本文選題：骨髓間充質(zhì)干細(xì)胞 + exosomes�。� 參考：《四川醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：肝臟缺血再灌注損傷(hepatic ischemia reperfusion injury,HIRI)是指肝臟因缺血、缺氧而造成功能障礙和結(jié)構(gòu)損傷,恢復(fù)血供后,功能障礙和結(jié)構(gòu)損傷不但不能完全恢復(fù),反而損傷進(jìn)一步加重,甚至發(fā)生不可逆性損傷的現(xiàn)象。HIRI常見于肝臟外科、肝移植以及低血容量性休克等過程中,尤其肝移植過程中難以避免。目前,HIRI在臨床上受到廣泛的關(guān)注,但對其防治還沒有理想的措施。近年來研究表明骨髓間充質(zhì)干細(xì)胞來源exosomes(bone-marrow mesenchymal stem cell-derived exosomes, BMSCs-exosomes)能通過傳遞具有生物學(xué)活性的蛋白質(zhì)、脂類、mRNA及mircoRNA等物質(zhì),激活受損傷組織細(xì)胞的自我保護(hù)與內(nèi)源性再生機(jī)制,同時還可調(diào)控機(jī)體免疫應(yīng)答反應(yīng),參與受損傷組織細(xì)胞的修復(fù)以及對器官功能的保護(hù)作用。本研究旨在探討B(tài)MSCs-exosomes對大鼠HIRI后肝臟組織的修復(fù)作用及可能的分子機(jī)制,為深入研究BMSCs-exosomes臨床防治HIRI提供理論依據(jù)。方法：(1)無菌條件下獲取大鼠骨髓間充質(zhì)干細(xì)胞,進(jìn)行原代細(xì)胞培養(yǎng)并傳代純化至3～6代,得到較為穩(wěn)定的骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)。(2)收集骨髓間充質(zhì)干細(xì)胞培養(yǎng)的上清液,采用ExoQuick-TC試劑盒分離提取培養(yǎng)上清液中微囊體顆粒,即BMSCs-exosomes,并采用負(fù)染透射電鏡進(jìn)行形態(tài)學(xué)鑒定。(3)將32只清潔級健康雄性SD大鼠隨機(jī)平均分為4組(即假手術(shù)組、PBS組、BMSCs組和exosomes組),通過建立SD大鼠HIRI模型,觀察各實驗組大鼠肝功能指標(biāo)ALT、炎癥因子TNF-α、 IL-1β和IL-10的表達(dá)水平及肝組織病理學(xué)、肝細(xì)胞的凋亡情況和肝組織中凋亡基因相關(guān)蛋白bcl-2、bax表達(dá)水平的變化。結(jié)果：(1) BMSCs原代細(xì)胞經(jīng)培養(yǎng)48 h后,鏡下可見細(xì)胞逐漸貼壁,9～10 d后可見細(xì)胞形態(tài)呈梭形或多邊形,14 d左右細(xì)胞貼滿整個培養(yǎng)瓶,經(jīng)傳代培養(yǎng)3代后,其形態(tài)逐漸表現(xiàn)為相對均一的梭形。(2)透射電子顯微鏡下可見BMSCs-exosomes呈具有明顯異質(zhì)性的圓形或橢圓形小囊泡,直徑30-100nm,包膜完整,且囊內(nèi)含有低電子密度物質(zhì)。(3)成功建立SD大鼠HIRI模型,各組相關(guān)指標(biāo)改變?nèi)缦拢孩傺錋LT水平PBS組大鼠血清ALT顯著高于假手術(shù)組,其差異有統(tǒng)計學(xué)意義(P0.05), BMSCs組和exosomes組血清ALT均顯著低于PBS組(P0.05),而BMSCs組與exosomes組間的血清ALT的差異無統(tǒng)計學(xué)意義(P0.05)。②血清促炎因子TNF-α、DL-1β和抑炎因子IL-10表達(dá)水平PBS組血清TNF-α和IL-1β表達(dá)水平顯著高于假手術(shù)組(P0.01), BMSCs組和exosomes組血清TNF-α和IL-1β表達(dá)水平顯著低于PBS組(P0.05),而BMSCs組與exosomes組間TNF-α和IL-1p表達(dá)水平的差異無統(tǒng)計學(xué)意義(P0.05)；假手術(shù)組和PBS組血清IL-10水平均較低,BMSCs組和exosomes組血清IL-10水平均顯著高于PBS組(P0.05),而BMSCs組與exosomes組間血清IL-10水平的差異無統(tǒng)計學(xué)意義(P0.05)。③肝臟組織病理學(xué)PBS組可見明顯的肝細(xì)胞腫脹、部分肝細(xì)胞胞漿空泡化、肝細(xì)胞點狀壞死、肝竇充血和炎性細(xì)胞浸潤；而與PBS組相比,BMSCs組和exosomes組肝臟的病理損傷明顯減輕,而二者之間肝臟損傷情況無明顯差異。④肝細(xì)胞凋亡指數(shù)肝細(xì)胞呈現(xiàn)棕褐色者為TUNEL陽性細(xì)胞。PBS組肝細(xì)胞凋亡指數(shù)顯著高于假手術(shù)組(P0.05)；BMSCs組和exosomes組肝細(xì)胞凋亡指數(shù)均顯著低于PBS組(P0.05)；但BMSCs組和exosomes組間肝細(xì)胞凋亡指數(shù)無明顯差異(P0.05)。⑤肝組織中bcl-2、bax表達(dá)水平PBS組肝組織中bcl-2的表達(dá)水平顯著低于假手術(shù)組(P0.05),bax表達(dá)水平顯著高于假手術(shù)組(P0.05),BMSCs組和exosomes組肝組織中bcl-2表達(dá)水平顯著高于PBS組(P0.05),bax表達(dá)水平顯著低于PBS組(P0.05),而BMSCs組與exosomes組間肝組織bcl-2、bax表達(dá)水平的差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論：本結(jié)果表明BMSCs-exosomes可通過抗凋亡機(jī)制,抑制HIRI時肝細(xì)胞凋亡；同時通過調(diào)控促炎因子與抑炎因子的平衡,減輕炎癥對肝組織細(xì)胞的損傷,對大鼠肝臟缺血再灌注損傷發(fā)揮修復(fù)作用。還發(fā)現(xiàn)BMSCs和BMSCs-exosomes在大鼠HIRI中的修復(fù)功能是等效的,這為BMSCs-exosomes可完全替代BMSCs移植防治HIRI提供堅實的理論基礎(chǔ)
[Abstract]:Objective: liver ischemia reperfusion injury (hepatic ischemia reperfusion injury (HIRI)) refers to the liver dysfunction and structural damage caused by ischemia and hypoxia. After the recovery of blood supply, the dysfunction and structural damage can not be completely recovered, but the damage is further aggravated, even the phenomenon of irreversible damage is common in the liver. In the process of surgery, liver transplantation and hypovolemic shock, especially liver transplantation, it is difficult to avoid it. At present, HIRI has been widely concerned in clinic, but there are no ideal measures for its prevention and control. In recent years, the study showed that bone marrow mesenchymal stem cells (exosomes) derived from bone-marrow mesenchymal stem cell-derived exosomes, BMSCs-exo Somes) can activate the mechanism of self protection and endogenous regeneration of damaged tissue cells by transferring biological active proteins, lipids, mRNA and mircoRNA, and also regulate the immune response response, repair the damaged tissue cells and protect the function of organs. This study aims to explore BMSCs-ex. The effect of osomes on the repair and possible molecular mechanism of liver tissue after HIRI in rats provides a theoretical basis for the in-depth study of BMSCs-exosomes clinical prevention and control of HIRI. Methods: (1) the rat bone marrow mesenchymal stem cells were obtained under aseptic conditions, and the primary cells were cultured and purified to 3~6 generations to obtain more stable mesenchymal stem cells (MSCs). Bone marrow mesenchymal stem cells, BMSCs). (2) collect the supernatant of bone marrow mesenchymal stem cells culture, use ExoQuick-TC reagent box to separate and extract microcapsule particles in the supernatant, that is BMSCs-exosomes, and use negative transmission electron microscope for morphological identification. (3) 32 healthy male SD rats were randomly divided into 4 groups. That is, sham operation group, group PBS, group BMSCs and group exosomes), by establishing HIRI model of SD rats, the expression level of hepatic function index ALT, inflammatory factor TNF- alpha, the expression level of IL-1 beta and IL-10, the liver histopathology, the apoptosis of hepatocytes and the changes of Bcl-2, Bax expression of apoptosis gene related proteins in liver tissues were observed. (1) After 48 h cells were cultured for 48 h, the cells were gradually adhered to the wall. After 9~10 D, the cell morphology was found to be shuttle or polygon, and 14 d cells were filled with the whole culture bottle. After the passage culture, the morphology gradually showed a relatively homogeneous shuttle shape. (2) the BMSCs-exosomes showed obvious heterogeneity under the transmission electron microscopy. Round or oval vesicles with a diameter of 30-100nm, a complete capsule and a low electron density substance in the capsule. (3) the HIRI model of SD rats was successfully established. The changes of the correlation indices of each group were as follows: (1) the serum ALT of the serum ALT level in the PBS group was significantly higher than that of the sham operation group (P0.05), and the serum ALT of the BMSCs group and the exosomes group showed a significant difference. There was no significant difference in serum ALT between group BMSCs and group exosomes (P0.05). (P0.05), there was no significant difference in serum ALT between group BMSCs and exosomes group (P0.05). The expression level of TNF- alpha and IL-1 beta in serum of serum proinflammatory factor TNF- a, DL-1 beta and anti inflammatory factors was significantly higher than that in sham operation group. There was no significant difference in the level of TNF- alpha and IL-1p expression between group BMSCs and exosomes group (P0.05), but the level of serum IL-10 in the sham operation group and the PBS group was lower than that in the BMSCs group and the exosomes group. The level of serum IL-10 in the BMSCs and exosomes groups was significantly higher than that in the BMSCs group and exosomes group, but there was no significant difference in the level of serum levels between the group and the group. (P0.05). (3) liver histopathological group PBS group showed obvious liver cell swelling, partial hepatocyte vacuolation, hepatic cell punctate necrosis, hepatic sinusoidal hyperemia and inflammatory cell infiltration, but compared with group PBS, the pathological damage of liver in group BMSCs and exosomes group was obviously reduced, and there was no significant difference between the two groups. 4. The apoptosis index of hepatocyte in group.PBS of dead index hepatocyte and TUNEL positive cell was significantly higher than that in sham operation group (P0.05), and the apoptosis index of hepatocyte in group BMSCs and exosomes group was significantly lower than that in group PBS (P0.05), but there was no significant difference between BMSCs group and exosomes group (P0.05). The expression level of Bcl-2 in the liver tissue of group PBS was significantly lower than that in sham operation group (P0.05), and the expression level of Bax was significantly higher than that of sham operation group (P0.05). The level of bcl-2 expression in the liver tissues of group BMSCs and exosomes was significantly higher than that in the PBS group (P0.05), and the level of Bax expression was significantly lower than that in the PBS group. The difference was not statistically significant (P0.05). Conclusion: this result shows that BMSCs-exosomes can inhibit the apoptosis of liver cells by anti apoptosis mechanism and reduce the damage of inflammation to liver tissue cells by regulating the balance of proinflammatory factors and anti inflammatory factors, and remediates the liver ischemia-reperfusion injury in rats. Also, BMSCs and BMSCs- are also found. The repair function of exosomes in rat HIRI is equivalent, which provides a solid theoretical foundation for BMSCs-exosomes to completely replace BMSCs transplantation to prevent HIRI.
【學(xué)位授予單位】：四川醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R657.3

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