【作者】 陈悦；

【导师】 李慕军；

【作者基本信息】 广西医科大学 ， 儿科学， 2014， 博士

【摘要】 第一部分孕期不同HAART方案暴露对HIV感染母亲及HIV未感染新生儿的影响目的:了解孕期接受高效抗逆转录病毒治疗（HAART）方案预防HIV母婴传播的安全性和有效性，探讨两种不同HAART方案对妊娠结局、孕妇和胎儿造血及免疫功能的影响。方法:71名单胎妊娠HIV感染孕妇按照HAART方案的不同分为含齐多夫定（AZT）组40人、不含AZT组31人，以40名健康孕妇为对照组。HIV阳性孕妇自妊娠14周及14周以后开始抗病毒治疗，含AZT组的HAART方案为齐多夫定+拉米夫定（AZT+3TC）联合克力芝（LPV/r）或依非那仑（EFV）或奈韦拉平（NVP），不含AZT组的HAART方案为替诺福韦（TDF）+3TC联合LPV/r或EFV或NVP。采集开始抗病毒治疗时和分娩前孕妇肘静脉血、脐带血，进行血细胞参数测定和CD4+、CD8+T淋巴细胞测定，同时收集孕妇和新生儿基本信息进行分析，婴儿出生后4～6周及4个月进行HIV-1-DNA核酸测定。结果:1HAART治疗的两组孕妇当中，无妊娠期高血压疾病和妊娠期糖尿病的发生；HIV阳性母亲分娩新生儿无HIV感染发生，未观察到新生儿出生缺陷的发生，无死胎和死产发生。2三组均无新生儿重度窒息发生，轻度窒息发生率无显著差异（P＞0.05）。两HAART暴露组之间新生儿脐带血T淋巴细胞相关指标均无显著性差异（P＞0.05）。两HAART组出生体重低于对照组（P＜0.05）。三组低出生体重发生率分别为15.00%、16.13%、5.00%，两治疗组高于对照组（P＜0.05）。3含AZT组的中位治疗时间为20周，不含AZT组中位治疗时间为21周。两治疗组开始治疗时CD4+T淋巴细胞计数、CD4/CD8无差异；分娩前含AZT组孕妇CD4+T淋巴细胞计数560.32±226.10个/mm3，高于不含AZT组的483.38±246.50个/mm3，含AZT组CD4/CD8显著高于不含AZT组（P＜0.05）。分娩前含AZT组CD4+T淋巴细胞较开始治疗时均数上升了129.79个/mm3，升幅达30.15%，CD4/CD8升高，CD8+T淋巴细胞均数降低415.97个/mm3，降幅为29.57%，P＜0.05；而不含AZT组上述指标虽有改变但没有统计学意义。4分娩前含AZT组与正常对照组相比，RBC、Hb、HCT、N显著降低，MCV、MCH、RDW显著增高（P＜0.05）；两HAART组相比，含AZT组RBC降低、L显著降低，MCV、MCH、RDW显著增高（P＜0.05）；各组间WBC和PLT没有统计学差异。三组孕妇轻度贫血的发生率没有统计学差异（P＞0.05），含AZT组孕妇中度贫血发生率高于其他两组（P＜0.05）。5新生儿脐带血含AZT组较对照组RBC、Hb、HCT、N显著降低，MCV、MCH、RDW、L、PLT显著增高（P＜0.05）；两HAART组相比，含AZT组RBC、Hb、HCT显著降低，但MCV、RDW显著增高（P＜0.05）；各组间WBC没有统计学差异。HAART暴露的两组轻度贫血发生率较对照组高（P＜0.05）；含AZT组新生儿轻度、中度贫血较不含AZT组更多见（P＜0.05）。结论：1、长疗程HAART治疗后，HIV母婴传播率接近为零，孕期HAART治疗安全、有效；2、孕期采用长疗程含AZT的HAART方案有助于孕妇的免疫重建；3、孕期含AZT的HAART暴露导致孕妇及新生儿巨幼红细胞性贫血和中性粒细胞减少，新生儿血小板增多。第二部分妊娠期齐多夫定干预对子代造血干/祖细胞抑制效应的研究目的：系统观察和阐明齐多夫定对新生儿、新生鼠造血干/祖细胞活性抑制效应的变化规律。方法：针对齐多夫定干预造血干/祖细胞活性的影响，进行三方面研究：1在临床研究方面，HAART暴露的新生儿脐血分离单个核细胞，流式细胞术检测CD34+细胞比例，培养粒单系祖细胞(CFU-GM)、红系祖细胞(BFU-E)以及巨核系祖细胞(CFU-Meg)并计算克隆形成集落数；2细胞学层次研究：取脐血造血干/祖细胞、新生小鼠骨髓单个核细胞，在CFU-GM、BFU-E和CFU-Meg体系和造血干/祖细胞悬浮培养体系中，进行体外AZT干预，浓度分别为0.1μM、0.5μM、1.0μM、2.0μM、4.0μM，观察与计算成集落数，流式细胞术检测干预后脐血造血干/祖细胞、小鼠骨髓单个核细胞的凋亡率；3Balb/c孕小鼠宫内暴露，AZT剂量为5.0mg/日/只和10.0mg/日/只，干预时间为孕期第10天至分娩，检测新生鼠CFU-GM、BFU-E和CFU-Meg成集落数。结果：临床15例HAART暴露脐血单个核细胞中，CD34+细胞阳性率为22.6±9.6%以及CFU-GM、 BFU-E和CFU-Meg成集落数显著低于正常脐血水平；在体外AZT干预体系中，新生鼠骨髓单个核细胞和脐血造血干/祖细胞，在0.5μM以上浓度，CFU-GM、BFU-E和CFU-Meg成集落数显著降低，且凋亡率明显升高，而在0.1μM浓度，凋亡率变化不明显，CFU-GM成集落数明显降低，但BFU-E和CFU-Meg成集落数只有降低趋势。在不同剂量AZT宫内暴露新生小鼠骨髓单个核细胞，CFU-GM、BFU-E和CFU-Meg成集落数明显低于正常对照新生小鼠。结论：妊娠期齐多夫定对造血干/祖细胞活性表现为明显的抑制效应，造血干/祖细胞凋亡，估计与齐多夫定线粒体毒性有关。第三部分齐多夫定干预对子代造血干细胞自我更新信号通路调控的实验研究目的：探索齐多夫定（AZT）对造血干细胞自我更新相关信号通路PI3K/Akt/mTOR和相关分子Bmi-1的变化规律，明确其对造血干细胞影响的分子机制。方法：Balb/c孕小鼠AZT干预，AZT剂量分别为5.0mg/日/只和10.0mg/日/只，暴露时间为孕期第10天至分娩；取新生鼠骨髓细胞，Ficoll分离后，采用qRT-PCR与Western blot方法检测PI3K/Akt/mTOR和相关分子Bmi-1、PTEN的表达水平变化。对照组分别为正常新生鼠与成体小鼠和老年小鼠。结果：宫内AZT暴露的新生小鼠的PI3K/Akt/mTOR信号通路中，mTOR水平表达有升高趋势，与成体小鼠无明显区别，但明显低于老年小鼠，而PTEN基因的表达无显著变化；Bmi-1基因表达水平有较正常对照新生鼠和成体小鼠高，但明显低于老年对照小鼠。AZT宫内暴露新生小鼠P70S6、4EBP1D较正常新生小鼠升高但低于老年小鼠（P <0.05），老年小鼠上述蛋白表达低于成年小鼠（P <0.05）。结论：AZT对新生鼠造血干细胞自我更新相关信号通路关键分子mTOR与Bmi-1存在有限影响，仍可维持在较为正常调控水平范畴。更多还原

【Abstract】 PART ICLINICAL OBSERVATION OF DIFFERENT HAARTREGIMENS ON HIV-INFECTED PREGNANTWOMEN AND HIV-UNINFECTED NEWBORNSObjective: To explore the effectiveness and safety of pregnant women andnewborns exposed to highly active antiretroviral therapy (HAART) duringpregnancy; to observe the pregnant results and changes of maternal or fetalhematopoiesis and immune function in two different regimen of HAART.Methods: According to the different HAART regimens,71HIV-infectedpregnant women with single birth were divided into2groups,40ofcontaining zidovudine (AZT) group（AZT group）,31of AZT-free group,40healthy pregnant women in the control group. The antiretroviral therapy wasbegun in or after the14th pregnant week. The HAART regimens of the AZTgroup were AZT+3TC+LPV/r or+EFV or+NVP, the regimens of theAZT-free group were TDF+3TC+LPV/r or+EFV or+NVP. The samples of venous blood of the maternal and umbilical cord blood were collected indifferent points of time respectively, before antiretroviral treatment, beforedelivery and after delivery. The clinical research information as fellows:①the basic clinical information of the pregnant women and newborns;②hematologic parameters;③CD4+, CD8+T lymphocytes in peripheral blood;④the index of HIV nucleic acid was detected in4to6weeks and4monthsafter birth. Results:(1) Among two groups of the HAART, there were nogestational hypertension and gestational diabetes, and there were no birthdefects, stillbirth and MTCT in newborns.(2) No severe neonatal asphyxiaoccurred, and there were not statistically significant difference in mildasphyxia among the three groups (P>0.05). There were not significantdifferences between two therapy groups in umbilical cord blood levels of Tlymphocytes. The birth weights of newborns exposed to HAART were lowerthan the control group (P <0.05). The low birth weight rate were15.00%,16.13%and5.00%in the three groups, the rates of the two therapy groupswere higher than the control group (P <0.05).(3) The median duration oftreatment in AZT group was20weeks, while in the AZT-free group was21weeks; before the treatment, there were no significant differences between twotreatment groups in CD4+T lymphocyte count and CD4/CD8rate. But beforedelivery, CD4+T lymphocyte count and CD4/CD8in the AZT group weresignificantly higher than the AZT-free group (P <0.05), CD4+was560.32±226.10cells/mm3and CD4/CD8rate was0.63±0.29in AZT grouprespectively. Before delivery, the CD4+T lymphocyte of the AZT group wassignificantly higher than that before therapy, the mean increased and reachedto129.79cells/mm3, the increase was30.15%(P <0.05). While CD8+Tlymphocyte was significant lower than that before therapy， the mean decreased and reached to415.97cells/mm3，the decrease was29.57%(P <0.05). In addition, the rate of CD4/CD8increased significantly (P＜0.05). Inthe AZT-free group, there were no such significant changes of lymphocyteindexes in the two points of time.(4) Before delivery, the hematologicparameters of pregnant women with megaloblastic anemia in the AZT groupcompared with it of the control group, the statistical results as fellows: RBC,Hb, HCT and N decreased (P <0.05), MCV, MCH and RDW increased (P <0.05). Among the three groups, there were no significant differences in WBC,PLT and mild anemia (P>0.05); but in AZT group, the rate of moderateanemia reached to17.50%, it was more significant higher than other groups (P<0.05).(5) Among three groups, there were significant differences in RBC,Hb, HCT, MCV, MCH, RDW, N, L and PLT of umbilical cord blood ofnewborns (P <0.01), but there was no significant difference in WBC. In theAZT group, Megaloblastic anemia of newborns occurred easily; comparedwith the control group, the changes of hematologic parameters: RBC, Hb,HCT and N decreased (P <0.05), but MCV, MCH, RDW and L increased (P<0.05). In the two groups exposed to HAART, the rates of mild anemia were52.50%and29.03%and more significant higher than that of the control group(P <0.05). In the AZT group，the rate of moderate anemia was17.50%, andmore significant higher than that in the AZT-free group (P <0.05).Conclusion:①The MTCT rate of HIV was the lower-level because of a longterm HAART treatment, it show that there are enough effectiveness and safetyin HAART.②In the state of immune reconstruction, the long term AZ-containing HAART regimen is better than AZT-free regimen.③Duringpregnancy, maternal and neonatal megaloblastic anemia occur easily innewborns and pregnancy women with AZT-containing HAART exposure. PART ⅡRESEARCH ON INHIBITIONS OF OFFSPRINGHEMATOPOIETIC STEM/PROGENITOR CELLACTIVITY CAUSED BY ZIDOVUDINE DURINGPREGNANCYObjectives: To observe the suppressions of hematopoietic stem/progenitorcell activity of newborn and newborn mice caused by the exposure ofzidovudine(AZT). Methods: The research of zidovudine intervention onhematopoietic stem/progenitor cell activity:①The clinical research: thesamples of umbilical cord blood of newborns exposed to HAART werecollected；the indexes of hematopoietic stem/progenitor cell activity asfellows: the proportion of CD34+cells detected by flow cytometer (FCM) andthe colony-forming colonies of granulocytes monophyletic progenitor cells(CFU-GM), erythroid progenitor cells (BFU-E) and megakaryocyte progenitorcells (CFU-Meg).②AZT intervention in vitro: umbilical cord bloodhematopoietic stem/progenitor cells and bone marrow cells of newborn micewere isolated and cultured in vitro. The concentrations of AZT were0.1μM,0.5μM,1.0μM,2.0μM and4.0μM respectively. The indexes of hematopoieticstem/progenitor cell activity as fellows: the colony-forming colonies ofCFU-GM, BFU-E, CFU-Meg and apoptosis rates of hematopoietic stem/ progenitor cells detected by FCM.③The pregnancy Balb/c mice wereexposed to AZT with the dose of5.0mg/day and10.0mg/day, exposureperiods, from the10thday of pregnancy to birth. The colony-forming coloniesof CFU-GM, BFU-E, CFU-Meg were measured in monocytes of bone marrowcell of newborn mice. Results: In15cases of cord blood of newborn ofpregnancy women exposed to HAART, the positive rate of CD34+cells was22.6±9.6%and the CFU-GM, BFU-E and CFU-Meg were significantlylower than normal levels (P<0.05). Above the concentrations of0.5μM AZT,the number of colonies in the CFU-GM, BFU-E and CFU-Meg of bonemarrow cells of newborn mice and hematopoietic stem/progenitor cells ofumbilical cord blood were significantly reduced, while the rates of apoptosiswas significantly higher than control in vitro. In the concentration of0.1μMAZT, the rate of apoptosis was not significantly changed, the CFU-GMcolonies were significantly reduced, and the BFU-E and the CFU-Megcolonies decreased, but the differences were not significant. In different dosesof AZT, the CFU-GM, the BFU-E and the CFU-Meg colonies of newbornswere significant less than normal levels in vivo. Conclusions: In thenewborns of pregnancy mice exposed to AZT, the activity and apoptosis ofhematopoietic stem/progenitor cells are inhibited and increased respectively.It suggests that mitochondrial toxicity of hematopoietic stem/progenitor cellscaused by AZT. PART ⅢRESEARCH ON SELF-RENEWAL SIGNALINGPATHWAY OF OFFSPRING HEMATOPOIETIC STEMCELL REGULATED BY ZIDOVUDINEObjectives: To investigate the regulation of self-renewal signaling pathwayPI3K-AKT-mTOR and Bmi-1gen of hematopoietic stem/progenitor cells, innewborns of pregnancy mice exposed to Zidovudine (AZT). Methods: Thepregnancy Balb/c mice were exposed to AZT with the dose of5.0mg/day and10.0mg/day, exposure periods, from the10thday of pregnancy to birth. Theoffspring hematopoietic stem/progenitor cells of bone marrows of wereisolated by Ficoll. The self-renewal signaling pathways PI3K/Akt/mTOR,Bmi-1and PTEN gene expression were measured by qRT-PCR and Westernblot. In addition, the control group was normal newborn mice, adult mice andaged mice. Results: In newborn mice of pregnancy Balb/c mice exposed toAZT, the mTOR expression levels of offspring hematopoietic stem/progenitor cells increased, but had no significant differences with adult mice,and were significantly less than the aged mice; while PTEN gene expressionwere not significant changes; Bmi-1gene expression level were more higherthan the normal newborn mice and adult mice, but were significantly less thanthe aged mice. The changes of protein expressions of P70S6,4EBP1D ofoffspring hematopoietic stem/progenitor cells as follows: newborn mice <newborn mice-AZT≈adult mice <aged mice. Conclusions: In offspringhematopoietic stem/progenitor cells，the limited up-regulation of mTOR ofself-renewal signaling pathway and self-renewal gen Bmi-1are caused by pregnancy mice exposed to AZT, but it can locate in normal range.更多还原