【摘要】：[目的]系統(tǒng)性紅斑狼瘡(Systemic Lupus Erythematosus,SLE)是一種病因及發(fā)病機(jī)制上不明確,累及多器官?gòu)?fù)雜的自身免疫性疾病。SLE最常見(jiàn)的臨床表現(xiàn)是狼瘡性腎炎(lupus nephritis,LN),在SLE死亡病例中占到60%～70%,狼瘡性腎炎的病因和發(fā)病機(jī)制尚不十分清楚,目前認(rèn)為細(xì)胞因子網(wǎng)絡(luò)失衡或細(xì)胞因子異常表達(dá)是其發(fā)病的重要因素之一。間充質(zhì)干細(xì)胞(MSC)治療LN有一定效果。多項(xiàng)實(shí)驗(yàn)提示血或尿中骨橋蛋白(OPN)的表達(dá)和LN損傷程度及病情變化有著密切關(guān)系,對(duì)LN有重要的診斷和療效判斷價(jià)值。本課題的目的將探討臍帶組織來(lái)源間充質(zhì)干細(xì)胞(UC-MSC)治療LN的臨床療效和相關(guān)作用機(jī)制;探究骨橋蛋白(OPN)在UC-MSC治療LN過(guò)程中的作用及相關(guān)機(jī)制。[方法]1.將13例確診為L(zhǎng)N且WHO分型為Ⅲ/Ⅳ型的患者給予HUC-MSC或安慰劑替代治療(方案通過(guò)了倫理委員會(huì)),應(yīng)用ELISA測(cè)定兩組LN患者治療后2、6月血清中OPN、AP-1、Sp-1、NF-KB、MMP-2和MMP-9在蛋白水平的變化。2. (1)將24只22周齡雌性MRL/lpr小鼠隨機(jī)分為實(shí)驗(yàn)組(輸注標(biāo)記多聚賴(lài)氨酸修飾的超順磁性氧化鐵 SPIO-PLL (super paramagnetic iron oxide polylysine)的HUC-MSCs)和對(duì)照組(未輸注HUC-MSCs), MRI掃描觀察治療后UC-MSCs分布情況;(2)觀察兩組MRL/lpr小鼠在處理前后不同時(shí)間點(diǎn)體重、尿蛋白及相關(guān)免疫學(xué)指標(biāo)變化;(3)用攜帶目的基因綠色熒光蛋白GFP (green fluorescent protein)的慢病毒感染靶細(xì)胞,進(jìn)行人臍帶干細(xì)胞標(biāo)記,注射進(jìn)入MRL/lpr狼瘡小鼠體內(nèi),觀察UC-MSCs移植對(duì)MRL/lpr小鼠腎臟OPN表達(dá)影響。3.將24只22周齡雌性MRL/lpr小鼠隨機(jī)分為兩組:對(duì)照組和UC-MSCs治療組(治療組第18周進(jìn)行1X106 P3代UC-MSCs尾靜脈注射,對(duì)照組注射PBS)。12只22周齡的正常C57BL/6小鼠作為正常對(duì)照組。應(yīng)用RT-qPCR,western-blot,免疫組化的方法檢測(cè)HUC-MSCs移植治療MRL/lpr狼瘡小鼠前后與對(duì)照組腎臟組織中OPN、AP-1和Sp-1的表達(dá)水平,探索OPN的表達(dá)與AP-1和Sp-1相關(guān)性,同時(shí)檢測(cè)NF-KB,MMP-2和MMP-9的mRNA水平和蛋白水平,并探索與OPN表達(dá)的相關(guān)性。[結(jié)果]1. HUC-MSC移植治療LN患者2個(gè)月后,血清中OPN,MMP-2, MMP-9的表達(dá)水平與對(duì)照組相比均表現(xiàn)出降低。HUC-MSC移植治療LN患者6個(gè)月后,治療組OPN,MMP-2和MMP-9的表達(dá)水平與對(duì)照組相比,顯著降低,差異均具有顯著性(P0. 05)。2.血清中轉(zhuǎn)錄因子AP-1, SP-1和NF-κB/P65的表達(dá)水平:治療后6個(gè)月,與治療后2個(gè)月相比,治療組的AP-1, SP-1和NF-κB的表達(dá)水平均有下降趨勢(shì)。3. MRL/lpr狼瘡小鼠HUC-MSC移植治療后對(duì)比發(fā)現(xiàn),治療組小鼠24 h尿蛋白量,血清中抗ds-DNA抗體滴度,抗核抗體(ANA)滴度均顯著降低,體重有明顯增加。4. HUC-MSC移植能顯著降低OPN、MMP-2和MMP-9在MRL/lpr小鼠血清中的表達(dá)(P0. 05)。血清中轉(zhuǎn)錄因子AP-1、SP-1和NF-κ B/p65的表達(dá)水平顯著降低(P0. 05)。5. HUC-MSC移植可顯著降低MRL/lpr狼瘡小鼠腎臟組織中OPN,MMP-2, MMP-9及轉(zhuǎn)錄因子AP-1,SP-1和NF- κ B在蛋白及RNA的表達(dá)水平。6. HUC-MSC移植治療顯著降低MRL/lpr狼瘡小鼠在腎小球系膜細(xì)胞、腎小管中OPN,MMP-2, MMP-9及轉(zhuǎn)錄因子AP-1,SP-1和NF-κB的表達(dá)水平。[結(jié)論]HUC-MSC 移植可有效降低 LN 患者血清中 OPN, AP-1, SP-1,NF-κB/P65, MMP-2和MMP-9的表達(dá)水平,對(duì)LN患者治療有一定作用;HUC-MSC移植治療MRL/lpr狼瘡性小鼠腎炎有一定療效,治療組血清中OPN的表達(dá)量顯著降低,血清中轉(zhuǎn)錄因子AP-1、SP-1、MMP-2、MMP-9和NF-κB/p65的表達(dá)在蛋白和RNA水平降低,在狼瘡小鼠在腎小球系膜細(xì)胞、腎小管中表達(dá)也顯著降低。提示HUC-MSC移植治療狼瘡腎炎可能是通過(guò)降低轉(zhuǎn)錄因子AP-1、SP-1和NF-κB的表達(dá),抑制OPN的表達(dá),OPN表達(dá)的減少抑制了 NF-κB通路的活化,進(jìn)而降低通路下游MMP-2和MMP-9的表達(dá),減少腎臟的損傷。該研究為HUC-MSC移植治療LN提供了一定的依據(jù)。
[Abstract]:[Objective] Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease involving multiple organs with unclear etiology and pathogenesis. The most common clinical manifestation of SLE is lupus nephritis (LN), which accounts for 60% to 70% of SLE deaths. The etiology and pathogenesis of lupus nephritis are still unknown. It is not clear that the imbalance of cytokine network or abnormal expression of cytokines is one of the important factors in the pathogenesis of LN. Mesenchymal stem cells (MSC) have a certain effect on the treatment of LN. A number of experiments have suggested that the expression of osteopontin (OPN) in blood or urine is closely related to the degree of LN injury and the changes of LN condition. It is important for the diagnosis and treatment of LN. Objective: To investigate the clinical efficacy and mechanism of umbilical cord tissue-derived mesenchymal stem cells (UC-MSC) in the treatment of LN, and to explore the role and mechanism of osteopontin (OPN) in the treatment of LN by UC-MSC. [Methods] 1. HUC-MSC or placebo replacement were given to 13 patients with LN and WHO type III/IV. The levels of OPN, AP-1, Sp-1, NF-KB, MMP-2 and MMP-9 in serum of two groups of LN patients were measured by ELISA. 2. UC-MSCs of IC iron oxide polylysine group and control group (not infused with HUC-MSCs), the distribution of UC-MSCs was observed by MRI scan after treatment; (2) The changes of body weight, urinary protein and related immunological indexes were observed before and after treatment in two groups of MRL/lpr mice; (3) The slowness of GFP carrying the target gene green fluorescent protein (GFP) was observed. Human umbilical cord stem cells were labeled and injected into MRL/lpr lupus mice to observe the effect of UC-MSCs transplantation on OPN expression in kidney of MRL/lpr mice. Twelve 22-week-old normal C57BL/6 mice were injected with PBS as normal control group.The expression of OPN,AP-1 and Sp-1 in renal tissues of HUC-MSCs transplantation treated MRL/lpr mice and control group were detected by RT-qPCR,Western-blot and immunohistochemistry. The expression levels of OPN, MMP-2 and MMP-9 in serum of patients with LN after HUC-MSC transplantation were lower than those of the control group 2 months later. After 6 months of HUC-MSC transplantation, the expression levels of OPN, MMP-2 and MMP-9 in the treatment group were significantly lower than those in the control group. Serum levels of transcription factors AP-1, SP-1 and NF-kappa B/P65: 6 months after treatment, compared with 2 months after treatment, the expression levels of AP-1, SP-1 and NF-kappa B in the treatment group showed a downward trend. 3. Compared with HUC-MSC transplantation in lupus mice treated with MRL/lpr, 24 hours urinary protein and serum protein levels in the treatment group were found. HUC-MSC transplantation significantly reduced the expression of OPN, MMP-2 and MMP-9 in the serum of MRL/lpr mice (P 0.05). The expression levels of transcription factors AP-1, SP-1 and NF-kappa B/p65 in the serum were significantly decreased (P 0.05). 5. HUC-MSC transplantation significantly reduced the expression of MRL/lpr wolf. Expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in renal tissues of mice with lupus erythematosus. 6. HUC-MSC transplantation significantly decreased the expression of OPN, MMP-2, MMP-9 and transcription factors AP-1, SP-1 and NF-kappa B in glomerular mesangial cells, renal tubules of mice with lupus erythematosus. The expression levels of OPN, AP-1, SP-1, NF-kappa B/P65, MMP-2 and MMP-9 in LN patients have a certain effect on the treatment of LN patients; HUC-MSC transplantation has a certain effect on the treatment of lupus nephritis in MRL/lpr mice; the expression level of OPN in serum of treatment group is significantly reduced, and the expression of transcription factors AP-1, SP-1, MMP-2, MMP-9 and NF-kappa B/p65 in protein and RNA. These results suggest that HUC-MSC transplantation may reduce the expression of transcription factors AP-1, SP-1 and NF-kappa B, inhibit the expression of OPN. The decrease of OPN expression inhibits the activation of NF-kappa B pathway, thereby reducing the expression of MMP-2 and MMP-9 downstream of the pathway. This study provides a basis for HUC-MSC transplantation in the treatment of LN.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R593.242

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