【作者】 张晶；

【导师】 韩伟；

【作者基本信息】 上海交通大学 ， 基因工程药物与天然药物， 2008， 博士

【摘要】 本论文的研究目的,是探讨白介素1受体拮抗剂(interleukin 1 receptor antagonist, IL-1Ra)在小鼠骨髓造血系统中的作用及机制,并且对其进行了预防化疗导致的骨髓抑制的临床前研究。实验中采用的化疗小鼠模型是通过尾静脉一次性注射5-氟尿嘧啶(5-Fu),导致骨髓损伤和随后的再生修复。5-Fu注射后小鼠的骨髓变化分为两个阶段:首先是第1～7天的骨髓损伤阶段;接下来是第8～14天的骨髓再生修复阶段。为了研究细胞因子对这一损伤―修复过程的调控,我们抽提了5-Fu注射后0、3、7、11、14天5个时间点的小鼠骨髓细胞RNA,制备了基因芯片。通过基因芯片技术检测发现,在化疗后骨髓损伤和修复过程中许多调节因子在基因水平会呈现不同趋势的波动。其中IL-1Ra的表达呈现了先上升后下降的趋势。为了验证基因芯片的结果,我们对5-Fu注射后5个时间点的小鼠血清中的IL-1Ra水平进行了ELISA检测,得到了与芯片相一致的表达趋势。这种表达量剧烈的波动使我们认为IL-1Ra可能作为一个内源性的造血调控因子参与了骨髓损伤的修复过程。为了进行体内的功能研究,我们首先利用原核表达pET系统诱导表达了重组小鼠的IL-1Ra蛋白,并且利用阴离子交换层析的方法进行纯化,得到了纯度为96%以上的纯化产物,经内毒素检测合格;并通过细胞学实验证明该蛋白具有良好的生物学活性,可以用于下一步的体内功能实验。本论文中通过IL-1Ra在正常小鼠的在体表达和重组蛋白注射两个方法研究了IL-1Ra对小鼠骨髓造血系统的作用。对于在体表达IL-1Ra的实验,我们构建了真核表达的重组质粒pcDNA-IL-1Ra,使用电穿孔的方式,将质粒转染到小鼠的胫前肌。用ELISA的方法检测转染后不同时间点IL-1Ra在小鼠体内的表达水平。证明在转染后的第5天小鼠血清中的IL-1Ra水平最高,随后逐渐下降。较高水平的表达可以维持10天左右。转染后进行pcDNA-IL-1Ra质粒组和pcDNA3.1质粒对照组小鼠的外周血和骨髓细胞数的比较。发现在第5天IL-1Ra可以降低骨髓细胞总数,并且在第10天和第17天pcDNA-IL-1Ra质粒组的外周血白细胞和血小板数均低于pcDNA3.1质粒对照组。通过该实验我们认为,IL-1Ra可以影响小鼠骨髓的造血平衡。质粒转染作为基因治疗的一种,虽然能够有效的导入外源基因,但是对机体的损伤较大,并且表达时间和表达量都难以控制。因此我们采用有活性的重组小鼠IL-1Ra蛋白(rMuIL-1Ra)皮下注射的给药方式进行该基因在小鼠骨髓造血系统中的功能研究。在正常小鼠中的量效实验结果显示,连续注射rMuIL-1Ra (1mg/kg) 5天后,除单核细胞外,IL-1Ra显著降低小鼠骨髓细胞(BM cells)、外周血白细胞(WBC)、粒细胞(GR)、淋巴细胞(LY)的数量,且减少的细胞数量在5到10天恢复到rMuIL-1Ra注射前水平。结果证明IL-1Ra对正常小鼠造血功能具有可逆性抑制作用。我们选取1mg/kg体重的剂量在正常小鼠中进行rMuIL-1Ra蛋白分别连续注射1天、3天、5天、7天、9天的时效实验。连续注射rMuIL-1Ra蛋白1天、3天、5天的实验组的骨髓细胞数逐渐下降,而继续注射rMuIL-1Ra 7天至9天骨髓细胞数虽然仍低于对照组,但是比连续注射5天组有所上升。实验结果表明,连续注射rMuIL-1Ra蛋白5天对正常小鼠骨髓细胞数的抑制最大。通过对该实验中几个时间点小鼠外周血血清中IL-1β水平的ELISA检测,发现外源rMuIL-1Ra蛋白的注射可以影响小鼠体内IL-1β水平的变化。连续注射rMuIL-1Ra蛋白5天时,小鼠体内的IL-1β水平显著上升,持续注射该蛋白5天以上小鼠体内IL-1β水平会回落至正常范围。接下来进一步研究rMuIL-1Ra对5-Fu化疗小鼠造血系统的作用。我们进行了五个部分的实验:(1) 5-Fu前2天或者5天预防性应用rMuIL-1Ra对化疗小鼠骨髓损伤的保护作用; (2) 5-Fu后5天治疗性应用rMuIL-1Ra对化疗小鼠骨髓损伤的缓解作用; (3) 5-Fu前后各2天连续注射rMuIL-1Ra对小鼠血象的观察比较; (4) 5-Fu后第7～10天连续注射rMuIL-1Ra对小鼠血象的观察比较; (5)注射rMuIL-1Ra对5-Fu反复化疗小鼠的骨髓保护作用。所有实验组都设一个相应的无热源PBS对照组,并且rMuIL-1Ra蛋白都是每天一次皮下注射。实验结果显示,5-Fu化疗前的预防性应用和5-Fu化疗后的治疗性应用rMuIL-1Ra蛋白均对化疗产生的骨髓损伤起到缓解作用。前四组实验相对于对照组比较均在不同程度上促进了外周血血小板和骨髓细胞的恢复;5-Fu (280mg/kg)前2天连续注射rMuIL-1Ra可以使小鼠的存活率从70%提高到95%;亚致死剂量5-Fu (300mg/kg)前5天连续注射rMuIL-1Ra可以使小鼠的存活率从30%提高到75%;亚致死剂量300mg/kg的5-Fu后5天连续注射rMuIL-1Ra使小鼠存活率从20%提高到90%;5-Fu后第7～10天连续注射rMuIL-1Ra蛋白在第11天的外周血血小板的水平统计学上显著的低于PBS对照组。rMuIL-1Ra对5-Fu反复化疗的小鼠造血系统仍然能够起到保护作用,体现在对外周血淋巴细胞,血小板恢复的促进和小鼠体重的恢复上。关于IL-1Ra可以保护化疗造成的骨髓损伤的机制研究,我们首先取正常小鼠连续5天皮下给药rMuIL-1Ra后24h内处死小鼠,应用流式细胞仪检测骨髓中不同造血细胞亚群细胞周期数据。骨髓单个核细胞(MNC)被逐级分为:未成熟细胞(Lin-,细胞不表达成熟血细胞表面标志);造血祖细胞(KL细胞,Lin- -c-kit);造血干细胞(SKL细胞,Sca-1+-c-kit+- Lin-)。结果显示,处于S和G2/M期的4类细胞亚群均显著减少;处于G1期的KL和SKL细胞均显著减少,而G0期细胞相应显著增多。我们的结论是:IL-1Ra作用的靶细胞是骨髓造血干细胞和造血祖细胞;rMuIL-1Ra注射小鼠后,驱使小鼠骨髓造血干/祖细胞进入细胞静止期G0期,表现为G0期细胞增多,G1、S和G2/M期细胞减少。也就是说IL-1Ra的注射减缓了细胞周期的循环和细胞的增殖。这从机理上解释了预防性应用rMuIL-1Ra于细胞周期特异性的化疗药物5-Fu之前,能够减少骨髓细胞进入S期的数量从而对小鼠骨髓起到保护作用,缓解化疗对骨髓的损伤。其次,我们获取5-Fu化疗前预防性应用rMuIL-1Ra的小鼠骨髓,检测其对造血祖细胞集落形成的影响。结果显示,和对照组相比,正常小鼠连续5天注射MuIL-1Ra重组蛋白可以显著的减少小鼠每条腿的骨髓造血祖细胞的集落数量。而化疗之后的第3天和第7天,蛋白组造血祖细胞集落的数量都显著高于对照组。体内IL-1Ra的含量和IL-1β的水平是一个动态平衡的过程,如果IL-1在体内含量过高,机体会分泌更多IL-1Ra来拮抗IL-1,以恢复二者的平衡状态。因此,我们运用ELISA的手段对化疗小鼠不同时间连续注射rMuIL-1Ra蛋白后各个时间点小鼠体内的IL-1β的水平进行了检测。结果显示,在5-Fu后连续注射5天的实验组中,小鼠外周血中的IL-1β的水平的升高相对于对照组和其它两种给药组明显提前。其它几组都是在第11天IL-1β的水平达到最高,而5-Fu后连续注射5天的实验组的IL-1β的水平在第7天达到最高。这更确切地说明外源的rMuIL-1Ra蛋白的连续注射可以引起小鼠体内IL-1β水平的升高。这也解释了在5-Fu后治疗性应用rMuIL-1Ra蛋白可以对小鼠的血小板和骨髓的恢复起到促进作用。并且IL-1β水平的升高更可能是促进血小板恢复的重要原因。综合以上的研究结果,我们可以得出结论:应用rMuIL-1Ra蛋白可以促进化疗引起的小鼠骨髓损伤的恢复;在5-Fu化疗之前的预防性应用和之后的治疗性应用rMuIL-1Ra蛋白都可以促进骨髓的恢复、显著加快外周血血小板的恢复还大幅度的提高大剂量化疗下小鼠的存活率。人和小鼠的IL-1Ra蛋白有高达78%的同源性,而且功能相似。因此本文的工作为开发基于IL-1Ra的人的化疗保护药物奠定了实验基础。更多还原

【Abstract】 Myelosuppression is the most common adverse effect of cytotoxic chemotherapy and is a major limiting factor in the clinical treatment of cancer. Therefore, promotion of hematopoiesis remains an extremely important challenge in cancer therapy. This paper studies the effects and mechanism of interleukin 1 receptor antagonist in bone marrow hematopoietic system of mice and preclinical research on prevention of chemotherapy-induced myelosuppression. We used 5-fluorouracil (5-Fu) mouse chemotherapy model to identify bone marrow (BM) damage and regeneration. After 5-Fu injection, the mouse BM is severely damaged and regeneration is initiated. Within 14 days, the damaged BM is fully recovered through active hematopoiesis. For Affymetrix Gene Chip analysis, five total RNA samples were extracted from bone marrow cells collected at days 0, 3, 7, 11, and 14 days post-5-Fu treatment. The chip data shows that IL-1Ra is upregulated after 5-Fu treatment on day 7 and then downregulated to the baseline on day 14. To validate the gene chip results, protein expression was evaluated using an ELISA kit for IL-1Ra. ELISA analysis of mouse serum after 5-Fu treatment showed that IL-1Ra protein level in serum was increased obviously at day 7 after 5-Fu injection, which is consistent with the gene expression data. Both ELISA and microarray data indicated that IL-1Ra may be a hematopoietic inhibitor and the overexpression of endogenous IL-1Ra is likely responsible for the observed suppression of hematopoiesis after chemotherapy.The function of IL-1Ra in regulation of BM regeneration was examined by two methods: (1) transient overexpression of the protein through plasmid muscle electroporation in normal mice; (2) injection of the recombinant murine IL-1Ra in normal and 5-Fu treated mice. The effects of IL-1Ra on BM regeneration were analyzed by measuring the peripheral white blood cell, platelet and BM cell counts, cell cycle analysis, hematopoietic colony assay, and mouse survival rate after 5-Fu.We constructed the eukaryote expression plasmid pcDNA-IL-1Ra, which contain the cDNA sequence of the mouse IL-1Ra gene. The plasmid was introduced to the mouse tibia muscle by electroporation. The expression level of the mouse IL-1Ra in serum after electroporation was measured by ELISA method. We found that IL-1Ra expression lasted for about 10 days. The mice with pcDNA-IL-1Ra showed some significant differences in peripheral blood and BM cell counts compared to the control pcDNA3.1 mice. There were significant decreased in bone marrow cell counts in the pcDNA-IL-1Ra mice on day 5. At day 10 and 17, pcDNA-IL-1Ra group showed lower peripheral blood cell counts than control group. Hematological data analysis suggests that IL-1Ra overexpression may have some effects on bone marrow hematopoietic system in mice.To further study the role of IL-1Ra in protection of BM from chemotherapy damage, we produced the recombinant murine IL-1Ra protein. The murine IL-1Ra is a low molecular protein of 17KDa with no glycosylation. The prokaryotic expression system was chosen to make the protein using the pET expression system. The vector was pET28a and the host strain was E.coli BL21. IPTG was used to induce the expression of recombinant protein. We constructed the prokaryotic expressing plasmid pET28-IL-1Ra, which contained the coding sequence of mature murine IL-1Ra protein. Then we induced the expression of the rMuIL-1Ra by 1mM IPTG. The collected bacteria went through sonication, Urea denaturation, dilution before the final anion-exchange chromatography by Q Sepharose. When the conductivity reached 60mS/cm, rMuIL-1Ra was eluted from the colume. SDS-PAGE showed the purity was above 96%. The bioactivity assay demonstrated that the rMuIL-1Ra had good activity and may be used to do next functional experiments.We first examined the role of rMuIL-1Ra in normal mice. We tried 3 doses of rMuIL-1Ra 0.01, 0.1 and 1mg/kg. PBS of equal volume was injected in control group. All mice of the 4 groups were injected for 5 days and once a day. The date starting the injection was noted as day 0 and data of the mice were collected at day 0, 5, 10 and 15. At day 5, rMuIL-1Ra groups of the highest doses showed decreased cells counts compared to the control group, but that was recovered rapidly at day 10. These results indicate that the administration of rMuIL-1Ra at various dosages results in decreased BM and peripheral WBC production in mice.Next, we studied the time-effect of rMuIL-1Ra in normal mice. This study shows the effects of rMuIL-1Ra on the number of bone marrow cells in response to the injection of rMuIL-1Ra for different time periods. Mean bone marrow cells counts show a steady decrease over the treated time period, compared with the PBS group. These differences achieved statistical significance at day5.We further studied the effect of rMuIL-1Ra on the 5-Fu treated mice. Four parts of experiments were taken: (1) injection of rMuIL-1Ra for two or five days prior to 5-Fu treatment; (2) injection of rMuIL-1Ra for five days after chemotherapy; (3) administration of rMuIL-1Ra for two days before and two days after 5-Fu treatment; (4) administration of rMuIL-1Ra at 7～10 days after 5-Fu treatment. In cohort 1, platelets data shows that rMuIL-1Ra groups recovered better than the control PBS group at day 3, 7 and 11. The control group had lower BM cells counts than rMuIL-1Ra group at 3, 7, and 11. The survival data demonstrate that pretreatment of mice for 2 days with rMuIL-1Ra could protect 95% of the mice from the subsequent sub-lethal dose of 5-Fu treatment. In cohort 2, platelets data shows that at days 7, 11, and 14, the rMuIL-1Ra groups recovered better than the control group. A significant difference appeared between the rMuIL-1Ra group and the control group in BM cells counts at day 11, 14 and 21. Survival was scored daily for 14 days. Survival rate was increased to 90% of the mice compared to the PBS group which was 20%. This chemoprotection by rMuIL-1Ra may allow an increased doses of 5-Fu without adverse effects on survival. In cohort 3, data demonstrated that the use of rMuIL-1Ra before and after chemotherapy has no much effect on bone marrow hematopoiesis. To investigate the interaction between IL-1Ra and IL-1β,rMuIL-1Ra was injected at 7～10 days after 5-Fu treatment to block the IL-1βaction. The increase of platelet was completely suppressed. The data indicated that endogenous upregulation of IL-1βis responsible for the recovery (rebound) of platelet counts after 5-Fu treatment.To further study the mechanism of the protection of effect of rMuIL-1Ra, we administrated rMuIL-1Ra to normal mice for 5 days and killed these animals within 24h. We analyzed the cell cycle of BMN、Lin-、Lin--c-kit+ (KL) and Lin--c-kit+-Sca-1+ (SKL) cells through FACS. The results showed that rMuIL-1Ra can reduce the BMN and Lin- cells into S and G2/M phase. For more primitive KL and SKL cell cycle analysis, we found that in addition to S phase and G2 / M phase of the ratio decreased, KL and SKL cells has a significant increase in G0 of the G0/G1 phase. Other words, the injection of IL-1Ra can slow down the cell cycle and proliferation. This mechanism can explain the preventive application of rMuIL-1Ra before 5-Fu (cell cycle specific drug) was able to reduce the number of bone marrow cells into S phase. Therefore, the bone marrow damage induced by chemotherapy can be eased.The expression levels between IL-1Ra and IL-1βis a dynamic process. Plasma samples from mice of several cohorts following 5-Fu administration were assayed for IL-1Ra and IL-1βexpression. The serum of mice was analyzed at 3, 7, 11 and 14 days post injection using ELISA analysis. In cohort 1, 3 and control group, the level of IL-1βexpression was steady until day 11, after which IL-1βexpression increased―reaching a peak at day 11. However, in cohort 2, the level of IL-1βexpression reached the peak at day 7 and then decreased the baseline. Therefore, these results suggest that the use of IL-1Ra after chemotherapy would allow for greater doses of chemotherapeutic agents to be administratered.In summary, this work purified the recombinant murine IL-1Ra protein, studied the function of IL-1Ra in regeneration of bone marrow after chemotherapy, which suggest that IL-1Ra may be an important BM protector against chemotherapy induced myelosuppression.更多还原