姜黄素调控分型介导的巨噬细胞对创面愈合的影响

发布时间：2018-07-03 07:52

本文选题：巨噬细胞 + 姜黄素　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:本课题利用单核细胞定向诱导分化技术获得不同表型巨噬细胞(M1、M2),用不同浓度姜黄素对M1型巨噬细胞进行诱导干预,通过细胞悬液创周局部注射的手段对大鼠创伤模型进行差异化处理,并通过若干愈合指标、细胞因子的测定,讨论不同浓度姜黄素诱导的M1型巨噬细胞后对创面愈合的影响。方法:提取大鼠股骨骨髓细胞,并通过差速贴壁、M-CSF刺激等方法纯化、激活获得实验所需的巨噬细胞,应用流式细胞技术分析培养细胞结果。使用LPS刺激巨噬细胞M0向M1转化,IL-4诱导巨噬细胞M0向M2转化,并应用流式细胞技术分析验证M1、M2诱导的有效性。应用不同浓度姜黄素诱导M1型巨噬细胞向M2型巨噬细胞极化并获得相应细胞混悬液;应用流式细胞技术分析姜黄素对M1诱导的极化效率,大鼠创伤模型的制备及其可用性的评价。实验大鼠背部制作6枚直径6mm全层皮肤贯通伤模型,通过创面愈合率、HE染色分析评价所制模型的可用性。创面的实验条件干预及评价:实验动物分1天组、4天组、7天组、10天组、14天组5组,每组5只。按照随机双盲的原则分别对每只大鼠的六枚创面做如下处理:(1)局部(皮下、真皮层及创基浸润,同下)注射注射灭菌PBS;(2)局部注射M1+Cur0组细胞悬液;(3)(6)不做任何处理;(4)局部注射M1+Cur25组细胞悬液;(5)局部注射M2+Cur0组细胞悬液。到达实验天数后,处死实验鼠取标本进行愈合相关指标的检测、分析。通过创面未愈面积宏观描述愈合速率的差异,HE染色、MASSON染色、免疫组化微观分析导致愈合速率差异存在的原因。结果:1.大鼠骨髓细胞培养CD45、CD68双阳性细胞比率可达86.5%。2.姜黄素刺激M1型巨噬细胞向M2型巨噬细胞极化的最佳作用浓度为25μmol/L,极化效率为29.59%。3.大鼠背部不同解剖位置的六枚创面愈合速率无统计学差异,HE染色结果无明显差异。相对于空白对照组,M1+Cur0组创面可观察到愈合延迟,HE染色及MASSON染色提示创面存在炎细胞浸润时间长、肉芽组织生长缓慢;创面愈合率、免疫组化(VEGF、b FGF、TGF-b1)定量分析提示相关指标较空白对照组存在统计学差异。M2+Cur0组及M1+Cur25组创面可观察到愈合提前,上述指标分析结果与M1+Cur0组相反且存在统计学意义。PBS组与空白对照组间无统计学差异。结论:1.M-CSF可体外诱导大鼠骨髓细胞形成巨噬细胞。2.LPS、IL-4分别可诱导巨噬细胞向M1、M2极化。3.姜黄素可诱导M1型巨噬细胞向M2型极化,且最佳极化效率29.59%。4.打孔器法制备大鼠背部创伤愈合模型的方法可用。5.姜黄素调控分型介导的巨噬细胞促进创面愈合。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R641

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