發(fā)布時(shí)間：2018-06-12 07:30

  本文選題：DDB2基因 + 重型痤瘡�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]探討中國漢族重型痤瘡易感基因DDB2在重型痤瘡患者及健康對(duì)照人群血清中的表達(dá)差異,同時(shí)探討DDB2對(duì)人HaCaT細(xì)胞增殖、凋亡的影響及初步探討DDB2對(duì)NF- κ B信號(hào)通路的影響。[方法]1、利用ELISA對(duì)重型痤瘡患者和健康對(duì)照人群血清中DDB2的濃度進(jìn)行檢測(cè)。2、構(gòu)建DDB2基因的過表達(dá)慢病毒載體PGMLV-PA6-DDB2及DDB2 基因的干擾慢病毒載體 PGMLV-SC5-DDB2-Sh1、PGMLV-SC5-DDB2-Sh2、PGMLV-SC5-DDB2-Sh3。293T細(xì)胞中包裝并產(chǎn)生DDB2過表達(dá)慢病毒及干擾慢病毒,然后分別感染至人的HaCaT細(xì)胞,利用嘌呤霉素進(jìn)行篩選,構(gòu)建穩(wěn)定表達(dá)的細(xì)胞株。3、采用Cell Counting Kit-8 (CCK-8)法和EdU實(shí)驗(yàn)檢測(cè)感染慢病毒前后細(xì)胞增殖情況、流式細(xì)胞術(shù)實(shí)驗(yàn)檢測(cè)感染前后細(xì)胞凋亡情況。4、在細(xì)胞水平通過免疫熒光及Western blot實(shí)驗(yàn)證實(shí)DDB2可能的靶基因NF- κB。[結(jié)果]1、ELISA結(jié)果顯示,與健康對(duì)照人群相比,重型痤瘡患者血清中DDB2濃度明顯降低(P0.05)。2、經(jīng)Westernblot實(shí)驗(yàn)證實(shí),在穩(wěn)定感染DDB2基因的過表達(dá)慢病毒的HaCaT細(xì)胞中,能夠高水平表達(dá)DDB2的蛋白;在穩(wěn)定感染DDB2基因的干擾慢病毒的HaCaT細(xì)胞中,能夠低水平表達(dá)DDB2的蛋白。3、實(shí)驗(yàn)分為三組(HaCaT細(xì)胞正常對(duì)照(NC)組、DDB2過表達(dá)組和DDB2干擾組)。(1) CCK8法檢測(cè)細(xì)胞增殖實(shí)驗(yàn):分別在(12h、24h、36h、48h、72h)5個(gè)時(shí)間點(diǎn)行CCK8檢測(cè)HaCaT細(xì)胞的OD值,結(jié)果顯示,相對(duì)于NC組細(xì)胞,DDB2過表達(dá)組HaCaT細(xì)胞增殖活性降低(P0.05),DDB2干擾組HaCaT細(xì)胞增殖活性增加(P0.05)。(2) EdU實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力:EdU結(jié)果顯示,相對(duì)于組細(xì)胞,DDB2過表達(dá)組增殖期HaCaT細(xì)胞減少(P0.01),DDB2干擾組增殖期HaCaT細(xì)胞增加(P0.01)。(3)流式細(xì)胞術(shù)實(shí)驗(yàn)檢測(cè)細(xì)胞凋亡:利用流式細(xì)胞術(shù)檢測(cè)接種24h后的HaCaT細(xì)胞細(xì)胞凋亡情況,結(jié)果顯示,與NC組比較,DDB2過表達(dá)組凋亡率明顯升高(P0.05)。4、通過Western blot,與NC組比較,DDB2過表達(dá)組HaCaT細(xì)胞NF- κ B/p65蛋白表達(dá)明顯減少,DDB2干擾組HaCaT細(xì)胞NF- κ B/p65蛋白表達(dá)明顯增加。與NC組比較,DDB2過表達(dá)組IκBα的表達(dá)明顯升高,DDB2干擾組IκBα的表達(dá)明顯明顯降低。5、通過免疫熒光檢測(cè)NF-κB/p65表達(dá)情況,與NC組比較,DDB2過表達(dá)組HaCaT細(xì)胞NF- κ B/p65表達(dá)明顯減少,DDB2干擾組HaCaT細(xì)胞NF-κ B/p65表達(dá)明顯增加。[結(jié)論]1、易感基因DDB2在重型痤瘡患者血清中表達(dá)水平較健康對(duì)照人群降低。2、建立DDB2過表達(dá)慢病毒及干擾慢病毒。3、DDB2可能通過調(diào)控HaCaT細(xì)胞增殖、凋亡,參與痤瘡的發(fā)病過程。此外,DDB2可能靶向NF-κB信號(hào)通路,參與痤瘡的炎癥反應(yīng),促進(jìn)痤瘡的發(fā)生及發(fā)展,這為痤瘡的發(fā)病機(jī)制的研究及治療提供新思路。
[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 偽 in DDB2 overexpression group was significantly higher than that in NC group. The expression of I 魏 B 偽 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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R758.733

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫素姣;楊浩然;楊智;劉玲;涂穎;鄒勇莉;何黎;;云南漢族痤瘡表型與家族史相關(guān)性的研究[J];中華皮膚科雜志;2007年11期

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本文編號(hào)：2008884