發(fā)布時(shí)間：2018-03-14 17:09

  本文選題：白細(xì)胞介素-10　切入點(diǎn)：樹(shù)突狀細(xì)胞　出處：《遼寧醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 研究CD86及NF-κB在IL-10-DC誘導(dǎo)大鼠同種異體角膜移植免疫耐受中的作用。 方法 1. DC培養(yǎng)與鑒定:參照經(jīng)典Hermans方案直接分離誘生法進(jìn)行細(xì)胞培養(yǎng),分三組,分別為6-DC組、12-DC組和IL-10-DC組。在經(jīng)典分離誘導(dǎo)方案(GM-CSF+IL-4)的基礎(chǔ)上培養(yǎng)至第6天為6-DC;第二組細(xì)胞繼續(xù)培養(yǎng)至12天,即為12-DC;第三組在第6天加入IL-10繼續(xù)培養(yǎng)至12天,為IL-10-DC。光鏡觀察DC形態(tài),用流式細(xì)胞儀(FCM)檢測(cè)所得DC表型。 2.用培養(yǎng)的供體來(lái)源的6-DC及IL-10-DC預(yù)處理受體,建立Wistar-SD大鼠穿透性角膜移植模型。供體Wistar大鼠21只;受體SD大鼠42只,受體鼠隨機(jī)分為3組,每組均為14只,角膜移植前3d,每只受體鼠經(jīng)尾靜脈注射不同物質(zhì)。A組:注射PBS;B組:注射6-DC,細(xì)胞數(shù)為2×10~6個(gè)/只;C組:注射IL-10-DC,細(xì)胞數(shù)為2×10~6個(gè)/只。以混濁、水腫和新生血管3項(xiàng)指標(biāo)作為臨床評(píng)估標(biāo)準(zhǔn),觀察術(shù)后各組受體角膜植片的存活時(shí)間。術(shù)后第14天各組隨機(jī)抽取4只鼠取術(shù)眼角膜行組織病理學(xué)檢查及免疫組化法檢測(cè)CD86及NF-κb的表達(dá)。 結(jié)果 1.DC的生長(zhǎng)情況、形態(tài)和表型:新分離的細(xì)胞呈圓形,胞體小。培養(yǎng)至第2天,細(xì)胞開(kāi)始集落生長(zhǎng);第4天集落最大,隨著培養(yǎng)時(shí)間的延長(zhǎng),細(xì)胞體積增加呈現(xiàn)典型的樹(shù)突狀細(xì)胞形態(tài)。流式細(xì)胞儀檢測(cè)DC的表型:IL-10-DC組CD83、CD86表達(dá)最低,而12-DC組表達(dá)最高。三組比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。 2.各組大鼠角膜植片存活情況:平均存活時(shí)間為A組(10.10±1.66)d,B組(17.80±1.81)d,C組(26.40±1.65)d。術(shù)后第14d,A組角膜植片均發(fā)生排斥,而B(niǎo)、C兩組角膜植片仍保持透明。B、C兩組植片存活時(shí)間與A組比較顯著延長(zhǎng)(P0.01);B、C組間比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 植片組織病理學(xué)檢查:A組角膜植片顯著水腫、增厚,出現(xiàn)大量的淋巴細(xì)胞和單核細(xì)胞浸潤(rùn),角膜基質(zhì)排列紊亂。B、C兩組植片炎性細(xì)胞浸潤(rùn)均顯著減少,角膜厚度及結(jié)構(gòu)基本正常。CD86及NF-κB,在對(duì)照組中呈高表達(dá)(P0.05),B,C組間比較無(wú)顯著性差異(P0.05)。 結(jié)論 1.直接分離誘生法聯(lián)合應(yīng)用細(xì)胞因子GM-CSF、IL-4可以從大鼠骨髓中獲得大量較高純度imDC,滿足了進(jìn)一步實(shí)驗(yàn)對(duì)細(xì)胞量的要求。IL-10可有效抑制DC成熟。 2.攝取供體的imDC能顯著延長(zhǎng)角膜植片的存活時(shí)間,成功誘導(dǎo)了同種異體大鼠角膜移植的免疫耐受。CD86及NF-κB參與了同種異體角膜移植排斥反應(yīng)的調(diào)控。IL-10-DC可抑制CD86及NF-κB的表達(dá)水平,從而抑制角膜排斥反應(yīng)的發(fā)生。
[Abstract]:Purpose. To investigate the role of CD86 and NF- 魏 B in IL-10-DC induced immune tolerance in rat corneal allograft. Method. 1. DC culture and identification: according to the classical Hermans protocol, the cells were cultured by direct isolation and induction, and were divided into three groups. 6-DC group (12-DC group) and IL-10-DC group (6-DC group) were cultured on the 6th day, the second group continued to culture to 12 days, the third group added IL-10 to 12 days, and the third group was cultured to 12 days after the addition of IL-10 on the 6th day, the second group continued to culture to 12 days, and the third group was cultured to 12 days after the addition of IL-10 on the 6th day. The morphology of DC was observed by light microscope, and the phenotype of DC was detected by flow cytometry (FCM). 2. The penetrating corneal transplantation model of Wistar-SD rats was established by preconditioning the recipients with 6-DC and IL-10-DC from cultured donors. 21 Wistar donor rats and 42 SD recipients were randomly divided into 3 groups, with 14 rats in each group. 3 days before corneal transplantation, each recipient rat was injected with different substances through caudal vein. Group A: group B: group B: group B: injected with 6-DC.The number of cells in group C was 2 脳 10 ~ (-6) cells injected with IL-10-DC2 脳 10 ~ (-6) cells per mouse. Edema and neovascularization were used as clinical evaluation criteria. The survival time of corneal grafts was observed and the expression of CD86 and NF- 魏 b were detected by immunohistochemistry and histopathological examination. Results. 1. The growth, morphology and phenotype of DC: the newly isolated cells were round and small. At the second day of culture, the colony began to grow, and on the 4th day, the colony was the largest, with the extension of culture time. The increase of cell volume showed typical dendritic cell morphology. The expression of CD83 + CD86 was the lowest in the DC group with flow cytometry and the highest in the 12-DC group. The difference among the three groups was statistically significant (P 0.01). 2. Survival status of corneal grafts in each group: the mean survival time was 10.10 鹵1.66 days in group A and 17.80 鹵1.81 days in group C, and the corneal graft rejection occurred in group A on the 14th day after operation. However, the survival time of corneal grafts in group B and C was significantly longer than that in group A, and there was a significant difference between group A and group A (P 0.01). Histopathological examination of grafts: in group 1, corneal grafts showed significant edema, thickening, large number of lymphocytes and monocytes infiltrating, and inflammatory cell infiltration in corneal grafts decreased significantly in both groups. The corneal thickness and structure were basically normal. CD86 and NF- 魏 B were almost normal. There was no significant difference between the two groups (P 0.05). Conclusion. 1. A large number of imDCs with high purity can be obtained from rat bone marrow by direct isolation and induction combined with cytokine GM-CSF- IL-4, which meets the requirements of further experiments. IL-10 can effectively inhibit DC maturation. 2. The survival time of corneal grafts was significantly prolonged by uptake of donor imDC, and the immune tolerance. CD86 and NF- 魏 B involved in the regulation of corneal allograft rejection. IL-10-DC could inhibit the expression of CD86 and NF- 魏 B. Thus inhibiting the occurrence of corneal rejection.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R779.65

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