本文選題：異種移植　切入點(diǎn)：心肌細(xì)胞凋亡　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:在心臟移植后,急性血管性排斥反應(yīng)導(dǎo)致心肌細(xì)胞間質(zhì)出血、水腫,周圍中性粒細(xì)胞浸潤(rùn),血管內(nèi)皮細(xì)胞死亡,最終細(xì)胞凋亡。本實(shí)驗(yàn)擬研究Toll樣受體通路及其下游通路核因子-κB(NF-κB)在心肌細(xì)胞凋亡中的作用。方法:在體外培養(yǎng)C57BL6小鼠的心肌細(xì)胞及SD大鼠和BALB/C小鼠的脾細(xì)胞。在體外建立C57BL6小鼠到BALB/C小鼠和SD大鼠的心臟移植細(xì)胞模型。將實(shí)驗(yàn)分為六組,A組:C57BL6小鼠心肌細(xì)胞+PBS液(PBS組);B組:C57BL6小鼠心肌細(xì)胞+TNF-α(TNF-α組);C組:C57BL6小鼠心肌細(xì)胞+BALB/C小鼠脾細(xì)胞(Mouse SC組);D組:C57BL6小鼠心肌細(xì)胞+BALB/C小鼠脾細(xì)胞+BALB/C小鼠血清(Mouse SC+S組);E組:C57BL6小鼠心肌細(xì)胞+SD大鼠脾細(xì)胞(Rat SC組);F組:C57BL6小鼠心肌細(xì)胞+SD大鼠脾細(xì)胞+SD大鼠血清(Rat SC+S組),共培養(yǎng)時(shí)間為108h。運(yùn)用Annexin-V/PI法檢測(cè)各組C57BL6小鼠心肌細(xì)胞凋亡,比較同種和異種免疫原對(duì)心肌細(xì)胞刺激效應(yīng)的差別。RT-PCR法檢測(cè)A組、C組、D組、E組、F組心肌細(xì)胞TLR-1、TLR-2、TLR-3、TLR-4、TLR-5、TLR-7、TLR-9的表達(dá),篩選出被激活的Toll樣受體。利用相應(yīng)的Toll樣受體的阻斷抗體抑制激活的Toll樣受體,Annexin-V/PI法檢測(cè)心肌細(xì)胞凋亡,Western-blot法檢測(cè)其下游通道NF-κB的表達(dá)變化。結(jié)果:運(yùn)用酶消化法培養(yǎng)C57BL6小鼠的心肌細(xì)胞,細(xì)胞活性好,純度達(dá)80-90%。供心心肌細(xì)胞凋亡檢測(cè)實(shí)驗(yàn)中,TNF-α組、Rat SC組、Rat SC+S組的心肌細(xì)胞凋亡比例達(dá)71.31±7.71%、70.05±5.73%、68.61±4.71%,與PBS組、Mouse SC組、Mouse SC+S組相比,心肌細(xì)胞凋亡程度有明顯差異(P0.001)。檢測(cè)未經(jīng)處理的C57BL6小鼠心肌細(xì)胞的各Toll樣受體信號(hào)的表達(dá),均在相對(duì)較低水平,呈靜息狀態(tài)。各實(shí)驗(yàn)組中,在Rat SC組和Rat SC+S組心肌細(xì)胞的TLR-2和TLR-3的表達(dá)明顯增高(P0.05),其中TLR-3的增高更加明顯(P0.05)。運(yùn)用TLR-3抗體來(lái)抑制心肌細(xì)胞TLR-3信號(hào)通路后,供心心肌細(xì)胞的凋亡比例減少(P0.05),NF-κB p65蛋白的表達(dá)量降低(P0.05)。結(jié)論:1.在小鼠-大鼠心臟移植細(xì)胞模型中,TLR-2和TLR-3是異種免疫原誘導(dǎo)的心肌細(xì)胞凋亡過(guò)程中所激活的主要Toll受體種類,而TLR-3是受激活最明顯的受體。2.通過(guò)抑制TLR-3受體信號(hào)通路的激活,可抑制下游的NF-κB表達(dá),減輕心肌細(xì)胞凋亡。
[Abstract]:Objective: after cardiac transplantation, acute vascular rejection resulted in interstitial hemorrhage, edema, neutrophil infiltration and vascular endothelial cell death. The purpose of this study was to investigate the role of nuclear factor-魏 B-魏-魏 B in cardiomyocyte apoptosis. Methods: cardiomyocytes of C57BL6 mice and spleen cells of SD rats and BALB/C mice were cultured in vitro. The model of heart transplantation cells from C57BL6 mice to BALB/C mice and SD rats was established in vitro. The experiment was divided into six groups: group A, group 1: C57BL6 mice cardiomyocytes, PBS solution group, group B, group B: TNF- 偽 + TNF- 偽 group, group C, group C, group C, group C, group BALB/C, cardiomyocytes of C57BL6 mouse cardiomyocytes: TNF- 偽, TNF- 偽, TNF- 偽, TNF- 偽, TNF- 偽 and TNF- 偽. Murine splenocytes / mice / mice / mice / mice / / D / / BALB/C / C57BL6 / BALB/C mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / mice / / / mice / mice / mice / mice / mice /. The co-culture time of SD rat SC S group was 108 h. Myocardial cell apoptosis was detected by Annexin-V/PI assay in C57BL6 mice. RT-PCR was used to detect the expression of TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-7 and TLR-9 in cardiomyocytes of group A, C, C, D, E and F, respectively, by comparing the effects of allogeneic and xenogeneic immunogen on cardiomyocytes. The activated Toll like receptors were screened. The expression of NF- 魏 B in the downstream channel of cardiomyocytes was detected by using the Annexin-V / Pi method to detect the expression of NF- 魏 B in the downstream channels of the activated Toll like receptors by blocking antibodies against the corresponding Toll like receptors. Results: the enzyme digestion method was used to detect the expression of NF- 魏 B. Cultured cardiomyocytes of C57BL6 mice, The percentage of cardiomyocyte apoptosis in TNF- 偽 group was 71.31 鹵7.71 and 70.05 鹵5.731.61 鹵4.71 in rat SC group, which was higher than that in PBS group, compared with mouse SC group in TNF- 偽 group, and the percentage of apoptosis in rat SC group was 71.31 鹵7.71 in TNF- 偽 group, and 68.61 鹵4.71 in mouse SC group in TNF- 偽 group. There was a significant difference in the degree of cardiomyocyte apoptosis. The expression of Toll like receptors in untreated C57BL6 mouse cardiomyocytes was detected at a relatively low level and in a resting state. In Rat SC group and Rat SC S group, the expression of TLR-2 and TLR-3 in cardiomyocytes was significantly higher than that in Rat SC group, especially in TLR-3 group. TLR-3 antibody was used to inhibit the TLR-3 signal pathway in cardiomyocytes. The ratio of apoptosis in donor cardiomyocytes decreased the expression of P0.05- 魏 B p65 protein. Conclusion: 1. TLR-2 and TLR-3 are the main types of Toll receptors activated by xenogen-induced cardiomyocyte apoptosis in murine rat heart transplantation cell model. By inhibiting the activation of TLR-3 receptor signaling pathway, TLR-3 can inhibit the expression of NF- 魏 B downstream and attenuate cardiomyocyte apoptosis.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R654.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李溫斌,陳寶田,石鏡,趙莉敏,潘桂榮,董冉;心臟移植后急性排異反應(yīng)體外模型的建立及其應(yīng)用研究[J];中國(guó)免疫學(xué)雜志;1997年02期

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本文編號(hào)：1586228