慢性间歇性低压低氧抗大鼠再生障碍性贫血作用及其机制的研究
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摘要
再生障碍性贫血（再障, aplastic anemina,AA）是由化学、物理、生物、遗传及不明原因引起的一种获得性骨髓造血功能衰竭综合征，是一种严重危害人类健康的恶性血液疾病。AA的病理机制复杂，呈高度异质性。临床上主要表现为贫血、出血倾向和感染症状。目前AA尚无特异有效的治疗方法。因此，有关AA机制及其防治措施的研究具有重要的理论和实用意义。
大量研究表明，慢性间歇性低压低氧（chronic intermittent hypobarichypoxia, CIHH）对机体多种组织、器官具有保护作用。例如，CIHH可增强机体对缺血、缺氧的耐受性，对抗缺血/再灌注所致心功能损伤及心律失常，保护脑、肝脏、肾脏等器官组织，调节免疫功能，抗细胞凋亡，抗氧化。此外，CIHH可降低血压、调节血管舒缩功能、易化压力感受性反射等。我们以往研究表明，CIHH对基础心功能无影响，但可对抗缺血/再灌注和急性缺氧/复氧对心脏的损伤，发挥心脏保护作用。近来，我们研究结果显示，CIHH对肾血管性高血压大鼠、胶原诱导关节炎大鼠和果糖诱导代谢综合征大鼠均具有明显的防治作用。
众所周知，高原低氧可促进红细胞的生成。研究表明，低氧能显著影响造血过程，促进造血祖细胞（hemopoietic progenitor cells, HPCs）或造血干细胞（hemopoietic stem cells, HSCs）、骨髓间质干细胞（bone marrowmesenchymal stem cells, BMMSCs）增殖并抑制其分化，以及改善骨髓微环境（hematopoietic microenviroment, HIM）。但有关CIHH对血液系统及造血机能的影响尚未明了，也未见CIHH对AA作用的报道。
本研究旨在利用5-氟尿嘧啶（5-fluorouracil,5-FU）联合白消安（busulfan, BU）诱导的AA动物模型，应用实验动物学、细胞生物学、形态学和分子生物学等方法，从整体、组织和细胞分子不同水平、系统动态地观察CIHH处理对AA的影响，并探讨其机制。研究分为四部分：（1）探讨CIHH对5-FU和BU联合诱导的AA大鼠的防治作用；（2）通过观察CIHH对AA大鼠外周血T淋巴细胞亚群的影响，探讨CIHH抗大鼠AA作用的细胞免疫学机制；（3）通过观察CIHH对AA大鼠骨髓细胞凋亡的影响，探讨CIHH抗大鼠AA作用的细胞凋亡机制；（4）通过观察CIHH对AA大鼠BMMSCs及其粘附性的影响，探讨CIHH抗大鼠AA作用的粘附分子机制。
第一部分慢性间歇性低压低氧抗大鼠再生障碍性贫血的作用
目的：初步探讨CIHH对5-FU联合BU诱导的的AA大鼠的防治作用。
方法:雄性成年SD大鼠随机分为6组：对照组（Con）、CIHH组（CIHH）、CIHH预处理组（Pre-T）、预处理再障组（Pre-AA）、CIHH后处理组（Post-T）和后处理再障组（Post-AA）。Pre-AA和Post-AA大鼠给予5-FU联合BU腹腔注射诱导AA；Pre-T和Post-T大鼠分别在AA造模前、后接受28天、模拟海拔3,000米（大气压：525mmHg，PO2：108.8mmHg）、每天5小时的低压低氧处理；CIHH大鼠仅给予模拟海拔3,000米（大气压：525mmHg，PO2：108.8mmHg）、每天5小时的低压低氧处理; Con大鼠不给予AA造模和间歇性低压低氧处理。H-E染色观察骨髓形态学变化；甲基纤维素半固体培养法检测骨髓粒-单系集落形成单位（colony-forming unit-granulocyte-macrophage, CFU-GM）、红系集落形成单位（colony-forming unit-erythroid, CFU-E)、红系爆式集落形成单位（burst-forming unit-erythroid, BFU-E）和巨核系集落形成单位（colony-forming unit-megakaiyocyte, CFU-MK）产率；酶联免疫吸附试验（enzymelinked immunosorbent assay, ELISA）检测血清白介素-3（interleukin-3,IL-3）、促红细胞生成素（erythropoietin, EPO）、肿瘤坏死因子-α （tumornecrosis factor-alpha, TNF-α）和白介素-2（interleukin-2, IL-2）水平。
结果：1.与Con大鼠相比，CIHH和Pre-T大鼠体重无显著差异（P<0.05）；Pre-AA组、Post-AA和Post-T大鼠体重显著下降（P<0.01）。
2. Pre-T大鼠AA发生率（25%）显著低于Pre-AA大鼠（80%, P <0.01）；Pre-T首次发病时间（24.9±4.4天）显著迟于Pre-AA大鼠（16.2±5.3天, P<0.01）；Post-T大鼠死亡率（30%）显著低于Post-AA大鼠（70%, P <0.01）。
3.与Con大鼠相比，CIHH和Pre-T大鼠外周血红细胞（red blood cell,RBC）、白细胞（white blood cell, WBC）、血小板（platelet, PLA）、血红蛋白（hemoglobin, HGB）、红细胞比容（hematocrit, HCT）、网织红细胞（retieulocyte, Ret）绝对值、中性粒细胞（neutrophils, NEUT）百分比、淋巴细胞（lymphocyte, LYM）百分比和单核细胞（monocyte, MON）百分比无显著差异（P>0.05）；Pre-AA和Post-AA大鼠除LYM%显著增多（P<0.01）外，其余各血象指标均明显减少（P<0.01）；Post-T大鼠除RBC、WBC、PLA、HGB、HCT和Ret绝对值显著减少（P<0.01）外，其它血象指标无显著差异（P>0.05）。
4.与Con大鼠相比，Pre-AA和Post-AA大鼠骨髓组织明显脂肪化，造血细胞减少，非造血细胞增多，骨髓间质血窦扩张明显，毛细血管网明显减少，造血功能显著低下。Pre-T和Post-T大鼠骨髓的各种改变明显改善。
5.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠的骨髓单个核细胞(bone marrow mononucleated cells, BMMNCs)和骨髓各类细胞均无显著变化（P>0.05）；Pre-AA和Post-AA大鼠的BMMNCs数、原粒细胞、分叶粒细胞、碱性早幼粒细胞、早红细胞、中红细胞、晚红细胞和巨核细胞显著减少（P<0.01），而其中幼粒细胞、晚幼粒细胞、杆状粒细胞、酸性中幼粒细胞、单核细胞和淋巴细胞显著增多（P<0.01）,其他骨髓细胞无显著差异（P>0.05）。
6.与Con大鼠相比，CIHH和Pre-T大鼠的CFU-GM、CFU-E、BFU-E和CFU-MK产率无显著差异（P>0.05）；Pre-AA、Post-AA和Post-T大鼠的CFU-GM、CFU-E、BFU-E和CFU-MK集落产率显著减少（P<0.01）。
7.与Con大鼠相比，CIHH大鼠血清IL-3、EPO、TNF-α和IL-2水平均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠血清IL-3和EPO水平显著减少（P<0.01），而其TNF-α和IL-2水平显著增多（P<0.05）；Pre-T和Post-T大鼠血清IL-3和EPO水平显著增多（P<0.01），而其TNF-α和IL-2水平无显著差异（P>0.05）。
小结: CIHH预处理可预防大鼠AA的发生，CIHH后处理有抗AA的作用，可减轻AA大鼠造血功能障碍，改善骨髓组织病理损伤。此作用与CIHH升高血清正性造血调控因子IL-3和EPO，降低造负性血调控因子TNF-α和IL-2有关。
第二部分慢性间歇性低压低氧对再生障碍性贫血大鼠T淋巴细胞的影响
目的:通过观察CIHH对AA大鼠外周血T淋巴细胞亚群的影响，探讨CIHH抗大鼠AA作用的细胞免疫机制。
方法:实验分组、AA模型制备和CIHH处理和同本研究第一部分。流式细胞术（flow cytometry, FCM）检测大鼠外周血T淋巴细胞及其亚群CD3~+、CD4~+、CD8~+、CD4~+HLA-DR~+和CD8~+HLA-DR~+；ELISA法检测大鼠血清干扰素-γ（interferon, IFN-γ）、白介素-4（interleukin-4, IL-4）和白介素-9（interleukin-9, IL-9）（分别代表Th1、Th2、Th9细胞表达水平）；免疫组化法检测大鼠骨髓组织IFN-γ、IL-4和IL-9蛋白。
结果：1.与Con大鼠相比，CIHH大鼠外周血T淋巴细胞亚群CD3~+、CD4~+、CD8~+、CD4~+HLA-DR~+、CD8~+HLA-DR~+百分比和CD4~+/CD8~+值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠外周血T淋巴细胞亚群CD3~+、CD8~+、CD8~+HLA-DR~+百分比显著升高（P<0.01），CD4~+百分比和CD4~+/CD8~+值显著降低（P<0.01）； Pre-T大鼠外周血T淋巴细胞亚群各参数无显著差异（P<0.01）；Post-T大鼠外周血T淋巴细胞亚群CD3~+、CD8~+和CD4~+HLA-DR~+无显著差异（P>0.05），而CD4~+、CD4~+/CD8~+和CD8~+HLA-DR~+显著升高（P<0.05）。
2.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠血清IFN-γ、IL-4、IL-9水平和IFN-γ/IL-4（Th1/Th2）值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠血清促炎因子IFN-γ、IL-9水平和IFN-γ/IL-4（Th1/Th2）值显著升高（P<0.01），抗炎因子IL-4水平显著降低（P<0.05）。
3.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠骨髓组织IFN-γ、IL-9、IL-4水平和IFN-γ/IL-4（Th1/Th2）值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓组织促炎因子IFN-γ、IL-9水平和IFN-γ/IL-4（Th1/Th2）值显著升高（P<0.01），抗炎因子IL-4水平显著降低（P<0.01）。
小结: CIHH处理对大鼠基础状态外周血T淋巴细胞亚群、血清和骨髓组织中炎性因子IFN-γ、IL-9及IL-4无影响，但可有效对抗AA大鼠外周血辅助性T淋巴细胞（CD4~+）的降低，细胞毒性T淋巴细胞（CD8~+）和活化的细胞毒性T淋巴细胞（CD8~+HLA-DR~+）的升高，从而维持T淋巴细胞亚群的平衡，促炎因子和抗炎因子间的平衡。CIHH的细胞免疫调节作用可能是CIHH抗大鼠AA的作用机制之一。
第三部分慢性间歇性低压低氧对再生障碍性贫血大鼠骨髓细胞凋亡的影响
目的:通过观察CIHH对AA大鼠骨髓细胞凋亡的影响，探讨CIHH抗大鼠AA作用的细胞凋亡机制。
方法:实验分组、AA模型制备和CIHH处理和同本研究第一部分。FCM和原位末端标记法（terminal dUTP nick end labeling, TUNEL）检测大鼠骨髓细胞凋亡；免疫组化法检测大鼠骨髓细胞凋亡相关蛋白Fas、Bax、Bcl-2、Caspase-3和Caspase-8表达；Western blot法检测大鼠骨髓细胞低氧诱导因子-1α（hypoxia inducible factor-1alpha, HIF-1α）和核转录因子-κB（nuclear factor-kappa B, NF-κB）表达。
结果：1. FCM和TUNEL结果均显示，与Con大鼠相比，CIHH、Pre-T和Post-T大鼠骨髓细胞凋亡率无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞凋亡率显著升高（P<0.01）。
2.与Con大鼠相比，CIHH，Pre-T和Post-T大鼠骨髓细胞凋亡蛋白Fas、Bax、Bcl-2、Caspase-3、Caspase-8表达和Bax/Bcl-2值无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞促凋亡蛋白Fas、Bax、Caspase-3、Caspase-8和Bax/Bcl-2值显著升高（P<0.05-P<0.01），而抑凋亡蛋白Bcl-2显著降低（P<0.05）。
3.与Con大鼠相比，CIHH和Pre-T大鼠骨髓细胞HIF-1α和NF-κB蛋白表达无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞HIF-1α和NF-κB蛋白表达显著升高（P<0.05-P<0.01）；Post-T大鼠骨髓细胞HIF-1α蛋白表达显著升高（P<0.01），而其NF-κB蛋白表达无显著差异（P>0.05）。
小结: CIHH处理对基础状态下大鼠骨髓细胞凋亡和凋亡无影响，但可对抗AA大鼠HIF-1α和NF-κB蛋白过度表达，下调促凋亡蛋白Fas、Bax、Caspase-3和Caspase-8表达，上调抑凋亡蛋白Bcl-2表达，最终抗骨髓细胞凋亡。CIHH抗骨髓细胞凋亡可能也是CIHH抗大鼠AA的作用机制之一。
第四部分慢性间歇性低压低氧对再生障碍性贫血大鼠骨髓间质干细胞粘附性的影响
目的:通过观察CIHH对AA大鼠BMMSCs及其粘附性的影响，探讨CIHH抗大鼠AA作用的粘附分子机制。
方法:实验分组、CIHH处理和AA模型制备同本研究第一部分。体外长期骨髓植入培养（long-term bone marrow explant cultures, LTBMC-Ex）动态观察骨髓贴壁细胞形态和骨髓成纤维细胞集落形成单位（colonyforming unit-fibroblast, CFU-F）的变化；差速贴壁法培养并纯化BMMSCs；FCM检测BMMSCs极晚期抗原-4（very late antigen-4, VLA-4）、血管细胞间粘附分子-1（vascular cell adhesion molecule-1, VCAM-1）、细胞间粘附分子-1（intercellular adhesion molecule-1, ICAM-1）、CD162和CD164；Western blot法检测BMMSCs表达p38丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）。
结果：1.体外骨髓长期植入培养过程中，Pre-AA和Post-AA大鼠骨髓基质细胞层发育不良，细胞多为不成熟的大基质前体细胞和成纤维样细胞。CIHH处理对基础状态下大鼠骨髓基质细胞层无影响，但可有效对抗AA大鼠骨髓基质细胞层发育不良，重新形成有效支持造血的骨髓基质细胞层。
2.与Con大鼠相比，CIHH和Pre-T大鼠骨髓的CFU-F集落数无显著变化（P>0.05）；Pre-AA和Post-AA大鼠骨髓的CFU-F集落数显著减少（P<0.01）； Post-T大鼠骨髓的CFU-F集落数显著减少（P<0.01）。
3.与Con大鼠相比，CIHH大鼠BMMSCs表达VLA-4、VCAM-1、ICAM-1、CD162和CD164无显著差异（P>0.05）; Pre-AA和Post-AA大鼠BMMSCs表达VLA-4、VCAM-1和ICAM-1显著减少（P<0.01），而表达CD162和CD164显著增多（P<0.01）；Pre-T大鼠BMMSCs表达VLA-4、VCAM-1、ICAM-1和CD164无显著差异（P>0.05），但表达CD162显著增多（P<0.05）;Post-T大鼠BMMSCs表达VCAM-1和ICAM-1无显著差异（P>0.05），但表达VLA-4显著减少（P<0.01），表达CD162和CD164显著增多（P<0.01）。
4.与Con大鼠相比，CIHH和Pre-T大鼠BMMSCs表达p38MAPK无显著差异（P>0.05）；Pre-AA、Post-AA和Post-T大鼠BMMSCs表达p38MAPK显著升高（P<0.01）。
小结：CIHH处理对基础状态下骨髓基质细胞发育和BMMSCs粘附性无显著影响；但可有效修复AA大鼠发育不良的骨髓基质细胞，明显增强AA大鼠BMMSCs减弱的粘附性。CIHH的粘附分子调节机制也是CIHH抗大鼠AA的作用机制之一。
结论： CIHH处理可通过抑制AA大鼠信号通路HIF-1α、NF-κB和p38MAPK蛋白过度表达及下游信号分子的异常变化，维持造血调控因子间、T淋巴细胞亚群间、炎性因子间和粘附分子间的平衡，从而减轻骨髓造血组织损伤和造血功能抑制、减少造血细胞凋亡，增强骨髓造血细胞与造血微环境间的粘附性，发挥防治大鼠AA作用。
Aplastic anemia (AA), a common clinical malignant blood disease, is anacquired bone marrow failure syndrome caused by chemical, physics, biologi-cal, heredity and some unknown causes. The main clinical manifestations areanemia, bleeding tendency and infection symptoms. The pathogenesis of AA iscomplicated and heterogeneity. Up till now, there are no effective methods forAA treatment. So it is significant to explore the mechanism and developtherapeutic methods theoretically and practically.
A great number of researches have shown that chronic intermittenthypobaric hypoxia (CIHH) has protective effect on tissue and organ of the body.For example, CIHH enhances the tolerance of body to ischemia and hypoxia,protects heart, brain, liver and kidney against ischemia/reperfusion injury, andantagonizes apoptosis and oxidation. Also it was reported that CIHH regulatesimmune function and vasomotoricity, keeps blood pressure stable throughfacilitating baroreflex. Our previous studies showed that CIHH had no effectson basic cardiac function, but protected heart against ischemia/reperfusioninjury. Recently, our research demonstrated that CIHH had effective preventiveand therapeutic effects on the renal vascular hypertension, collagen-inducedarthritis and fructose feeding-induced metabolic syndrome rats.
It is well known that the plateau hypoxia promotes the production of redblood cells. Researches in vitro showed that hypoxia could promote theproliferation of hemopoietic progenitor cells (HPCs) or hemopoietic stem cells(HSCs), bone marrow mesenchymal stem cells (BMMSCs), and could inhibitthe differentiation of HSCs and BMMSCs. Hypoxia has been proved toimprove hematopoietic microenviroment (HIM) and affect the hematopoieticprocess. However, the effect of CIHH on blood system and hemopoiesis is notclear yet and the effect of CIHH on AA has not been reported.
The objective of this study was to investigate the effect of CIHH on AArats induced by combination of5-fluorouracil (5-FU) and busulfan (BU)systemically in whole body, tissue, cellular and molecular levels usingexperimental zoology, cellular biology, morphology and molecular biologymethods, and explore the underlying mechanism. The study consists of fourparts:(1) To confirm the preventive and therapeutic effect of CIHH on AA ratswith hematopoietic stem cell failure induced by combination of5-FU and BU;(2) To investigate the immunological mechanism for protective effect of CIHHagainst AA through observing the effect of CIHH on T lymphocyte subpopula-tions of peripheral blood in AA rats;(3) To investigate the anti-apoptosismechanism for protective effect of CIHH against AA through observing effectof CIHH on bone marrow cells apoptosis in AA rats;(4) To investigate thehematopoietic HIM for protective effect of CIHH against AA throughobserving effect of CIHH on BMMSCs and its adhesiveness.
PartⅠThe protective effects of chronic intermittent hypobaric hypoxiaagainst aplastic anemia rats
Objective: To investigate the preventive and therapeutic effect of CIHHon AA rats with hematopoietic stem cell failure induced by combination of5-FU and BU.
Methods: The adult male Sprague-Dawley rats were randomly dividedinto the six groups: control group (Con), CIHH treatment group (CIHH), CIHHpre-treatment group (Pre-T), pre-aplastic anemia group (Pre-AA), CIHH post-treatment group (Post-T), post-aplastic anemia group (Post-AA). Pre-AA和Post-AA rats were given an intraperitoneal injection with combination of5-FUand BU to induce AA; Pre-T and Post-T rats were exposed to hypobarichypoxia simulating3,000m altitude(barometric pressure:525mmHg, PO2:108.8mmHg) in a hypobaric chamb-er for28days,5hours per day before andafter AA induction, respectively. CIHH rats accepted intermittent hypobarichypoxia treatment only and Con rats were not experienced neither AAinduction nor CIHH treatment. The pathologic morphology of bone marrowwas observed by hematoxylin-eosin (H-E) staining. The HPCs of colony-forming unit-granulocyte-macrophage (CFU-GM), colony-formingunit-erythroid (CFU-E), burst-forming unit-eryth-roid (BFU-E) andcolony-forming forming unit-megakaiyocyte (CFU-MK) were assayed by themethod of methylcellulose-based semisolid cultures. The serum interleukin-3(IL-3), erythropoietin (EPO), tumor necrosis factor-alpha (TNF-α) andinterleukin-2(IL-2) was measured by enzyme linked immuno-sorbent assay(ELISA).
Results:1. Compared with Con rats, the body weight of CIHH and Pre-Trats were not significantly different (P>0.05);The body weight of Pre-AA andPost-AA rats significantly decreased (P<0.01); The body weight of Post-T ratssignificantly decreased (P<0.01).
2. The incidence rate of AA in Pre-T rats (25%) was significantly lowerthan that in Pre-AA rats (80%, P<0.01).The initial onset time of AA in Pre-Trats (24.9±4.4d) was significantly later than that in Pre-AA rats (16.2±5.3d,P<0.01).The mority in Post-T rats (30%) was significantly lower than that inPost-AA rats (70%, P<0.01).
3. Compared with Con rats, the value of red blood cells (RBCs), whiteblood cells (WBCs), platelets (PLAs), retieulocytes (Rets), hemoglobin (HGB),hematocrit (HCT), the percentage of neutrophils (NEU), lymphocyte (LYM)and monocyte (MON) in peripheral blood of CIHH and Pre-T rats were notsignificantly different (P>0.05); Except percentage of LYM was significantlyincreased (P<0.01), other indexes of peripheral blood in Pre-AA and Post-AArats were obviously decreased (P<0.01); Except the value of RBCs, WBCs,PLA, Rets, HGB and HCT were significantly decreased (P<0.01), otherindexes of peripheral blood in Post-T rats were not significantly different (P>0.05).
4. Compared with Con rats, the bone marrow tissue in Pre-AA and Post-AA rats displayed a pinelosis, hematopoietic cells decrease, non-hematopoieticcells were significantly increase, and the percentage of hematopeitic cells weresignificantly decreased in Pre-AA and Post-AA rats. The number of bonemarrow capillary significantly reduced and bone marrow mesenchymal blood sinus was dilated in Pre-AA and Post-AA rats. The changes of pathologicmorphology in bone marrow tissue of Pre-T and Post-T rats were significantlyimproved.
5. Compared with Con rats, the value of bone marrow mononucleatedcells (BMMNCs) and other bone marrow cells in CIHH, Pre-T and Post-T ratswere not significantly different (P>0.05);The value of BMMNCs andmyeloblasts, segmented granulocytes, basophilic promyelocytes, basophilicerythroblasts, polychromatophilic erythroblasts, normoblasts and megakaryo-cytes in AA rats were significantly decreased (P<0.01), and the percentage ofmyelocytes, metamylocytes, stabform granulocytes, eosinophilic myelocyteslymphocytes and monocytes in Pre-AA and Post-AA rats were significantlyincreased (P<0.01), which the other cells in Pre-AA and Post-AA rats bonemarrow were not significantly different (P>0.05).
6. Compared with Con rats, the colonies of CFU-GM, CFU-E, BFU-E andCFU-MK in CIHH and Pre-T rats were not significantly different (P>0.05).The colonies of CFU-GM, CFU-E, BFU-E and CFU-MK in Pre-AA, Post-AAand Post-T rats were significantly decreased (P<0.01).
7. Compared with Con rats, the serum concentration of IL-3, EPO, TNF-αand IL-2were not significantly different in CIHH rats (P>0.05); The serumconcentration of IL-3and EPO were significantly decreased, while TNF-α andIL-2were significantly increased in Pre-AA and Post-AA rats (P<0.01). Theserum concentration of IL-3and EPO were significantly increased (P<0.01),while TNF-α and IL-2were not different in Pre-T and Post-T rats (P>0.05).
Summary: This study confirmed for the first time that CIHH pre-treatment has preventive effect on incidence of AA, and CIHH post-treatmenthas protective effect on AA, lessening the functional disturbance and improvethe pathological damage of bone marrow tissue in AA rats. The protectiveeffect of CIHH on AA was related to the increase of serum IL-3and EPO, thepositive hematopoiesis regulatory factor, and to the decrease of serum TNF-αand IL-2, the negative hematopoiesis regulatory factor.
Part II Effects of chronic intermittent hypobaric hypoxia on T lympho-cytes in aplastic anemia rats
Objective: To investigate the immunological mechanism for protectiveeffect of CIHH against AA through observing the effect of CIHH on Tlymphocyte subpopulations of peripheral blood in AA rats.
Methods: Experimental grouping, preparation of AA and CIHH treatmentwere the same as Part. T lymphocytes and subpopulations of CD3~+, CD4~+,CD8~+, CD4~+HLA-DR~+and CD8~+HLA-DR~+in peripheral blood of rats weredetected by flow cytometry (FCM). Serum interferon-gamma (IFN-γ),interleukin-4(IL-4) and interleukin-9(IL-9) in rats were assayed by ELISA.Protein expression of IFN-γ, IL-4and IL-9in bone marrow of rats wasmeasured by immunohistochemistry method.
Results:1. Compared with Con rats, the percentage of CD3~+, CD4~+, CD8~+,CD4~+HLA-DR~+and CD8~+HLA-DR~+T lymphocyte subpopulations were notsignificantly different in CIHH and Pre-T rats (P>0.05); The percentages ofCD3~+T lymphocytes, CD8~+and CD8~+HLA-DR~+T lymphocyte subpopulationswere significantly increased and the percentages of CD4~+T lymphocytesubpopulations and the ratio of CD4~+/CD8~+were significantly decreased inPre-AA and Post-AA rats (P<0.01); The percentages of CD3~+, CD8~+and CD4~+HLA-DR~+T lymphocyte subpopulations were not significantly different (P>0.05), while the percentages of CD4~+, CD8~+HLA-DR~+and the ratio of CD4~+/CD8~+were significantly increased in Post-T rats (P<0.05).
2. Compared with Con rats, serum IFN-γ, IL-4, IL-9and the ratio ofIFN-γ/IL-4(Th1/Th2) were not significantly different CIHH, Pre-T and Post-Trats (P>0.05); Serum IFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) inPre-AA and Post-AA rats were significantly increased (P<0.01), and serumIL-4were significantly decreased in Pre-AA and Post-AA rats (P<0.05).
3. Compared with Con rats, protein expression of IFN-γ, IL-4, IL-9andthe ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow tissue were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The protein expression ofIFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow weresignificantly increased (P<0.01), while the protein expression of IL-4of bone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01).
Summary: Under physiological condition， CIHH treatment had nosignificant effects on T lymphocyte subpopulations and the levels of IFN-γ, IL-9and IL-4in peripheral blood，serum and bone marrow，but effectivelyantagonized the reduction of helper T lymphocytes (CD4~+) and the increase ofcytotoxic T lymphocytes (CD8~+) and activated cytotoxic T lymphocytes (CD8~+HLA-DR~+), keeping the balance of peripheral blood T lymphocyte subsets andthe balance between proinflammatory cytokines and anti-inflammatory cyto-kines. The regulation of CIHH on cellular immunity may be one of themechanisms of anti-AA effect of CIHH.
Part III Effects of chronic intermittent hypobaric hypoxia on apoptosis ofbone marrow cells in aplastic anemia rats
Objective: To investigate the anti-apoptosis mechanism for protectiveeffect of CIHH against AA through observing effect of CIHH on bone marrowcells apoptosis in AA rats.
Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The apoptosis of bone marrow cells wasexamined by FCM and terminal dUTP nick end labeling (TUNEL). Theexpression of apoptosis associated protein Fas, Bax, Bcl-2, Caspase-3andCaspase-8was measured by immunohistochemisty method. The expression ofhypoxia inducible factor-1alpha (HIF-1α) and nuclear factor-kappa B (NF-κB)protein was analyzed by western blot.
Results:1. The results of FCM and TUNEL showed that, compared withCon rats, the apoptosis rate of bone marrow cells were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The apoptosis rate in thebone marrow cells of Pre-AA and Post-AA rats were significantly increased (P<0.01).
2. Compared with Con rats, the protein expressions of Fas, Bax, Bcl-2,Caspase-3, Caspase-8and the radio of Bax/Bcl-2of bone marrow cells werenot significantly different in CIHH, Pre-T and Post-T rats (P>0.05); The proteinexpressions of Fas, Bax, Caspase-3, Caspase-8and the ratio of Bax/Bcl-2were significantly increased (P<0.05-P<0.01), while the protein expression of Bcl-2of bone marrow cells were significantly decreased in Pre-AA and Post-AA rats(P<0.05).
3. Compared with Con rats, the protein expressions of HIF-1α and NF-κBof bone marrow cells were not significantly different in CIHH and Pre-T rats (P>0.05); The protein expression of HIF-1α and NF-κB of bone marrow cellswere significantly increased in Pre-AA and Post-AA rats (P<0.05-P<0.01); Theprotein expression of HIF-1α of bone marrow cells were significantly increasedin Post-T rats (P<0.01),and the protein expression of NF-κB of bone marrowcells in the bone marrow cells were not significantly different in Post-T rats (P>0.05).
Summary: Under physiological condition, CIHH treatment had nosignificant effect on apoptosis and apoptosis associated protein of bone marrowcells, but effectively antagonized the overexpression of HIF-1α and NF-κBproteins, down-regulated the expression of positive apoptosis proteins Fas, Bax,Caspase-3and Caspase-8, up-regulated the expression of negative apoptosisprotein Bcl-2, resulting in anti-apoptosis eventually. The anti-apoptosis ofCIHH on bone marrow cells might be one of the mechanisms of anti-AA effectof CIHH.
Part IV Effects of chronic intermittent hypobaric hypoxia on adhesivenessof bone marrow mesenchymal stem cells in aplastic anemia rats
Objective: To investigate the hematopoietic HIM for protective effect ofCIHH against AA through observing effect of CIHH on BMMSCs and itsadhesiveness.
Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The morphology of adhension cell andcolony-forming unit-fibroblast (CFU-F) were observed by long-term bonemarrow explant cultures (LTBMC-Ex) dynamically. BMMSCs were culturedand purifucated in vitro by the method of differential velocity adherent cellsculture.Very late antigen-4(VLA-4), vascular cell adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1), CD162and CD164 of BMMSCs were analyzed by FCM. The protein expression of p38mitogen-activated protein kinase (p38MAPK) was assayed by western blot.
Results:1. During LTBMC-Ex, the bone marrow stromal cells in Pre-AAand Post-AA rats were dysplasia, and the most cells were immature precursorcells and fibroblast-like cells. Under the basic physiological condition, CIHHtreatment had no effect on development of bone marrow stromal cells layer, butantagonized effectively the dysplasia of bone marrow stromal cells layer andrenewed a valid layer to support hematopoiesis.
2. Compared with Con rats, the CFU-F colonies of bone marrow had nosignificantly changes in CIHH and Pre-T rats (P>0.05); The CFU-F colonies ofbone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01); The CFU-F colonies of bone marrow were significantly decreased inPost-T rats (P<0.01).
3. Compared with Con rats, the protein expressions of VLA-4, VCAM-1,ICAM-1, CD162and CD164of BMMSCs were not significantly different inCIHH rats (P>0.05). The protein expressions of VLA-4, VCAM-1and ICAM-1of BMMSCs were significantly decreased (P<0.01), while CD162and CD164of BMMSCs were significantly increased in Pre-AA and Post-AA rats (P<0.01). The protein expression of VLA-4, VCAM-1, ICAM-1and CD164ofBMMSCs were not significantly different (P>0.05), but CD162of BMMSCswas significantly increased in Pre-T rats (P<0.01). The protein expressions ofVCAM-1and ICAM-1of BMMSCs were not significantly different in (P>0.05), while VLA-4of BMMSCs was significantly decreased in Post-T rats (P<0.01) and the protein expressions of CD162and CD164of BMMSCs weresignificantly increased in Post-T rats (P<0.01).
4. Compared with Con rats, the protein expression of p38MAPK ofBMMSCs was not significantly different in CIHH and Pre-T rats (P>0.05). Theprotein expression of p38MAPK of BMMSCs was significantly increased inPre-AA, Post-AA and Post-T rats (P<0.01).
Summary: Under physiological condition,CIHH treatment had nosignificant effect on development of bone marrow stromal cells layer and the adhensiveness of BMMSCs, but effectively antagonized the dysplasia of bonemarrow stromal cells layer to support hematopoietsis and the adhensiveness ofBMMSCs in AA rats. This demonstrates that CIHH treatment improves theadhesion of BMMSCs and enhance nutritional support to HSCs in AA rats,which might be one of mechanism of anti-AA effect of CIHH.
Conclusion: CIHH has protective effect agains aplastic anemia of ratsthrough inhibit-ing the overexpression of HIF-1α, NF-κB, p38MAPK anddownstream signal molecules, maintaining homeostasis of hematopoieticregulatory factors, keeping balance of T lymphocyte subpopulations,proinflammatory/anti-inflammatory factors,and adhensive molecules, lesseningthe damage of hematopoietic tissue and inhibition of hematopoietic function inbone marrow, decreasing hematopoietic cell apoptosis, and enhancing theadhesiveness and nutritional support between the bone marrow hematopoieticcells and hematopoietic mic-roenvironment.
大量研究表明，慢性间歇性低压低氧（chronic intermittent hypobarichypoxia, CIHH）对机体多种组织、器官具有保护作用。例如，CIHH可增强机体对缺血、缺氧的耐受性，对抗缺血/再灌注所致心功能损伤及心律失常，保护脑、肝脏、肾脏等器官组织，调节免疫功能，抗细胞凋亡，抗氧化。此外，CIHH可降低血压、调节血管舒缩功能、易化压力感受性反射等。我们以往研究表明，CIHH对基础心功能无影响，但可对抗缺血/再灌注和急性缺氧/复氧对心脏的损伤，发挥心脏保护作用。近来，我们研究结果显示，CIHH对肾血管性高血压大鼠、胶原诱导关节炎大鼠和果糖诱导代谢综合征大鼠均具有明显的防治作用。
众所周知，高原低氧可促进红细胞的生成。研究表明，低氧能显著影响造血过程，促进造血祖细胞（hemopoietic progenitor cells, HPCs）或造血干细胞（hemopoietic stem cells, HSCs）、骨髓间质干细胞（bone marrowmesenchymal stem cells, BMMSCs）增殖并抑制其分化，以及改善骨髓微环境（hematopoietic microenviroment, HIM）。但有关CIHH对血液系统及造血机能的影响尚未明了，也未见CIHH对AA作用的报道。
本研究旨在利用5-氟尿嘧啶（5-fluorouracil,5-FU）联合白消安（busulfan, BU）诱导的AA动物模型，应用实验动物学、细胞生物学、形态学和分子生物学等方法，从整体、组织和细胞分子不同水平、系统动态地观察CIHH处理对AA的影响，并探讨其机制。研究分为四部分：（1）探讨CIHH对5-FU和BU联合诱导的AA大鼠的防治作用；（2）通过观察CIHH对AA大鼠外周血T淋巴细胞亚群的影响，探讨CIHH抗大鼠AA作用的细胞免疫学机制；（3）通过观察CIHH对AA大鼠骨髓细胞凋亡的影响，探讨CIHH抗大鼠AA作用的细胞凋亡机制；（4）通过观察CIHH对AA大鼠BMMSCs及其粘附性的影响，探讨CIHH抗大鼠AA作用的粘附分子机制。
第一部分慢性间歇性低压低氧抗大鼠再生障碍性贫血的作用
目的：初步探讨CIHH对5-FU联合BU诱导的的AA大鼠的防治作用。
方法:雄性成年SD大鼠随机分为6组：对照组（Con）、CIHH组（CIHH）、CIHH预处理组（Pre-T）、预处理再障组（Pre-AA）、CIHH后处理组（Post-T）和后处理再障组（Post-AA）。Pre-AA和Post-AA大鼠给予5-FU联合BU腹腔注射诱导AA；Pre-T和Post-T大鼠分别在AA造模前、后接受28天、模拟海拔3,000米（大气压：525mmHg，PO2：108.8mmHg）、每天5小时的低压低氧处理；CIHH大鼠仅给予模拟海拔3,000米（大气压：525mmHg，PO2：108.8mmHg）、每天5小时的低压低氧处理; Con大鼠不给予AA造模和间歇性低压低氧处理。H-E染色观察骨髓形态学变化；甲基纤维素半固体培养法检测骨髓粒-单系集落形成单位（colony-forming unit-granulocyte-macrophage, CFU-GM）、红系集落形成单位（colony-forming unit-erythroid, CFU-E)、红系爆式集落形成单位（burst-forming unit-erythroid, BFU-E）和巨核系集落形成单位（colony-forming unit-megakaiyocyte, CFU-MK）产率；酶联免疫吸附试验（enzymelinked immunosorbent assay, ELISA）检测血清白介素-3（interleukin-3,IL-3）、促红细胞生成素（erythropoietin, EPO）、肿瘤坏死因子-α （tumornecrosis factor-alpha, TNF-α）和白介素-2（interleukin-2, IL-2）水平。
结果：1.与Con大鼠相比，CIHH和Pre-T大鼠体重无显著差异（P<0.05）；Pre-AA组、Post-AA和Post-T大鼠体重显著下降（P<0.01）。
2. Pre-T大鼠AA发生率（25%）显著低于Pre-AA大鼠（80%, P <0.01）；Pre-T首次发病时间（24.9±4.4天）显著迟于Pre-AA大鼠（16.2±5.3天, P<0.01）；Post-T大鼠死亡率（30%）显著低于Post-AA大鼠（70%, P <0.01）。
3.与Con大鼠相比，CIHH和Pre-T大鼠外周血红细胞（red blood cell,RBC）、白细胞（white blood cell, WBC）、血小板（platelet, PLA）、血红蛋白（hemoglobin, HGB）、红细胞比容（hematocrit, HCT）、网织红细胞（retieulocyte, Ret）绝对值、中性粒细胞（neutrophils, NEUT）百分比、淋巴细胞（lymphocyte, LYM）百分比和单核细胞（monocyte, MON）百分比无显著差异（P>0.05）；Pre-AA和Post-AA大鼠除LYM%显著增多（P<0.01）外，其余各血象指标均明显减少（P<0.01）；Post-T大鼠除RBC、WBC、PLA、HGB、HCT和Ret绝对值显著减少（P<0.01）外，其它血象指标无显著差异（P>0.05）。
4.与Con大鼠相比，Pre-AA和Post-AA大鼠骨髓组织明显脂肪化，造血细胞减少，非造血细胞增多，骨髓间质血窦扩张明显，毛细血管网明显减少，造血功能显著低下。Pre-T和Post-T大鼠骨髓的各种改变明显改善。
5.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠的骨髓单个核细胞(bone marrow mononucleated cells, BMMNCs)和骨髓各类细胞均无显著变化（P>0.05）；Pre-AA和Post-AA大鼠的BMMNCs数、原粒细胞、分叶粒细胞、碱性早幼粒细胞、早红细胞、中红细胞、晚红细胞和巨核细胞显著减少（P<0.01），而其中幼粒细胞、晚幼粒细胞、杆状粒细胞、酸性中幼粒细胞、单核细胞和淋巴细胞显著增多（P<0.01）,其他骨髓细胞无显著差异（P>0.05）。
6.与Con大鼠相比，CIHH和Pre-T大鼠的CFU-GM、CFU-E、BFU-E和CFU-MK产率无显著差异（P>0.05）；Pre-AA、Post-AA和Post-T大鼠的CFU-GM、CFU-E、BFU-E和CFU-MK集落产率显著减少（P<0.01）。
7.与Con大鼠相比，CIHH大鼠血清IL-3、EPO、TNF-α和IL-2水平均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠血清IL-3和EPO水平显著减少（P<0.01），而其TNF-α和IL-2水平显著增多（P<0.05）；Pre-T和Post-T大鼠血清IL-3和EPO水平显著增多（P<0.01），而其TNF-α和IL-2水平无显著差异（P>0.05）。
小结: CIHH预处理可预防大鼠AA的发生，CIHH后处理有抗AA的作用，可减轻AA大鼠造血功能障碍，改善骨髓组织病理损伤。此作用与CIHH升高血清正性造血调控因子IL-3和EPO，降低造负性血调控因子TNF-α和IL-2有关。
第二部分慢性间歇性低压低氧对再生障碍性贫血大鼠T淋巴细胞的影响
目的:通过观察CIHH对AA大鼠外周血T淋巴细胞亚群的影响，探讨CIHH抗大鼠AA作用的细胞免疫机制。
方法:实验分组、AA模型制备和CIHH处理和同本研究第一部分。流式细胞术（flow cytometry, FCM）检测大鼠外周血T淋巴细胞及其亚群CD3~+、CD4~+、CD8~+、CD4~+HLA-DR~+和CD8~+HLA-DR~+；ELISA法检测大鼠血清干扰素-γ（interferon, IFN-γ）、白介素-4（interleukin-4, IL-4）和白介素-9（interleukin-9, IL-9）（分别代表Th1、Th2、Th9细胞表达水平）；免疫组化法检测大鼠骨髓组织IFN-γ、IL-4和IL-9蛋白。
结果：1.与Con大鼠相比，CIHH大鼠外周血T淋巴细胞亚群CD3~+、CD4~+、CD8~+、CD4~+HLA-DR~+、CD8~+HLA-DR~+百分比和CD4~+/CD8~+值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠外周血T淋巴细胞亚群CD3~+、CD8~+、CD8~+HLA-DR~+百分比显著升高（P<0.01），CD4~+百分比和CD4~+/CD8~+值显著降低（P<0.01）； Pre-T大鼠外周血T淋巴细胞亚群各参数无显著差异（P<0.01）；Post-T大鼠外周血T淋巴细胞亚群CD3~+、CD8~+和CD4~+HLA-DR~+无显著差异（P>0.05），而CD4~+、CD4~+/CD8~+和CD8~+HLA-DR~+显著升高（P<0.05）。
2.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠血清IFN-γ、IL-4、IL-9水平和IFN-γ/IL-4（Th1/Th2）值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠血清促炎因子IFN-γ、IL-9水平和IFN-γ/IL-4（Th1/Th2）值显著升高（P<0.01），抗炎因子IL-4水平显著降低（P<0.05）。
3.与Con大鼠相比，CIHH、Pre-T和Post-T大鼠骨髓组织IFN-γ、IL-9、IL-4水平和IFN-γ/IL-4（Th1/Th2）值均无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓组织促炎因子IFN-γ、IL-9水平和IFN-γ/IL-4（Th1/Th2）值显著升高（P<0.01），抗炎因子IL-4水平显著降低（P<0.01）。
小结: CIHH处理对大鼠基础状态外周血T淋巴细胞亚群、血清和骨髓组织中炎性因子IFN-γ、IL-9及IL-4无影响，但可有效对抗AA大鼠外周血辅助性T淋巴细胞（CD4~+）的降低，细胞毒性T淋巴细胞（CD8~+）和活化的细胞毒性T淋巴细胞（CD8~+HLA-DR~+）的升高，从而维持T淋巴细胞亚群的平衡，促炎因子和抗炎因子间的平衡。CIHH的细胞免疫调节作用可能是CIHH抗大鼠AA的作用机制之一。
第三部分慢性间歇性低压低氧对再生障碍性贫血大鼠骨髓细胞凋亡的影响
目的:通过观察CIHH对AA大鼠骨髓细胞凋亡的影响，探讨CIHH抗大鼠AA作用的细胞凋亡机制。
方法:实验分组、AA模型制备和CIHH处理和同本研究第一部分。FCM和原位末端标记法（terminal dUTP nick end labeling, TUNEL）检测大鼠骨髓细胞凋亡；免疫组化法检测大鼠骨髓细胞凋亡相关蛋白Fas、Bax、Bcl-2、Caspase-3和Caspase-8表达；Western blot法检测大鼠骨髓细胞低氧诱导因子-1α（hypoxia inducible factor-1alpha, HIF-1α）和核转录因子-κB（nuclear factor-kappa B, NF-κB）表达。
结果：1. FCM和TUNEL结果均显示，与Con大鼠相比，CIHH、Pre-T和Post-T大鼠骨髓细胞凋亡率无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞凋亡率显著升高（P<0.01）。
2.与Con大鼠相比，CIHH，Pre-T和Post-T大鼠骨髓细胞凋亡蛋白Fas、Bax、Bcl-2、Caspase-3、Caspase-8表达和Bax/Bcl-2值无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞促凋亡蛋白Fas、Bax、Caspase-3、Caspase-8和Bax/Bcl-2值显著升高（P<0.05-P<0.01），而抑凋亡蛋白Bcl-2显著降低（P<0.05）。
3.与Con大鼠相比，CIHH和Pre-T大鼠骨髓细胞HIF-1α和NF-κB蛋白表达无显著差异（P>0.05）；Pre-AA和Post-AA大鼠骨髓细胞HIF-1α和NF-κB蛋白表达显著升高（P<0.05-P<0.01）；Post-T大鼠骨髓细胞HIF-1α蛋白表达显著升高（P<0.01），而其NF-κB蛋白表达无显著差异（P>0.05）。
小结: CIHH处理对基础状态下大鼠骨髓细胞凋亡和凋亡无影响，但可对抗AA大鼠HIF-1α和NF-κB蛋白过度表达，下调促凋亡蛋白Fas、Bax、Caspase-3和Caspase-8表达，上调抑凋亡蛋白Bcl-2表达，最终抗骨髓细胞凋亡。CIHH抗骨髓细胞凋亡可能也是CIHH抗大鼠AA的作用机制之一。
第四部分慢性间歇性低压低氧对再生障碍性贫血大鼠骨髓间质干细胞粘附性的影响
目的:通过观察CIHH对AA大鼠BMMSCs及其粘附性的影响，探讨CIHH抗大鼠AA作用的粘附分子机制。
方法:实验分组、CIHH处理和AA模型制备同本研究第一部分。体外长期骨髓植入培养（long-term bone marrow explant cultures, LTBMC-Ex）动态观察骨髓贴壁细胞形态和骨髓成纤维细胞集落形成单位（colonyforming unit-fibroblast, CFU-F）的变化；差速贴壁法培养并纯化BMMSCs；FCM检测BMMSCs极晚期抗原-4（very late antigen-4, VLA-4）、血管细胞间粘附分子-1（vascular cell adhesion molecule-1, VCAM-1）、细胞间粘附分子-1（intercellular adhesion molecule-1, ICAM-1）、CD162和CD164；Western blot法检测BMMSCs表达p38丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）。
结果：1.体外骨髓长期植入培养过程中，Pre-AA和Post-AA大鼠骨髓基质细胞层发育不良，细胞多为不成熟的大基质前体细胞和成纤维样细胞。CIHH处理对基础状态下大鼠骨髓基质细胞层无影响，但可有效对抗AA大鼠骨髓基质细胞层发育不良，重新形成有效支持造血的骨髓基质细胞层。
2.与Con大鼠相比，CIHH和Pre-T大鼠骨髓的CFU-F集落数无显著变化（P>0.05）；Pre-AA和Post-AA大鼠骨髓的CFU-F集落数显著减少（P<0.01）； Post-T大鼠骨髓的CFU-F集落数显著减少（P<0.01）。
3.与Con大鼠相比，CIHH大鼠BMMSCs表达VLA-4、VCAM-1、ICAM-1、CD162和CD164无显著差异（P>0.05）; Pre-AA和Post-AA大鼠BMMSCs表达VLA-4、VCAM-1和ICAM-1显著减少（P<0.01），而表达CD162和CD164显著增多（P<0.01）；Pre-T大鼠BMMSCs表达VLA-4、VCAM-1、ICAM-1和CD164无显著差异（P>0.05），但表达CD162显著增多（P<0.05）;Post-T大鼠BMMSCs表达VCAM-1和ICAM-1无显著差异（P>0.05），但表达VLA-4显著减少（P<0.01），表达CD162和CD164显著增多（P<0.01）。
4.与Con大鼠相比，CIHH和Pre-T大鼠BMMSCs表达p38MAPK无显著差异（P>0.05）；Pre-AA、Post-AA和Post-T大鼠BMMSCs表达p38MAPK显著升高（P<0.01）。
小结：CIHH处理对基础状态下骨髓基质细胞发育和BMMSCs粘附性无显著影响；但可有效修复AA大鼠发育不良的骨髓基质细胞，明显增强AA大鼠BMMSCs减弱的粘附性。CIHH的粘附分子调节机制也是CIHH抗大鼠AA的作用机制之一。
结论： CIHH处理可通过抑制AA大鼠信号通路HIF-1α、NF-κB和p38MAPK蛋白过度表达及下游信号分子的异常变化，维持造血调控因子间、T淋巴细胞亚群间、炎性因子间和粘附分子间的平衡，从而减轻骨髓造血组织损伤和造血功能抑制、减少造血细胞凋亡，增强骨髓造血细胞与造血微环境间的粘附性，发挥防治大鼠AA作用。
Aplastic anemia (AA), a common clinical malignant blood disease, is anacquired bone marrow failure syndrome caused by chemical, physics, biologi-cal, heredity and some unknown causes. The main clinical manifestations areanemia, bleeding tendency and infection symptoms. The pathogenesis of AA iscomplicated and heterogeneity. Up till now, there are no effective methods forAA treatment. So it is significant to explore the mechanism and developtherapeutic methods theoretically and practically.
A great number of researches have shown that chronic intermittenthypobaric hypoxia (CIHH) has protective effect on tissue and organ of the body.For example, CIHH enhances the tolerance of body to ischemia and hypoxia,protects heart, brain, liver and kidney against ischemia/reperfusion injury, andantagonizes apoptosis and oxidation. Also it was reported that CIHH regulatesimmune function and vasomotoricity, keeps blood pressure stable throughfacilitating baroreflex. Our previous studies showed that CIHH had no effectson basic cardiac function, but protected heart against ischemia/reperfusioninjury. Recently, our research demonstrated that CIHH had effective preventiveand therapeutic effects on the renal vascular hypertension, collagen-inducedarthritis and fructose feeding-induced metabolic syndrome rats.
It is well known that the plateau hypoxia promotes the production of redblood cells. Researches in vitro showed that hypoxia could promote theproliferation of hemopoietic progenitor cells (HPCs) or hemopoietic stem cells(HSCs), bone marrow mesenchymal stem cells (BMMSCs), and could inhibitthe differentiation of HSCs and BMMSCs. Hypoxia has been proved toimprove hematopoietic microenviroment (HIM) and affect the hematopoieticprocess. However, the effect of CIHH on blood system and hemopoiesis is notclear yet and the effect of CIHH on AA has not been reported.
The objective of this study was to investigate the effect of CIHH on AArats induced by combination of5-fluorouracil (5-FU) and busulfan (BU)systemically in whole body, tissue, cellular and molecular levels usingexperimental zoology, cellular biology, morphology and molecular biologymethods, and explore the underlying mechanism. The study consists of fourparts:(1) To confirm the preventive and therapeutic effect of CIHH on AA ratswith hematopoietic stem cell failure induced by combination of5-FU and BU;(2) To investigate the immunological mechanism for protective effect of CIHHagainst AA through observing the effect of CIHH on T lymphocyte subpopula-tions of peripheral blood in AA rats;(3) To investigate the anti-apoptosismechanism for protective effect of CIHH against AA through observing effectof CIHH on bone marrow cells apoptosis in AA rats;(4) To investigate thehematopoietic HIM for protective effect of CIHH against AA throughobserving effect of CIHH on BMMSCs and its adhesiveness.
PartⅠThe protective effects of chronic intermittent hypobaric hypoxiaagainst aplastic anemia rats
Objective: To investigate the preventive and therapeutic effect of CIHHon AA rats with hematopoietic stem cell failure induced by combination of5-FU and BU.
Methods: The adult male Sprague-Dawley rats were randomly dividedinto the six groups: control group (Con), CIHH treatment group (CIHH), CIHHpre-treatment group (Pre-T), pre-aplastic anemia group (Pre-AA), CIHH post-treatment group (Post-T), post-aplastic anemia group (Post-AA). Pre-AA和Post-AA rats were given an intraperitoneal injection with combination of5-FUand BU to induce AA; Pre-T and Post-T rats were exposed to hypobarichypoxia simulating3,000m altitude(barometric pressure:525mmHg, PO2:108.8mmHg) in a hypobaric chamb-er for28days,5hours per day before andafter AA induction, respectively. CIHH rats accepted intermittent hypobarichypoxia treatment only and Con rats were not experienced neither AAinduction nor CIHH treatment. The pathologic morphology of bone marrowwas observed by hematoxylin-eosin (H-E) staining. The HPCs of colony-forming unit-granulocyte-macrophage (CFU-GM), colony-formingunit-erythroid (CFU-E), burst-forming unit-eryth-roid (BFU-E) andcolony-forming forming unit-megakaiyocyte (CFU-MK) were assayed by themethod of methylcellulose-based semisolid cultures. The serum interleukin-3(IL-3), erythropoietin (EPO), tumor necrosis factor-alpha (TNF-α) andinterleukin-2(IL-2) was measured by enzyme linked immuno-sorbent assay(ELISA).
Results:1. Compared with Con rats, the body weight of CIHH and Pre-Trats were not significantly different (P>0.05);The body weight of Pre-AA andPost-AA rats significantly decreased (P<0.01); The body weight of Post-T ratssignificantly decreased (P<0.01).
2. The incidence rate of AA in Pre-T rats (25%) was significantly lowerthan that in Pre-AA rats (80%, P<0.01).The initial onset time of AA in Pre-Trats (24.9±4.4d) was significantly later than that in Pre-AA rats (16.2±5.3d,P<0.01).The mority in Post-T rats (30%) was significantly lower than that inPost-AA rats (70%, P<0.01).
3. Compared with Con rats, the value of red blood cells (RBCs), whiteblood cells (WBCs), platelets (PLAs), retieulocytes (Rets), hemoglobin (HGB),hematocrit (HCT), the percentage of neutrophils (NEU), lymphocyte (LYM)and monocyte (MON) in peripheral blood of CIHH and Pre-T rats were notsignificantly different (P>0.05); Except percentage of LYM was significantlyincreased (P<0.01), other indexes of peripheral blood in Pre-AA and Post-AArats were obviously decreased (P<0.01); Except the value of RBCs, WBCs,PLA, Rets, HGB and HCT were significantly decreased (P<0.01), otherindexes of peripheral blood in Post-T rats were not significantly different (P>0.05).
4. Compared with Con rats, the bone marrow tissue in Pre-AA and Post-AA rats displayed a pinelosis, hematopoietic cells decrease, non-hematopoieticcells were significantly increase, and the percentage of hematopeitic cells weresignificantly decreased in Pre-AA and Post-AA rats. The number of bonemarrow capillary significantly reduced and bone marrow mesenchymal blood sinus was dilated in Pre-AA and Post-AA rats. The changes of pathologicmorphology in bone marrow tissue of Pre-T and Post-T rats were significantlyimproved.
5. Compared with Con rats, the value of bone marrow mononucleatedcells (BMMNCs) and other bone marrow cells in CIHH, Pre-T and Post-T ratswere not significantly different (P>0.05);The value of BMMNCs andmyeloblasts, segmented granulocytes, basophilic promyelocytes, basophilicerythroblasts, polychromatophilic erythroblasts, normoblasts and megakaryo-cytes in AA rats were significantly decreased (P<0.01), and the percentage ofmyelocytes, metamylocytes, stabform granulocytes, eosinophilic myelocyteslymphocytes and monocytes in Pre-AA and Post-AA rats were significantlyincreased (P<0.01), which the other cells in Pre-AA and Post-AA rats bonemarrow were not significantly different (P>0.05).
6. Compared with Con rats, the colonies of CFU-GM, CFU-E, BFU-E andCFU-MK in CIHH and Pre-T rats were not significantly different (P>0.05).The colonies of CFU-GM, CFU-E, BFU-E and CFU-MK in Pre-AA, Post-AAand Post-T rats were significantly decreased (P<0.01).
7. Compared with Con rats, the serum concentration of IL-3, EPO, TNF-αand IL-2were not significantly different in CIHH rats (P>0.05); The serumconcentration of IL-3and EPO were significantly decreased, while TNF-α andIL-2were significantly increased in Pre-AA and Post-AA rats (P<0.01). Theserum concentration of IL-3and EPO were significantly increased (P<0.01),while TNF-α and IL-2were not different in Pre-T and Post-T rats (P>0.05).
Summary: This study confirmed for the first time that CIHH pre-treatment has preventive effect on incidence of AA, and CIHH post-treatmenthas protective effect on AA, lessening the functional disturbance and improvethe pathological damage of bone marrow tissue in AA rats. The protectiveeffect of CIHH on AA was related to the increase of serum IL-3and EPO, thepositive hematopoiesis regulatory factor, and to the decrease of serum TNF-αand IL-2, the negative hematopoiesis regulatory factor.
Part II Effects of chronic intermittent hypobaric hypoxia on T lympho-cytes in aplastic anemia rats
Objective: To investigate the immunological mechanism for protectiveeffect of CIHH against AA through observing the effect of CIHH on Tlymphocyte subpopulations of peripheral blood in AA rats.
Methods: Experimental grouping, preparation of AA and CIHH treatmentwere the same as Part. T lymphocytes and subpopulations of CD3~+, CD4~+,CD8~+, CD4~+HLA-DR~+and CD8~+HLA-DR~+in peripheral blood of rats weredetected by flow cytometry (FCM). Serum interferon-gamma (IFN-γ),interleukin-4(IL-4) and interleukin-9(IL-9) in rats were assayed by ELISA.Protein expression of IFN-γ, IL-4and IL-9in bone marrow of rats wasmeasured by immunohistochemistry method.
Results:1. Compared with Con rats, the percentage of CD3~+, CD4~+, CD8~+,CD4~+HLA-DR~+and CD8~+HLA-DR~+T lymphocyte subpopulations were notsignificantly different in CIHH and Pre-T rats (P>0.05); The percentages ofCD3~+T lymphocytes, CD8~+and CD8~+HLA-DR~+T lymphocyte subpopulationswere significantly increased and the percentages of CD4~+T lymphocytesubpopulations and the ratio of CD4~+/CD8~+were significantly decreased inPre-AA and Post-AA rats (P<0.01); The percentages of CD3~+, CD8~+and CD4~+HLA-DR~+T lymphocyte subpopulations were not significantly different (P>0.05), while the percentages of CD4~+, CD8~+HLA-DR~+and the ratio of CD4~+/CD8~+were significantly increased in Post-T rats (P<0.05).
2. Compared with Con rats, serum IFN-γ, IL-4, IL-9and the ratio ofIFN-γ/IL-4(Th1/Th2) were not significantly different CIHH, Pre-T and Post-Trats (P>0.05); Serum IFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) inPre-AA and Post-AA rats were significantly increased (P<0.01), and serumIL-4were significantly decreased in Pre-AA and Post-AA rats (P<0.05).
3. Compared with Con rats, protein expression of IFN-γ, IL-4, IL-9andthe ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow tissue were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The protein expression ofIFN-γ, IL-9and the ratio of IFN-γ/IL-4(Th1/Th2) of bone marrow weresignificantly increased (P<0.01), while the protein expression of IL-4of bone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01).
Summary: Under physiological condition， CIHH treatment had nosignificant effects on T lymphocyte subpopulations and the levels of IFN-γ, IL-9and IL-4in peripheral blood，serum and bone marrow，but effectivelyantagonized the reduction of helper T lymphocytes (CD4~+) and the increase ofcytotoxic T lymphocytes (CD8~+) and activated cytotoxic T lymphocytes (CD8~+HLA-DR~+), keeping the balance of peripheral blood T lymphocyte subsets andthe balance between proinflammatory cytokines and anti-inflammatory cyto-kines. The regulation of CIHH on cellular immunity may be one of themechanisms of anti-AA effect of CIHH.
Part III Effects of chronic intermittent hypobaric hypoxia on apoptosis ofbone marrow cells in aplastic anemia rats
Objective: To investigate the anti-apoptosis mechanism for protectiveeffect of CIHH against AA through observing effect of CIHH on bone marrowcells apoptosis in AA rats.
Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The apoptosis of bone marrow cells wasexamined by FCM and terminal dUTP nick end labeling (TUNEL). Theexpression of apoptosis associated protein Fas, Bax, Bcl-2, Caspase-3andCaspase-8was measured by immunohistochemisty method. The expression ofhypoxia inducible factor-1alpha (HIF-1α) and nuclear factor-kappa B (NF-κB)protein was analyzed by western blot.
Results:1. The results of FCM and TUNEL showed that, compared withCon rats, the apoptosis rate of bone marrow cells were not significantlydifferent in CIHH, Pre-T and Post-T rats (P>0.05); The apoptosis rate in thebone marrow cells of Pre-AA and Post-AA rats were significantly increased (P<0.01).
2. Compared with Con rats, the protein expressions of Fas, Bax, Bcl-2,Caspase-3, Caspase-8and the radio of Bax/Bcl-2of bone marrow cells werenot significantly different in CIHH, Pre-T and Post-T rats (P>0.05); The proteinexpressions of Fas, Bax, Caspase-3, Caspase-8and the ratio of Bax/Bcl-2were significantly increased (P<0.05-P<0.01), while the protein expression of Bcl-2of bone marrow cells were significantly decreased in Pre-AA and Post-AA rats(P<0.05).
3. Compared with Con rats, the protein expressions of HIF-1α and NF-κBof bone marrow cells were not significantly different in CIHH and Pre-T rats (P>0.05); The protein expression of HIF-1α and NF-κB of bone marrow cellswere significantly increased in Pre-AA and Post-AA rats (P<0.05-P<0.01); Theprotein expression of HIF-1α of bone marrow cells were significantly increasedin Post-T rats (P<0.01),and the protein expression of NF-κB of bone marrowcells in the bone marrow cells were not significantly different in Post-T rats (P>0.05).
Summary: Under physiological condition, CIHH treatment had nosignificant effect on apoptosis and apoptosis associated protein of bone marrowcells, but effectively antagonized the overexpression of HIF-1α and NF-κBproteins, down-regulated the expression of positive apoptosis proteins Fas, Bax,Caspase-3and Caspase-8, up-regulated the expression of negative apoptosisprotein Bcl-2, resulting in anti-apoptosis eventually. The anti-apoptosis ofCIHH on bone marrow cells might be one of the mechanisms of anti-AA effectof CIHH.
Part IV Effects of chronic intermittent hypobaric hypoxia on adhesivenessof bone marrow mesenchymal stem cells in aplastic anemia rats
Objective: To investigate the hematopoietic HIM for protective effect ofCIHH against AA through observing effect of CIHH on BMMSCs and itsadhesiveness.
Methods: Experimental grouping, preparation of AA and CIHHtreatment were the same as Part. The morphology of adhension cell andcolony-forming unit-fibroblast (CFU-F) were observed by long-term bonemarrow explant cultures (LTBMC-Ex) dynamically. BMMSCs were culturedand purifucated in vitro by the method of differential velocity adherent cellsculture.Very late antigen-4(VLA-4), vascular cell adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1), CD162and CD164 of BMMSCs were analyzed by FCM. The protein expression of p38mitogen-activated protein kinase (p38MAPK) was assayed by western blot.
Results:1. During LTBMC-Ex, the bone marrow stromal cells in Pre-AAand Post-AA rats were dysplasia, and the most cells were immature precursorcells and fibroblast-like cells. Under the basic physiological condition, CIHHtreatment had no effect on development of bone marrow stromal cells layer, butantagonized effectively the dysplasia of bone marrow stromal cells layer andrenewed a valid layer to support hematopoiesis.
2. Compared with Con rats, the CFU-F colonies of bone marrow had nosignificantly changes in CIHH and Pre-T rats (P>0.05); The CFU-F colonies ofbone marrow were significantly decreased in Pre-AA and Post-AA rats (P<0.01); The CFU-F colonies of bone marrow were significantly decreased inPost-T rats (P<0.01).
3. Compared with Con rats, the protein expressions of VLA-4, VCAM-1,ICAM-1, CD162and CD164of BMMSCs were not significantly different inCIHH rats (P>0.05). The protein expressions of VLA-4, VCAM-1and ICAM-1of BMMSCs were significantly decreased (P<0.01), while CD162and CD164of BMMSCs were significantly increased in Pre-AA and Post-AA rats (P<0.01). The protein expression of VLA-4, VCAM-1, ICAM-1and CD164ofBMMSCs were not significantly different (P>0.05), but CD162of BMMSCswas significantly increased in Pre-T rats (P<0.01). The protein expressions ofVCAM-1and ICAM-1of BMMSCs were not significantly different in (P>0.05), while VLA-4of BMMSCs was significantly decreased in Post-T rats (P<0.01) and the protein expressions of CD162and CD164of BMMSCs weresignificantly increased in Post-T rats (P<0.01).
4. Compared with Con rats, the protein expression of p38MAPK ofBMMSCs was not significantly different in CIHH and Pre-T rats (P>0.05). Theprotein expression of p38MAPK of BMMSCs was significantly increased inPre-AA, Post-AA and Post-T rats (P<0.01).
Summary: Under physiological condition,CIHH treatment had nosignificant effect on development of bone marrow stromal cells layer and the adhensiveness of BMMSCs, but effectively antagonized the dysplasia of bonemarrow stromal cells layer to support hematopoietsis and the adhensiveness ofBMMSCs in AA rats. This demonstrates that CIHH treatment improves theadhesion of BMMSCs and enhance nutritional support to HSCs in AA rats,which might be one of mechanism of anti-AA effect of CIHH.
Conclusion: CIHH has protective effect agains aplastic anemia of ratsthrough inhibit-ing the overexpression of HIF-1α, NF-κB, p38MAPK anddownstream signal molecules, maintaining homeostasis of hematopoieticregulatory factors, keeping balance of T lymphocyte subpopulations,proinflammatory/anti-inflammatory factors,and adhensive molecules, lesseningthe damage of hematopoietic tissue and inhibition of hematopoietic function inbone marrow, decreasing hematopoietic cell apoptosis, and enhancing theadhesiveness and nutritional support between the bone marrow hematopoieticcells and hematopoietic mic-roenvironment.
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