TGF-β抑制自然杀伤细胞在细粒棘球蚴免疫逃逸作用中的初步探讨

发布时间：2018-05-06 08:06

本文选题：细粒棘球蚴 + TGF-β1　；参考：《石河子大学》2014年硕士论文

【摘要】：目的通过建立细粒棘球蚴感染小鼠动物模型，探讨细粒棘球蚴是否通过促进脾细胞分泌抑制性分子TGF-β1而促使CD4+CD25+T调节细胞分化，从而下调NK细胞活性受体NKG2D的表达，最终抑制NK细胞对原头蚴的杀伤，而形成对宿主的免疫逃避。方法 1.建立小鼠细粒棘球蚴感染模型，实验组用原头蚴感染BALB/c小鼠，对照组小鼠腹腔注射同体积的PBS；分别于第1、3、5、7、9、12天处死后，用流式细胞仪检测脾细胞中T淋巴细胞亚群CD4+/CD8+T细胞比值及CD4+CD25+T细胞的含量及NK细胞的活性受体NKG2D的表达；qRT-PCR法检测Foxp3mRNA和TGF-β1mRNA的相对表达量；Yac-1作为靶细胞与小鼠脾细胞共培养，用LDH法检测小鼠NK细胞对Yac-1的裂解率，观察脾细胞的杀伤活性。 2.建立小鼠细粒棘球蚴感染模型，并用抑制剂特异性阻断TGF-β1受体，于第9天处死后用流式细胞仪检测T淋巴细胞亚群CD4+/CD8+T细胞比值及CD4+CD25+T细胞的含量及NK细胞的活性受体NKG2D的表达；用LDH法检测脾细胞的杀伤活性；Western Bolting检测细粒棘球蚴对TGF-β/Smad的信号通路的影响。结果 1.在细粒棘球蚴感染早期，CD4+CD25+Foxp3+T比值增高：与对照组相比小鼠CD4+/CD8+T细胞比值逐渐下降；随着感染时间的延长，CD4+CD25+T细胞数量呈逐渐增高；Foxp3在基因水平上的表达呈增高的趋势；与对照组相比，除感染后第1天无统计学差异外，其他的均有统计学差异（P0.05）。 2.在细粒棘球蚴感染早期，外周血中TGF-β1的含量增高：ELISA结果显示外周血细胞因子TGF-β1水平显著高于对照组（P0.05），并随着感染时间的延长而增高；qRT-PCR法检测到TGF-β1在mRNA水平的表达量也高于对照组（P0.05），随着感染时间的延长，呈增高的趋势。 3.在细粒棘球蚴感染早期，，小鼠NK细胞数量及杀伤活性均下降：小鼠NK细胞对Yac-1细胞的裂解率与对照组比较均有统计学意义（P0.05）；随着时间的延长NK细胞的活性受体NKG2D的表达量呈下降的趋势，与对照组比较，感染后1、3、9、12天NKG2D的表达量都有统计学意义（P0.05）。随着时间的延长NK细胞数量呈下降的趋势，与感染前比较，感染后第1、3、9、12天NK数量差异都有统计学意义（P0.05）。细粒棘球蚴早期，感染小鼠NK细胞的杀伤活性与其活性受体NKG2D的表达呈正相关，R2=0.679。 4.细粒棘球蚴早期感染小鼠，通过抑制剂SB-525334阻断TGF-β1受体后，上述所有结果均逆转：其下游分子Smad2/3磷酸化蛋白表达下降，NK细胞活性受体NKG2D表达增加，NK细胞对靶细胞的裂解率恢复，小鼠NK细胞的活性与其活性受体NKG2D的相关性分析的表达呈正相关，CD4+/CD8+T细胞比值升高，CD4+CD25+T细胞数量减少。结论 1.通过建立细粒棘球蚴感染小鼠动物模型，观察到：细粒棘球蚴通过促进脾细胞分泌抑制性分子TGF-β1而促使CD4+CD25+T调节细胞分化，从而下调NK细胞活性受体NKG2D的表达，最终导致NK细胞对原头蚴的杀伤减弱，而形成对宿主的免疫逃避。 2. TGF-β1受体阻断剂SB-525334可能在临床上预防包虫病的复发有一定实际意义。
[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.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.32

【参考文献】