未成熟樹突狀細胞誘導糖尿病小鼠異種胰島細胞移植免疫耐受的研究
發(fā)布時間：2018-10-08 20:10
【摘要】： 糖尿病是嚴重威脅人類生命與健康的全球性多發(fā)病,胰島素替代治療不能阻止其并發(fā)癥的出現,而β細胞替代治療(包括胰腺移植和胰島細胞移植)能達到生理性調控胰島素分泌的目的,并維持正常血糖。但是移植物排斥反應始終影響移植效果,甚至導致移植胰島失功。樹突狀細胞(dendritic cell,DC)的外周致耐受作用通常由未成熟樹突狀細胞(immature dendritic cell,imDC)介導,由于imDC不表達共刺激分子,當imDC攜帶自身抗原移行至外周淋巴組織后不能使T細胞活化,從而誘導T細胞無能,導致自身耐受。基于這一理論,建立小鼠糖尿病模型,經外周循環(huán)注射負載供體抗原的小鼠imDC誘導異種免疫耐受后,移植大鼠胰島細胞,觀察糖尿病小鼠移植物存活時間和移植物排斥反應。 方法:1.不同STZ劑量建立糖尿病動物模型:將36只雄性,6～8周SPF級的BALB/c小鼠完全隨機分為G_1(對照組)、G_2、G_3和G_4組,進行糖尿病小鼠建模試驗,分別用檸檬酸-檸檬酸鈉緩沖液和80mg/kg、200mg/kg、240mg/kg三種濃度的STZ進行腹腔注射,監(jiān)測小鼠血糖濃度,分析建模成功率和生存率,確定最佳建模劑量。 2.imDC提取和培養(yǎng):將20只雄性,6～8周SPF級的BALB/c小鼠,取股骨、脛骨沖洗骨髓,沖洗液經淋巴細胞分離液離心,提取單核細胞。單核細胞經洗滌后置入含20%胎牛血清的RPMI1640培養(yǎng)基中,在37℃,5%CO2條件下培養(yǎng),第1、3、5天分別加入細胞因子IL-4、GM-CSF。第7天收取貼壁細胞,標定單克隆抗體,經流式細胞儀鑒定細胞數量及imDC比例。 3.胰島細胞提取和純化移植:30只雌性,8～10周SPF級Wistar大鼠,無菌取胰腺并經膠原酶適度消化,消化液經密度梯度離心分離純化出胰島細胞。用DTZ和AO-PI染色分別鑒定胰島細胞的形態(tài)和存活率。 4.imDC預處理和胰島細胞移植:將40只BALB/c小鼠完全隨機分為T_1、T_2、T_3、T_4組,建立糖尿病模型。其中T_1組為糖尿病對照組,腎被膜下注射等量RPMI1640液;T_2組為單純胰島細胞移植組,腎被膜下移植異種胰島細胞;T_3組和T_4組為經imDC預處理后異種胰島細胞移植組,外周靜脈注射供體骨髓細胞培養(yǎng)擴增的imDC后經腎被膜下胰島細胞移植。監(jiān)測各組糖尿病小鼠實驗干預后血糖、體重及皮毛等變化。 結果:1.小鼠經腹腔注射不同劑量(80mg/kg、200mg/kg、240mg/kg)STZ誘導糖尿病模型的成功率分別為:0%,77.78%和71.43%;200mg/kg和240mg/kg STZ腹腔注射組誘導糖尿病模型小鼠實驗觀察期內的生存率分別為:85.71%和40%。 2.小鼠骨髓來源細胞經低劑量GM-CSF聯合IL-4擴增出的細胞具有典型DC的形態(tài)特征,并且高度表達imDC的表面分子,基本不表達成熟DC的表面分子。 3.每只Wistar大鼠胰腺可分離純化出胰島細胞約533.33個。胰島細胞的純化達到80%,存活率為95%。 4.T_1組糖尿病小鼠的血糖濃度始終維持在糖尿病水平,體重隨時間延長而下降,且部分小鼠病程中出現皮毛污穢粘連、高滲性昏迷,感染等表現;T_2組小鼠經腎被膜下異種胰島細胞移植后血糖濃度很快恢復至正常水平,體重上升,但是術后5天血糖開始再次升高,體重隨之下降,表明單純胰島細胞移植排斥反應強烈,胰島細胞短期內失功;T_3組和T_4組小鼠經外周靜脈注射經供體抗原修飾的自身骨髓細胞培養(yǎng)分化的imDC后行腎被膜下異種胰島細胞移植,移植后小鼠血糖濃度在術后第2天左右恢復正常,并且血糖濃度維持在正常水平的時間較T_2組明顯延長,體重未見明顯下降。 結論:1.一次性腹腔注射STZ 200mg/kg誘導BALB/c小鼠的糖尿病模型成功率和生存率明顯優(yōu)于STZ 80mg/kg和STZ 240mg/kg注射組。 2.小鼠骨髓細胞經體外低劑量細胞因子誘導,可擴增分化充足的imDC。 3.異種胰島細胞移植術前注射imDC可明顯延長糖尿病小鼠移植術后血糖濃度維持正常水平的時間,和單純胰島細胞移植組相比可明顯減輕移植排斥反應,延長移植物存活時間。
[Abstract]:Diabetes is a worldwide multi-morbidity which seriously threatens human life and health, and insulin replacement therapy can not prevent the occurrence of complications, while the replacement therapy of pancreatic islet cells (including pancreas transplantation and islet cell transplantation) can achieve the purpose of physiological regulation of insulin secretion. and maintain normal blood glucose. However, graft rejection will always affect the graft effect, and may even lead to graft loss. The peripheral induced tolerance of dendritic cells (DC) is usually mediated by immature dendritic cells (imDC). Since imDC does not express co-stimulatory molecules, T cells can not be activated after imDC carries its own antigen to peripheral lymphoid tissue, thus inducing T cell incompetence. resulting in self-tolerance. Based on this theory, the model of diabetic mice was established, and the islet cells were transplanted to observe the graft survival time and graft rejection in diabetic mice. Fang Methods: 1. Diabetic animal models were established with different STZ dosages: 36 male, 6-8 week SPF BALB/ c mice were randomly divided into G _ 1 (control group), G _ 2, G _ 3 and G _ 4 groups, and the model test of diabetic mice was carried out. citric acid-sodium citrate buffer and 80mg/ kg, 200mg were used respectively. Three concentrations of STZ were injected intraperitoneally to monitor the blood glucose concentration of mice, analyze the modeling success rate and survival rate, and determine the best construction. Module dose: 2. imDC extraction and culture: 20 male, 6-8 week SPF BALB/ c mice, femur, tibia flush bone marrow, rinse liquid was centrifuged by lymphocyte separation solution extracting monocytes and monocytes, washing, placing in RPMI1640 culture medium containing 20% fetal bovine serum, culturing at 37 & deg; C, 5% CO2, adding cytokines IL-4 respectively on Days 1, 3 and 5, GM-CSF, adherent cells were charged on day 7, monoclonal antibodies were calibrated, and the number of cells was identified by flow cytometry and imDC ratio. 3. Islet cell extraction and purification transplantation: 30 females, 8-10 weeks of SPF Wistar rats, aseptically removed the pancreas and moderately digested with collagenase, and the digestive juice was subjected to density gradient. Islet cells were purified by centrifugation and identified by DTZ and AO-PI staining respectively. Morphology and survival of islet cells. 4. imDC pretreatment and islet cell transplantation: 40 BALB/ c mice were randomly divided into T _ 1, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T 3, T _ 4 groups were used to establish the model of diabetes mellitus. The T _ 1 group was a diabetic control group and the renal was injected with the same amount of RPMI1640 liquid under the membrane; the T _ 2 group was a simple islet cell transplantation group; the kidney was transplanted with different islet cells under the membrane; the T _ 3 group and the T _ 4 group were im DC pre-treated xenogeneic islet cell transplantation group, peripheral vein injection donor bone marrow cell culture amplification i After mDC, the islet cells were transplanted through the kidney under the membrane, and the mice of each group were monitored. blood after intervention Results: 1. The success rate of STZ-induced diabetic model induced by different doses (80mg/ kg, 200mg/ kg, 240mg/ kg) was 0%, 77. 78% and 71. 43%, respectively. The experimental observation period of diabetic model mice was induced by intraperitoneal injection of 200mg/ kg and 240mg/ kg STZ. The survival rates were 85. 71% and 40%, respectively. The cells of bone marrow derived from mouse were characterized by low dose of GM-CSF and IL-4, and highly expressed i a surface molecule of mDC that does not substantially express the surface molecules of mature DC. 3. Each W Isolation and purification of pancreatic islet cells from pancreatic islet cells in Sprague-Dawley rats 33. 33. The purification of islet cells reached 80%, and the survival rate was 95%. The blood glucose concentration of diabetic mice in group T _ 1 was always maintained at the level of diabetes, and the body weight prolonged with time. In addition, the blood glucose concentration in T _ 2 group was restored to normal level and body weight increased, but the blood glucose began to increase again at 5 days after operation, and the body weight increased again. The results showed that islet cell transplantation rejection was very strong and islet cells lose power in the short term; T _ 3 group and T _ 4 group mice were injected with donor antigen modified autologous bone marrow cells to culture differentiated imDC after intravenous injection of imDC. After transplantation of islet cells, the blood glucose concentration in mice returned to normal at about 2 days after transplantation and blood sugar concentration maintenance Conclusion: 1. The diabetic model of BALB/ c mice was induced by STZ 200mg/ kg in one-time intraperitoneal injection. Power and survival rates were significantly better than STZ 80mg/ kg and STZ 240mg/ kg injection Group. 2. Bone marrow cells of mice were induced by low dose cytokines in vitro, and sufficient imDC could be amplified. The injection of imDC before transplantation of xenogeneic islet cells could significantly prolong the blood glucose concentration after transplantation in diabetic mice.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392.4
本文編號：2258108
[Abstract]:Diabetes is a worldwide multi-morbidity which seriously threatens human life and health, and insulin replacement therapy can not prevent the occurrence of complications, while the replacement therapy of pancreatic islet cells (including pancreas transplantation and islet cell transplantation) can achieve the purpose of physiological regulation of insulin secretion. and maintain normal blood glucose. However, graft rejection will always affect the graft effect, and may even lead to graft loss. The peripheral induced tolerance of dendritic cells (DC) is usually mediated by immature dendritic cells (imDC). Since imDC does not express co-stimulatory molecules, T cells can not be activated after imDC carries its own antigen to peripheral lymphoid tissue, thus inducing T cell incompetence. resulting in self-tolerance. Based on this theory, the model of diabetic mice was established, and the islet cells were transplanted to observe the graft survival time and graft rejection in diabetic mice. Fang Methods: 1. Diabetic animal models were established with different STZ dosages: 36 male, 6-8 week SPF BALB/ c mice were randomly divided into G _ 1 (control group), G _ 2, G _ 3 and G _ 4 groups, and the model test of diabetic mice was carried out. citric acid-sodium citrate buffer and 80mg/ kg, 200mg were used respectively. Three concentrations of STZ were injected intraperitoneally to monitor the blood glucose concentration of mice, analyze the modeling success rate and survival rate, and determine the best construction. Module dose: 2. imDC extraction and culture: 20 male, 6-8 week SPF BALB/ c mice, femur, tibia flush bone marrow, rinse liquid was centrifuged by lymphocyte separation solution extracting monocytes and monocytes, washing, placing in RPMI1640 culture medium containing 20% fetal bovine serum, culturing at 37 & deg; C, 5% CO2, adding cytokines IL-4 respectively on Days 1, 3 and 5, GM-CSF, adherent cells were charged on day 7, monoclonal antibodies were calibrated, and the number of cells was identified by flow cytometry and imDC ratio. 3. Islet cell extraction and purification transplantation: 30 females, 8-10 weeks of SPF Wistar rats, aseptically removed the pancreas and moderately digested with collagenase, and the digestive juice was subjected to density gradient. Islet cells were purified by centrifugation and identified by DTZ and AO-PI staining respectively. Morphology and survival of islet cells. 4. imDC pretreatment and islet cell transplantation: 40 BALB/ c mice were randomly divided into T _ 1, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T _ 2, T 3, T _ 4 groups were used to establish the model of diabetes mellitus. The T _ 1 group was a diabetic control group and the renal was injected with the same amount of RPMI1640 liquid under the membrane; the T _ 2 group was a simple islet cell transplantation group; the kidney was transplanted with different islet cells under the membrane; the T _ 3 group and the T _ 4 group were im DC pre-treated xenogeneic islet cell transplantation group, peripheral vein injection donor bone marrow cell culture amplification i After mDC, the islet cells were transplanted through the kidney under the membrane, and the mice of each group were monitored. blood after intervention Results: 1. The success rate of STZ-induced diabetic model induced by different doses (80mg/ kg, 200mg/ kg, 240mg/ kg) was 0%, 77. 78% and 71. 43%, respectively. The experimental observation period of diabetic model mice was induced by intraperitoneal injection of 200mg/ kg and 240mg/ kg STZ. The survival rates were 85. 71% and 40%, respectively. The cells of bone marrow derived from mouse were characterized by low dose of GM-CSF and IL-4, and highly expressed i a surface molecule of mDC that does not substantially express the surface molecules of mature DC. 3. Each W Isolation and purification of pancreatic islet cells from pancreatic islet cells in Sprague-Dawley rats 33. 33. The purification of islet cells reached 80%, and the survival rate was 95%. The blood glucose concentration of diabetic mice in group T _ 1 was always maintained at the level of diabetes, and the body weight prolonged with time. In addition, the blood glucose concentration in T _ 2 group was restored to normal level and body weight increased, but the blood glucose began to increase again at 5 days after operation, and the body weight increased again. The results showed that islet cell transplantation rejection was very strong and islet cells lose power in the short term; T _ 3 group and T _ 4 group mice were injected with donor antigen modified autologous bone marrow cells to culture differentiated imDC after intravenous injection of imDC. After transplantation of islet cells, the blood glucose concentration in mice returned to normal at about 2 days after transplantation and blood sugar concentration maintenance Conclusion: 1. The diabetic model of BALB/ c mice was induced by STZ 200mg/ kg in one-time intraperitoneal injection. Power and survival rates were significantly better than STZ 80mg/ kg and STZ 240mg/ kg injection Group. 2. Bone marrow cells of mice were induced by low dose cytokines in vitro, and sufficient imDC could be amplified. The injection of imDC before transplantation of xenogeneic islet cells could significantly prolong the blood glucose concentration after transplantation in diabetic mice.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392.4
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