未成熟树突状细胞诱导糖尿病小鼠异种胰岛细胞移植免疫耐受的研究
发布时间：2018-10-08 20:10
【摘要】： 糖尿病是严重威胁人类生命与健康的全球性多发病,胰岛素替代治疗不能阻止其并发症的出现,而β细胞替代治疗(包括胰腺移植和胰岛细胞移植)能达到生理性调控胰岛素分泌的目的,并维持正常血糖。但是移植物排斥反应始终影响移植效果,甚至导致移植胰岛失功。树突状细胞(dendritic cell,DC)的外周致耐受作用通常由未成熟树突状细胞(immature dendritic cell,imDC)介导,由于imDC不表达共刺激分子,当imDC携带自身抗原移行至外周淋巴组织后不能使T细胞活化,从而诱导T细胞无能,导致自身耐受。基于这一理论,建立小鼠糖尿病模型,经外周循环注射负载供体抗原的小鼠imDC诱导异种免疫耐受后,移植大鼠胰岛细胞,观察糖尿病小鼠移植物存活时间和移植物排斥反应。 方法:1.不同STZ剂量建立糖尿病动物模型:将36只雄性,6～8周SPF级的BALB/c小鼠完全随机分为G_1(对照组)、G_2、G_3和G_4组,进行糖尿病小鼠建模试验,分别用柠檬酸-柠檬酸钠缓冲液和80mg/kg、200mg/kg、240mg/kg三种浓度的STZ进行腹腔注射,监测小鼠血糖浓度,分析建模成功率和生存率,确定最佳建模剂量。 2.imDC提取和培养:将20只雄性,6～8周SPF级的BALB/c小鼠,取股骨、胫骨冲洗骨髓,冲洗液经淋巴细胞分离液离心,提取单核细胞。单核细胞经洗涤后置入含20%胎牛血清的RPMI1640培养基中,在37℃,5%CO2条件下培养,第1、3、5天分别加入细胞因子IL-4、GM-CSF。第7天收取贴壁细胞,标定单克隆抗体,经流式细胞仪鉴定细胞数量及imDC比例。 3.胰岛细胞提取和纯化移植:30只雌性,8～10周SPF级Wistar大鼠,无菌取胰腺并经胶原酶适度消化,消化液经密度梯度离心分离纯化出胰岛细胞。用DTZ和AO-PI染色分别鉴定胰岛细胞的形态和存活率。 4.imDC预处理和胰岛细胞移植:将40只BALB/c小鼠完全随机分为T_1、T_2、T_3、T_4组,建立糖尿病模型。其中T_1组为糖尿病对照组,肾被膜下注射等量RPMI1640液;T_2组为单纯胰岛细胞移植组,肾被膜下移植异种胰岛细胞;T_3组和T_4组为经imDC预处理后异种胰岛细胞移植组,外周静脉注射供体骨髓细胞培养扩增的imDC后经肾被膜下胰岛细胞移植。监测各组糖尿病小鼠实验干预后血糖、体重及皮毛等变化。 结果:1.小鼠经腹腔注射不同剂量(80mg/kg、200mg/kg、240mg/kg)STZ诱导糖尿病模型的成功率分别为:0%,77.78%和71.43%;200mg/kg和240mg/kg STZ腹腔注射组诱导糖尿病模型小鼠实验观察期内的生存率分别为:85.71%和40%。 2.小鼠骨髓来源细胞经低剂量GM-CSF联合IL-4扩增出的细胞具有典型DC的形态特征,并且高度表达imDC的表面分子,基本不表达成熟DC的表面分子。 3.每只Wistar大鼠胰腺可分离纯化出胰岛细胞约533.33个。胰岛细胞的纯化达到80%,存活率为95%。 4.T_1组糖尿病小鼠的血糖浓度始终维持在糖尿病水平,体重随时间延长而下降,且部分小鼠病程中出现皮毛污秽粘连、高渗性昏迷,感染等表现;T_2组小鼠经肾被膜下异种胰岛细胞移植后血糖浓度很快恢复至正常水平,体重上升,但是术后5天血糖开始再次升高,体重随之下降,表明单纯胰岛细胞移植排斥反应强烈,胰岛细胞短期内失功;T_3组和T_4组小鼠经外周静脉注射经供体抗原修饰的自身骨髓细胞培养分化的imDC后行肾被膜下异种胰岛细胞移植,移植后小鼠血糖浓度在术后第2天左右恢复正常,并且血糖浓度维持在正常水平的时间较T_2组明显延长,体重未见明显下降。 结论:1.一次性腹腔注射STZ 200mg/kg诱导BALB/c小鼠的糖尿病模型成功率和生存率明显优于STZ 80mg/kg和STZ 240mg/kg注射组。 2.小鼠骨髓细胞经体外低剂量细胞因子诱导,可扩增分化充足的imDC。 3.异种胰岛细胞移植术前注射imDC可明显延长糖尿病小鼠移植术后血糖浓度维持正常水平的时间,和单纯胰岛细胞移植组相比可明显减轻移植排斥反应,延长移植物存活时间。
[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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.4
本文编号：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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.4
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