本文選題：膿毒癥 + 微小RNA-21��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討烏司他丁在脂多糖誘導(dǎo)的RAW264.7細(xì)胞的保護(hù)作用及對mi R-21表達(dá)影響。方法:(1)用不同濃度(100,250,500 ng/m L)LPS刺激RAW264.7細(xì)胞,RT-PCR法檢測mi R-21的表達(dá)、腫瘤壞死因子-α(Tumor necrosis factor-α,TNF-α)m RNA、白介素-6(interleukin-6,IL-6)m RNA的表達(dá);(2)采用500ng/m L LPS刺激RAW264.7細(xì)胞6h、12h、24h,采用RT-PCR檢測TNF-αm RNA;(3)四甲基偶氮唑(Methyl thiazolyl tetrazolium,MTT)法檢測不同濃度(10,100,1000U/m L)UTI對RAW264.7細(xì)胞活性的影響;設(shè)置正常組、模型組(LPS 500ng/m L剌激組)、實(shí)驗(yàn)組(LPS 500ng/m L+UTI 1000U/m L組)和陰性實(shí)驗(yàn)對照組(UTI 1000U/m L),觀察RAW264.7細(xì)胞生長狀態(tài);(4)用不同濃度(10,100,1000 U/m L)UTI預(yù)處理2h后,LPS刺激小鼠RAW264.7細(xì)胞誘導(dǎo)炎癥模型,RT-PCR檢測腫瘤壞死因子TNF-αm RNA、IL-6 m RNA、mi R-21的表達(dá),Western Blot法檢測UTI(1000 U/m L)時(shí)PTEN蛋白表達(dá)水平。(5)設(shè)置空白組、模型組(LPS 500 ng/m L剌激組)、實(shí)驗(yàn)組(LPS+mi R-21模擬劑、LPS+mi R-21抑制劑組):RT-PCR法檢測mi R-21的表達(dá),Western Blot法檢測PTEN蛋白表達(dá)水平。結(jié)果:1.UTI對細(xì)胞的保護(hù)作用1 UTI對RAW264.7細(xì)胞的毒性作用:UTI濃度小于1000U/m L時(shí)對RAW264.7細(xì)胞無毒性作用,各組細(xì)胞OD值比較差異無統(tǒng)計(jì)學(xué)意義(P=0.117);2.不同濃度(10,100,1000U/m L)UTI能有效降低TNF-αm RNA表達(dá)及IL-6 m RNA表達(dá)(P10.05,P20.05)。2.不同LPS濃度下TNF-αm RNA、IL-6 m RNA及mi R-21的表達(dá)情況與正常組相比,在LPS(100,250,500ng/m L)刺激RAW264.7細(xì)胞后,mi R-21、TNF-αm RNA及IL-6 m RNA的分泌量與基礎(chǔ)分泌量相比均明顯升高(P0.05),并隨LPS濃度升高而增加(P0.05)。3.UTI對mi R-21表達(dá)的影響不同濃度(10,100,1000U/m L)UTI能有效降低mi R-21的表達(dá),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.mi R-21對PTEN蛋白表達(dá)的影響與空白組相比較,在LPS組中,mi R-21表達(dá)上調(diào),PTEN蛋白表達(dá)下調(diào);在加入.mi R-21抑制劑組中,mi R-21表達(dá)明顯下降,PTEN蛋白表達(dá)明顯升高;mi R-21模擬劑組中,mi R-21表達(dá)明顯升高,PTEN蛋白表達(dá)明顯下降(P0.05)。5.UTI對PTEN蛋白表達(dá)的影響基于上述UTI下調(diào)mi R-21表達(dá)水平的實(shí)驗(yàn)結(jié)果,進(jìn)一步觀察UTI對mi R-21靶基因PTEN蛋白的影響。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),與空白組比較,PTEN蛋白表達(dá)水平在LPS組中明顯下降,在UTI組中表達(dá)水平明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.UTI可降低LPS誘導(dǎo)的RAW264.7細(xì)胞中TNF-α、IL-6 m RNA的表達(dá)。2.LPS可誘導(dǎo)RAW264.7細(xì)胞mi R-21的高表達(dá),且呈濃度依賴性;mi R-21高表達(dá)可抑制PTEN蛋白的表達(dá)。3.UTI可顯著下調(diào)mi R-21的表達(dá),并上調(diào)靶基因PTEN蛋白表達(dá)水平。綜上所述,UTI可抑制LPS誘導(dǎo)RAW264.7細(xì)胞的炎癥反應(yīng),對細(xì)胞起著保護(hù)作用,其機(jī)制可能與UTI引起mi R-21表達(dá)下調(diào)有關(guān)。
[Abstract]:Aim: to investigate the protective effect of ulinastatin on lipopolysaccharide-induced RAW264.7 cells and its effect on the expression of miR-21. Methods the expression of miR-21 was detected by RT-PCR in RAW264.7 cells stimulated with different concentrations of 100250500 ng/m L)LPS. TNF- 偽 -Tumor necrosis factor- 偽 -TNF- 偽 -mRNAs, interleukin-6h6 interleukin-6tr (IL-6m RNA expression) were used to stimulate RAW264.7 cells for 6 h and 12 h to 24 h by 500ng/m L LPS. TNF- 偽 m RNA-3) tetrazolium L)UTI (MTT) assay was used to detect the effect of different concentrations of 10 100 渭 m L)UTI on RAW264.7 cell activity. In normal group, TNF- 偽 m RNA-3) was used to detect the effect of TNF- 偽 necrosis factor- 偽 on the activity of RAW264.7 cells with different concentrations of TNF- 偽 necrosis factor- 偽 (TNF- 偽 necrosis factor- 偽 -TNF- 偽 -TNF- 偽 -TNF- 偽 -TNF- 偽). The model group was stimulated by 500ng/m L, the experimental group was treated with 500ng/m L UTI 1000U/m L) and the negative control group was used to observe the growth state of RAW264.7 cells. The RAW264.7 cells were pretreated with different concentrations of 10100U / m L)UTI for 2 hours. The model of RAW264.7 cells was induced by RT-PCR after pretreatment with different concentrations of 10100U / m L)UTI for 2 hours. The expression of TNF- 偽 m RNA-IL-6 m RNAi R-21 was detected. Western Blot assay was used to detect the expression level of PTEN protein in UTI(1000 UP / mL. (5) blank group was set up. The expression of miR-21 in the model group was detected by ng/m RT-PCR and the expression level of PTEN protein was detected by Western Blot method in the LPS-mi R-21 mimic of the experimental group and the LPS-mi R-21 inhibitor group. Results 1. The cytotoxic effect of UTI on RAW264.7 cells. When the concentration of UTI was lower than that of 1000U/m L, there was no toxic effect on RAW264.7 cells. There was no significant difference in OD value between the two groups. The expression of TNF- 偽 m RNA and the expression of IL-6 m RNA were significantly decreased by different concentrations of 10100U ~ 1000Um L)UTI. The expression of IL-6 m RNA and miR-21 in TNF- 偽 m RNAs at different concentrations of LPS was significantly higher than that in normal controls. After stimulation of RAW264.7 cells with LPS(100250500ng/m L, the secretion of Mi R-21 TNF- 偽 m RNA and IL-6 m RNA increased significantly compared with the basal secretion, and increased with the increase of LPS concentration. 3. The effect of UTI on the expression of miR-21 was observed. The difference was statistically significant (P 0.05) .4.mi R-21 had a significant effect on the expression of PTEN protein compared with the control group. In the LPS group, the expression of PTEN R-21 up-regulated the expression of PTEN protein. The expression of PTEN protein decreased significantly in the group treated with .mi R-21 inhibitor; the expression of PTEN protein increased significantly in the mimic group of miR-21; the expression of PTEN protein decreased significantly in the group of mi-R-21 mimics; the effect of P0.05. 5.UTI on the expression of PTEN protein was based on the down-regulation of miR-21 protein by the above-mentioned UTI. Expression level of the experimental results, To investigate the effect of UTI on PTEN protein of miR-21 target gene. The results showed that compared with the blank group, the expression level of PTEN protein in the LPS group was significantly decreased, and the expression level in the UTI group was significantly increased. The difference was statistically significant (P 0.05). Conclusion: 1. UTI can decrease the expression of TNF- 偽 IL-6 m RNA in RAW264.7 cells induced by LPS. 2.LPs can induce the high expression of miR-21 in RAW264.7 cells, and the high expression of PTEN protein in RAW264.7 cells can be inhibited in a dose-dependent manner. 3. UTI can significantly down-regulate the expression of miR-21. The expression level of target gene PTEN protein was upregulated. In conclusion, LPS can inhibit the inflammatory response of RAW264.7 cells induced by UTI, which may be related to the down-regulation of miR-21 expression induced by UTI.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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