体外活化TLR3对HBV宫内感染新生儿免疫状况的影响

Effect of in vitro activation of TLR3 on immune state of neonates with HBV intrauterine infection

目的:通过体外活化Toll样受体3(TLR3),探索将Poly I:C(免疫佐剂)作为TLR3配体提高HBV宫内感染者TLR3蛋白含量,进而通过改变HBV宫内感染者DCs细胞及B细胞百分比,提供改善机体免疫状态的可能性。方法:采用面对面问卷调查方法连续收集152对HBsAg阳性孕妇及其新生儿流行病学资料,并采集新生儿脐血及股静脉血。分别采用酶联免疫吸附试验(ELISA)及实时荧光定量PCR检测新生儿出生24 h内外周血乙肝病毒标志物及HBV DNA含量,密度梯度离心法分离脐血单个核细胞(CBMCs)以进行体外培养,经HBV与Poly I:C刺激后,流式细胞术(FCM)检测新生儿CBMCs的TLR3蛋白含量及DCs细胞、B细胞的百分比。结果:HBV非宫内感染的TLR3蛋白含量在经HBV以及PolyI:C+HBV后呈现下降趋势。而HBV宫内感染组呈现先下降后升高的趋势。HBV非宫内感染组与宫内感染组CBMCs经PolyI:C+HBV刺激活化后,两组TLR3蛋白含量、mDC、pDC比例差异均无统计学意义(t=0.11,P=0.91;t=0.46,P=0.64;t=0.21,P=0.23);而两组B细胞百分比差异有统计学意义(t=2.36,P=0.02)。HBV宫内感染组经Poly I:C+HBV刺激后与非宫内感染组经HBV刺激后的B细胞百分比比较差异亦有统计学意义(t=2.45,P=0.02)。结论:Poly I:C作为免疫佐剂一定程度上提高HBV宫内感染者的TLR3蛋白水平,使宫内感染组的TLR3蛋白水平逐渐接近于非宫内感染组,进而改变CBMCs中DCs细胞及B细胞百分比含量,为改善HBV宫内感染新生儿的免疫状况使其接近非宫内感染者水平提供可能。

Objective：To explore the possibility of Poly I：C （ a kind of immunologic adjutants） as a ligand of Toll - like receptor （TLR3） to improve TLR3 protein content, change proportions of DCs and B cells in patients with HBV intrauterine infection, and improve immune state by in vitro activation of TLR3. Methods：The epidemiological data of 152 pregnant women with positive HBsAg and their neo- nates were collected continuously by face - to - face question,laire investigation method, the umbilical cord blood samples and femoral venous blood samples of neonates were abstracted. ELISA and real - time fluorescent quantitative PCR were used to detect HBV biomarkers and HBV DNA content in peripheral blood of neonates within 24 hours after birth, density gradient centrifugation was used to isolate cord blood mononuclear cells （CBMCs） for in vitro culture, after stimulation with HBV and Poly I：C, flow cytometry was used to detect TLR3 protein content, proportions of DCs and B cells of neonates. Results： After treated with HBV and PolyI ： C ＋ HBV, the content of TLR3 protein in non - intrauterine infection group showed a decreasing trend ; while in intrauterine infection group, the content of TLR3 protein increased at first and then decreased. After stimulation and activation with PolyI ： C ＋ HBV, there was no statistically significant difference in TLR3 pro- tein content, mDC proportion, and pDC proportion in CBMCs between intrauterine infection group and non - intrauterine infection group （t = 0. 11, P =0. 91 ; t =0. 46, P =0. 64; t =0. 21, P =0. 23） ; but there was statistically significant difference in proportion of B ceils between the two groups （ t = 2. 36, P = 0. 02）. There was statistically significant difference in proportion of B cells between intrauterine infection group after stimulation with Poly I ： C ＋ HBV and non - intrauterine infection group ＇after stimulation with HBV （ t = 2. 45, P = 0. 02）. Con- clusion：As a kind of immunologic adjutant, poly I：C improves TLR3 protein level of patients with HBV intrauterine infection to a certain de- gree, makes TLR3 protein level of intrauterine infection group approaching to that of non - intrauterine infection group gradually, changes the proportions of DCs and B cells in CBMCs, and provides a way to improve immune state of neonates with HBV intrauterine infection.