兰州大学机构库 >基础医学院
结核分枝杆菌抗原致骨髓造血细胞增殖分化异常实验研究暨烯醇化酶-1单克隆抗体制备与作用初步研究
Alternative TitleExperimental study on the abnormal proliferation and differentiation of bone marrow hematopoietic cells induced by Mycobacterium tuberculosis antigen and preliminary effect of enolase-1 monoclonal antibody
李菲
Thesis Advisor祝秉东
2019-10-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Degree Discipline遗传学
Keyword结核病 骨髓造血细胞 Lin- c-Kit+(LK)细胞 抗原持续刺激 IL-2
Abstract 第一章  结核分枝杆菌抗原持续刺激致骨髓造血细胞增殖分化异常实验研究
结核分枝杆菌感染机体后主要激活CD4+ T细胞和CD8+ T细胞,CD4+ T细胞分化为Th1型细胞,分泌IFN-γ、TNF-α、IL-2等细胞因子,激活巨噬细胞等细胞免疫应答,CD8+ T细胞分化为CTL细胞直接杀伤靶细胞,发挥清除结核分枝杆菌的作用。然而耐药结核、痰菌阳性的纤维空洞型肺结核等重症结核病患者体内结核分枝杆菌长期慢性感染会导致机体免疫功能低下。我们实验室前期应用结核分枝杆菌抗原持续刺激模拟临床结核分枝杆菌持续感染的状态,发现结核分枝杆菌抗原持续刺激会导致T细胞耗竭,表现为T细胞受特异性抗原刺激后增殖能力下降、产生细胞因子IFN-γ和IL-2减少。在该结核分枝杆菌抗原持续刺激致T细胞耗竭的动物模型上,进一步对T细胞耗竭进行治疗研究,发现给予IL-2干预可逆转其耗竭状态。在上述研究基础上,我们进而拟研究抗原持续刺激导致T细胞耗竭过程中骨髓造血功能的变化。维持骨髓造血干细胞(HSC)及前体细胞稳态是机体维持正常造血功能及建立有效抗结核免疫应答的基础。骨髓HSC的分化增殖受GM-CSF、IL-2、IFN-γ等细胞因子的调节。研究发现淋巴细胞脉络丛脑膜炎病毒(LCMV)等病毒及细菌感染过程中,细胞因子IFN-γ持续分泌,可引起造血功能障碍。重症结核病出现免疫耗竭及骨髓造血功能障碍的研究较少,相关机制尚不清楚。目的:我们推测结核分枝杆菌抗原持续刺激情况下,刺激产生的细胞因子持续作用于骨髓,造成骨髓HSC及造血前体细胞功能异常。本实验继续应用前期我们实验室建立的结核分枝杆菌抗原持续刺激引起T细胞耗竭的动物模型,研究结核分枝杆菌抗原持续刺激对骨髓造血功能的影响。方法:为了研究结核分枝杆菌抗原持续刺激对骨髓造血及T细胞免疫功能的影响,本课题应用结核分枝杆菌抗原持续刺激小鼠,观察结核分枝杆菌抗原持续刺激过程中骨髓造血细胞增殖能力的变化,并分析与造血细胞分化相关转录因子表达水平的改变,评价结核抗原刺激诱导的免疫应答对骨髓造血功能的影响。在此基础上进一步研究IL-2治疗对骨髓造血功能的影响。(1)抗原刺激及IL-2治疗程序。考虑到生物安全等因素,本课题应用结核分枝杆菌抗原持续刺激模拟临床结核分枝杆菌持续感染对免疫系统和骨髓造血功能的影响。本研究首先用卡介苗(Bacillus Calmette-Guérin, BCG)免疫C57BL/6小鼠,然后用结核分枝杆菌抗原ESAT-6、CFP-10联合融合蛋白Mtb10.4-HspX(MH)与佐剂DDA和Poly(I:C)持续刺激,每周一次,反复皮下注射。抗原反复刺激12周后检测到骨髓造血细胞(Lin- c-Kit+,LK细胞)增殖能力降低,抗原刺激22次后检测到明显的T细胞功能障碍。采用IL-2对T细胞及骨髓造血细胞增殖分化异常的小鼠进行干预治疗,研究IL-2治疗后骨髓造血细胞增殖及分化能力的改变。研究中,我们设立蛋白抗原强化免疫2次作为抗原短暂刺激组对照。同时设立佐剂对照组排除佐剂对T细胞及造血细胞功能的影响。(2)T细胞功能分析。通过流式细胞术、细胞因子胞内染色法检测脾脏CD4+ T淋巴细胞分泌IFN-γ的水平;应用MHC I类分子-抗原表位肽五聚体染色技术检测TB10.4 4-12特异性的记忆性CD8+ T细胞的数量。
(3)骨髓造血细胞的功能分析。无菌分离骨髓细胞,应用EdU增殖试验检测造血细胞的增殖能力;用ELISA方法检测骨髓造血微环境中IFN-γ和IL-2的水平;磁珠分选造血细胞,并应用实时荧光定量RT-PCR检测造血细胞分化相关转录因子Batf2、IRF8、GATA2和NOTCH1的表达水平,分析抗原刺激对造血细胞分化的影响。(4)IL-2对骨髓造血细胞的治疗作用及机制分析。IL-2干预后,检测上述指标的变化,分析IL-2的治疗效果及机制。结果:(1)结核分枝杆菌抗原持续刺激对T细胞功能的影响。应用结核分枝杆菌蛋白抗原持续刺激小鼠,对细胞因子的检测发现,与抗原短暂刺激组相比,抗原持续刺激12周时,脾脏CD4+ T细胞受特异性抗原刺激分泌较高水平的IFN-γ(p<0.05),但IFN-γ在持续抗原刺激22周后下降;应用MHC抗原表位肽五聚体技术检测发现持续抗原刺激12~22周,脾脏中TB10.4 4-12特异性记忆性CD8+ T细胞的数量相比抗原短暂刺激组明显减少(p<0.01)。(2)结核分枝杆菌抗原持续刺激对造血细胞功能的影响。应用结核分枝杆菌蛋白抗原持续刺激小鼠,抗原持续刺激12周时,骨髓造血微环境中IFN-γ的水平明显升高(p<0.05),但IFN-γ在抗原持续刺激后期下降。EdU增殖试验表明,与抗原短暂刺激组相比,抗原持续刺激12周及22周后LK细胞的增殖能力降低(p<0.05)。骨髓c-Kit+的细胞群中转录因子的表达水平与佐剂对照组相比也发生了明显变化:决定髓系分化的转录因子Batf2和IRF8表达上调,而促进淋巴系细胞分化的转录因子NOTCH1表达下调。(3)IL-2的治疗作用。在给予小鼠结核分枝杆菌蛋白抗原持续刺激的同时,我们应用IL-2进行干预治疗。结果发现,IL-2干预后,骨髓造血微环境中IL-2的水平升高。相比抗原持续刺激组,IL-2处理可下调Batf2和IRF8的表达,上调GATA2和NOTCH1的表达,LK细胞的增殖能力回升(p<0.05)。同时,T细胞耗竭也得到逆转:CD4+ T细胞分泌IFN-γ的水平升高(p<0.05),脾脏TB10.4 4-12特异性记忆性CD8+ T细胞的数量显著增加(p<0.05)。(4)JAK3-STAT5信号通路的研究。JAK3-STAT5信号转导通路对造血细胞分化增殖起调控作用。相比佐剂组,抗原持续刺激组中JAK3和STAT5 mRNA表达水平较低。IL-2治疗后,JAK3和STAT5表达明显增强。
结论:结核分枝杆菌抗原刺激诱导IFN-γ分泌为主的Th1型免疫应答。结核分枝杆菌抗原持续刺激诱导IFN-γ水平在一定时间内增高造成骨髓LK细胞增殖能力降低,并上调髓系分化相关的转录因子Batf2和IRF8的表达;补充IL-2可增强LK细胞的增殖能力,上调淋巴系分化相关转录因子GATA2、NOTCH1的表达,维持骨髓造血的稳态。此外,IL-2上调JAK3和STAT5的表达,推测IL-2也通过上调JAK3-STAT5途径分子表达调节造血细胞的增殖分化。因此,结核分枝杆菌抗原持续刺激会影响造血作用,导致造血细胞增殖能力降低,髓系分化相关的转录因子表达增高,而IL-2治疗可维持骨髓造血的稳态。本实验首次研究了结核分枝杆菌抗原持续刺激对骨髓造血细胞发育分化的影响,揭示了结核分枝杆菌持续感染状态下结核病患者免疫功能低下的机制及骨髓造血功能的改变,为进一步研究重症肺结核的精准诊治提供了思路。第二章  粟粒性肺结核的临床病例分析和BCG感染诱导骨髓造血细胞增殖分化异常机制研究 粟粒性肺结核是由于大量结核分枝杆菌在短时间内进入血液循环所引起的急性全身血行播散型结核病。其病情急且凶险、病死率较高。粟粒性肺结核患者由于体内结核分枝杆菌大量感染会导致机体造血功能障碍和免疫功能低下,但相关机制尚不清楚。目的:我们推测粟粒性肺结核是由大量结核分枝杆菌入血后,诱发T细胞过强的免疫反应,产生的细胞因子进一步作用于骨髓造血细胞,影响骨髓造血细胞的发育分化,淋巴细胞生成减少,最终引起机体免疫功能降低。为验证上述假设,本实验应用卡介苗(BCG)入血感染模拟临床粟粒性肺结核感染的免疫病理变化,研究其对骨髓造血系统和免疫系统的影响。方法:首先,调取临床粟粒性肺结核患者的病例资料,分析研究粟粒性肺结核临床病例外周血血细胞数量变化,研究骨髓造血功能的变化特点;其次,应用不同剂量BCG经尾静脉注射或滴鼻的方式感染小鼠,观察T细胞功能和骨髓造血功能的变化,并分析与造血细胞分化相关转录因子表达水平的改变,评价BCG感染对骨髓造血功能的影响和免疫应答特点,建立应用BCG感染模拟粟粒性结核造血功能受损的动物模型。(1)临床病例资料分析。分析2015年至2019年兰州市肺科医院收治入院的肺结核病例28,047例,其中粟粒性肺结核病例共118例。通过回顾性分析,研究粟粒性肺结核患者外周血血细胞数量变化。(2)BCG感染及IL-2治疗程序。根据文献报道及考虑到生物安全等因素,本课题应用大剂量BCG替代结核分枝杆菌感染小鼠来模拟临床粟粒性肺结核感染,首先用大剂量BCG(5×107 CFU/ml)分别以尾静脉注射和滴鼻的方式感染小鼠,分别于感染后3周、5周和8周,检测外周血血细胞数量、T细胞功能和骨髓造血细胞(LK细胞)增殖分化能力。研究中,设立低剂量BCG(5×105 CFU/ml)滴鼻感染小鼠作为对照,同时设PBS对照组排除外界因素和年龄对T细胞和骨髓造血细胞功能的影响。(3)外周血细胞数量、血清IFN-γ的水平和脏器指数的变化。将新鲜采集的小鼠血液加入抗凝管中,通过动物血液分析仪检测不同组血细胞数量的变化。未抗凝的外周血凝固后离心收集血清,用ELISA检测血清中IFN-γ的水平;通过称量脾脏、肝脏、肺脏及小鼠重量,观察BCG感染对脾脏指数、肝脏指数和肺脏指数的影响。(4)T细胞功能分析。通过ELISA检测脾脏淋巴细胞受BCG蛋白纯化衍生物(PPD)刺激后分泌IFN-γ和IL-2的水平;应用流式细胞术检测脾脏CD4+和CD8+ T淋巴细胞表面表达抑制性受体PD-1的水平;应用EdU增殖试验检测脾脏CD4+和CD8+ T淋巴细胞受PPD刺激后的增殖能力。(5)骨髓造血细胞的功能分析。无菌分离骨髓细胞并对其进行磁珠分选,得到LK细胞,用EdU增殖试验检测LK细胞的增殖能力;用ELISA的方法检测骨髓造血微环境中IFN-γ、IL-2、TNF-α、IL-6和GM-CSF的水平;提取LK细胞的总RNA,并应用实时荧光定量RT-PCR检测LK细胞中转录因子Batf2、IRF8、GATA2和NOTCH1的表达水平,分析BCG感染对LK细胞分化的影响。
(6)IL-2治疗作用分析。在大剂量BCG入血感染后,应用IL-2对发生T细胞耗竭及骨髓造血细胞增殖分化异常的小鼠进行干预治疗。IL-2干预后,检测上述指标的变化,分析IL-2的治疗效果及机制。结果:(1)临床病例资料分析。我们回顾性分析了2015年至2019年兰州市肺科医院收治的结核病病例28,047例,其中粟粒性肺结核病例共118例。根据患者外周血血细胞检测结果分析患者各类血细胞变化,发现118例粟粒性肺结核患者中89%发生淋巴细胞减少(105例)、23%发生白细胞减少(27例)、8.5%发生中性粒细胞减少(10例)、49.2%发生血红蛋白减少(58例)、11%发生红细胞减少(13例)、8.5%发生血小板减少(10例)、全血细胞减少6例,占4.2%。以上数据表明粟粒性肺结核常继发淋巴细胞减少,其次可继发白细胞减少、血红蛋白减少、血小板减少等各类血细胞减少症。
(2)BCG感染对外周血血细胞数量和脏器指数的影响。在BCG感染后5周,与PBS组相比,大剂量BCG尾静脉注射组小鼠外周血白细胞、淋巴细胞和血小板减少,小鼠脾脏指数、肝脏指数和肺脏指数明显升高,提示脾脏、肝脏和肺脏明显肿大。(3)BCG感染对T细胞功能的影响。在BCG感染后3周、5周和8周,与PBS组、低剂量BCG滴鼻组和高剂量BCG滴鼻组相比,高剂量BCG尾静脉注射组脾脏CD8+ T细胞明显高表达抑制性受体PD-1(p<0.05)。EdU增殖试验表明:低剂量BCG滴鼻组脾脏CD8+ T细胞增殖能力明显强于其他组;相对于PBS组、低剂量BCG滴鼻组和高剂量BCG滴鼻组,高剂量BCG尾静脉注射组脾脏CD8+ T细胞的增殖能力明显降低(p<0.05)。ELISA检测脾脏淋巴细胞受特异性抗原PPD刺激分泌IFN-γ和IL-2的水平,结果如下:在3-5周时高剂量BCG尾静脉注射组较低剂量BCG滴鼻组及PBS组产生较高水平的IFN-γ(p<0.05),8周后产生IFN-γ的能力明显降低;在感染3-5周低剂量BCG感染组产生IL-2的水平高于PBS组和高剂量BCG尾静脉注射组(p<0.05)。(4)BCG感染对骨髓造血细胞功能的影响。在BCG感染后3周,高剂量BCG尾静脉注射组LK细胞增殖能力明显高于PBS组(p<0.01),随后逐渐降低。骨髓上清液中细胞因子的变化如下:在BCG感染后5周,相比PBS组,高剂量BCG尾静脉注射组产生较高水平的IFN-γ和TNF-α(p<0.05),IL-2、IL-6和GM-CSF的水平无明显变化,但低剂量BCG滴鼻组IL-2、IL-6和GM-CSF明显增高(p<0.05)。高剂量BCG尾静脉注射组骨髓LK细胞群中转录因子的表达水平与PBS对照组相比也发生了明显变化:髓系分化相关的转录因子Batf2和IRF8表达上调,而淋巴系分化相关的转录因子GATA2和NOTCH1表达下调。(5)IL-2的治疗作用。在大剂量BCG入血感染小鼠后,用IL-2进行干预治疗。结果发现,IL-2干预后,T细胞耗竭得以逆转。结论:回顾性分析临床粟粒性肺结核患者的病例资料,结果发现粟粒性肺结核患者会出现淋巴细胞减少、血红蛋白减少、血小板减少等造血功能受损表现,和文献报道一致。应用小鼠模型研究发现,大剂量BCG入血感染可致外周血淋巴细胞和血小板减少,诱导骨髓造血微环境中细胞因子IFN-γ和TNF-α的水平增高,导致骨髓LK细胞增殖能力降低,并上调髓系分化相关的转录因子Batf2和IRF8的表达,下调淋巴系分化相关转录因子GATA2和NOTCH1的表达,最终导致T细胞耗竭。补充IL-2后T细胞耗竭有所逆转。本研究结合临床病例资料和动物实验研究,从骨髓造血细胞发育分化的角度揭示粟粒性肺结核免疫功能低下的机制及骨髓造血功能的改变,为粟粒性肺结核防治提供了参考。第三章  糖酵解酶ENO1单克隆抗体制备及其作用初步研究T细胞发育分化过程需要糖酵解和氧化磷酸化提供能量,尤其是效应T细胞的糖酵解代谢更为活跃。上一章研究我们发现大剂量BCG入血感染会使T细胞过度活化,导致T细胞耗竭的发生。我们推测抑制糖酵解,减轻T细胞的活化,有可能阻断T细胞耗竭的发生。糖酵解也是肿瘤代谢的重要特征。我们实验室前期通过蛋白质组学筛选到宫颈癌组织高表达糖酵解酶烯醇化酶1(ENO1)。用ShRNA干扰沉默ENO1基因的表达可明显降低宫颈癌Hela和SiHa细胞的成瘤能力及侵袭转移能力,说明抑制ENO1表达可以阻断糖酵解,从而起到治疗肿瘤的作用。目的:虽然应用shRNA干扰ENO1表达可抑制肿瘤生长,但RNA干扰技术受到病毒载体和运输系统的限制。为了促进临床应用,本课题拟制备ENO1的单克隆抗体,进一步研究其抗肿瘤作用。
此外,前两章的研究发现结核分枝杆菌抗原持续刺激和大剂量BCG入血感染会导致T细胞功能低下甚至耗竭,如何扭转耗竭对于结核病防治有重要意义。细胞代谢变化可决定T细胞功能,因而细胞代谢被认为是T细胞功能和分化的关键调节因子。本研究制备ENO1的单克隆抗体,后期将进一步通过干预T细胞代谢,研究治疗T细胞耗竭的新方法和策略。方法:本课题拟用真核表达系统表达ENO1蛋白,纯化ENO1蛋白,制备ENO1单克隆抗体。预期ENO1的单克隆抗体通过干扰肿瘤细胞表面ENO1可以降低肿瘤细胞迁移侵袭能力。我们筛选有较强抗宫颈癌SiHa细胞迁移侵袭能力的ENO1单克隆抗体。对编码ENO1单克隆抗体的基因进行测序,将其可变区基因序列重组构建在腺相关病毒上。后续我们将对腺相关病毒表达抗体可变区蛋白多肽的功能进行鉴定,对ENO1单克隆抗体对糖酵解的抑制作用展开深入研究。
(1)ENO1的克隆、表达和纯化 方案一:以pET30a-ENO1质粒为模板,将ENO1基因克隆入真核表达载体pcDNA中,构建重组质粒pcDNA3.1-ENO1,并将pcDNA3.1-ENO1以瞬时转染的方式成功转入人胚肾细胞(HEK-293T细胞)中,表达出ENO1蛋白,并用Ni-NTA介质进行纯化。方案二:将pcDNA3.1-ENO1以稳定转染的方式成功转染入中国仓鼠卵巢细胞(CHO-K1细胞)中,表达和纯化ENO1蛋白。方案三:将ENO1基因克隆入杆状病毒表达载体pFastBac1中,转入昆虫细胞Sf9中,表达和纯化ENO1蛋白。
(2)ENO1单克隆抗体的制备。用ENO1蛋白反复免疫BALB/c小鼠,取出抗体滴度较高的小鼠脾脏细胞,与骨髓瘤细胞SP2/0进行融合。对阳性杂交瘤细胞进行反复筛选,获得效价较高的杂交瘤细胞株,诱导腹水产生,纯化得到ENO1单克隆抗体。(3)ENO1单克隆抗体的抗肿瘤作用评价。采用细胞划痕实验和Transwell小室实验检测五个ENO1单克隆抗体对宫颈癌SiHa细胞迁移、侵袭能力的影响,筛选出效果最佳的ENO1单克隆抗体。(4)ENO1单克隆抗体可变区基因测序。分别提取五株杂交瘤细胞的RNA,通过RT-PCR获得相应的cDNA。基于抗体重链和轻链恒定区的已知序列设计3’-末端基因特异性引物,联合5’-末端的通用引物用于cDNA基因的PCR扩增,通过对扩增产物进行测序,获得ENO1单克隆抗体重链和轻链可变区基因序列。(5)腺相关病毒包装ENO1抗体的可变区基因。选择对宫颈癌细胞的迁移、侵袭能力有明显抑制效果的ENO1单克隆抗体H1的轻链和重链可变区基因,中间用Linker--(GGGGS)3连接,克隆在pAAV-MCS载体上,由公司合成,简称rAAV-H1。对AAV辅助病毒系统中的pHelper、pAAV-RC和rAAV-H1菌液进行复苏、保种和扩增,并大量抽提质粒,将其转染入AAV-293细胞中,观察其转染效率、收集病毒并进行滴度测定。同时,将带有绿色荧光蛋白的pAAV-IRES-ZsGreen1转入AAV293细胞中,作为空载体对照(rAAV-GFP)。结果:(1)ENO1蛋白表达与纯化。首先将pcDNA3.1-ENO1以瞬时转染的方式成功转染入HEK-293T细胞中,表达纯化ENO1蛋白,但蛋白得率太低;将pcDNA3.1-ENO1以稳定转染的方式成功转染入CHO-K1细胞中,表达纯化目的蛋白,同样蛋白得率太低;最后将ENO1基因克隆入pFastBac1中,转入Sf9细胞中,蛋白表达量高,纯化出高纯度的ENO1蛋白。(2)ENO1单克隆抗体制备。ENO1蛋白免疫小鼠后,通过杂交瘤技术成功筛选到5株效价较高的阳性杂交瘤细胞株,纯化获得相应的单克隆抗体。(3)ENO1单克隆抗体对宫颈癌细胞迁移、侵袭作用的影响。应用细胞划痕实验和Transwell小室实验,初步评价5个ENO1单克隆抗体的抗肿瘤细胞迁移、侵袭作用。检测结果示五个ENO1单克隆抗体对宫颈癌SiHa细胞迁移、侵袭能力均有抑制作用,其中H1的抑制作用最强。(4)ENO1单克隆抗体可变区基因测序。在得到单克隆抗体的基础上,利用通用引物从杂交瘤细胞的RNA钓取抗体的重链和轻链可变区基因,对获得的抗体可变区基因进行测序,成功获得可变区基因序列。(5)腺相关病毒包装ENO1抗体的可变区基因。将得到的ENO1抗体重链和轻链可变区基因构建在腺相关病毒载体上,得到rAAV-H1。大量抽提得到浓度较高的pHelper、pAAV-RC和rAAV-H1质粒,将其转染入AAV293细胞中,结果示转染成功,且转染效率达到90%。结论:应用杆状病毒表达载体,成功表达和纯化出高纯度的ENO1蛋白;应用ENO1蛋白免疫小鼠,通过杂交瘤技术成功制备和筛选到5个高效价的ENO1单克隆抗体;通过ENO1单克隆抗体对宫颈癌细胞的迁移、侵袭能力的抑制作用评价,筛选出有明显抑制效果的ENO1单克隆抗体H1;成功获得ENO1单克隆抗体的可变区基因序列。将ENO1抗体的可变区基因重组到腺相关病毒载体上,得到rAAV-H1。ENO1单克隆抗体的制备和相关研究为下一步研究ENO1单克隆抗体抑制糖酵解抗肿瘤和T细胞耗竭作用奠定了基础。
Other AbstractPart One: Experimental study on the abnormal proliferation and differentiation of bone marrow hematopoietic cells induced by persistent Mycobacterium tuberculosis antigen stimulationMycobacterium tuberculosis (M. tuberculosis) infection mainly activates CD4+ T cells and CD8+ T cells. CD4+ T cells were induced to differentiate into Th1 type cells, which produce cytokines such as IFN-γ, TNF-α, and IL-2, and activate cell-mediated immunity, CD8+ T cells are differentiated into CTL cells to directly kill target cells, which play protective roles in clearing M. tuberculosis. However, long-term chronic infection of M. tuberculosis in patients with severe tuberculosis such as drug-resistant tuberculosis and sputum positive cavitary tuberculosis could lead to low immune response. In our previous studies, applying persistent M. tuberculosis antigen stimulation to mimic the status of M. tuberculosis persistent infection, we found that persistent stimulation with M. tuberculosis antigen could lead to T cells exhaustion, in which the proliferative ability of T cells decreased, and the production of cytokines IFN-γ and IL-2 also decreased. On the animal model of T cells exhaustion induced by persistent M. tuberculosis antigen stimulation, further therapeutic studies on T cells exhaustion had found that IL-2 supplementation could reverse the exhaustion. Based on the above research, we further want to know whether persistent antigen stimulation could lead to the changes of bone marrow hematopoiesis during T cell exhaustion. Maintaining the steady state of bone marrow hematopoietic stem cells (HSCs) and precursor cells are the basis for the establishment of an effective anti-tuberculosis immune response. The differentiation and proliferation of HSC in bone marrow are regulated by cytokines such as GM-CSF, IL-2 and IFN-γ. It was reported that in the process of persistent viral and bacterial infections, such as lymphocytic choriomeningitis virus (LCMV) infection, cytokine IFN-γ was continuously secreted, which might cause hematopoiesis dysfunction. There are few studies on immune exhaustion and bone marrow hematopoiesis dysfunction in severe tuberculosis (TB), and the relevant mechanisms are still unclear.  Objective:  We speculated that under the continuous stimulation with M. tuberculosis antigen, high levels of cytokines were induced, which would cause the abnormal function of bone marrow HSC and hematopoietic precursor cells. This experiment continued to use the animal model of T cell exhaustion induced by persistent M. tuberculosis antigen stimulation, which previously established in our laboratory, and study the effect of persistent M. tuberculosis antigen stimulation on bone marrow hematopoietic function.Methods: To study the effects of persistent M. tuberculosis antigen stimulation on bone marrow hematopoiesis and T cell immune function, M. tuberculosis antigen was used to persistently stimulate mice, in order to observe the changes in the proliferative capacity of bone marrow hematopoietic cells during persistent stimulation of M. tuberculosis antigen, and analyze the changes in the expression levels of transcription factors related to the differentiation of hematopoietic cells. The effect of immune response induced by M. tuberculosis antigen stimulation on hematopoietic function in bone marrow was evaluated. On this basis, the effect of IL-2 on bone marrow hematopoietic function was further studied.  (1) Antigen stimulation and IL-2 treatment procedure. Taking the factors such as biosafety into account, this study applied M. tuberculosis antigen persistent stimulation to mimic the effects of continuous M. tuberculosis infection in clinic on the immune system and bone marrow hematopoietic function. In our study, using Bacillus Calmette - Guérin (BCG) to immunize C57BL/6 mice for the first time, and repeated subcutaneous injection with M. tuberculosis antigen ESAT-6, CFP-10 combined with Mtb10.4-HspX (MH) and adjuvants DDA and Poly (I:C) weekly. After repeated antigen stimulation for 12 weeks, the proliferating ability of bone marrow hematopoietic cells (Lin- c-Kit+, LK cells) was decreased, and obvious T cell dysfunction was detected after antigen stimulation for 22 times. IL-2 was used to treat T cells and bone marrow hematopoiesis dysfunction mice to study the changes of bone marrow hematopoietic cell proliferation and differentiation after IL-2 treatment. In the study, boosting BCG with the antigens twice was used as a control of transient antigen stimulation. At the same time, an adjuvant control group was established to exclude the effects of adjuvant on T cell and hematopoiesis functions. (2) Analysis of T cell function. The level of IFN-γ secreted by spleen CD4+ T lymphocytes was detected by flow cytometry and cytokine intracellular staining. The number of TB10.4 4-12 specific memory CD8+ T cells was detected by the technique of MHC class I peptide pentamer staining technique.
(3) Functional analysis of bone marrow hematopoietic function. The bone marrow cells were aseptically separated, and the proliferating activity of bone marrow hematopoietic cells was detected by EdU proliferation assay. The secretion levels of IFN-γ and IL-2 in the bone marrow hematopoietic inductive microenvironment was detected by ELISA. The hematopoietic cells were sorted by magnetic beads, and the expression levels of transcription factors Batf2, IRF8, GATA2 and NOTCH1 in hematopoietic cells were detected by Real-time PCR, to analyze the effect of antigen stimulation on the differentiation of hematopoietic cells.  (4) Treatment effect and mechanism of IL-2 on bone marrow hematopoietic cells. After IL-2 intervention, the changes in the above indicators were tested to measure the therapeutic effect and mechanism of IL-2.Results:  (1) Effect of persistent stimulation with M. tuberculosis antigen on T cell function. Applying M. tuberculosis protein antigen to persistently stimulate mice, the detection of cytokines indicated that compared with transient antigen stimulation group, when antigen persistently stimulated for 12 weeks, higher levels of IFN-γ were secreted by antigen-specific spleen CD4+ T cells (p<0.05), however, IFN-γ decreased after antigen persistently stimulated for 22 weeks. MHC antigenic epitope pentamer detection showed that after persistent antigen stimulation from 12 to 22 weeks, the number of TB10.4 4-12-specific memory CD8+ T cells in the spleen significantly decreased (p<0.01).  (2) Persistent stimulation with M. tuberculosis antigen does effects on hematopoiesis function. Applying M. tuberculosis protein antigen to persistently stimulate mice, when antigen persistently stimulated for 12 weeks, higher levels of IFN-γ in the bone marrow hematopoietic inductive microenvironment were detected (p<0.05), but eventually showed a downward trend. EdU proliferation test showed that compared with transient antigen stimulation group, after persistent antigen stimulated for 12 and 22 weeks, the proliferating activity of LK cells decreased (p<0.05). The expression levels of transcription factors in c-Kit+ cells in bone marrow also changed: compared with the adjuvant group, the expressions of Batf2 and IRF8, which involved in myeloid differentiation, were up-regulated, while the expressions of NOTCH1, which governing lymphoid commitment, were down-regulated.  (3) The therapeutic effect of IL-2. While persistently stimulated mice with M. tuberculosis protein antigen, IL-2 were used to treat mice. The results showed that, the level of IL-2 in the hematopoietic inductive microenvironment was increased after IL-2 intervention. Compared with antigen persistence group, IL-2 treatment could down-regulate the expression of Batf2 and IRF8, up-regulate the expression of GATA2 and NOTCH1, and restore the proliferating activity of LK cells (p<0.05). At the same time, T cell exhaustion was also reversed: the levels of IFN-γ secreted by spleen CD4+ T lymphocytes increased (p<0.05), the number of splenic TB10.4 4-12 specific memory CD8+ T cells was significantly increased (p<0.05).  (4) Study of JAK3-STAT5 signaling pathway. JAK3-STAT5 signal pathway regulates the differentiation and proliferation of hematopoietic cells. The expression levels of JAK3 and STAT5 mRNA in antigen persistence group were lower than the adjuvant group. And the expression levels of JAK3 and STAT5 were significantly up-regulated after IL-2 treatment.Conclusion: M. tuberculosis antigen persistently stimulates a Th1-type immune response with IFN-γ secretion. M. tuberculosis antigen persistent stimulation decreased the proliferating activity of LK cells, up-regulated the expression of transcription factors Batf2 and IRF8, which promoted myeloid differentiation as the consequence of elevated IFN-γ production. IL-2 supplementation enhanced the proliferating activity of LK cells, up-regulated the expression of transcription factors GATA2 and NOTCH1 involved in lymphoid commitment, and contributed to maintaining the homeostasis of hematopoiesis. In addition, IL-2 could up-regulate the expression levels of JAK3 and STAT5, and presumably enhanced the proliferation and differentiation of hematopoietic cells by JAK3-STAT5 pathway. Thus, persistent stimulation with M. tuberculosis antigens affected hematopoiesis, resulting in decreasing of the proliferating activity of hematopoietic cells, upregulating the expression of transcription factors that promote myeloid differentiation. However, IL-2 treatment maintained the homeostasis of hematopoiesis. This study is the first to analyze the effect of M. tuberculosis antigen stimulation on the development and differentiation of bone marrow hematopoietic cells. Our study contributes to reveal the mechanism of immune dysfunction and the changes of bone marrow hematopoietic function in patients with tuberculosis under M. tuberculosis continuous infection, which provides a way to further study the precise diagnosis and treatment of severe pulmonary tuberculosis.Part Two:  Analysis of miliary tuberculosis cases and mechanism research on the abnormal proliferation and differentiation of bone marrow hematopoietic cells induced by BCG infection Miliary tuberculosis is caused by a large amount of M. tuberculosis entering into blood circulation in a short period of time. Its onset is acute, the condition is dangerous, and the mortality rate is high. The immune responses in miliary tuberculosis patients declined due to long-term chronic infection with M. tuberculosis. The relevant mechanisms are still unclear.  Objective: We hypothesized that miliary tuberculosis, which is caused by a large number of tuberculosis entering into blood, might induce excessive immune response of T cells at early stage, and the produced cytokines act on bone marrow hematopoietic cells, affect the development and differentiation of immune cells, at last the development of lymphocytes would be reduced, and ultimately caused immune dysfunction. In order to verify the above hypothesis, in this study, BCG entering into blood infection was used to mimic the immunopathological changes of clinical miliary tuberculosis infection, and its effects on the bone marrow hematopoietic system and immune system were investigated.Methods: First, through investigating the data of clinical cases with miliary tuberculosis, the changes of peripheral blood cells count in miliary tuberculosis were analyzed to reflect the changes of bone marrow hematopoiesis function. Then, different doses of BCG were applied via intravenous injection or intranasal route to infect mice. The changes of T cell function and bone marrow hematopoietic function were analyzed. Meanwhile, we analyzed the changes of expression levels of transcription factors related to the differentiation of hematopoietic cells, and evaluated the effect of BCG infection on bone marrow hematopoietic function. After infecting mice with different doses of BCG via different routes, the immune response characteristics and the effects on the function of bone marrow hematopoietic cells were compared. By this way we used BCG infection to establish an animal model to mimic the impaired hematopoiesis of miliary tuberculosis.  (1) Analysis of clinical case data. We analyzed 28,047 cases of tuberculosis from Lanzhou Pulmonary Hospital during 2015 to 2019, including 118 cases of miliary tuberculosis. Through retrospective analysis, the number of peripheral blood cells in clinical cases of miliary tuberculosis was investigated.  (2) BCG infection and IL-2 treatment program. According to literature reports and taking the factors such as biosafety into account, we applied high-dose BCG replacing M. tuberculosis to infect mice to mimic the infection of clinical miliary tuberculosis. In this study, mice were first infected with high-dose BCG (5 × 107 CFU/ml) via intravenous injection or intranasal route, peripheral blood cells count, T cell function and the proliferation and differentiation characteristics of bone marrow hematopoietic cells (LK cells) were detected respectively at 3 weeks, 5 weeks and 8 weeks after infection. The mice infected by a low-dose of BCG (5 × 105 CFU/ml) via intranasal route were established as a control, and the PBS control group was designed to exclude the influence of external factors and age on the function of T cells and bone marrow hematopoietic cells.  (3) Changes of peripheral blood cells counts, the levels of IFN-γ in serum and organ index. Adding the fresh blood from mice into the anti-coagulation tube, and the changes of blood cells count from different groups were measured by an animal blood analyzer. Peripheral blood without anti-coagulation was centrifuged, and the level of IFN-γ in serum was measured by ELISA. The effects of BCG infection on spleen index, liver index and lung index were observed by weighing the spleen, liver, lung and mouse.
(4) Analysis of T cell function. The levels of IFN-γ and IL-2 secreted by spleen lymphocytes after stimulated with BCG purified protein derivative (PPD) were detected by ELISA. The expression levels of inhibitory receptor PD-1 on CD4+ and CD8+ T lymphocytes were detected by flow cytometry. The proliferative capacity of CD4+ and CD8+ T lymphocytes after PPD stimulation were detected by EdU proliferation assay.
(5) Analysis of bone marrow hematopoietic function. The bone marrow cells were aseptically separated and subjected to magnetic bead sorting to obtain LK cells. The proliferating activity of bone marrow hematopoietic cells was detected by EdU proliferation assay. The secretion levels of IFN-γ, IL-2, TNF-α, IL-6 and GM-CSF in the bone marrow hematopoietic inductive microenvironment was detected by ELISA. The RNA was extraction from LK cells and the expression levels of transcription factors Batf2, IRF8, GATA2 and NOTCH1 in LK cells were detected by Real-time PCR, to analyze the effect of BCG infection on the differentiation of hematopoietic cells.
(6) Analysis of IL-2 treatment. After high-dose BCG entering into blood infection, IL-2 was used to treat mice with T cells exhaustion and abnormal proliferation and differentiation of bone marrow hematopoietic cells. After IL-2 intervention, the changes in the above indicators were tested to measure the therapeutic effect and mechanism of IL-2.Results:  (1) Analysis of clinical case data. According to the test results of peripheral blood cells from patients, the changes in many kinds of blood cells count were analyzed. It was found that including 118 cases of miliary tuberculosis, 89% of them had lymphopenia (105 cases), 23% of cases had leucopenia (27 cases), 8.5% of cases had neutropenia (10 cases), 49.2% of cases had hemoglobin reduction (58 cases), 11% of cases had erythrocyte reduction (13 cases), 8.5% of cases had thrombocytopenia (10 cases), and 4.2% of cases had pancytopenia. The above data indicated that miliary tuberculosis often had lymphopenia first, followed by various types of cytopenia such as leukopenia, hemoglobin reduction, and thrombocytopenia.  (2) The effect of BCG infection on peripheral blood cells count and organ index. 5 weeks after BCG infection, compared with PBS group, leukopenia, lymphopenia, and thrombocytopenia occurred in peripheral blood, and the spleen index, liver index, and lung index of mice were significantly increased in high BCG i.v. group, which indicated that the spleen, liver, and lung enlarged.  (3) The effect of BCG infection on T cell function. At 3 weeks, 5 weeks and 8 weeks after BCG infection, spleen CD8+ T cells in high BCG i.v. group showed high expression level of inhibitory receptors PD-1 compared with PBS group, low BCG i.n. group and high BCG i.n. group (p<0.05). EdU proliferation test showed that the proliferation of CD8+ T cells in low-dose BCG i.n. group was significantly stronger than other groups. Compared with PBS group, low BCG i.n. group and high BCG i.n. group, the proliferation of CD8+ T cells in high BCG i.v. group was significantly decreased (p<0.05). The levels of IFN-γ secreted by spleen lymphocytes stimulated by specific antigen PPD were detected by ELISA. The results showed that at 3-5 weeks, the levels of IFN-γ in high BCG i.v. group were higher than that in low BCG i.n. group and PBS group (p<0.05), after 8 weeks, the ability of producing IFN-γ was significantly reduced; IL-2 levels were higher in low BCG i.n. group at 3-5 weeks than that in PBS group and high BCG i.v. group (p<0.05).  (4) The effect of BCG infection on the function of bone marrow hematopoietic cells. At 3 weeks after BCG infection, the proliferative capacity of LK cells in high BCG i.v. group was significantly higher than that in PBS group (p<0.01), and then gradually decrease. The level of cytokines in bone marrow supernatant were as follow: At 5 weeks after BCG infection, high BCG i.v. group induced higher levels of IFN-γ and TNF-α than that in PBS group (p<0.05), while the levels of IL-2, IL-6 and GM-CSF were not significantly different from those in PBS group, but the levels of IL-2, IL-6 and GM-CSF significantly higher than those in PBS group (p<0.05). The expression level of transcription factors in LK cells from high BCG i.v. group also changed significantly compared with PBS control group: the expression levels of transcription factors Batf2 and IRF8 involved in myeloid differentiation, were up-regulated, while the expression levels of GATA2 and NOTCH1, which governing lymphoid commitment, were down-regulated.  (5) Therapeutic effects of IL-2. After high-dose BCG entering into blood infected mice, IL-2 was used to treat mice. The results showed that the T cell exhaustion was reversed.Conclusion: Retrospective analysis of cases from patients with clinical miliary tuberculosis showed that impaired hematopoiesis function such as lymphopenia, thrombocytopenia and pancytopenia were found in the patients with miliary tuberculosis, being consistent with the literature reports. Using mouse model, we found that high-dose BCG entering into blood infection could cause peripheral lymphopenia and thrombocytopenia, and induce the increasing expression of cytokines IFN-γ and TNF-α, which reduce the proliferation activity of LK cells, and up-regulate the expression levels of transcription factors Batf2 and IRF8 invoved in myeloid differentiation, and down-regulate the expression levels of transcription factors GATA2 and NOTCH1 involved in lymphoid commitment, eventually leading to T cells exhaustion. IL-2 supplementation could reverse T cells exhaustion. This study combines clinical case data with animal experiments, from the perspectives of the development and differentiation of bone marrow hematopoietic cells to reveal the mechanism of low immune function in miliary tuberculosis and the changes of bone marrow hematopoietic function, which provides a reference for the prevention and treatment of miliary tuberculosis.Part Three: Preparation of monoclonal antibody against glycolytic enzyme ENO1 and the preliminary study on its effectsT cell development and differentiation processes require glycolysis and oxidative phosphorylation to provide energy, especially effector T cells with enhanced glycolysis processes. In the previous chapter, we found that high-dose BCG entering into blood infection could cause the over-activation of T cells, leading to the occurrence of T cell exhaustion. We hypothesized that inhibiting glycolysis and mitigating the activation of T cells, might block the immunopathological process of T cell exhaustion.  Glycolysis is an important characteristic of tumor metabolism. In our laboratory, we found enolase 1 (ENO1) highly expressed in cervical cancer tissue by proteomics. ENO1 is an enzyme in glycolysis process. Silencing ENO1 gene expression by ShRNA interference could significantly reduce the tumorigenic ability and invasion and metastasis ability of cervical cancer Hela and SiHa cells, indicating that inhibition of ENO1 expression could block glycolysis, and thereby play a role in treating tumor.  Objective: Although the use of shRNA to interfere with ENO1 expression could inhibit tumor growth, the application of RNA interference technology in clinic is limited by viral vectors and transportation systems. In order to promote clinical applications, this study intends to prepare the monoclonal antibody of ENO1, to further evaluate the effect of the monoclonal antibody against tumor.  In addition, the first two chapters found that both persistent M. tuberculosis antigen stimulation and large number of BCG entering into blood infection could lead to low immune response or even exhausted. How to reverse the exhaustion is of great significance for the prevention and treatment of tuberculosis. Changes in cellular metabolism can determine T cell function, and thus cellular metabolism considered to be a key regulator of T cell function and differentiation. This study intends to prepare ENO1 monoclonal antibodies for further study new methods and strategies for the treatment of T cell exhaustion by intervening T cell metabolism.Methods: This study intented to use the eukaryotic expression system to express and purify the ENO1 protein, and prepare ENO1 monoclonal antibodies. It was expected that ENO1 monoclonal antibodies could reduce the migration and invasion of tumor cell by interfering with ENO1 on the surface of tumor cells. For this reason, we screened the ENO1 monoclonal antibody with strong anti-cervical cancer SiHa cell migration and invasion ability. The gene encoding the ENO1 monoclonal antibody was sequenced, and the variable region gene sequence was recombined into an adeno-associated virus. In the follow-up study, we will identify the function of the protein polypeptide from antibody variable region expressed by adeno-associated virus, and conduct an in-depth study on the intervention of ENO1 monoclonal antibody on inhibition effect of glycolysis.  (1) Cloning, expression and purification of ENO1  Method one: Using pET30a-ENO1 plasmid as template, the ENO1 gene was cloned into the expression vector pcDNA3.1 to construct the recombinant plasmid pcDNA3.1-ENO1; the pcDNA3.1-ENO1 was successfully transfected into Human embryonic kidney cells (HEK-293T cells) by transient transfection; the target protein was expressed; and the target protein was purified by Ni-NTA column.  Method two: The pcDNA3.1-ENO1 was successfully transfected into Chinese hamster ovary cells (CHO-K1 cells) in a stable transfection manner to express the target protein.  Method three: The ENO1 gene was cloned into the baculovirus expression vector pFastBac1, transfected into insect cells Sf9, and the target protein was expressed and purified.  (2) Preparation of ENO1 monoclonal antibody. BALB/c mice were repeatedly immunized with ENO1 protein, and spleen cells of mice with higher antibody titer were taken out and fused with mouse myeloma cells SP2/0. The hybridoma cells were repeatedly screened to obtain a hybridoma cell strain with high antibody titer. ENO1 monoclonal antibody was purified. (3) Evaluation of the inhibitory effect of ENO1 monoclonal antibody on migration and invasion of cervical cancer cells. The effect of five ENO1 monoclonal antibodies on the migration and invasion of cervical cancer SiHa cells was examined by cell scratch test and Transwell chamber assay, and the ENO1 monoclonal antibody with best effects was selected.  (4) The variable region gene sequencing of ENO1 monoclonal antibody. RNA of five hybridoma cells were extracted separately, and the corresponding cDNA was obtained by RT-PCR. The 3'-end gene-specific primers were designed based on the known sequences of the heavy and light chain constant regions of antibody, and the universal primers of the 5'-end were used to PCR-amplify the cDNA gene. The amplified product was sequenced to obtain the variable region gene sequence encoding the heavy and light chain of ENO1 monoclonal antibody.  (5) The variable region gene of ENO1 antibody packaged into the adeno-associated virus. The light chain and heavy chain variable region genes of ENO1 monoclonal antibody H1, which had a significant inhibitory effect on the migration and invasion of cervical cancer cells, were ligated with linker--(GGGGS)3 and cloned on pAAV-MCS vector, which synthesized by company, referred as rAAV-H1. The pHelper, pAAV-RC bacteria in AAV helper virus system and rAAV-H1 bacterium were activated, preserved and amplified, and the plasmid was extracted in large quantities, transfected into AAV-293 cells, the transfection efficiency was observed and the virus was collected. At the same time, pAAV-IRES-ZsGreen1 with green fluorescent protein was transferred into AAV293 cells as a vector control (rAAV-GFP).Result:  (1) The expression and purification of ENO1 protein. First, pcDNA3.1-ENO1 was successfully transfected into HEK-293T cells by transient transfection to express the target protein. However, the results showed that the protein yield was too low. Then, the pcDNA3.1-ENO1 was successfully transfected into CHO-K1 cells in a stable transfection manner. The protein yield was also very low. At last, the ENO1 gene was cloned into the baculovirus expression vector pFastBac1, and transfected into insect cell Sf9. The protein yield was very high. The ENO1 protein with high purity was successfully purified.
(2) The preparation of ENO1 monoclonal antibody. After immunization of mice with ENO1 protein, 5 positive hybridoma cell lines with higher titer and related ENO1 monoclonal antibodies were successfully screened by hybridoma technology.  (3) The effect of ENO1 monoclonal antibody on the migration and invasion of cervical cancer cells. The anti-tumor migration and invasion effects of five monoclonal antibodies were initially evaluated using cell scratch test and Transwell chamber. The results showed that five ENO1 monoclonal antibodies could inhibit the migration and invasion of cervical cancer SiHa cells. Among them, H1 had the strongest inhibitory effect.  (4) The variable region gene sequencing of ENO1 monoclonal antibody. On basis of the monoclonal antibody, the genes encoding heavy and light chain variable region of the antibody were extracted from the hybridoma cell RNA using universal primers. By gene sequencing, the variable region gene was successfully obtained.  (5) The variable region gene of ENO1 antibody packaged into the adeno-associated virus. The obtained heavy and light chain variable region genes of ENO1 antibody were constructed on an adeno-associated virus vector to obtain rAAV-H1. A large concentration of pHelper, pAAV-RC and rAAV-H1 plasmids were obtained and transfected into AAV293 cells. The results showed that the transfection was successful and the transfection efficiency reached 90%.Conclusion: ENO1 protein with high concentration and purity was successfully purified by baculovirus expression vector. Using ENO1 protein to immune mice, 5 ENO1 monoclonal antibodies with higher titer were successfully prepared by hybridoma technology. By evaluation of inhibiting the migration and invasion of cervical cancer cells by ENO1 monoclonal antibody, we found that the ENO1 monoclonal antibody H1 had the significant inhibition effect. The variable region genes of ENO1 monoclonal antibodies were successfully obtained, then the variable region gene of ENO1 antibody was recombined into an adeno-associated virus vector to obtain rAAV-H1. The preparation and related research of ENO1 monoclonal antibody also lay the foundation for further research on the effect of ENO1 monoclonal antibody on anti-tumor and anti-T cell exhaustion through inhibiting glycolysis.
Pages196
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/340875
Collection基础医学院
Affiliation基础医学院
First Author AffilicationSchool of Basic Medical Sciences
Recommended Citation
GB/T 7714
李菲. 结核分枝杆菌抗原致骨髓造血细胞增殖分化异常实验研究暨烯醇化酶-1单克隆抗体制备与作用初步研究[D]. 兰州. 兰州大学,2019.
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