中国汉族重型痤疮易感基因DDB2的功能研究

发布时间：2018-06-12 07:30

  本文选题：DDB2基因 + 重型痤疮　； 参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]探讨中国汉族重型痤疮易感基因DDB2在重型痤疮患者及健康对照人群血清中的表达差异,同时探讨DDB2对人HaCaT细胞增殖、凋亡的影响及初步探讨DDB2对NF- κ B信号通路的影响。[方法]1、利用ELISA对重型痤疮患者和健康对照人群血清中DDB2的浓度进行检测。2、构建DDB2基因的过表达慢病毒载体PGMLV-PA6-DDB2及DDB2 基因的干扰慢病毒载体 PGMLV-SC5-DDB2-Sh1、PGMLV-SC5-DDB2-Sh2、PGMLV-SC5-DDB2-Sh3。293T细胞中包装并产生DDB2过表达慢病毒及干扰慢病毒,然后分别感染至人的HaCaT细胞,利用嘌呤霉素进行筛选,构建稳定表达的细胞株。3、采用Cell Counting Kit-8 (CCK-8)法和EdU实验检测感染慢病毒前后细胞增殖情况、流式细胞术实验检测感染前后细胞凋亡情况。4、在细胞水平通过免疫荧光及Western blot实验证实DDB2可能的靶基因NF- κB。[结果]1、ELISA结果显示,与健康对照人群相比,重型痤疮患者血清中DDB2浓度明显降低(P0.05)。2、经Westernblot实验证实,在稳定感染DDB2基因的过表达慢病毒的HaCaT细胞中,能够高水平表达DDB2的蛋白;在稳定感染DDB2基因的干扰慢病毒的HaCaT细胞中,能够低水平表达DDB2的蛋白。3、实验分为三组(HaCaT细胞正常对照(NC)组、DDB2过表达组和DDB2干扰组)。(1) CCK8法检测细胞增殖实验:分别在(12h、24h、36h、48h、72h)5个时间点行CCK8检测HaCaT细胞的OD值,结果显示,相对于NC组细胞,DDB2过表达组HaCaT细胞增殖活性降低(P0.05),DDB2干扰组HaCaT细胞增殖活性增加(P0.05)。(2) EdU实验检测细胞增殖能力:EdU结果显示,相对于组细胞,DDB2过表达组增殖期HaCaT细胞减少(P0.01),DDB2干扰组增殖期HaCaT细胞增加(P0.01)。(3)流式细胞术实验检测细胞凋亡:利用流式细胞术检测接种24h后的HaCaT细胞细胞凋亡情况,结果显示,与NC组比较,DDB2过表达组凋亡率明显升高(P0.05)。4、通过Western blot,与NC组比较,DDB2过表达组HaCaT细胞NF- κ B/p65蛋白表达明显减少,DDB2干扰组HaCaT细胞NF- κ B/p65蛋白表达明显增加。与NC组比较,DDB2过表达组IκBα的表达明显升高,DDB2干扰组IκBα的表达明显明显降低。5、通过免疫荧光检测NF-κB/p65表达情况,与NC组比较,DDB2过表达组HaCaT细胞NF- κ B/p65表达明显减少,DDB2干扰组HaCaT细胞NF-κ B/p65表达明显增加。[结论]1、易感基因DDB2在重型痤疮患者血清中表达水平较健康对照人群降低。2、建立DDB2过表达慢病毒及干扰慢病毒。3、DDB2可能通过调控HaCaT细胞增殖、凋亡,参与痤疮的发病过程。此外,DDB2可能靶向NF-κB信号通路,参与痤疮的炎症反应,促进痤疮的发生及发展,这为痤疮的发病机制的研究及治疗提供新思路。
[Abstract]:[objective] to investigate the expression of severe acne susceptibility gene DDB2 in serum of patients with severe acne and healthy controls, and to explore the proliferation of human HaCaT cells by DDB2. Effects of DDB2 on NF- 魏 B signaling pathway and apoptosis. [methods] 1. The concentration of DDB2 in serum of patients with severe acne and healthy controls was detected by Elisa, and the lentivirus vector PGMLV-PA6-DDB2 and the interfering lentivirus vector PGMLV-SC5-DDB2-Sh2MLV-SC5-DDB2-Sh2MLV-SC5-DDB2-Sh3.293 T cells were constructed. DDB2 overexpresses lentiviruses and interferes with lentiviruses, Then human HaCaT cells were infected by purine mycin to construct stable expression cell line. Cell Counting Kit-8 CCK-8 method and EdU experiment were used to detect the proliferation of lentivirus cells before and after infection. Flow cytometry was used to detect apoptosis before and after infection. Immunofluorescence and Western blot assay were used to confirm the possible target gene NF- 魏 B of DDB2. [results] 1the results of Elisa showed that the serum DDB2 concentration of patients with severe acne was significantly lower than that of healthy controls. Western blot showed that DDB2 protein could be expressed at a high level in HaCaT cells with stable overexpression of DDB2 gene. In HaCaT cells with stable infection with DDB2 gene interfering with lentivirus, The protein. 3, which can express DDB2 at low level, was divided into three groups: the OD value of HaCaT cells was detected by CCK8 method at 24 h / 24 h / 36 h / 48 h / 72 h), respectively. Compared with NC group, the proliferation activity of HaCaT cells in DDB2 overexpression group was lower than that in control group. The proliferation activity of HaCaT cells increased in P0.05DDB2 interference group. Compared with DDB2 overexpression group, the number of HaCaT cells decreased in proliferative phase compared with that in DDB2 overexpression group. In DDB2 interference group, the number of HaCaT cells increased in proliferating phase.) flow cytometry (FCM) was used to detect the apoptosis of HaCaT cells after 24 hours inoculation. The results showed that the apoptosis of HaCaT cells was detected by flow cytometry. Compared with NC group, the apoptotic rate of DDB2 overexpression group was significantly higher than that of NC group. The NF- 魏 B / p65 protein expression of HaCaT cells in DDB2 overexpression group was significantly lower than that in NC group, and the NF- 魏 B / p65 protein expression in HaCaT cells in DDB2 overexpression group was significantly higher than that in NC group. The expression of I 魏 B 伪 in DDB2 overexpression group was significantly higher than that in NC group. The expression of I 魏 B 伪 in DDB2 interference group was significantly lower than that in NC group. The expression of NF- 魏 B / p65 was detected by immunofluorescence. Compared with NC group, the expression of NF- 魏 B / p65 in HaCaT cells significantly decreased in DDB2 overexpression group. [conclusion] 1. The expression level of susceptible gene DDB2 in serum of severe acne patients is lower than that of healthy controls. The establishment of DDB2 overexpression lentivirus and interfering lentivirus. 3 DDB2 may be involved in the pathogenesis of acne by regulating the proliferation and apoptosis of HaCaT cells. In addition, DDB2 may target NF- 魏 B signaling pathway, participate in the inflammatory response of acne, promote the occurrence and development of acne, which provides a new idea for the study and treatment of acne.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.733

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相关期刊论文 前1条

1 孙素姣;杨浩然;杨智;刘玲;涂颖;邹勇莉;何黎;;云南汉族痤疮表型与家族史相关性的研究[J];中华皮肤科杂志;2007年11期

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