本文選題：白瘢風(fēng) + siRNA　； 參考：《天津醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的 通過對(duì)新發(fā)現(xiàn)的白癜風(fēng)相關(guān)基因CLEC2B進(jìn)行功能學(xué)研究以揭示該基因可能的相關(guān)致病靶點(diǎn)。建立淋巴細(xì)胞瞬時(shí)轉(zhuǎn)染體系,沉默淋巴細(xì)胞中CLEC2B基因表達(dá),觀察其對(duì)淋巴細(xì)胞增殖及其細(xì)胞因子IL-4mRNA、sIL-2RmRNA、 IL-6mRNA表達(dá)的調(diào)節(jié)作用,觀察CLEC2B基因沉默后淋巴細(xì)胞上清液對(duì)黑素細(xì)胞增殖活性的影響從而揭示CLEC2B基因參與白癜風(fēng)發(fā)病的免疫學(xué)機(jī)制。 方法 本實(shí)驗(yàn)選用Jurkat淋巴細(xì)胞及B16黑素瘤細(xì)胞為研究對(duì)象,應(yīng)用RNA干擾技術(shù)下調(diào)淋巴細(xì)胞CLEC2BmRNA的表達(dá),實(shí)時(shí)熒光定量PCR法分析CLEC2B基因沉默后淋巴細(xì)胞細(xì)胞因子IL-4mRNA、sIL-2RmRNA、IL-6mRNA表達(dá)情況,MTT法檢測(cè)CLEC2B基因沉默后淋巴細(xì)胞增殖情況和淋巴細(xì)胞上清液對(duì)黑素細(xì)胞增殖的影響。 結(jié)果 1. DMRIE-C脂質(zhì)體與CLEC2BsiRNA按合理配比(2μl:80nM)可進(jìn)行對(duì)淋巴細(xì)胞的瞬時(shí)轉(zhuǎn)染并最終實(shí)現(xiàn)CLEC2B基因沉默。 2. CLEC2B基因沉默后淋巴細(xì)胞增殖變化無顯著統(tǒng)計(jì)學(xué)差異；淋巴細(xì)胞細(xì)胞因子表達(dá)變化：IL-4mRNA表達(dá)上調(diào),sIL-2RmRNA表達(dá)下降,IL-6mRNA變化無統(tǒng)計(jì)學(xué)差異。 3. CLEC2B基因沉默后淋巴細(xì)胞上清液對(duì)黑素細(xì)胞增殖活性抑制減少,有利于黑素細(xì)胞存活。 結(jié)論 淋巴細(xì)胞中存在CLEC2B基因表達(dá),且該基因沉默后可直接調(diào)節(jié)淋巴細(xì)胞多種細(xì)胞因子IL-4、sIL-2RmRNA的轉(zhuǎn)錄表達(dá),但對(duì)淋巴細(xì)胞本身的增殖可能無明顯影響。CLEC2B基因也可通過淋巴細(xì)胞上清液影響黑素細(xì)胞增殖活性,提示CLEC2B基因在淋巴細(xì)胞中的表達(dá)水平對(duì)調(diào)節(jié)其功能有重要作用并可通過淋巴細(xì)胞改變黑素細(xì)胞的生存環(huán)境。揭示了CLEC2B基因可通過調(diào)節(jié)細(xì)胞因子水平及間接影響黑素增殖參與白癜風(fēng)發(fā)病的免疫學(xué)機(jī)制,為CLEC2B信號(hào)傳導(dǎo)途徑研究提供了新的實(shí)驗(yàn)依據(jù)。
[Abstract]:Purpose A novel vitiligo associated gene (CLEC2B) was studied by functional analysis to reveal its possible pathogenicity targets. Lymphocyte transient transfection system was established to silence the expression of CLEC2B gene in lymphocytes and to observe its regulatory effect on lymphocyte proliferation and cytokine IL-4mRNAsIL-2RmRNAs, IL-6mRNA expression. To observe the effect of lymphocyte supernatant of CLEC2B gene silencing on the proliferation of melanocytes, so as to reveal the immunological mechanism of CLEC2B gene involved in the pathogenesis of vitiligo. Method In this study, Jurkat lymphocytes and B16 melanoma cells were used to down-regulate the expression of CLEC2BmRNA in lymphocytes by RNA interference technique. The expression of IL-4mRNAsIL-2RmRNA-6 mRNA after CLEC2B gene silencing was analyzed by real-time fluorescence quantitative PCR. The lymphocyte proliferation after CLEC2B gene silencing and the effect of lymphocyte supernatant on the proliferation of melanocytes were detected by MTT assay. Result 1. DMRIE-C liposomes and CLEC2BsiRNA can transfect lymphocytes at a reasonable ratio of 2 渭 l: 80nM and finally achieve CLEC2B gene silencing. 2. There was no significant difference in lymphocyte proliferation after CLEC2B gene silencing, while there was no significant difference in the expression of cytokines and the up-regulation of IL-4 mRNA expression and the decrease of IL-2R mRNA expression. There was no significant difference in the changes of IL-6 mRNA expression. 3. After CLEC2B gene silencing, the lymphocyte supernatant inhibited the proliferation of melanocytes, which was beneficial to the survival of melanocytes. Conclusion There is CLEC2B gene expression in lymphocytes, and the silencing of this gene can directly regulate the transcription expression of IL-4 / sIL-2R mRNA in lymphocytes. However, the proliferation of melanocytes may not be affected by the proliferation of lymphocytes. CLEC2B gene can also affect the proliferation activity of melanocytes through the supernatant of lymphocytes. It is suggested that the expression of CLEC2B gene in lymphocytes plays an important role in regulating its function and can change the living environment of melanocytes through lymphocytes. It is revealed that CLEC2B gene can play an important role in the pathogenesis of vitiligo by regulating cytokine level and indirectly affecting melanin proliferation, which provides a new experimental basis for the study of CLEC2B signal transduction pathway.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R758.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 陳振東;韋曉新;吳永官;;RNAi技術(shù)及其應(yīng)用[J];安徽農(nóng)業(yè)科學(xué);2008年35期

2 王慶紅;王曉娟;甘成軍;彭旭;羅高興;吳軍;;CD4~+CD25~+T細(xì)胞對(duì)NK細(xì)胞活性的影響[J];重慶醫(yī)學(xué);2007年11期

3 周婧,栗玉珍,楊士華,于淞,趙育楨;槲皮素對(duì)鼠B16黑素瘤細(xì)胞酪氨酸酶的影響[J];臨床皮膚科雜志;2005年10期

相關(guān)博士學(xué)位論文 前1條

1 謝建輝;C型凝集素樣受體Dectin-1與CLEC-2的相關(guān)研究[D];復(fù)旦大學(xué);2008年

，

本文編號(hào)：1815697