本文選題：子宮內(nèi)膜再生細(xì)胞 + 缺血再灌注損傷��； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：背景:腎臟缺血再灌注損傷(Renal Ischemia Reperfusion Injury,IRI)是影響臨床急性腎損傷的主要病因,經(jīng)常發(fā)生在腎移植、腎臟手術(shù)、慢性腎疾病、腎外傷中,在臨床上十分常見。目前已進(jìn)行了大量相關(guān)研究,但是發(fā)病率、死亡率仍然很高,尚無有效的預(yù)防及治療手段。間充質(zhì)干細(xì)胞(Mesenchymal stem cells,MSCs)是來源于中胚層的具有高度自我更新能力和多向分化潛能的多能干細(xì)胞,存在于多種組織,可在體外培養(yǎng)擴(kuò)增,并可在特定條件下誘導(dǎo)分化為神經(jīng)細(xì)胞、成骨細(xì)胞、軟骨細(xì)胞、心肌細(xì)胞、脂肪細(xì)胞等,是細(xì)胞替代治療和組織工程的種子細(xì)胞,具有廣闊的臨床應(yīng)用前景。大量研究表明,MSCs能降低腎損傷,保護(hù)腎功能。人子宮內(nèi)膜再生細(xì)胞(ERC)是一種新型來源的MSC,目前研究發(fā)現(xiàn)其具有保護(hù)缺血壞死心肌、促進(jìn)燒傷組織修復(fù)及減輕潰瘍性結(jié)腸炎腸道病變,而關(guān)于ERC對(duì)腎缺血再灌注損傷的影響尚未見報(bào)道,以及ERC是否通過何種途徑發(fā)揮抑炎及免疫調(diào)控,進(jìn)而有效減輕腎缺血再灌注損傷,保護(hù)腎功能還有待于進(jìn)一步研究。目的:利用小鼠腎缺血再灌注損傷模型,探討子宮內(nèi)膜再生細(xì)胞對(duì)腎缺血再灌注損傷的治療作用及機(jī)理。方法:(1)應(yīng)用密度梯度離心法分離培養(yǎng)人子宮內(nèi)膜再生細(xì)胞,觀察細(xì)胞形態(tài)。(2)選取C57BL/6小鼠,雄性,8周齡,體重約20-25g,隨機(jī)分為3組,每組6只:假手術(shù)組(Sham組)、缺血再灌注損傷組(IRI-untreated組)、ERC治療組(ERC-treated組),缺血再灌注前2小時(shí)經(jīng)尾靜脈輸注1×106 ERCs(0.25mlPBS重懸細(xì)胞)。建立腎IRI模型,行雙側(cè)腎蒂微血管夾夾閉30min,去除微血管夾關(guān)腹,再灌注48h。(3)術(shù)后48h處死小鼠,留取全血、腎組織、脾臟,保存待檢測(cè)。全自動(dòng)生化分析儀檢測(cè)血清肌酐(Cr)和尿素氮(BUN)水平,評(píng)價(jià)腎功能;HE染色觀察腎臟組織病理改變,評(píng)價(jià)腎病理損傷程度;免疫組化染色觀察腎臟CD3+T細(xì)胞和中性粒細(xì)胞(Ly6G+)浸潤(rùn)情況;ELISA檢測(cè)全血細(xì)胞因子TNF-α,IFN-γ,IL-4和IL-6水平,以評(píng)估炎癥反應(yīng)情況;流式細(xì)胞分析檢測(cè)脾臟調(diào)節(jié)性T細(xì)胞(Tregs)、CD4+和CD8+T細(xì)胞;流式檢測(cè)腎臟CD3+T細(xì)胞、F4/80+巨噬細(xì)胞、CD206+細(xì)胞、髓系抑制細(xì)胞(CD11B+Ly6C+和CD11B+Ly6G+),評(píng)估ERC對(duì)腎臟細(xì)胞群的影響。結(jié)果:1.應(yīng)用密度梯度離心法分離培養(yǎng)的ERC,顯微鏡下呈細(xì)胞呈梭形、紡錘形、成纖維細(xì)胞樣,體積大,貼壁生長(zhǎng),細(xì)胞生長(zhǎng)速度快,呈集落樣。2.iri-untreated組與sham組比較,血清肌酐、尿素氮水平顯著升高,腎病理損傷嚴(yán)重,腎結(jié)構(gòu)紊亂、腎小管壞死、間質(zhì)充血水腫;erc-treated組與iri-untreated組相比,血清肌酐、尿素氮水平明顯下降,腎功能顯著改善,腎病理損傷明顯減輕,腎小管壞死減少,無明顯間質(zhì)充血水腫。3.iri-untreated組與sham組比較,腎臟cd3+t細(xì)胞和ly6g+細(xì)胞浸潤(rùn)增多;erc-treated組與iri-untreated組相比,cd3+t細(xì)胞和ly6g+細(xì)胞浸潤(rùn)減少。4.iri-untreated組與sham組比較,流式檢測(cè)分析示:腎臟cd3+t細(xì)胞增多,ki67升高,t細(xì)胞增殖明顯增加;erc-treated組與iri-untreated組相比,cd3+t細(xì)胞顯著減少,ki67降低,t細(xì)胞增殖減弱,p0.05。5.iri-untreated組與sham組相比,脾臟、腎臟cd4+和cd8+t細(xì)胞比例顯著上升;erc-treated組與iri-untreated組相比,cd4+和cd8+t細(xì)胞比例明顯下降,p0.05。6.erc-treated組與iri-untreated組相比,脾臟cd4+cd25+tregs細(xì)胞比例顯著上調(diào),p0.05;iri-untreated組與sham組相比,cd4+cd25+tregs細(xì)胞水平相當(dāng),p0.05,差異無統(tǒng)計(jì)學(xué)意義。7.erc-treated組與iri-untreated組相比,cd11b+ly6c+和cd11b+ly6g+細(xì)胞比例顯著升高;iri-untreated組與sham組相比,cd11b+ly6c+和cd11b+ly6g+細(xì)胞輕度升高。8.流式檢測(cè)分析腎組織中巨噬細(xì)胞,iri-untreated組與sham組相比,f4/80+巨噬細(xì)胞浸潤(rùn)增加;erc-treated組與iri-untreated組比較,腎組織中f4/80+巨噬細(xì)胞浸潤(rùn)減少,但具有修復(fù)作用的cd206+細(xì)胞(m2巨噬細(xì)胞)比例升高,p0.05差異有統(tǒng)計(jì)學(xué)意義。9.erc-treated組與iri-untreated組相比,細(xì)胞因子tnf-α、ifn-γ和il-6明顯下降,而il-4水平上升p0.05;iri-untreated組與sham組相比,tnf-α、ifn-γ和il-6水平升高,p0.05差異有統(tǒng)計(jì)學(xué)意義。結(jié)論:1.成功利用小鼠腎缺血再灌注損傷模型,證實(shí)erc能夠有效改善腎功能,降低腎病理損傷。2.ercs減輕小鼠腎缺血再灌注損傷與下調(diào)腎臟cd3+、cd4+、cd8+t細(xì)胞和脾臟cd4+、cd8+t細(xì)胞比例有關(guān)。3.ercs上調(diào)脾臟tregs細(xì)胞繼而降低損傷程度促進(jìn)腎損傷修復(fù)。4.ERCs保護(hù)腎組織與下調(diào)腎臟F4/80+巨噬細(xì)胞,同時(shí)上調(diào)CD206+細(xì)胞(M2巨噬細(xì)胞)相關(guān)。5.ERCs減輕腎病理損傷,改善腎功能,與提高腎臟髓系抑制細(xì)胞CD11b+Ly6C+和CD11b+Ly6G+比例比例有關(guān)。6.ERCs通過影響體內(nèi)炎癥因子(TNF-α、IFN-γ、IL-4和IL-6)水平,降低炎癥反應(yīng),參與腎損傷修復(fù)。
[Abstract]:Background: Renal Ischemia Reperfusion Injury (IRI) is the main cause of clinical acute renal injury. It often occurs in kidney transplantation, kidney surgery, chronic renal disease, and renal trauma, which is very common in clinic. A large number of related studies have been carried out, but the incidence and mortality are still high and there is no effective. The means of prevention and treatment. Mesenchymal stem cells (MSCs) is a multipotent stem cell derived from the mesoderm with high self renewal and pluripotent differentiation potential. It exists in a variety of tissues and can be cultured in vitro, and can be induced into nerve cells, osteoblasts, chondrocytes, and myocardium under specific conditions. Cells and adipocytes, which are the seed cells of cell replacement therapy and tissue engineering, have broad prospects for clinical application. A large number of studies have shown that MSCs can reduce renal damage and protect renal function. Human endometrial regenerative cells (ERC) are a new source of MSC. The research has found that it has the protection of ischemic necrosis myocardium and the promotion of burn tissue. The effect of ERC on renal ischemia-reperfusion injury has not been reported, and the effect of ERC on anti-inflammatory and immune regulation is not yet reported, and the renal ischemia reperfusion injury is effectively alleviated, and the protection of renal function is still to be studied. The therapeutic effect and mechanism of endometrial regenerative cells on renal ischemia reperfusion injury were studied. Methods: (1) the density gradient centrifugation was used to isolate and culture human endometrium regenerative cells and observe cell morphology. (2) C57BL/6 mice were selected, male, 8 weeks old, weight about 20-25g, and were randomly divided into 3 groups: 6 group of sham operation group (group Sham), ischemia. Reperfusion injury group (group IRI-untreated), ERC treatment group (group ERC-treated), 1 x 106 ERCs (0.25mlPBS heavy cell) infusion through tail vein 2 hours before ischemia-reperfusion. Establish renal IRI model, bilateral renal pedicle microvascular clip clipping 30min, remove microvascular clips, and reperfusion after 48h. (3) operation 48h to kill mice, leaving whole blood, kidney tissue, spleen, preserving. The total automatic biochemical analyzer was used to detect serum creatinine (Cr) and urea nitrogen (BUN) levels and evaluate renal function; HE staining was used to observe pathological changes of kidney tissue and to evaluate the degree of renal pathological injury; immunohistochemical staining was used to observe the renal CD3+T cells and neutrophils (Ly6G+) immersion; ELISA was used to detect whole blood cytokines TNF- a, IFN- y, IL-4 and IL-6. Level to assess the inflammatory response; flow cytometry was used to detect spleen regulatory T cells (Tregs), CD4+ and CD8+T cells; flow cytometry was used to detect renal CD3+T cells, F4/80+ macrophages, CD206+ cells, myeloid suppressor cells (CD11B+Ly6C+ and CD11B+Ly6G+), and to evaluate the effect of ERC on renal cell groups. Results: 1. the density gradient centrifugation method was used to separate the cultured cells. Under the microscope, the ERC was spindle shaped, spindle shaped, fibroblast like, large, wall growing, and fast growing. The colony.2.iri-untreated group was compared with the sham group, serum creatinine, urea nitrogen level increased significantly, renal pathological injury was serious, renal structure disorder, renal tubular necrosis, interstitial hyperemia edema, and erc-treated group and iri-u Compared with group ntreated, serum creatinine, urea nitrogen level decreased significantly, renal function improved significantly, renal pathological damage was significantly reduced, renal tubular necrosis was reduced, and no interstitial congestion edema was found in group.3.iri-untreated and sham group, and renal cd3+t cell and ly6g+ cell infiltration increased; erc-treated group and iri-untreated group were compared with cd3+t cells and ly6g+. Compared with the sham group, flow cytometry analysis showed that the number of cd3+t cells in the kidney increased, the Ki67 increased and the proliferation of T cells increased obviously, and the cd3+t cells decreased significantly, Ki67 decreased, and the proliferation of T cells decreased in the erc-treated group compared with the iri-untreated group, and the p0.05.5.iri-untreated group was compared with the sham group, the spleen, kidney and kidney. Compared with the iri-untreated group, the proportion of cd4+ and cd8+t cells decreased significantly in the erc-treated group and the p0.05.6.erc-treated group compared with the iri-untreated group. The proportion of cd4+cd25+tregs cells in the spleen was significantly up, P0.05, and the iri-untreated group was comparable to the sham group, and there was no significant difference in the level of the cd4+ cd25+tregs cells. Compared with group iri-untreated, the proportion of cd11b+ly6c+ and cd11b+ly6g+ cells increased significantly in group.Erc-treated, and in iri-untreated and sham groups, cd11b+ly6c+ and cd11b+ly6g+ cells slightly increased.8. flow cytometry to analyze macrophages in renal tissue. Iri-untreated and sham groups increased the infiltration of f4/80+ macrophages. Compared with the ntreated group, the infiltration of f4/80+ macrophages in the renal tissue decreased, but the proportion of the cd206+ cells (M2 macrophages) with the repair effect increased. The difference in P0.05 was statistically significant between the.9.erc-treated group and the iri-untreated group, and the cytokine tnf- a, ifn- gamma and IL-6 decreased obviously, while the IL-4 level increased. The ratio of tnf- alpha, ifn- gamma and IL-6 increased and the difference of P0.05 was statistically significant. Conclusion: 1. the renal ischemia reperfusion injury model was successfully used in mice. It was proved that ERC could effectively improve renal function and reduce renal pathological injury.2.ercs to reduce renal ischemia-reperfusion injury in mice and down regulation of cd3+, cd4+, cd8+t cells and spleen cd4+, cd8+t cells. .3.ercs up regulation of spleen Tregs cells and reducing the degree of injury, promote renal injury to repair the renal tissue and reduce the renal F4/80+ macrophage, and increase the CD206+ cell (M2 macrophage) related.5.ERCs to alleviate renal pathological damage, improve renal function, and improve the proportion of CD11b+Ly6C+ and CD11b+Ly6G+ ratio of renal myeloid cells. 6.ERCs can reduce the inflammatory reaction and participate in the repair of renal injury by affecting the levels of TNF-, IFN-, IL-4 and IL-6 in vivo.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R692

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Immunosuppressive effects of rat mesenchymal stem cells:involvement of CD4~+ CD25~+ regulatory T cells[J];Hepatobiliary & Pancreatic Diseases International;2008年06期

，

本文編號(hào)：1824877