發(fā)布時(shí)間：2018-04-28 22:39

  本文選題：間充質(zhì)干細(xì)胞 + 移植　； 參考：《暨南大學(xué)》2014年碩士論文

【摘要】：目的：利用間充質(zhì)干細(xì)胞（Mesenchymal stem cells，MSCs）免疫調(diào)節(jié)作用和低免疫原性的特性，探討其對(duì)腦死亡大鼠腎臟免疫調(diào)節(jié)及移植后腎臟長期功能的影響。 方法：（1）近交系、雄性F344大鼠骨髓來源的間充質(zhì)干細(xì)胞貼壁分離、培養(yǎng)、純化及流式細(xì)胞儀鑒定、傳代、凍存。（2）將近交系、雄性F344大鼠（體質(zhì)量200g~250g）作為供體，近交系、雄性Lewis大鼠（體質(zhì)量200g~250g）作為受體。分3組：①正常對(duì)照組（G1，n=5）：取F344大鼠左腎，原位植入已切除左腎的Lewis大鼠；②腦死亡組（G2，n=5）：將F344大鼠誘導(dǎo)腦死亡，6小時(shí)候后取其左腎，原位植入已切除左腎的Lewis大鼠；③腦死亡+MSCs組（G3，n=5）：將F344大鼠誘導(dǎo)腦死亡后，輸注MSCs，6小時(shí)候后取左腎，再植入已切除左腎的Lewis大鼠；（3）所有Lewis大鼠腎移植手術(shù)當(dāng)天起連續(xù)10天每天給予肌注環(huán)孢素A（0.15mg/100g大鼠體重），術(shù)后第10天切除右腎，術(shù)后第14天、21天、28天、35天分別抽血，檢測(cè)血清肌酐（Scr）;（4）將術(shù)后35天獲取的移植腎標(biāo)本和術(shù)后第10天切除的右腎標(biāo)本行①免疫組化檢測(cè)；②移植腎病理組織學(xué)檢測(cè)。 結(jié)果：本研究將貼壁分離法獲得的細(xì)胞傳至第三代，經(jīng)流式細(xì)胞儀檢測(cè)具有低表達(dá)CD34、CD45，高表達(dá)CD29、CD105和CD44的細(xì)胞特征，符合MSCs表型特點(diǎn)。這些細(xì)胞光鏡下呈成纖維樣、梭形，形態(tài)較均一，另外，它們可誘導(dǎo)分化成為脂肪細(xì)胞，證實(shí)從近交系、雄性F344大鼠骨髓分離、培養(yǎng)、純化的細(xì)胞是MSCs。所有建立的腦死亡模型大鼠均符合腦死亡供體要求：（深昏迷，自主呼吸停止，無條件反射，瞳孔對(duì)光反射消失，角膜反射消失，觀察6小時(shí)大鼠平均動(dòng)脈壓仍大于80mmHg）。檢測(cè)腎移植術(shù)后不同時(shí)間三組大鼠Scr來評(píng)估移植腎的功能，G2組大鼠的Scr均比G1、G3組的高，，且差異具有統(tǒng)計(jì)學(xué)意義（P0.01），G3組大鼠的Scr略高于G1組，差異無統(tǒng)計(jì)學(xué)意義（P0.05）。病理學(xué)檢測(cè)取各組腎組織行HE染色，在光鏡下觀察，發(fā)現(xiàn)G2組出現(xiàn)了較明顯的單核細(xì)胞浸潤及腎小管上皮炎癥（P0.05），G3組和G1組無明顯差異（P0.05）；三組移植腎的動(dòng)脈血管都有明顯炎癥改變，且G2組的比G1和G3組的嚴(yán)重，但三組差異無統(tǒng)計(jì)學(xué)意義（P0.05）。免疫組化檢測(cè)顯示G2組移植腎腎小球、腎小管及間質(zhì)細(xì)胞IL-1β、TNF-α表達(dá)呈陽性，G1、G3組的表達(dá)均低于G2組（P0.05）。 結(jié)論：MSCs可調(diào)節(jié)腦死亡供體大鼠腎臟免疫狀態(tài)，減輕供腎的炎癥損害，移植后對(duì)受體腎臟具有保護(hù)作用，減少移植腎組織IL-1β、TNF-α炎癥因子產(chǎn)生，移植腎能獲得良好的長期功能，。
[Abstract]:Aim: to investigate the effects of mesenchymal stem cells (MSCs) on renal immunomodulation and long-term renal function after transplantation in brain-dead rats. Methods the bone marrow-derived mesenchymal stem cells from male F344 rats were isolated, cultured, purified and identified by flow cytometry. The inbred lines and male F344 rats (200g / 250g) were used as inbred lines. Male Lewis rats (200g / kg) were used as receptors. The rats were divided into 3 groups: 1 normal control group: the left kidney of F344 rats was taken from the left kidney and implanted into the brain dead group of Lewis rats with resected left kidney in situ. The left kidney of F344 rats was harvested after brain death induced 6 hours later and implanted into the Lewis rats with resected left kidney in situ. 3 brain death group (MSCs group): after inducing brain death in F344 rats, the left kidney was taken after 6 hours of infusion. All Lewis rats were given intramuscular injection of cyclosporine (A(0.15mg/100g) daily for 10 days from the day of renal transplantation. The right kidney was resected on the 10th day after operation. The blood was taken from the 14th day to the 21st day and from the 28th day to the 35th day after operation. Serum creatinine (creatinine) the grafted kidney specimens obtained 35 days after operation and the right kidney specimens removed on the 10th day after operation were examined by immunohistochemistry. Results: the cells obtained by adherent method were transferred to the third generation in this study. Flow cytometry was used to detect the cells with low expression of CD34, CD45, high expression of CD29, CD105 and CD44, which were in line with the phenotypic characteristics of MSCs. These cells were fibroblast-like, fusiform and homogenous under light microscope. In addition, they could be induced to differentiate into adipocytes. It was confirmed that the cells isolated, cultured and purified from the bone marrow of inbred and male F344 rats were MSCs. All of the established brain death model rats were in accordance with the brain death donor requirement: (deep coma, spontaneous respiratory arrest, no conditioned reflex, pupil light reflex disappeared, corneal reflex disappeared. The mean arterial pressure of 6 hours was still more than 80 mm HgN. The Scr of group G _ 2 was higher than that of group G _ 3, and the Scr of group G _ 3 was higher than that of group G _ 1 after renal transplantation, and the difference was not statistically significant (P 0.05). The renal tissues in each group were stained with HE and observed under light microscope. It was found that there were obvious monocyte infiltration and tubular epithelial inflammation in G2 group and there was no significant difference between G3 group and G1 group, and there were obvious inflammatory changes in artery vessels of grafts in all three groups, and the severity of inflammation in G2 group was more serious than that in G1 and G3 groups. However, there was no significant difference among the three groups (P 0.05). Immunohistochemical staining showed that the expression of IL-1 尾 -TNF- 偽 in glomeruli, tubules and interstitial cells in G2 group was lower than that in G2 group (P 0.05). Conclusion\
【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】：2014
【分類號(hào)】：R699.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳潔;曾耀英;曾慧蘭;陳孝銀;蘇澤軒;劉嘉雯;王喬峰;李偉;吳文燕;;補(bǔ)陽還五湯預(yù)先干預(yù)對(duì)大鼠腦死亡后腎臟炎癥細(xì)胞因子表達(dá)的影響[J];中藥材;2009年12期

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