細(xì)胞組及供體壞死細(xì)胞組IL-4水平均無明顯改變。 13、輸注供體凋亡細(xì)胞后7天，糖尿病大鼠外周血和脾臟組織IL-10水平顯著升高；直至移植后7天、14天仍明顯高于其它各組，有顯著性差異(P＜0.050)；移植物排斥后則降低。同時，接受Hank's液預(yù)輸注組、供體正常細(xì)胞組及供體壞死細(xì)胞組無明顯變化。 14、輸注供體凋亡細(xì)胞后7天，，糖尿病大鼠血清TGF-β1水平明顯升高，并在胰島移植后7，14天仍可維持較高水平(P＜0.050)；移植物排斥后TGF-β1水平下降。而其余Hank's液預(yù)輸注組、供體正常細(xì)胞組及供體壞死細(xì)胞組無明顯變化。 結(jié)論 1、直線加速器高能X線照射能在體外簡便安全有效誘導(dǎo)大鼠脾細(xì)胞凋亡。 2、采用膠原酶P胰管灌注消化，短期低溫培養(yǎng)后Ficoll-400不連續(xù)梯度純化能分離、純化出較大數(shù)量且功能良好的大鼠胰島細(xì)胞。 3、預(yù)輸注供體凋亡細(xì)胞能顯著延長同種異體大鼠胰島移植物的存活時間。應(yīng)用供體凋亡細(xì)胞誘導(dǎo)胰島移植免疫耐受是一條可行的途徑。 4、以單向混合淋巴細(xì)胞培養(yǎng)實驗證實，預(yù)輸注供體凋亡細(xì)胞的受體鼠淋巴細(xì)胞對供體淋巴細(xì)胞刺激的增殖反應(yīng)具有明顯抑制作用。 5、供體凋亡細(xì)胞預(yù)輸注能顯著升高胰島移植糖尿病大鼠外周血和脾臟CD4~+CD25~+T細(xì)胞比例。 6、供體凋亡細(xì)胞預(yù)輸注可使胰島移植糖尿病大鼠血清和脾臟IL-2、IFN-γ呈現(xiàn)較低水平，而顯著升高IL-10、TGF-β水平，IL-4則無明顯改變，提示供體凋亡細(xì)胞輸注誘導(dǎo)胰島移植免疫耐受可能包含調(diào)節(jié)Th1／Th2、Th3平衡，糾正免疫偏離的作用；并且更為重要的機(jī)制可能是，Tr、Tr1等尤其是CD4~+CD25~+T調(diào)節(jié)性淋巴細(xì)胞的參與。
[Abstract]:Research background
Islet transplantation to reconstruct the insulin secretion system is a promising cure for diabetes in.2000 years. The success of the Edmonton scheme marks a breakthrough in islet transplantation, leading to extensive clinical trials of islet transplantation in the world. However, immune rejection and immunosuppressive agents have been carried out. The side effects and potential risks are still difficult to overcome. It is widely believed that the key to the solution of allograft rejection is to induce the receptor to produce immune tolerance to the cells, tissues or organs of the donor. The mechanism of the transplantation immune tolerance is very complex, although there are many methods to induce immune tolerance, but they have not been achieved. Cell apoptosis is a common physiological and pathological phenomenon in living organisms. It is a necessary link in normal organs and tissue development, eliminating harmful, excessive and non functional cells and maintaining homeostasis. The existing study shows that apoptosis is an important role in maintaining balance and stability of the immune state of the body. The apoptotic cells can regulate the immune system actively. The apoptotic cells can secrete the chemotactic factor of lipid, change the cell membrane structure to express "eat-me" signal and induce the phagocyte to scavenging it. At the same time, the apoptotic cells are phagocyted by the antigen presenting cells and through the phagocytic cells secreting the inhibitory immune factors such as TGF- beta, PG E_2, IL-10 and so on, cause a special antigen recognition microenvironment, which can induce the related lymphocyte to produce immune tolerance against apoptotic antigen, without causing inflammatory response. Accordingly, sun Erwei put forward the theory of using donor apoptotic cell infusion receptor to induce donor specific immune tolerance. In fact, the apoptotic cells have a direct inhibitory effect on the proliferation of T lymphocytes in vitro, and the preinfusion of apoptotic cells can significantly prolong the rat model of heart transplantation and the survival time of the liver transplantation model. Therefore, we should use the diabetic rat model for allogenic islet transplantation with the method of intravenous infusion of donor apoptotic splenic lymphocytes. To investigate the survival time of the islet graft, understand the lymphocyte types, cytokines and molecular basis of the apoptotic cells regulating the immune response process, and explore the methods of prolonging the survival of the islet grafts.
objective
In order to find a new way to induce the immune tolerance of islet transplantation, this paper provides a new basis for finding the immune tolerance of islet transplantation induced by apoptotic cell vein infusion.
Method
1, the effect of linear accelerator irradiation on the apoptosis of splenocytes of rats in vitro: the splenic lymphocyte suspension of Wistar rats was obtained by grinding method. Splenic cell suspension was added into cell culture bottle, four groups were added, group A was used as control group, B, C, and D group were irradiated with Varian medical linear accelerator. The absorption doses of each group were 1.5Gy, 2.0Gy and 3.0Gy. in each group, respectively. The cells were incubate at 37 C and incubate in 5%CO_2 incubator. The apoptosis rate was detected by flow cytometry after 4h, 8h and 12h respectively.
2, isolation and purification of rat islet cells: using collagenase P pancreatic duct perfusion and digestion, after short-term hypothermia culture, Ficoll-400 was purified from Wistar rat islets in discontinuous gradient.
3, establish an animal model of diabetes: an experimental diabetic model of SD rats (2 consecutive blood glucose > 16.7mmol / L) was prepared by intraperitoneal injection of STZ (56mg / kg / weight):
4, the effect of donor apoptotic cell infusion on diabetic islet graft: male Wistar rats as donors and male SD rats as receptors. The diabetic SD rats were randomly divided into Hank's liquid injection (A group), normal donor splenocytes preinfusion (group B), donor apoptotic splenocytes preinfusion (C group) and donor necrotic splenocytes pre infusion (D group) in each group (group D). Each group was divided into four groups. The islet transplantation was performed 7 days after pretreatment, and the islet cells of 1000IEQ were transplanted into the renal cysts of each group of diabetic rats. The differences in the survival time of each group were compared. At the same time, the experimental groups were seventh days after pretreatment (before the islet transplantation), 7 days after the islet transplantation, 14 days after the islet transplantation, and the radioimmunoassay was used to determine the serum of rats after rejection. Insulin level. SD rats with donor apoptotic splenocytes pretransfused to diabetic rats during the different period of transplantation (including the normal period of blood glucose, two periods of rejection) to remove the graft carrier kidney; the rest of the experimental groups were taken out of the transplant carrier kidney for 2 days after the graft rejection of diabetic rats, and made the immunohistochemical detection of insulin.
5, the effect of donor apoptotic cells infusion on the mixed lymphocyte reaction in diabetic rats: diabetic SD rats were grouped according to the methods mentioned above, seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, spleenes were isolated from rats after rejection, and the donor Wis treated with mitomycin C Unidirectional mixed lymphocyte culture was carried out on tar rats and spleen lymphocytes from F344 / N rats without the third strain, and the proliferation of recipient lymphocytes was observed by MTT.
6, the effect of donor apoptotic cells infusion on T lymphocyte subsets and CD4~+CD25~+T cells in diabetic rats: diabetic SD rats were grouped according to the methods mentioned above, seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, and lymphocytic suspension prepared from peripheral blood and rat spleen. The ratio of CD8~+, CD4~+, CD4~+CD25~+ / CD4~+T lymphocyte was detected by flow cytometry.
7, the effect of donor apoptotic cells infusion on the microenvironment of cytokines in diabetic rats: groups of diabetic SD rats were grouped according to the above methods, each group was seventh days after preconditioning (before islet transplantation), 7 days after islet transplantation, 14 days after islet transplantation, after rejection, the peripheral blood and rat spleen tissue specimens were taken, and Luminex multifunction flow point was applied. The contents of IL-2, IFN-, IL-4 and IL-10 were detected by liquid phase microarray, and the content of TGF- beta 1 in peripheral serum was detected by enzyme-linked immunosorbent assay.
Result
1, the splenic cells of Wistar rats were irradiated by high energy X-ray in the linear accelerator. After the absorption of 1.5Gy group, there was obvious apoptosis in the group of 4h, and then the apoptosis rate increased in the.2.0Gy group after 4h, and reached the peak (61.17 + 3.70)% (P < 0.050) to the peak of apoptosis (P < 0.050), but the apoptosis rate of 12h decreased slightly in the.3.0Gy group. The rate of necrosis was significantly increased and the necrosis rate increased significantly.
2, the pancreas of each Wistar rat was separated, and the average number of islet cell masses was (1063.91 + 84.74) IEQ after purification, and the purity of DTZ staining showed (70.51 + 6.20)%. The release of insulin in islet B cells was 2.72 times as high as that of low glucose stimulation.
3, STZ (120mg / kg / body weight) by intraperitoneal injection can successfully induce experimental diabetes in SD rats, and the success rate of modeling is 94%.
4, the survival time of pancreatic islet graft in SD rats was significantly different from that of the other three groups (P < 0.050), the median survival time (MST) was (31 + 6.41) days and the longest was 42 days. The survival time of the allograft in the normal splenocytes infusion group was also prolonged, and MST was (12 + 2.97) days (P < 0.050). Pre infusion Hank There was no such effect in's solution and necrotic cell group, MST was (6 + 1.67), (6 + 1.10) days.
5, on the 7 day after donor pre infusion, the serum insulin level in the 14 day diabetic rats increased significantly (19.26 + 4.50) mIU / ml, (20.70 + 6.33) mIU / ml (P < 0.050) and the level of insulin after rejection. The insulin level in the normal cell group was also significantly higher (P < 0.050) on the 7 day after the transplantation (P < 0.050), and the level of insulin was obvious on the day of the transplantation. However, there was no significant change in pre infusion of Hank's and necrotic cells.
6, the pancreatic islet graft in the donor cell preinfusion group was taken out of the tissue section at the normal blood glucose level. The insulin positive islet cell group was found to be brown in the presence of Insulin immunohistochemical staining, and the insulin positive islet cells were not found in the graft after the rejection. In the Hank's solution group, the graft of the donor donor normal cell or the necrotic cell group was detected by Insulin immunohistochemical staining after being discharged, and no insulin positive cells existed.
7, the proliferation response of splenocytes in the spleen cells treated with mitomycin C on the spleen cells treated by mitomycin C was significantly lower than that of the Hank's liquid infusion group, the donor normal splenocytes group and the donor necrotic splenocytes group (P < 0.050) in the SD rats treated with mitomycin C (P < 0.050), and the inhibitory effect was still significant in the 14 day after the transplantation (P < < < < < < 0. 0.050) and compared with the proliferation reaction of spleen cells of F344 / N rats, the difference was obvious. Before transplantation, there were significant differences in each time point of 7,14 days after transplantation (P < 0.050).
8, the proportion of CD4~+T cells and CDS~+T cells in both peripheral and spleen tissues increased significantly (P < 0.050), and there was no significant difference between each group (P > 0.050).
9, the proportion of peripheral blood and spleen CD4~+CD25~+T cells in the diabetic rats was significantly higher than that of the pre infusion group 7 days after the apoptosis of the donor cells. The donor normal cell group and the donor necrotic cell group accounted for (11.49 + 1.29)% of the CD4~+T cells and (12.44 + 1.63)% (P < 0.050), respectively, and still kept a high proportion on the 14 day after the transplantation, and on the 14 day after the transplantation. Then it fell down.
10, there was no significant difference in the IL-2 level between the peripheral blood and the spleen in the experimental groups before the islet transplantation (P > 0.050). All groups were significantly higher after transplantation, but the IL-2 level of the donor apoptotic cell infusion group was significantly lower than that of the Hank's infusion group at 7 days after the transplantation, and the donor normal cell group and the donor necrotic cell group were significant. The difference was (P < 0.050), and the difference was significantly increased after rejection.
11, there was no significant difference in the level of IFN- gamma in the peripheral blood and spleen tissues of the experimental groups before the islet transplantation (P > 0.050). All groups were significantly higher after transplantation, but the donor group was compared with the Hank's liquid infusion group at 7 days and 14 days after transplantation, and there was a significant difference in the level of the donor normal cell group and the donor necrotic cell group. (P < 0.050). After rejection, the increase was significant.
12, there was no significant difference in the IL-4 level between the peripheral blood and the spleen tissues of the experimental groups before the islet transplantation (P > 0.050), and 7 days after the transplantation, 14 days after the transplantation and the pre transfusion group Hank's solution, the donor normal cell group, the donor apoptotic cell group and the donor necrotic cell group had no significant changes in the IL-4 water.
13, on the 7 day after infusion of donor apoptotic cells, the levels of IL-10 in peripheral blood and spleen tissues of diabetic rats were significantly higher than those of other groups until 7 days after transplantation. There was a significant difference (P < 0.050), while the graft rejection was reduced. At the same time, the group of donor normal cells and donor necrotic cells group were not clear. Change.
14, the level of serum TGF- beta 1 in diabetic rats was significantly increased at 7 days after infusion of donor cells, and still maintained a high level in 7,14 days after islet transplantation (P < 0.050), and the level of TGF- beta 1 decreased after graft rejection, but there was no significant change in the normal cell group and donor necrotic cell group in the rest of the Hank's solution group.
conclusion
1, high energy X-ray irradiation of linear accelerator can be used in vitro to induce apoptosis of rat spleen cells in a simple, safe and effective way.
2, the collagenase P pancreatic duct was perfused and digested. After short term low temperature culture, Ficoll-400 discontinuous gradient purification could be separated and the large number and well functioning rat islet cells were purified.
3, pre infusion of donor apoptotic cells can significantly prolong the survival time of allogeneic rat islet grafts.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R657.5;R392.4

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