本文選題：α1-抗胰蛋白酶 + β細(xì)胞移植��； 參考：《暨南大學(xué)》2013年碩士論文

【摘要】：目的：利用人AAT（α1-抗胰蛋白酶）基因?qū)胍葝uβ細(xì)胞構(gòu)建NIT-hAA系，將NIT-hAA移植到7周齡的雌性NOD小鼠左腎包膜下，利用環(huán)磷酰胺促進(jìn)糖尿病發(fā)病，觀察hAAT是否有效阻止胰島自身免疫破壞和對(duì)移植β細(xì)胞免疫保護(hù)的作用。 方法：已成功構(gòu)建NIT-hAAT細(xì)胞系，Western Blotting技術(shù)鑒定hAAT在NIT－1細(xì)胞能穩(wěn)定表達(dá)。將NIT-hAAT細(xì)胞和NIT－1細(xì)胞分別移植到7周齡未發(fā)病的雌性NOD小鼠左腎包膜下，腹腔注射環(huán)磷酰胺促進(jìn)糖尿病發(fā)病。首先，進(jìn)行血糖水平動(dòng)態(tài)監(jiān)測(cè)、葡萄糖耐量實(shí)驗(yàn)、統(tǒng)計(jì)糖尿病發(fā)病時(shí)間和發(fā)病率，評(píng)估NIT-hAAT移植能否有效阻止NOD小鼠糖尿病發(fā)��；其次，在不同時(shí)間點(diǎn)對(duì)NOD鼠胰腺、移植部位進(jìn)行病理觀察，并用TUNEL檢測(cè)凋亡胰島細(xì)胞，觀察NIT-hAAT移植對(duì)β細(xì)胞免疫保護(hù)的作用；最后，采用ELISA動(dòng)態(tài)檢測(cè)小鼠血清hAAT濃度、小鼠血清Th1細(xì)胞因子（TNF-α、IFN-γ）與Th2細(xì)胞因子（IL-4、IL-10）水平，流式細(xì)胞術(shù)分析小鼠脾淋巴細(xì)胞Treg/Th17水平，分析hAAT對(duì)移植的β細(xì)胞免疫保護(hù)的作用機(jī)制。 結(jié)果：在NIT-hAAT移植后21天內(nèi)，小鼠體內(nèi)hAAT水平維持在164.33-340.33μg/ml之間，hAAT可推遲NOD小鼠糖尿病發(fā)病時(shí)間和降低發(fā)病率。病理觀察發(fā)現(xiàn)hAAT明顯減輕自身胰島細(xì)胞炎癥浸潤(rùn)，降低胰島炎癥評(píng)級(jí)，減少胰島細(xì)胞凋亡；同時(shí)，，明顯減少移植部位β細(xì)胞炎癥浸潤(rùn)。在免疫保護(hù)機(jī)制方面，hAAT通過(guò)增加Treg細(xì)胞數(shù)量和減少Th17細(xì)胞數(shù)量；上調(diào)Th2細(xì)胞因子和下調(diào)Th1細(xì)胞因子，減輕NOD小鼠體內(nèi)炎癥反應(yīng)。hAAT可抑制自身免疫對(duì)自體胰島免疫破壞和移植β細(xì)胞免疫排斥，具有雙重免疫保護(hù)作用。但遺憾的是，NIT-hAAT細(xì)胞分泌hAAT的量在移植14天后開始下降，49天時(shí)已無(wú)法檢測(cè)。移植后期，自體胰島與移植β細(xì)胞被免疫破壞，細(xì)胞凋亡數(shù)量增加，移植部位炎性細(xì)胞浸潤(rùn)逐漸增加，同時(shí)，Treg細(xì)胞數(shù)量與Th2細(xì)胞因子水平逐漸下降，Th17細(xì)胞數(shù)量與Th1細(xì)胞因子水平逐漸上升，在移植后49d后自體胰島細(xì)胞和移植β細(xì)胞被免疫完全破壞，血糖水平和糖尿病發(fā)病率近似NIT-1組與糖尿病水平。 結(jié)論：NIT-hAAT移植后可表達(dá)hAAT，在短期內(nèi)明顯改善NOD小鼠體內(nèi)炎癥反應(yīng)，抑制自身免疫對(duì)自體胰島細(xì)胞和移植β細(xì)胞的破壞，發(fā)揮對(duì)自身胰島和移植細(xì)胞的雙重免疫保護(hù)作用。但在移植后期由于hAAT表達(dá)量不足，無(wú)法誘導(dǎo)形成長(zhǎng)期免疫耐受，不能完全阻止自身免疫對(duì)胰島細(xì)胞破壞和機(jī)體對(duì)移植β細(xì)胞免疫排斥雙重作用。
[Abstract]:Objective: to construct a NIT-hAA cell line by introducing human AAT- 偽 1-antitrypsin gene into islet 尾 cells and transplant NIT-hAA into the left renal capsule of 7-week-old female NOD mice. Cyclophosphamide (cyclophosphamide) was used to promote the pathogenesis of diabetes mellitus. To observe whether hAAT can effectively prevent islet autoimmune damage and protect 尾-cells from transplantation. Methods: NIT-hAAT cell line was successfully constructed to identify the stable expression of hAAT in NIT-1 cells by Western Blotting. NIT-hAAT cells and NIT-1 cells were transplanted into the left renal capsule of 7 week-old female NOD mice, and cyclophosphamide was injected intraperitoneally to promote the onset of diabetes. First, blood glucose levels were dynamically monitored, glucose tolerance test was performed to calculate the onset time and incidence of diabetes, and to evaluate whether NIT-hAAT transplantation could effectively prevent diabetes in NOD mice. Secondly, pancreas of NOD mice was treated at different time points. The transplantation site was observed pathologically, the apoptotic islet cells were detected by TUNEL, and the protective effect of NIT-hAAT transplantation on 尾 -cell immunity was observed. Finally, the serum hAAT concentration of mice was dynamically detected by ELISA. The levels of Th1 cytokine TNF- 偽 (IFN- 緯) and Th2 cytokine (IL-4 / IL-10) in serum of mice were determined by flow cytometry, and the level of Treg/Th17 in spleen lymphocytes of mice was analyzed by flow cytometry, and the mechanism of hAAT on the immunological protection of transplanted 尾 cells was analyzed. Results: within 21 days after NIT-hAAT transplantation, maintaining the level of hAAT between 164.33-340.33 渭 g/ml in mice could delay the onset of diabetes and reduce the incidence of diabetes in NOD mice. Pathological observation showed that hAAT could obviously reduce the inflammatory infiltration of islet cells, reduce the inflammatory grade of islet cells and decrease the apoptosis of islet cells, at the same time, the inflammatory infiltration of 尾 cells in transplantation site was significantly reduced. In immune protection mechanism, hAAT increased the number of Treg cells and reduced the number of Th17 cells, upregulated Th2 cytokines and down-regulated Th1 cytokines. Attenuating inflammation in NOD mice, hAAT can inhibit autoimmune damage to autoimmune islets and immune rejection of transplanted 尾 cells, which has double immune protective effect. Unfortunately, the amount of hAAT secreted by NIT-hAAT cells began to decrease 14 days after transplantation and could not be detected at 49 days after transplantation. In the late stage of transplantation, autogenous islets and transplanted 尾 cells were destroyed by immunity, the number of apoptotic cells increased, and the infiltration of inflammatory cells gradually increased. At the same time, the number of Treg cells and the level of Th2 cytokines decreased gradually. The number of Th17 cells and the level of Th1 cytokines increased gradually, and the autogenous islet cells and transplanted 尾 cells were completely destroyed after 49 days of transplantation. The level of blood sugar and the incidence of diabetes were similar to those of NIT-1 group and diabetes mellitus group. ConclusionWhen NIT-hAAT can express hAAT. in a short period of time, it can obviously improve the inflammatory reaction in NOD mice, inhibit the destruction of autologous islet cells and transplanted 尾 cells by autoimmunity, and play a dual protective effect on autoimmune islets and transplanted cells. However, at the late stage of transplantation, due to the insufficient expression of hAAT, it was unable to induce long-term immune tolerance and could not completely prevent the destruction of islet cells by autoimmunity and the immune rejection of transplanted 尾 cells.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392.11

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