本文選題：細粒棘球蚴 + TGF-β1　； 參考：《石河子大學》2014年碩士論文

【摘要】：目的 通過建立細粒棘球蚴感染小鼠動物模型，探討細粒棘球蚴是否通過促進脾細胞分泌抑制性分子TGF-β1而促使CD4+CD25+T調節(jié)細胞分化，從而下調NK細胞活性受體NKG2D的表達，最終抑制NK細胞對原頭蚴的殺傷，而形成對宿主的免疫逃避。 方法 1.建立小鼠細粒棘球蚴感染模型，實驗組用原頭蚴感染BALB/c小鼠，對照組小鼠腹腔注射同體積的PBS；分別于第1、3、5、7、9、12天處死后，用流式細胞儀檢測脾細胞中T淋巴細胞亞群CD4+/CD8+T細胞比值及CD4+CD25+T細胞的含量及NK細胞的活性受體NKG2D的表達；qRT-PCR法檢測Foxp3mRNA和TGF-β1mRNA的相對表達量；Yac-1作為靶細胞與小鼠脾細胞共培養(yǎng)，用LDH法檢測小鼠NK細胞對Yac-1的裂解率，觀察脾細胞的殺傷活性。 2.建立小鼠細粒棘球蚴感染模型，并用抑制劑特異性阻斷TGF-β1受體，于第9天處死后用流式細胞儀檢測T淋巴細胞亞群CD4+/CD8+T細胞比值及CD4+CD25+T細胞的含量及NK細胞的活性受體NKG2D的表達；用LDH法檢測脾細胞的殺傷活性；Western Bolting檢測細粒棘球蚴對TGF-β/Smad的信號通路的影響。 結果 1.在細粒棘球蚴感染早期，CD4+CD25+Foxp3+T比值增高：與對照組相比小鼠CD4+/CD8+T細胞比值逐漸下降；隨著感染時間的延長，CD4+CD25+T細胞數(shù)量呈逐漸增高；Foxp3在基因水平上的表達呈增高的趨勢；與對照組相比，除感染后第1天無統(tǒng)計學差異外，其他的均有統(tǒng)計學差異（P0.05）。 2.在細粒棘球蚴感染早期，外周血中TGF-β1的含量增高：ELISA結果顯示外周血細胞因子TGF-β1水平顯著高于對照組（P0.05），并隨著感染時間的延長而增高；qRT-PCR法檢測到TGF-β1在mRNA水平的表達量也高于對照組（P0.05），隨著感染時間的延長，呈增高的趨勢。 3.在細粒棘球蚴感染早期，，小鼠NK細胞數(shù)量及殺傷活性均下降：小鼠NK細胞對Yac-1細胞的裂解率與對照組比較均有統(tǒng)計學意義（P0.05）；隨著時間的延長NK細胞的活性受體NKG2D的表達量呈下降的趨勢，與對照組比較，感染后1、3、9、12天NKG2D的表達量都有統(tǒng)計學意義（P0.05）。隨著時間的延長NK細胞數(shù)量呈下降的趨勢，與感染前比較，感染后第1、3、9、12天NK數(shù)量差異都有統(tǒng)計學意義（P0.05）。細粒棘球蚴早期，感染小鼠NK細胞的殺傷活性與其活性受體NKG2D的表達呈正相關，R2=0.679。 4.細粒棘球蚴早期感染小鼠，通過抑制劑SB-525334阻斷TGF-β1受體后，上述所有結果均逆轉：其下游分子Smad2/3磷酸化蛋白表達下降，NK細胞活性受體NKG2D表達增加，NK細胞對靶細胞的裂解率恢復，小鼠NK細胞的活性與其活性受體NKG2D的相關性分析的表達呈正相關，CD4+/CD8+T細胞比值升高，CD4+CD25+T細胞數(shù)量減少。 結論 1.通過建立細粒棘球蚴感染小鼠動物模型，觀察到：細粒棘球蚴通過促進脾細胞分泌抑制性分子TGF-β1而促使CD4+CD25+T調節(jié)細胞分化，從而下調NK細胞活性受體NKG2D的表達，最終導致NK細胞對原頭蚴的殺傷減弱，而形成對宿主的免疫逃避。 2. TGF-β1受體阻斷劑SB-525334可能在臨床上預防包蟲病的復發(fā)有一定實際意義。
[Abstract]:objective
By establishing a mouse model of Echinococcus granulosus infection, it is explored whether Echinococcus granulosus promotes CD4+CD25+T to regulate cell differentiation by promoting the secretion of suppressor molecule TGF- beta 1 in spleen cells, thus reducing the expression of NK cell active receptor NKG2D, and ultimately inhibiting the killing of NK cells to the original cercariae, and forming immune escape to the host.
Method
1. the mice model of Echinococcus granulosus infection was established. The experimental group infected BALB/c mice with the original cercariae, and the control group was injected with the same volume of PBS. After the death on day 1,3,5,7,9,12, the ratio of CD4+/CD8+T cells of T lymphocyte subsets and the content of CD4+ CD25+T cells in the spleen cells and the NKG2 active receptor NKG2 in the NK cells were detected by flow cytometry. The expression of D and the relative expression of Foxp3mRNA and TGF- beta 1mRNA were detected by qRT-PCR; Yac-1 was co cultured with mouse spleen cells as target cells, and LDH method was used to detect the cracking rate of mice NK cells to Yac-1, and the killing activity of spleen cells was observed.
2. the mice model of Echinococcus granulosus infection was established and the TGF- beta 1 receptor was blocked by the inhibitor specificity. After ninth days of death, the ratio of CD4+/CD8+T cells in T lymphocyte subsets and the content of CD4+CD25+T cells and the expression of NKG2D in NK cells were detected by flow cytometry; LDH method was used to detect the killing activity of spleen cells; Western Bolting was used. The effects of Echinococcus granulosus on TGF- beta /Smad signaling pathway were examined.
Result
1. in the early stage of infection of Echinococcus granulosus, the ratio of CD4+CD25+Foxp3+T increased: the ratio of CD4+/CD8+T cells decreased gradually compared with the control group; the number of CD4+CD25+T cells increased gradually with the prolongation of the infection time; the expression of Foxp3 at the gene level was increasing; compared with the control group, there was no statistical difference between the two days after infection. The other were statistically different (P0.05).
2. in the early stage of infection of Echinococcus granulosus, the content of TGF- beta 1 in peripheral blood increased: ELISA results showed that the level of peripheral blood cell factor TGF- beta 1 was significantly higher than that of the control group (P0.05), and increased with the prolongation of the infection time, and the expression of TGF- beta 1 at mRNA level was also higher than that of the control group (P0.05), with the prolongation of the infection time, with the qRT-PCR method. The trend is increasing.
3. in the early stage of infection of Echinococcus granulosus, the number and killing activity of NK cells in mice decreased: the cracking rate of mouse NK cells to Yac-1 cells was significantly higher than that of the control group (P0.05); the expression of active receptor NKG2D in NK cells decreased with time, and compared with the control group, the 1,3,9,12 day NKG2D table after infection was compared with the control group. The amount of NK cells was statistically significant (P0.05). Compared with pre infection, the number of NK in 1,3,9,12 days after infection was statistically significant (P0.05). In the early stage of Echinococcus granulosus, the killing activity of NK cells in infected mice was positively related to the expression of the active receptor NKG2D, R2=0.679.
4. the early infection of Echinococcus granulosus in mice, after blocking the TGF- beta 1 receptor by the inhibitor SB-525334, all the results were reversed: the downstream molecule Smad2/3 phosphorylation protein expression decreased, the NK cell active receptor NKG2D expression increased, the NK cell lysis rate of the target cells was restored, the activity of NK cells in the rat NK and the correlation of the active receptor NKG2D The expression of CD4+/CD8+T was positively correlated, and the ratio of CD4+CD25+T cells increased.
conclusion
1. by establishing a mouse model of Echinococcus granulosus infection, it was observed that Echinococcus granulosus caused CD4+CD25+T to regulate cell differentiation by promoting the secretion of suppressor TGF- beta 1 in spleen cells, thus reducing the expression of NK cell active receptor NKG2D, which eventually led to the decrease of NK cells' killing of the original cercariae, and the immune escape to the host was formed.
2. the TGF- beta 1 receptor blocker SB-525334 may be clinically useful in preventing the recurrence of echinococcosis.

【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R532.32

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