本文選題：Orai1 + 小鼠��； 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：目的：構(gòu)建針對(duì)小鼠ORAI1基因進(jìn)行RNA干擾從而下調(diào)細(xì)胞內(nèi)Orai1蛋白表達(dá)的重組慢病毒Lenti-ORAI1-shRNA,并測(cè)定重組慢病毒的感染效率與干擾效率。建立Lenti-ORAI1-shRNA感染小鼠鼻腔的體內(nèi)模型,并測(cè)定其對(duì)小鼠鼻黏膜中ORAI1基因的轉(zhuǎn)錄抑制效率的動(dòng)態(tài)變化；建立Lenti-ORAI1-shRNA氣道內(nèi)干預(yù)變應(yīng)性鼻炎小鼠模型并觀察其對(duì)鼻炎癥狀、小鼠鼻腔局部與全身炎癥相關(guān)免疫細(xì)胞功能的影響并初步探尋其機(jī)制。 材料與方法：針對(duì)小鼠ORAI1基因序列設(shè)計(jì)RNA干擾靶序列,合成靶序列的寡聚DNA,與經(jīng)ClaI及MluI雙酶切的pLVTHM慢病毒載體連接,轉(zhuǎn)化大腸桿菌感受態(tài)細(xì)胞,產(chǎn)生重組RNA干擾慢病毒表達(dá)載體,將pRSV-REV質(zhì)粒、pMDlg-pRRE粒、pMD2.G與pLVTHM/ORAI1-shDNA重組質(zhì)�；旌细腥�293T細(xì)胞包裝慢病毒并測(cè)定病毒滴度。用包裝好的Lenti-ORAI1-shRNA感染PANC-1細(xì)胞,流式細(xì)胞分析測(cè)定其感染效率,熒光定量PCR檢測(cè)其對(duì)ORAI1基因的轉(zhuǎn)錄抑制效率。將BALB/c小鼠隨機(jī)分為7組,一組為正常對(duì)照組,另外六組為L(zhǎng)enti-ORAI1-shRNA干預(yù)組。將干預(yù)組小鼠麻醉后,鼻腔滴入8μl0.3%溶血卵磷脂,1小時(shí)后鼻腔滴入20μl滴度為1×109tu/ml的Lenti-ORAI1-shRNA進(jìn)行感染。分別于感染1,3,7,14,21,28天后處死小鼠,測(cè)定其鼻黏膜ORAI1mRNA水平。將小鼠隨機(jī)分為5組：正常對(duì)照組(normal),正常小鼠Lenti-ORAI1-shRNA干預(yù)組(NLI),變應(yīng)性鼻炎組(AR),變應(yīng)性鼻炎小鼠Lenti-GFP干預(yù)組(AR-GFP),變應(yīng)性鼻炎小鼠Lenti-ORAI1-shRNA干預(yù)組(AR-LI)。后三組小鼠以).5mg/ml OVA/20mg/ml Al(OH)3溶液腹腔注射3次致敏,40mg/ml OVA鼻腔滴入8次激發(fā)建立變應(yīng)性鼻炎小鼠模型,對(duì)NLI組與AR-LI組小鼠于首次鼻腔激發(fā)前3天鼻腔滴入20gl滴度為1×109tu／ml的Lenti-ORAI1-shRNA進(jìn)行干預(yù),對(duì)AR-GFP小鼠滴入相同劑量及滴度的Lenti-GFP進(jìn)行干預(yù)。計(jì)數(shù)各組小鼠末次鼻腔激發(fā)后10分鐘內(nèi)噴嚏數(shù)及撓鼻數(shù),Real-time RT PCR法測(cè)定各組小鼠鼻黏膜及脾臟ORAI1、LTC4S、EAR3、 germline Cε與IL-4mRNA水平,Western Blotting法測(cè)定各組小鼠鼻黏膜Orai1、IL-33、IL-17E與TSLP及脾臟Orai1蛋白表達(dá)水平,免疫組化法觀察各組小鼠鼻黏膜、鼻黏膜相關(guān)淋巴與脾臟中Orai1表達(dá)分布情況。ELISA法測(cè)定各組小鼠鼻腔灌洗液與血清中LTC4、ECP、OVA-IgE與IL-4的濃度。 結(jié)果：PANC-1細(xì)胞經(jīng)Lenti-ORAI1-shRNA感染后可表達(dá)慢病毒所攜帶的GFP基因而發(fā)出綠色熒光,當(dāng)感染復(fù)數(shù)為10時(shí),Lenti-ORAI1-shRNA感染PANC-1細(xì)胞48小時(shí)后感染效率為98.28%,對(duì)細(xì)胞ORAI1基因的轉(zhuǎn)錄抑制效率為90.5%；而用于對(duì)照的空載慢病毒在相同條件下感染效率為98.75%,對(duì)ORAI1基因的轉(zhuǎn)錄抑制效率為-3%。小鼠鼻腔感染Lenti-ORAI1-shRNA后,鼻黏膜ORAI1mRNA水平隨感染時(shí)間逐漸降低,至第3天時(shí)達(dá)最低,隨后逐漸升高,感染后一月左右Orai1mRNA水平仍低于正常對(duì)照組水平。AR-LI組小鼠鼻黏膜與脾臟ORAI1mRNA水平均較AR組下降,其鼻黏膜Orai1蛋白表達(dá)水平也低于AR組小鼠,末次鼻腔激發(fā)后撓鼻數(shù)與噴嚏數(shù)較AR組小鼠也都明顯減少；AR-GFP組小鼠鼻黏膜與脾臟ORAI1mRNA水平,Orai1表達(dá)水平及末次激發(fā)后噴嚏數(shù)與撓鼻數(shù)與AR組小鼠相比均無(wú)明顯差異。免疫組化結(jié)果提示AR-LI組小鼠鼻黏膜上皮層與固有層、鼻黏膜相關(guān)淋巴組織、脾臟生發(fā)中心等部位Orai1表達(dá)均較AR組小鼠減少。AR-LI組小鼠鼻黏膜與脾臟中LTC4S、 EAR3、germline Cε與IL-4的mRNA水平均明顯低于AR組小鼠,其鼻腔灌洗液中LTC4、OVA-IgE與IL-4的濃度以及血清中LTC4、ECP、OVA-IgE與IL-4的濃度也均低于AR組小鼠,AR-GFP組小鼠鼻黏膜與脾臟中LTC4S、 EAR3、germline Cε與IL-4的mRNA水平與AR組小鼠無(wú)明顯差異,其鼻腔灌洗液與血清中LTC4、ECP、OVA-IgE與IL-4的濃度與AR組小鼠也無(wú)明顯差異。AR組小鼠鼻黏膜中IL-33、IL-17E與TSLP的表達(dá)水平均明顯高于正常對(duì)照組小鼠,AR-LI組小鼠鼻黏膜中IL-33與TSLP的表達(dá)水平較AR組有所下降,但I(xiàn)L-17E的表達(dá)水平與AR組相比無(wú)明顯差異。AR-GFP組小鼠鼻黏膜中IL-33、IL-17E與TSLP的表達(dá)水平與AR組小鼠相比均無(wú)明顯差異。 結(jié)論：成功構(gòu)建針對(duì)ORAI1基因進(jìn)行RNA干擾的重組慢病毒Lenti-ORAI1-shRNA; Lenti-ORAI1-shRNA能夠成功感染PANC-1細(xì)胞并有效抑制ORAI1基因的轉(zhuǎn)錄水平。Lenti-ORAI1-shRNA鼻腔滴入可成功感染小鼠鼻黏膜細(xì)胞,感染后第3天對(duì)ORAI1基因的轉(zhuǎn)錄抑制效率達(dá)到最高。變應(yīng)性鼻炎小鼠Lenti-ORAI1-shRNA鼻腔干預(yù)后可有效下調(diào)其鼻黏膜與脾臟中Orai1表達(dá)水平,并減輕其鼻炎癥狀。下調(diào)鼻黏膜中Orai1表達(dá)不僅可降低變應(yīng)性鼻炎小鼠鼻黏膜中肥大細(xì)胞、嗜酸性粒細(xì)胞、B細(xì)胞與Th2細(xì)胞中相關(guān)炎性介質(zhì)的轉(zhuǎn)錄與釋放,也可降低全身如脾臟與血清中上述免疫細(xì)胞炎性介質(zhì)的轉(zhuǎn)錄與釋放。Lenti-ORAI1-shRNA鼻腔干預(yù)對(duì)小鼠變應(yīng)性鼻炎炎癥的抑制作用可能與其阻礙了鼻黏膜上皮細(xì)胞中IL-33與TSLP蛋白的合成和表達(dá)有關(guān)
[Abstract]:Objective: to construct a recombinant lentivirus Lenti-ORAI1-shRNA that interferes with the RNA interference of the mouse ORAI1 gene and reduce the expression of Orai1 protein in the cell, and determine the infection efficiency and interference efficiency of the recombinant lentivirus. The model of the nasal cavity in the mice infected with Lenti-ORAI1-shRNA is established and the transcriptional inhibition of the ORAI1 gene in the nasal mucosa of the mice is determined. The dynamic changes of efficiency; establish the Lenti-ORAI1-shRNA model of allergic rhinitis in the airway of the mice and observe the effects on the symptoms of rhinitis, the local and systemic inflammation related immune cell function in the nasal cavity of mice and explore its mechanism.
Materials and methods: to design RNA interference target sequence of mouse ORAI1 gene sequence, synthesize oligomeric DNA of target sequence, connect with pLVTHM lentivirus by ClaI and MluI double enzyme, transform Escherichia coli receptive cells, produce recombinant RNA to interfere with lentivirus expression vector, and reorganize pRSV-REV plasmids, pMDlg-pRRE particles, pMD2.G and pLVTHM/ORAI1-shDNA. The plasmid mixed infection 293T cells packed the lentivirus and measured the virus titer. The infected PANC-1 cells were infected with the packed Lenti-ORAI1-shRNA, the flow cytometry was used to determine the infection efficiency. The fluorescence quantitative PCR was used to detect the transcriptional inhibition efficiency of the ORAI1 gene. The BALB/c mice were randomly divided into 7 groups, one group was the normal control group and the other six groups were Lenti-ORAI1. -shRNA intervention group. After the intervention group was anesthetized, the nasal cavity was dripped into the 8 u l0.3% hemolytic lecithin, and the nasal drip was 1 * 109tu/ml Lenti-ORAI1-shRNA after 1 hours. The mice were killed after 1,3,7,14,21,28 infection and the ORAI1mRNA level of the nasal mucosa was measured. The rats were randomly divided into 5 groups: normal control group (normal), positive. The normal mice Lenti-ORAI1-shRNA intervention group (NLI), allergic rhinitis group (AR), allergic rhinitis mice Lenti-GFP intervention group (AR-GFP), allergic rhinitis mice Lenti-ORAI1-shRNA intervention group (AR-LI). The latter three groups of mice with.5mg/ml OVA/20mg/ml Al (OH) 3 solution intraperitoneal injection 3 times, 40mg/ml 8 times to stimulate the establishment of allergic rhinitis The mouse model was used to intervene in the NLI and AR-LI mice at the 20gl drop of 1 x 109tu / ml 3 days before the first nasal excitation, and the AR-GFP mice were treated with the same dose and titer Lenti-GFP. The number of sneezes and the number of nose in 10 minutes after the last nasal stimulation of the mice in each group and the Real-time RT PCR method were measured. The nasal mucosa and spleen of each group were ORAI1, LTC4S, EAR3, germline C epsilon and IL-4mRNA level. Western Blotting method was used to determine the expression level of Orai1, IL-33, IL-17E, TSLP and spleen. The concentrations of LTC4, ECP, OVA-IgE and IL-4 in nasal lavage fluid and serum of mice were determined.
Results: PANC-1 cells can express the GFP gene carried by lentivirus after Lenti-ORAI1-shRNA infection and send out green fluorescence. When the complex number is 10, the infection efficiency of Lenti-ORAI1-shRNA infected PANC-1 cells after 48 hours is 98.28% and the transcriptional inhibition efficiency of ORAI1 gene is 90.5%. The infection efficiency was 98.75%, and the transcriptional inhibition efficiency of ORAI1 gene was -3%. mouse nasal infection Lenti-ORAI1-shRNA, the ORAI1mRNA level of nasal mucosa gradually decreased with the infection time, and reached the lowest at third days, and then gradually increased. The level of Orai1mRNA was still lower than that of normal control group.AR-LI mice after one month. The level of ORAI1mRNA in spleen was lower than that in group AR, and the expression level of Orai1 protein in nasal mucosa was also lower than that in group AR. The number of nose and sneeze in the last nasal cavity were also significantly lower than that of the AR group. The level of ORAI1mRNA in the nasal mucosa and spleen in the AR-GFP group, the level of Orai1 expression, the number of sneezes after the last excitation and the number of the nose were compared with the mice in the AR group. The results of immunohistochemistry showed that the expression of Orai1 in nasal mucosa epithelium and lamina propria, nasal mucosa associated lymphoid tissue and spleen germinal center of AR-LI mice was lower than that of group AR mice and the mRNA levels of LTC4S, EAR3, germline C epsilon and IL-4 in the nasal mucosa and spleen of group.AR-LI mice were significantly lower than those in AR group, and their nasal lavage was more than that of the mice. The concentration of LTC4, OVA-IgE and IL-4 in the liquid and the concentration of LTC4, ECP, OVA-IgE and IL-4 in the serum were also lower than that of the AR mice. There was no significant difference between the nasal mucosa and the spleen in the AR-GFP group. The expression level of IL-33, IL-17E and TSLP in the nasal mucosa of.AR mice was significantly higher than that in the normal control group. The expression level of IL-33 and TSLP in the nasal mucosa of the AR-LI group was lower than that in the AR group, but the expression level of IL-17E was not significantly different from that in the AR group. There was no significant difference between the AR group and the mice.
Conclusion: the recombinant lentivirus Lenti-ORAI1-shRNA was successfully constructed for the RNA interference of the ORAI1 gene, and Lenti-ORAI1-shRNA could successfully infect PANC-1 cells and effectively inhibit the transcriptional level of ORAI1 gene,.Lenti-ORAI1-shRNA instillation could successfully infect the nasal mucosa cells of mice, and the transcriptional inhibition efficiency of ORAI1 gene was reached third days after infection. The expression of Orai1 in nasal mucosa and spleen can be reduced effectively after Lenti-ORAI1-shRNA nasal intervention in allergic rhinitis mice. The expression of Orai1 in nasal mucosa can not only reduce the inflammatory mediators of mast cells, eosinophil cells, B cells and Th2 cells in nasal mucosa of allergic rhinitis mice. Transcription and release can also reduce the transcriptional and release of the systemic inflammatory mediators of the spleen and serum, such as the spleen and serum, and the inhibitory effect of.Lenti-ORAI1-shRNA nasal intervention on inflammation of allergic rhinitis in mice may be related to the inhibition of the synthesis and expression of IL-33 and TSLP protein in the epithelial cells of the nasal mucosa.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R765.21

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10 鞠梅;張青松;張小華;陳昆;常寶珠;顧恒;;不同劑量的UVA1輻射硬皮病鼠模型的實(shí)驗(yàn)研究[A];中華醫(yī)學(xué)會(huì)第14次全國(guó)皮膚性病學(xué)術(shù)年會(huì)論文匯編[C];2008年

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8 李雷;美構(gòu)建出成神經(jīng)管細(xì)胞瘤小鼠模型[N];中國(guó)醫(yī)藥報(bào);2006年

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10 李e灞頸ㄊ迪凹?xì)J，

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