基于造血干细胞研究妊娠期核苷类逆转录酶抑制剂暴露抑制子代造血功能的机制

发布时间：2018-04-05 12:53

本文选题：高效抗逆转录病毒治疗　切入点：血液毒性　出处：《广西医科大学》2014年博士论文

【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。
[Abstract]:The effect of different HAART program exposure on HIV infected mothers and HIV uninfected newborns in the first part of pregnancy
Objective: To investigate the pregnancy receiving highly active antiretroviral therapy (HAART) is safe and effective strategies for the prevention of mother to child transmission of HIV, a study of two different HAART schemes on pregnancy outcome, effects of maternal and fetal hematopoiesis and immune function. Methods: 71 singleton pregnancy HIV infection of pregnant women according to different HAART schemes for Hamzi Dov (AZT) group of 40 people, with 31 people in the AZT group and 40 healthy pregnant women as control group after the start of antiviral therapy of.HIV positive pregnant women from pregnancy 14 weeks and 14 weeks, the HAART program with AZT group was Zidorf + lamivudine (AZT+3TC) combined with Chris Chi (LPV/r) or by the non Lun (EFV) or nevirapine (NVP), HAART group without AZT scheme for Nuo Fuwei (TDF +3TC) combined with LPV/r or EFV or NVP. acquisition began to antiviral treatment before delivery and maternal elbow vein blood, umbilical cord blood, blood cell parameters measurement and determination of CD4+, CD8+T lymphocytes, At the same time, the basic information of pregnant women and newborns was collected, and the HIV-1-DNA nucleic acid was measured at 4~6 and 4 months after birth.
Among the two groups of pregnant women treated with 1HAART, there was no occurrence of gestational hypertension and gestational diabetes mellitus. There was no HIV infection in HIV positive mothers, no birth defects occurred, no stillbirth and stillbirth occurred.
2 the three groups had no severe neonatal asphyxia, mild asphyxia rate had no significant difference (P > 0.05). There were no significant between group of neonatal cord blood T lymphocyte related indexes were exposed to two HAART (P > 0.05). The two group HAART birth weight lower than that of the control group (P < 0.05). Three groups of low birth the incidence rate of weight were 15%, 16.13%, 5%, two higher than the treatment group (P < 0.05).
3 with AZT median duration of treatment was 20 weeks, without AZT group the median duration of therapy was 21 weeks. Two treatment group began treatment CD4+T lymphocyte count, CD4/CD8 had no difference with AZT group of pregnant women before delivery; CD4+T lymphocyte count 560.32 + 226.10 /mm3, higher than that of the group without AZT 483.38 + 246.50 /mm3, AZT group CD4/CD8 was significantly higher than that of the group without AZT (P < 0.05). Before delivery with CD4+T lymphocytes in group AZT than at the start of treatment were increased by 129.79 /mm3, an increase of 30.15%, increase of CD4/CD8, CD8+T lymphocytes were decreased 415.97 /mm3, a decline of 29.57%, P < 0.05; without the above indexes in group AZT although the change but not statistically significant.
4 before delivery with AZT group compared with normal control group, RBC, Hb, HCT, N, MCV, MCH decreased significantly, RDW increased significantly (P < 0.05); two in HAART group compared with AZT group, RBC decreased, L decreased significantly, MCV, MCH, RDW increased significantly (P < 0.05); group between WBC and PLT had no statistical difference between the three groups of pregnant women. The incidence of mild anemia was not statistically significant (P > 0.05), with moderate incidence of anemia in pregnant women with AZT was higher than the other two groups (P < 0.05).
5 neonatal umbilical cord blood containing AZT group than in the control group RBC, Hb, HCT, MCV, N decreased significantly, MCH, RDW, L, PLT increased significantly (P < 0.05); two in HAART group compared with AZT group, RBC, Hb, HCT decreased significantly, but MCV, RDW increased significantly (P < 0.05) among the groups of WBC; two groups no statistically significant difference in.HAART exposed to mild anemia incidence was higher than that in control group (P < 0.05); AZT group in mild, moderate anemia group was more common than that without AZT (P < 0.05).
Conclusion:
1, after a long course of HAART treatment, the transmission rate of HIV mother and infant is close to zero, and the treatment of HAART during pregnancy is safe and effective.
2, the adoption of a long course of AZT - containing HAART program during pregnancy helps pregnant women to be immune to reconstruction;
3, HAART exposure with AZT during pregnancy leads to megaloblastic anemia and neutrophils in pregnant and newborn infants, and more thrombocytopenia in newborns.
Study on the inhibitory effect of zidovudine on hematopoietic stem / progenitor cells in the second part of pregnancy
Objective: to systematically observe and elucidate the changes in the inhibitory effect of zidovudine on the activity inhibition of hematopoietic stem / progenitor cells in newborn rats.
Methods: the effects of Zidorf's intervention on the activity of hematopoietic stem / progenitor cells were studied in three aspects.
1, in clinical research, mononuclear cells isolated from umbilical cord blood of newborns exposed to HAART, the proportion of CD34+ cells was detected by flow cytometry, cultured granulocyte progenitor cells (CFU-GM), erythroid progenitor cells (BFU-E) and megakaryocyte progenitor cells (CFU-Meg), and colony forming number was calculated.
2 cytological level research: umbilical cord blood hematopoietic stem / progenitor cells, mouse bone marrow mononuclear cells in CFU-GM, BFU-E and CFU-Meg system and hematopoietic stem / progenitor cell suspension culture system in vitro, AZT intervention, the concentration were 0.1 M, 0.5 M, 1 M, 2 M, 4 M, observation and calculation of colony formation, flow cytometry after intervention of umbilical cord blood hematopoietic stem / progenitor cells, apoptosis of mice bone marrow mononuclear cell rate;
Intrauterine exposure of 3Balb/c pregnant mice was AZT daily dose of 5.0mg/ day / day and 10.0mg/ day / day. The intervention time was tenth days to delivery, and the colony numbers of CFU-GM, BFU-E and CFU-Meg were detected in neonatal rats.
Results: 15 cases of clinical HAART exposure of umbilical cord blood mononuclear cells, the positive rate of CD34+ cells was 22.6 + 9.6%, CFU-GM, BFU-E and CFU-Meg into the colony number was significantly lower than the normal level of umbilical cord blood; in vitro AZT intervention system in newborn rat bone marrow mononuclear cells and human umbilical cord blood hematopoietic stem / progenitor cells, CFU-GM in more than 0.5 mu M the concentration of BFU-E and CFU-Meg, the colony number decreased significantly, and the apoptosis rate was significantly increased, and in 0.1 M concentration, the apoptosis rate did not change significantly, CFU-GM colony number decreased significantly, but BFU-E and CFU-Meg into the colony number only decreased. In different doses of AZT intrauterine exposure of newborn mouse bone marrow mononuclear cells CFU-GM, BFU-E, and CFU-Meg into the colony number was lower than that of normal control mice. Conclusion: the pregnancy of zidovudine on hematopoietic stem / progenitor cell activity showed a significant inhibitory effect, hematopoietic stem / progenitor cells apoptosis, and mitochondrial zidovudine estimation Toxicity is related.
Experimental study on regulation of self renewal signaling pathway of hematopoietic stem cells in the third part of zidovudine
Objective: To explore the molecular mechanism of zidovudine (AZT) on the signaling pathways PI3K/Akt/mTOR and Bmi-1 related to self-renewal in hematopoietic stem cells, and to clarify the molecular mechanism of its influence on hematopoietic stem cells.
Methods: pregnant Balb/c mice AZT intervention, AZT dose was 5.0mg/ / day and 10.0mg/ / only, the exposure time was tenth days of pregnancy childbirth; newborn rat bone marrow cells after Ficoll separation, using qRT-PCR and Western method for the detection of blot PI3K/Akt/mTOR and Bmi-1 protein, the expression of PTEN in control group were normal. Neonatal rats and adult and old mice.
Results: the PI3K/Akt/mTOR signaling pathway in neonatal mice after AZT exposure in the level of mTOR expression increased, no obvious difference with the adult mice, but significantly lower than in aged mice, and the expression of PTEN gene had no significant change; the expression level of Bmi-1 gene were compared with normal control rats and adult mice of new high, but lower than the old control mice.AZT intrauterine exposure of P70S6,4EBP1D newborn mice compared with normal newborn mice increased but lower in aged mice (P 0.05), the expression of the aged mice was lower than that of adult mice (P 0.05).
Conclusion: AZT has limited effects on mTOR and Bmi-1, which are related to the key signaling molecules of self-renewal related signaling pathways in neonatal stem cells, and can still be maintained at a relatively normal level.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.251

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