本文選題：巨噬細(xì)胞 + 姜黃素��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:本課題利用單核細(xì)胞定向誘導(dǎo)分化技術(shù)獲得不同表型巨噬細(xì)胞(M1、M2),用不同濃度姜黃素對(duì)M1型巨噬細(xì)胞進(jìn)行誘導(dǎo)干預(yù),通過(guò)細(xì)胞懸液創(chuàng)周局部注射的手段對(duì)大鼠創(chuàng)傷模型進(jìn)行差異化處理,并通過(guò)若干愈合指標(biāo)、細(xì)胞因子的測(cè)定,討論不同濃度姜黃素誘導(dǎo)的M1型巨噬細(xì)胞后對(duì)創(chuàng)面愈合的影響。方法:提取大鼠股骨骨髓細(xì)胞,并通過(guò)差速貼壁、M-CSF刺激等方法純化、激活獲得實(shí)驗(yàn)所需的巨噬細(xì)胞,應(yīng)用流式細(xì)胞技術(shù)分析培養(yǎng)細(xì)胞結(jié)果。使用LPS刺激巨噬細(xì)胞M0向M1轉(zhuǎn)化,IL-4誘導(dǎo)巨噬細(xì)胞M0向M2轉(zhuǎn)化,并應(yīng)用流式細(xì)胞技術(shù)分析驗(yàn)證M1、M2誘導(dǎo)的有效性。應(yīng)用不同濃度姜黃素誘導(dǎo)M1型巨噬細(xì)胞向M2型巨噬細(xì)胞極化并獲得相應(yīng)細(xì)胞混懸液;應(yīng)用流式細(xì)胞技術(shù)分析姜黃素對(duì)M1誘導(dǎo)的極化效率,大鼠創(chuàng)傷模型的制備及其可用性的評(píng)價(jià)。實(shí)驗(yàn)大鼠背部制作6枚直徑6mm全層皮膚貫通傷模型,通過(guò)創(chuàng)面愈合率、HE染色分析評(píng)價(jià)所制模型的可用性。創(chuàng)面的實(shí)驗(yàn)條件干預(yù)及評(píng)價(jià):實(shí)驗(yàn)動(dòng)物分1天組、4天組、7天組、10天組、14天組5組,每組5只。按照隨機(jī)雙盲的原則分別對(duì)每只大鼠的六枚創(chuàng)面做如下處理:(1)局部(皮下、真皮層及創(chuàng)基浸潤(rùn),同下)注射注射滅菌PBS;(2)局部注射M1+Cur0組細(xì)胞懸液;(3)(6)不做任何處理;(4)局部注射M1+Cur25組細(xì)胞懸液;(5)局部注射M2+Cur0組細(xì)胞懸液。到達(dá)實(shí)驗(yàn)天數(shù)后,處死實(shí)驗(yàn)鼠取標(biāo)本進(jìn)行愈合相關(guān)指標(biāo)的檢測(cè)、分析。通過(guò)創(chuàng)面未愈面積宏觀描述愈合速率的差異,HE染色、MASSON染色、免疫組化微觀分析導(dǎo)致愈合速率差異存在的原因。結(jié)果:1.大鼠骨髓細(xì)胞培養(yǎng)CD45、CD68雙陽(yáng)性細(xì)胞比率可達(dá)86.5%。2.姜黃素刺激M1型巨噬細(xì)胞向M2型巨噬細(xì)胞極化的最佳作用濃度為25μmol/L,極化效率為29.59%。3.大鼠背部不同解剖位置的六枚創(chuàng)面愈合速率無(wú)統(tǒng)計(jì)學(xué)差異,HE染色結(jié)果無(wú)明顯差異。相對(duì)于空白對(duì)照組,M1+Cur0組創(chuàng)面可觀察到愈合延遲,HE染色及MASSON染色提示創(chuàng)面存在炎細(xì)胞浸潤(rùn)時(shí)間長(zhǎng)、肉芽組織生長(zhǎng)緩慢;創(chuàng)面愈合率、免疫組化(VEGF、b FGF、TGF-b1)定量分析提示相關(guān)指標(biāo)較空白對(duì)照組存在統(tǒng)計(jì)學(xué)差異。M2+Cur0組及M1+Cur25組創(chuàng)面可觀察到愈合提前,上述指標(biāo)分析結(jié)果與M1+Cur0組相反且存在統(tǒng)計(jì)學(xué)意義。PBS組與空白對(duì)照組間無(wú)統(tǒng)計(jì)學(xué)差異。結(jié)論:1.M-CSF可體外誘導(dǎo)大鼠骨髓細(xì)胞形成巨噬細(xì)胞。2.LPS、IL-4分別可誘導(dǎo)巨噬細(xì)胞向M1、M2極化。3.姜黃素可誘導(dǎo)M1型巨噬細(xì)胞向M2型極化,且最佳極化效率29.59%。4.打孔器法制備大鼠背部創(chuàng)傷愈合模型的方法可用。5.姜黃素調(diào)控分型介導(dǎo)的巨噬細(xì)胞促進(jìn)創(chuàng)面愈合。
[Abstract]:Aim: to obtain different phenotypic macrophages (M _ 1 / M _ 2) by monocyte differentiation technique and to induce M _ 1 macrophages with different concentrations of curcumin. The wound model of rats was treated by local injection of cell suspensions, and the effects of different concentrations of curcumin induced M1 macrophages on wound healing were discussed through several healing indexes and cytokines. Methods: rat femur bone marrow cells were extracted and purified by differential adherent M-CSF stimulation. The macrophages were activated and the cultured cells were analyzed by flow cytometry. LPS-stimulated M0 to M1 transformation of macrophages and IL-4 were used to induce M0 to M2 transformation. Flow cytometry was used to verify the effectiveness of M1-M2-induced transformation. Different concentrations of curcumin were used to induce the polarization of M1 macrophages to M2 macrophages and the corresponding cell suspensions were obtained, and the polarization efficiency of M1 induced by curcumin was analyzed by flow cytometry. Preparation and usability evaluation of rat trauma model. Six 6mm full-thickness skin penetrating injury models were made on the back of experimental rats. The usability of the model was evaluated by HE staining. Experimental condition intervention and evaluation: experimental animals were divided into 1 day group, 4 day group, 7 day group, 10 day group, 14 day group, 5 groups, 5 rats in each group. According to the principle of random double blindness, six wounds of each rat were treated as follows: (1) Local (subcutaneous, dermal and invasive infiltration), (2) local injection of M1 Cur0 cell suspension; (3) no treatment; (4) local injection of M1 Cur25 cell suspension; (5) local injection of M2 Cur0 cell suspension. After reaching the experimental days, the rats were killed to collect the specimens for the detection and analysis of the related indexes of healing. The difference of healing rate was described by macroscopic description of the unhealed area of the wound. The reason of the difference in healing rate was analyzed by immunohistochemistry. The result is 1: 1. The double positive rate of CD45 and CD68 in rat bone marrow cells can reach 86.5%. 2. The best concentration of curcumin to stimulate the polarization of M1 macrophages to M2 macrophages was 25 渭 mol / L, and the polarization efficiency was 29.59.3. There was no significant difference in the healing rate of six wounds at different anatomical locations in the back of rats and there was no significant difference in HE staining results. Compared with the blank control group (M 1 Cur0 group), delayed healing was observed by HE staining and MASson staining, which indicated that the inflammatory cell infiltration time was long, granulation tissue grew slowly and wound healing rate was higher. The quantitative analysis of VEGF FGFB TGF-b1 showed that there was a statistical difference between the two groups. M2-Cur0 group and M1 Cur25 group showed that the wound healing was earlier than that in the blank control group, and the wound healing was earlier in the M2-Cur0 group and M1 Cur25 group than in the blank control group. The results of above indexes were contrary to those of M1 Cur0 group and there was no statistical difference between PBS group and blank control group. Conclusion 1. M-CSF can induce macrophage formation in rat bone marrow cells in vitro. 2. LPSN IL-4 can induce macrophage to M 1 M 2 polarization. 3, respectively. Curcumin could induce M 1 macrophages to polarization to M 2, and the best polarization efficiency was 29.59.4. The method of perforator can be used to establish the model of rat back wound healing. Curcumin mediates macrophages to promote wound healing.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R641

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