本文選題：高效抗逆轉(zhuǎn)錄病毒治療　切入點(diǎn)：血液毒性　出處：《廣西醫(yī)科大學(xué)》2014年博士論文

【摘要】：第一部分孕期不同HAART方案暴露對(duì)HIV感染母親及HIV未感染新生兒的影響 目的:了解孕期接受高效抗逆轉(zhuǎn)錄病毒治療（HAART）方案預(yù)防HIV母嬰傳播的安全性和有效性，探討兩種不同HAART方案對(duì)妊娠結(jié)局、孕婦和胎兒造血及免疫功能的影響。方法:71名單胎妊娠HIV感染孕婦按照HAART方案的不同分為含齊多夫定（AZT）組40人、不含AZT組31人，以40名健康孕婦為對(duì)照組。HIV陽性孕婦自妊娠14周及14周以后開始抗病毒治療，含AZT組的HAART方案為齊多夫定+拉米夫定（AZT+3TC）聯(lián)合克力芝（LPV/r）或依非那侖（EFV）或奈韋拉平（NVP），不含AZT組的HAART方案為替諾福韋（TDF）+3TC聯(lián)合LPV/r或EFV或NVP。采集開始抗病毒治療時(shí)和分娩前孕婦肘靜脈血、臍帶血，進(jìn)行血細(xì)胞參數(shù)測定和CD4+、CD8+T淋巴細(xì)胞測定，同時(shí)收集孕婦和新生兒基本信息進(jìn)行分析，嬰兒出生后4～6周及4個(gè)月進(jìn)行HIV-1-DNA核酸測定。結(jié)果: 1HAART治療的兩組孕婦當(dāng)中，無妊娠期高血壓疾病和妊娠期糖尿病的發(fā)生；HIV陽性母親分娩新生兒無HIV感染發(fā)生，未觀察到新生兒出生缺陷的發(fā)生，無死胎和死產(chǎn)發(fā)生。 2三組均無新生兒重度窒息發(fā)生，輕度窒息發(fā)生率無顯著差異（P＞0.05）。兩HAART暴露組之間新生兒臍帶血T淋巴細(xì)胞相關(guān)指標(biāo)均無顯著性差異（P＞0.05）。兩HAART組出生體重低于對(duì)照組（P＜0.05）。三組低出生體重發(fā)生率分別為15.00%、16.13%、5.00%，兩治療組高于對(duì)照組（P＜0.05）。 3含AZT組的中位治療時(shí)間為20周，不含AZT組中位治療時(shí)間為21周。兩治療組開始治療時(shí)CD4+T淋巴細(xì)胞計(jì)數(shù)、CD4/CD8無差異；分娩前含AZT組孕婦CD4+T淋巴細(xì)胞計(jì)數(shù)560.32±226.10個(gè)/mm3，高于不含AZT組的483.38±246.50個(gè)/mm3，含AZT組CD4/CD8顯著高于不含AZT組（P＜0.05）。分娩前含AZT組CD4+T淋巴細(xì)胞較開始治療時(shí)均數(shù)上升了129.79個(gè)/mm3，升幅達(dá)30.15%，CD4/CD8升高，CD8+T淋巴細(xì)胞均數(shù)降低415.97個(gè)/mm3，降幅為29.57%，P＜0.05；而不含AZT組上述指標(biāo)雖有改變但沒有統(tǒng)計(jì)學(xué)意義。 4分娩前含AZT組與正常對(duì)照組相比，RBC、Hb、HCT、N顯著降低，MCV、MCH、RDW顯著增高（P＜0.05）；兩HAART組相比，含AZT組RBC降低、L顯著降低，MCV、MCH、RDW顯著增高（P＜0.05）；各組間WBC和PLT沒有統(tǒng)計(jì)學(xué)差異。三組孕婦輕度貧血的發(fā)生率沒有統(tǒng)計(jì)學(xué)差異（P＞0.05），含AZT組孕婦中度貧血發(fā)生率高于其他兩組（P＜0.05）。 5新生兒臍帶血含AZT組較對(duì)照組RBC、Hb、HCT、N顯著降低，MCV、MCH、RDW、L、PLT顯著增高（P＜0.05）；兩HAART組相比，含AZT組RBC、Hb、HCT顯著降低，但MCV、RDW顯著增高（P＜0.05）；各組間WBC沒有統(tǒng)計(jì)學(xué)差異。HAART暴露的兩組輕度貧血發(fā)生率較對(duì)照組高（P＜0.05）；含AZT組新生兒輕度、中度貧血較不含AZT組更多見（P＜0.05）。 結(jié)論： 1、長療程HAART治療后，HIV母嬰傳播率接近為零，孕期HAART治療安全、有效； 2、孕期采用長療程含AZT的HAART方案有助于孕婦的免疫重建； 3、孕期含AZT的HAART暴露導(dǎo)致孕婦及新生兒巨幼紅細(xì)胞性貧血和中性粒細(xì)胞減少，新生兒血小板增多。 第二部分妊娠期齊多夫定干預(yù)對(duì)子代造血干/祖細(xì)胞抑制效應(yīng)的研究 目的：系統(tǒng)觀察和闡明齊多夫定對(duì)新生兒、新生鼠造血干/祖細(xì)胞活性抑制效應(yīng)的變化規(guī)律。 方法：針對(duì)齊多夫定干預(yù)造血干/祖細(xì)胞活性的影響，進(jìn)行三方面研究： 1在臨床研究方面，HAART暴露的新生兒臍血分離單個(gè)核細(xì)胞，流式細(xì)胞術(shù)檢測CD34+細(xì)胞比例，培養(yǎng)粒單系祖細(xì)胞(CFU-GM)、紅系祖細(xì)胞(BFU-E)以及巨核系祖細(xì)胞(CFU-Meg)并計(jì)算克隆形成集落數(shù)； 2細(xì)胞學(xué)層次研究：取臍血造血干/祖細(xì)胞、新生小鼠骨髓單個(gè)核細(xì)胞，在CFU-GM、BFU-E和CFU-Meg體系和造血干/祖細(xì)胞懸浮培養(yǎng)體系中，進(jìn)行體外AZT干預(yù)，濃度分別為0.1μM、0.5μM、1.0μM、2.0μM、4.0μM，觀察與計(jì)算成集落數(shù)，流式細(xì)胞術(shù)檢測干預(yù)后臍血造血干/祖細(xì)胞、小鼠骨髓單個(gè)核細(xì)胞的凋亡率； 3Balb/c孕小鼠宮內(nèi)暴露，AZT劑量為5.0mg/日/只和10.0mg/日/只，干預(yù)時(shí)間為孕期第10天至分娩，檢測新生鼠CFU-GM、BFU-E和CFU-Meg成集落數(shù)。 結(jié)果：臨床15例HAART暴露臍血單個(gè)核細(xì)胞中，CD34+細(xì)胞陽性率為22.6±9.6%以及CFU-GM、 BFU-E和CFU-Meg成集落數(shù)顯著低于正常臍血水平；在體外AZT干預(yù)體系中，新生鼠骨髓單個(gè)核細(xì)胞和臍血造血干/祖細(xì)胞，在0.5μM以上濃度，CFU-GM、BFU-E和CFU-Meg成集落數(shù)顯著降低，，且凋亡率明顯升高，而在0.1μM濃度，凋亡率變化不明顯，CFU-GM成集落數(shù)明顯降低，但BFU-E和CFU-Meg成集落數(shù)只有降低趨勢。在不同劑量AZT宮內(nèi)暴露新生小鼠骨髓單個(gè)核細(xì)胞，CFU-GM、BFU-E和CFU-Meg成集落數(shù)明顯低于正常對(duì)照新生小鼠。結(jié)論：妊娠期齊多夫定對(duì)造血干/祖細(xì)胞活性表現(xiàn)為明顯的抑制效應(yīng)，造血干/祖細(xì)胞凋亡，估計(jì)與齊多夫定線粒體毒性有關(guān)。 第三部分齊多夫定干預(yù)對(duì)子代造血干細(xì)胞自我更新信號(hào)通路調(diào)控的實(shí)驗(yàn)研究 目的：探索齊多夫定（AZT）對(duì)造血干細(xì)胞自我更新相關(guān)信號(hào)通路PI3K/Akt/mTOR和相關(guān)分子Bmi-1的變化規(guī)律，明確其對(duì)造血干細(xì)胞影響的分子機(jī)制。 方法：Balb/c孕小鼠AZT干預(yù)，AZT劑量分別為5.0mg/日/只和10.0mg/日/只，暴露時(shí)間為孕期第10天至分娩；取新生鼠骨髓細(xì)胞，F(xiàn)icoll分離后，采用qRT-PCR與Western blot方法檢測PI3K/Akt/mTOR和相關(guān)分子Bmi-1、PTEN的表達(dá)水平變化。對(duì)照組分別為正常新生鼠與成體小鼠和老年小鼠。 結(jié)果：宮內(nèi)AZT暴露的新生小鼠的PI3K/Akt/mTOR信號(hào)通路中，mTOR水平表達(dá)有升高趨勢，與成體小鼠無明顯區(qū)別，但明顯低于老年小鼠，而PTEN基因的表達(dá)無顯著變化；Bmi-1基因表達(dá)水平有較正常對(duì)照新生鼠和成體小鼠高，但明顯低于老年對(duì)照小鼠。AZT宮內(nèi)暴露新生小鼠P70S6、4EBP1D較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達(dá)低于成年小鼠（P 0.05）。 結(jié)論：AZT對(duì)新生鼠造血干細(xì)胞自我更新相關(guān)信號(hào)通路關(guān)鍵分子mTOR與Bmi-1存在有限影響，仍可維持在較為正常調(diào)控水平范疇。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.251

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李太生;;艾滋病的免疫功能重建研究進(jìn)展[J];傳染病信息;2003年04期

2 龐俊;黃紹標(biāo);;HAART阻斷HIV母嬰傳播效果觀察[J];廣西醫(yī)學(xué);2009年09期

3 蒙春蓮;;HIV感染孕產(chǎn)婦HAART治療對(duì)母嬰傳播的影響[J];廣西醫(yī)學(xué);2011年11期

4 郭學(xué)鵬,劉玉峰,王家勤;兒童白細(xì)胞缺乏癥[J];實(shí)用兒科臨床雜志;1993年03期

5 劉冬梅;龐俊;韋淑珍;謝園春;班素梅;;不同孕期HIV/AIDS孕婦接受抗病毒治療的效果評(píng)估[J];中華全科醫(yī)學(xué);2013年07期

6 王臨虹;方利文;王前;蔣巖;龔雙燕;張麒;張偉;李燕;孫定勇;莫云;;我國艾滋病母嬰傳播水平傳播時(shí)期及干預(yù)效果研究[J];中國艾滋病性病;2008年05期

7 張時(shí)民;血常規(guī)中紅細(xì)胞參數(shù)的診斷價(jià)值和意義[J];中國臨床醫(yī)生;2001年11期

8 莫云;黃越華;李映;;2007～2011年廣西艾滋病及艾滋病病毒感染孕產(chǎn)婦死亡狀況調(diào)查分析[J];中國臨床新醫(yī)學(xué);2012年10期

9 李愛杰;汪永忠;;HIV感染婦女分娩嬰兒低出生體質(zhì)量情況及影響因素分析[J];中國皮膚性病學(xué)雜志;2013年02期

10 張璐;王臨虹;方利文;;分娩方式與艾滋病母嬰傳播[J];中國艾滋病性病;2012年04期

本文編號(hào)：1714810