Mig重组蛋白的制备及其预防化疗骨髓毒副作用的研究
摘要
本研究的目的,是开发具有骨髓保护作用的药物,以应对恶性肿瘤大剂量化疗或者血液恶性病的治疗过程中所带来的骨髓抑制副作用。我们的实验模型是通过5-氟尿嘧啶(5-Fu)一次性注射小鼠,导致骨髓抑制和随后的骨髓再生修复。5-Fu注射后小鼠外周血和骨髓细胞数量先是下降,到第7天降至最低,然后逐渐回升,到第14天恢复到用药前水平,其血象的变化与人5-Fu化疗后血象变化规律一致,是研究骨髓再生机理的经典动物模型。我们认为对骨髓再生具有调节作用的因子在化疗后骨髓恢复的过程中,其基因表达水平会出现一定的波动。根据该假设我们研究了5-Fu注射后14天之内骨髓细胞RNA的变化,发现IFN-γ诱导的单核因子(Mig)及其受体CXCR3的表达呈现先上升后下降的趋势。
我们又取5-Fu注射后0、3、7、11、14天的小鼠血清,用ELISA检测血清中Mig因子的水平,发现Mig在正常小鼠血清中含量非常低,但是在5-Fu注射后第7天其浓度是正常水平的10倍以上,与芯片数据相一致。综上,我们认为Mig很有可能在骨髓抑制恢复的过程中发挥重要作用。
我们首先利用原核表达pET系统表达了重组人和小鼠的Mig蛋白,并利用离子交换层析方法进行纯化,得到了纯度分别为99%和95%的产物,并检测证明具有生物学活性,为Mig的功能研究打下了基础。我们从两个方面研究Mig对骨髓系统的作用。一,我们通过在体表达和重组蛋白注射两个方法研究使用Mig因子对小鼠的骨髓的作用;二,我们制备了重组小鼠Mig蛋白的多克隆抗体血清,观察使用抗体中和小鼠自身的Mig能否促进受损骨髓的恢复。
为了进行第一部分的功能研究,我们构建了真核细胞表达的重组质粒pcDNA-Mig,使用电脉冲方法,将质粒转染到小鼠的胫前肌。用小鼠Mig的ELISA试剂盒检测质粒转染后小鼠的血清,证实转染能够产生维持7天左右的M ig的暂时高表达。检测电转染之后小鼠的血象和骨髓细胞数量,比较pcDNA-Mig质粒组和pcDNA3.1质粒对照组的小鼠,发现Mig能够降低骨髓细胞总数,这种现象第5天即可观察到,维持到第10天,第27天得到恢复。股骨切片HE染色显示,pcDNA-Mig组比pcDNA3.1对照组骨髓细胞数量较低的时期,pcDNA-Mig组的小鼠骨髓中髓窦数量较多,直径较大。这可能与Mig的脉管抑制作用有关。
进一步将此方法应用到小鼠5-Fu骨髓抑制模型上,在质粒转染之后7天,对小鼠尾静脉注射5-Fu,剂量为200μg/kg体重。以注射5-Fu当天作为第0天,我们在第0、3、7、11、14天共5个时间点小鼠的外周血白细胞和骨髓细胞,并作股骨切片。细胞计数显示,在第11天pcDNA-Mig组的白细胞和骨髓细胞数量皆高于对照组,第14天两组的白细胞和骨髓细胞数量都恢复到正常水平。骨髓切片显示,第7天pcDNA-Mig组的骨髓损伤情况较对照组轻,有核细胞数量多,排列较规则,第11、14天的骨髓细胞密度大,形态规则;相比之下,对照组的骨髓在相同时间点的细胞数量较少。综合细胞计数与骨髓切片,我们得出结论,在5-Fu注射之前转染表达Mig的质粒,能够降低小鼠骨髓的损伤,促进其恢复。
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,但是对机体造成的损伤较大,表达量难于控制,安全性较差。而有活性的重组小鼠Mig蛋白(rMuMig)尾静脉注射可发挥同样的作用。我们首先观察rMuMig蛋白对正常小鼠骨髓的影响。选用0.15、1.5、15μg/kg体重三个剂量的rMuMig尾静脉注射小鼠,对照组注射同样体积的PBS。四组小鼠每天注射一次,连续注射5天,以开始注射的时间作为第0天,分别检测了第0、5、10、15共4个时间点小鼠的外周血白细胞以及骨髓细胞总数的变化,结果显示15μg/kg组能够强烈抑制外周血白细胞、血小板与骨髓细胞数量,1.5μg/kg能够引起骨髓细胞的明显变化,而更低剂量对造血系统没有明显作用。15μg/kg组的小鼠股骨切片与对照组对比,第5天髓窦和血管明显扩张,与对照组的相同部位相比,同样大的视野下观察到的髓窦数量多,直径大;第10天这种现象没有改善;第15天髓窦数量有所回降,直径也缩小;第20天,骨髓恢复到与对照组相似水平,髓窦数量和大小皆接近正常。这些结果与质粒转染后小鼠的骨髓反应相似。
我们选取10μg/kg体重的剂量,进一步观察rMuMig对5-Fu化疗小鼠的作用。我们采取两组给药方法,一种是先注射rMuMig,然后注射5-Fu;一种是先注射5-Fu,8小时后开始注射rMuMig。对照组注射等体积PBS代替蛋白溶液。统计发现,在250mg/kg体重的5-Fu作用下,对照组的小鼠只有10%左右的存活率,而先注射rMuMig可以将存活率提高到50%以上,但是先注射5-Fu后注射rMuMig的组小鼠全部死亡。
检测5-Fu注射后第0、3、7、11、14共5个时间点小鼠的外周血以及骨髓细胞,发现提前注射rMuMig的小鼠,其外周血白细胞和骨髓细胞数量的恢复皆好于对照组。特别是第14天对照组的骨髓细胞数量只有正常水平的1/10左右,而提前注射rMuMig的小鼠已恢复至接近正常水平。我们取两组小鼠的股骨做了切片,HE染色观察其形态学变化,发现在第0天蛋白组小鼠髓窦较多,髓窦直径较大,这与前面实验的结果吻合;第3天两组细胞减少,但没有明显的组间差异;第7天可见对照组小鼠的骨髓损伤较大,出现大面积空腔,而蛋白组相对损伤较小,空腔的数量少面积小,骨髓细胞排列较对照组有规则;第11天的rMuMig组小鼠骨髓中可以观察到脂肪细胞的出现,但是在第14天脂肪细胞即消失,代之以造血细胞。该实验证实,提前注射rMuMig,能够对5-Fu化疗的小鼠骨髓起到保护作用,从而有利于化疗后骨髓的恢复。
我们利用rMuMig免疫大鼠得到anti-Mig的抗体血清,理论上能够中和小鼠Mig蛋白,从而拮抗Mig对骨髓所起的作用。因此在注射250μg/kg体重的5-Fu之后,从第1天开始,每天一次,连续十天对小鼠皮下注射抗体血清,以未经Mig免疫的血清作为对照,观察对小鼠骨髓抑制后恢复的影响。结果发现,注射对照血清的小鼠在11天之内全部死亡,而注射anti-Mig血清的小鼠存活率高达60%左右。股骨切片结果与提前注射rMuMig蛋白后注射5-Fu的小鼠相似,即第11、14天anti-Mig抗体血清组的恢复好于对照组。
先注射rMuMig蛋白,然后注射250mg/kg 5-Fu之后,分别给予相同剂量的对照血清和anti-Mig抗体血清,发现给予anti-Mig抗体血清的小鼠其恢复明显好于对照血清组,在第14天外周血白细胞和骨髓细胞数量都较高。该实验进一步证实在5-Fu之后使用anti-Mig抗体血清拮抗小鼠本身的Mig蛋白,能够促进骨髓和造血系统的恢复。
为了进一步探讨Mig保护骨髓的机制,我们对正常小鼠和化疗恢复期小鼠注射rMuMig,然后分离其骨髓单个核细胞,进行细胞周期检测,发现rMuMig能够降低单个核细胞处于S期的比例。取小鼠注射PBS或者rMuMig,然后分离骨髓细胞进行集落培养,发现注射rMuMig之后的小鼠其得到的集落总数降低。由此,我们推测rMuMig可能通过抑制造血祖细胞进入S期,从而避过S期敏感的化疗药物5-Fu的作用,使得化疗过程中更多的祖细胞存留下来并进入分裂增殖,加快骨髓的恢复。
综合以上实验结果,我们能够得出结论:在化疗之前应用rMuMig蛋白,以及在化疗之后拮抗小鼠本身的Mig蛋白,都能够对小鼠化疗损伤的骨髓起到正面作用。而根据blast结果,人与小鼠的Mig蛋白具有68%的同源性,并且有82%的序列相似性,在功能上二者也是相似的,因此我们推论,人Mig也可以应用在人的化疗中,起到与小鼠Mig相似的作用。本文工作为开发基于Mig的人化疗保护药物奠定了实验基础。
Bone marrow (BM) suppression is the most serious side-effect caused by chemotherapy. Chemotherapy leads to decrease of blood and BM cell numbers. In response to BM damage of chemotherapy, BM regeneration is initiated. Within 3-4 weeks, the damaged BM is repaired through hematopoiesis. We propose that the expression of BM regeneration regulatory genes (BM-TRRGs) determines the speed of BM regeneration after chemotherapy insults. We used 5-fluorouracil (5-Fu) mouse chemotherapy model to identify BM-TRRGs. After 5-Fu injection, like in humans, the mouse BM is severely damaged and regeneration is initiated. Within 2-3 weeks, the damaged BM is fully recovered through active hematopoiesis.
Using gene expression mirochips, we analyzed the expression patterns of 39000 genes using total mRNA obtained from mouse BM after 5-Fu treatment. The expression of CXCL9, also named monokine induced by IFN-γ(Mig) , and its receptor CXCR3 were identified with expression patterns fit with our hypothesis. ELISA analysis of mouse serum after 5-Fu treatment showed that Mig protein level in serum was increased by 10-fold at day 7 after 5-Fu injection, which is consistent with the gene expression data. The gene and protein expression studies indicate that CXCL9/Mig may be involved in regulation of BM regeneration.
The function of Mig in regulation of BM regeneration was examined by 1) transient overexpression of the protein through plasmid muscle electroporation in normal mice and 5-Fu treated mice; 2) systemically use of the recombinant murine Mig (rMuMig) in normal and 5-Fu treated mice; 3) anti-Mig blocking antibody injection to block endogenous Mig in 5-Fu treated mice. The effects of BM regeneration were analyzed by measuring the peripheral blood and BM cell counts, BM tissue section, cell cycle analysis, hematopoietic colony assay, and mouse survival rate after 5-Fu.
We constructed the eukaryote expressing plasmid pcDNA-Mig, which contain the cDNA sequence of the mouse Mig gene. The plasmid was introduced to the mouse tibia muscle by electroporation. The expression of the mouse Mig in sera after electroporation was measured by ELISA method. We found that Mig expression lasted for 7 days. During the entire experiment, the mice with pcDNA-Mig showed no significant difference in peripheral white blood compared to the control pcDNA3.1 mice. But there were significant decrease in bone marrow cell counts in the pcDNA-Mig mice. HE stained BM slices showed that at day 5 and 10 more sinus appeared in pcDNA-Mig group mice, with the average diameter larger than normal.
To study the BM protection role of Mig, we injected 5-Fu at 200 mg/kg 7 days after plasmid electroporation into normal mice. Note the day of 5-Fu injection as day 0, mice were sacrificed at day 0, 3, 7, 11, 14 to count the peripheral white blood and bone marrow cells, as well as tissue section of the femurs. The cell counts showed that at day 11 pcDNA-Mig group was higher than pcDNA3.1 group in both white blood and bone marrow cells. Histology showed that at day 11 pcDNA-Mig group had more hematopoietic cells than the control group. Hematological data analysis suggests that Mig overexpression before 5-Fu treatment accelerates the BM recovery after 5-Fu treatement, which indicate that Mig may function as BM protector in chemotherapy.
To further study the role of Mig in protection of BM from chemotherapy damage, we produced the recombinant mouse Mig protein (rMuMig). The murine Mig is a low molecular protein of 12 kDa 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-m, which contained the coding sequence of mature murine Mig protein. Then we optimized the expression of the rMuMig and decided that when the bacteria culture reached OD600=0.8 to start the induction by 1mM IPTG. The collected bacteria went through sonication, Urea denaturation, dilution before the final cation-exchange chromatography by S Sepharose. When the conductivity reached 75mS/cm, rMuMig was eluted from the column. SDS-PAGE showed the purity was above 99%. The chemotaxis assay demonstrated that the ED50 of our purified rMuMig was 30ng/ml. Then we immunized rat to get anti-Mig polyclonal antibody serum.
We first examined the role of rMuMig in normal mice. We tried 3 doses of rMuMig - 0.15, 1.5, and 15μg/kg. PBS of equal volume was injected in control group. All mice of the 4 groups were injected consecutively 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, 15. The difference between the rMuMig groups and the control group on peripheral white blood cells was only observed on day 5 between the 15μg/kg group and the control. For the bone marrow cells, the two groups of 1.5 and 15μg/kg had lower cell counts at day 5 and recovered at day 10 and 15. Histology of 15μg/kg group showed more sinus and larger diameters of vessels than that of the control group. The results were similar to that of mice after plasmid transfection.
We further studied the effect of rMuMig on the 5-Fu treated mice. Two ways of administration were taken: administration of rMuMig before and after the 5-Fu treatment. PBS was injected as control. After treated with 250mg/kg 5-Fu the mice in the control group had a survival rate of only 10%, while administration of rMuMig before 5-Fu could protect more than 50% of mice from dying. But if rMuMig was administrated after 5-Fu treatment, no mice survived. The peripheral white blood and bone marrow cells were counted at day 0, 3, 7, 11, and 14 after 5-Fu treatment. At ay 7, 11 and 14, rMuMig group mice had more cells than control. Especially at day 14, the bone marrow of rMuMig group mice had already recovered to near the normal level, while that of control group was only 1/10 of the normal level. Histology revealed that bone marrow of rMuMig group was suppressed less severe at day 7. At day 11 and 14, more hematopoietic cells and regular blood vessels were observed in the rMuMig treated mice than that of control group.
The anti-Mig antibody was used to neutralize elevated expression of endogenous Mig after 5-Fu treatment. The anti-rMuMig polyclonal antibody raised in rat was administrated to mice after 5-Fu treatment consecutively for 10 days once a day. At day 0, 3, 7, 11, and 14 mice were sacrificed to count the WBC and BM cells, and histology femur tissue sections were examined. The result was similar to that of administration of rMuMig before 5-Fu treatment. The survival rate was up-regulated to 60% compared with 10% of the control preimmune serum group. And the mice injected with polyclonal antibody serum had a better and more rapid BM recovery than the control.
From the experiments of rMuMig and its antibody we conclude that: prior administration of rMuMig before 5-Fu protects mouse bone marrow form the chemotherapy damage; using anti-Mig antibody after 5-Fu enhance the bone marrow recovery.
To further study the mechanism of the protection effect of rMuMig, we administrated rMuMig to mice during the BM regeneration phase after 5-Fu treatment. We found that rMuMig inhibited bone marrow cells transition to S-phase of cell cycle, suggesting that the protection role of rMuMig is mediated by slowing down the BM cell cycle rendering the cells less vulnerable to cell-cycle specific chemotherapy drugs, like 5-Fu.
Human Mig is 82% identical to mouse Mig. We propose that human Mig may play similar roles as mouse Mig. We expressed and purified the recombinant human Mig (rHuMig). The prokaryotic expressing plasmid pET28-h was constructed, which contained the coding sequence of mature human Mig protein. When the bacteria culture reached 0.8 at OD600, the protein expression was induced using 1mM IPTG. Protein purification process was carried out similar to rMuMig. SDS-PAGE showed the purity was above 95%.
In summary, this work purified the recombinant murine Mig, studied the function of Mig in regeneration of bone marrow after chemotherapy, which suggest that Mig may be an important BM protector against chemotherapy induced BM side effect. Moreover, we also expressed and purified recombinant human Mig, which should facilitate the studies of human Mig as a BM protector in new protein drug development.
我们又取5-Fu注射后0、3、7、11、14天的小鼠血清,用ELISA检测血清中Mig因子的水平,发现Mig在正常小鼠血清中含量非常低,但是在5-Fu注射后第7天其浓度是正常水平的10倍以上,与芯片数据相一致。综上,我们认为Mig很有可能在骨髓抑制恢复的过程中发挥重要作用。
我们首先利用原核表达pET系统表达了重组人和小鼠的Mig蛋白,并利用离子交换层析方法进行纯化,得到了纯度分别为99%和95%的产物,并检测证明具有生物学活性,为Mig的功能研究打下了基础。我们从两个方面研究Mig对骨髓系统的作用。一,我们通过在体表达和重组蛋白注射两个方法研究使用Mig因子对小鼠的骨髓的作用;二,我们制备了重组小鼠Mig蛋白的多克隆抗体血清,观察使用抗体中和小鼠自身的Mig能否促进受损骨髓的恢复。
为了进行第一部分的功能研究,我们构建了真核细胞表达的重组质粒pcDNA-Mig,使用电脉冲方法,将质粒转染到小鼠的胫前肌。用小鼠Mig的ELISA试剂盒检测质粒转染后小鼠的血清,证实转染能够产生维持7天左右的M ig的暂时高表达。检测电转染之后小鼠的血象和骨髓细胞数量,比较pcDNA-Mig质粒组和pcDNA3.1质粒对照组的小鼠,发现Mig能够降低骨髓细胞总数,这种现象第5天即可观察到,维持到第10天,第27天得到恢复。股骨切片HE染色显示,pcDNA-Mig组比pcDNA3.1对照组骨髓细胞数量较低的时期,pcDNA-Mig组的小鼠骨髓中髓窦数量较多,直径较大。这可能与Mig的脉管抑制作用有关。
进一步将此方法应用到小鼠5-Fu骨髓抑制模型上,在质粒转染之后7天,对小鼠尾静脉注射5-Fu,剂量为200μg/kg体重。以注射5-Fu当天作为第0天,我们在第0、3、7、11、14天共5个时间点小鼠的外周血白细胞和骨髓细胞,并作股骨切片。细胞计数显示,在第11天pcDNA-Mig组的白细胞和骨髓细胞数量皆高于对照组,第14天两组的白细胞和骨髓细胞数量都恢复到正常水平。骨髓切片显示,第7天pcDNA-Mig组的骨髓损伤情况较对照组轻,有核细胞数量多,排列较规则,第11、14天的骨髓细胞密度大,形态规则;相比之下,对照组的骨髓在相同时间点的细胞数量较少。综合细胞计数与骨髓切片,我们得出结论,在5-Fu注射之前转染表达Mig的质粒,能够降低小鼠骨髓的损伤,促进其恢复。
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,但是对机体造成的损伤较大,表达量难于控制,安全性较差。而有活性的重组小鼠Mig蛋白(rMuMig)尾静脉注射可发挥同样的作用。我们首先观察rMuMig蛋白对正常小鼠骨髓的影响。选用0.15、1.5、15μg/kg体重三个剂量的rMuMig尾静脉注射小鼠,对照组注射同样体积的PBS。四组小鼠每天注射一次,连续注射5天,以开始注射的时间作为第0天,分别检测了第0、5、10、15共4个时间点小鼠的外周血白细胞以及骨髓细胞总数的变化,结果显示15μg/kg组能够强烈抑制外周血白细胞、血小板与骨髓细胞数量,1.5μg/kg能够引起骨髓细胞的明显变化,而更低剂量对造血系统没有明显作用。15μg/kg组的小鼠股骨切片与对照组对比,第5天髓窦和血管明显扩张,与对照组的相同部位相比,同样大的视野下观察到的髓窦数量多,直径大;第10天这种现象没有改善;第15天髓窦数量有所回降,直径也缩小;第20天,骨髓恢复到与对照组相似水平,髓窦数量和大小皆接近正常。这些结果与质粒转染后小鼠的骨髓反应相似。
我们选取10μg/kg体重的剂量,进一步观察rMuMig对5-Fu化疗小鼠的作用。我们采取两组给药方法,一种是先注射rMuMig,然后注射5-Fu;一种是先注射5-Fu,8小时后开始注射rMuMig。对照组注射等体积PBS代替蛋白溶液。统计发现,在250mg/kg体重的5-Fu作用下,对照组的小鼠只有10%左右的存活率,而先注射rMuMig可以将存活率提高到50%以上,但是先注射5-Fu后注射rMuMig的组小鼠全部死亡。
检测5-Fu注射后第0、3、7、11、14共5个时间点小鼠的外周血以及骨髓细胞,发现提前注射rMuMig的小鼠,其外周血白细胞和骨髓细胞数量的恢复皆好于对照组。特别是第14天对照组的骨髓细胞数量只有正常水平的1/10左右,而提前注射rMuMig的小鼠已恢复至接近正常水平。我们取两组小鼠的股骨做了切片,HE染色观察其形态学变化,发现在第0天蛋白组小鼠髓窦较多,髓窦直径较大,这与前面实验的结果吻合;第3天两组细胞减少,但没有明显的组间差异;第7天可见对照组小鼠的骨髓损伤较大,出现大面积空腔,而蛋白组相对损伤较小,空腔的数量少面积小,骨髓细胞排列较对照组有规则;第11天的rMuMig组小鼠骨髓中可以观察到脂肪细胞的出现,但是在第14天脂肪细胞即消失,代之以造血细胞。该实验证实,提前注射rMuMig,能够对5-Fu化疗的小鼠骨髓起到保护作用,从而有利于化疗后骨髓的恢复。
我们利用rMuMig免疫大鼠得到anti-Mig的抗体血清,理论上能够中和小鼠Mig蛋白,从而拮抗Mig对骨髓所起的作用。因此在注射250μg/kg体重的5-Fu之后,从第1天开始,每天一次,连续十天对小鼠皮下注射抗体血清,以未经Mig免疫的血清作为对照,观察对小鼠骨髓抑制后恢复的影响。结果发现,注射对照血清的小鼠在11天之内全部死亡,而注射anti-Mig血清的小鼠存活率高达60%左右。股骨切片结果与提前注射rMuMig蛋白后注射5-Fu的小鼠相似,即第11、14天anti-Mig抗体血清组的恢复好于对照组。
先注射rMuMig蛋白,然后注射250mg/kg 5-Fu之后,分别给予相同剂量的对照血清和anti-Mig抗体血清,发现给予anti-Mig抗体血清的小鼠其恢复明显好于对照血清组,在第14天外周血白细胞和骨髓细胞数量都较高。该实验进一步证实在5-Fu之后使用anti-Mig抗体血清拮抗小鼠本身的Mig蛋白,能够促进骨髓和造血系统的恢复。
为了进一步探讨Mig保护骨髓的机制,我们对正常小鼠和化疗恢复期小鼠注射rMuMig,然后分离其骨髓单个核细胞,进行细胞周期检测,发现rMuMig能够降低单个核细胞处于S期的比例。取小鼠注射PBS或者rMuMig,然后分离骨髓细胞进行集落培养,发现注射rMuMig之后的小鼠其得到的集落总数降低。由此,我们推测rMuMig可能通过抑制造血祖细胞进入S期,从而避过S期敏感的化疗药物5-Fu的作用,使得化疗过程中更多的祖细胞存留下来并进入分裂增殖,加快骨髓的恢复。
综合以上实验结果,我们能够得出结论:在化疗之前应用rMuMig蛋白,以及在化疗之后拮抗小鼠本身的Mig蛋白,都能够对小鼠化疗损伤的骨髓起到正面作用。而根据blast结果,人与小鼠的Mig蛋白具有68%的同源性,并且有82%的序列相似性,在功能上二者也是相似的,因此我们推论,人Mig也可以应用在人的化疗中,起到与小鼠Mig相似的作用。本文工作为开发基于Mig的人化疗保护药物奠定了实验基础。
Bone marrow (BM) suppression is the most serious side-effect caused by chemotherapy. Chemotherapy leads to decrease of blood and BM cell numbers. In response to BM damage of chemotherapy, BM regeneration is initiated. Within 3-4 weeks, the damaged BM is repaired through hematopoiesis. We propose that the expression of BM regeneration regulatory genes (BM-TRRGs) determines the speed of BM regeneration after chemotherapy insults. We used 5-fluorouracil (5-Fu) mouse chemotherapy model to identify BM-TRRGs. After 5-Fu injection, like in humans, the mouse BM is severely damaged and regeneration is initiated. Within 2-3 weeks, the damaged BM is fully recovered through active hematopoiesis.
Using gene expression mirochips, we analyzed the expression patterns of 39000 genes using total mRNA obtained from mouse BM after 5-Fu treatment. The expression of CXCL9, also named monokine induced by IFN-γ(Mig) , and its receptor CXCR3 were identified with expression patterns fit with our hypothesis. ELISA analysis of mouse serum after 5-Fu treatment showed that Mig protein level in serum was increased by 10-fold at day 7 after 5-Fu injection, which is consistent with the gene expression data. The gene and protein expression studies indicate that CXCL9/Mig may be involved in regulation of BM regeneration.
The function of Mig in regulation of BM regeneration was examined by 1) transient overexpression of the protein through plasmid muscle electroporation in normal mice and 5-Fu treated mice; 2) systemically use of the recombinant murine Mig (rMuMig) in normal and 5-Fu treated mice; 3) anti-Mig blocking antibody injection to block endogenous Mig in 5-Fu treated mice. The effects of BM regeneration were analyzed by measuring the peripheral blood and BM cell counts, BM tissue section, cell cycle analysis, hematopoietic colony assay, and mouse survival rate after 5-Fu.
We constructed the eukaryote expressing plasmid pcDNA-Mig, which contain the cDNA sequence of the mouse Mig gene. The plasmid was introduced to the mouse tibia muscle by electroporation. The expression of the mouse Mig in sera after electroporation was measured by ELISA method. We found that Mig expression lasted for 7 days. During the entire experiment, the mice with pcDNA-Mig showed no significant difference in peripheral white blood compared to the control pcDNA3.1 mice. But there were significant decrease in bone marrow cell counts in the pcDNA-Mig mice. HE stained BM slices showed that at day 5 and 10 more sinus appeared in pcDNA-Mig group mice, with the average diameter larger than normal.
To study the BM protection role of Mig, we injected 5-Fu at 200 mg/kg 7 days after plasmid electroporation into normal mice. Note the day of 5-Fu injection as day 0, mice were sacrificed at day 0, 3, 7, 11, 14 to count the peripheral white blood and bone marrow cells, as well as tissue section of the femurs. The cell counts showed that at day 11 pcDNA-Mig group was higher than pcDNA3.1 group in both white blood and bone marrow cells. Histology showed that at day 11 pcDNA-Mig group had more hematopoietic cells than the control group. Hematological data analysis suggests that Mig overexpression before 5-Fu treatment accelerates the BM recovery after 5-Fu treatement, which indicate that Mig may function as BM protector in chemotherapy.
To further study the role of Mig in protection of BM from chemotherapy damage, we produced the recombinant mouse Mig protein (rMuMig). The murine Mig is a low molecular protein of 12 kDa 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-m, which contained the coding sequence of mature murine Mig protein. Then we optimized the expression of the rMuMig and decided that when the bacteria culture reached OD600=0.8 to start the induction by 1mM IPTG. The collected bacteria went through sonication, Urea denaturation, dilution before the final cation-exchange chromatography by S Sepharose. When the conductivity reached 75mS/cm, rMuMig was eluted from the column. SDS-PAGE showed the purity was above 99%. The chemotaxis assay demonstrated that the ED50 of our purified rMuMig was 30ng/ml. Then we immunized rat to get anti-Mig polyclonal antibody serum.
We first examined the role of rMuMig in normal mice. We tried 3 doses of rMuMig - 0.15, 1.5, and 15μg/kg. PBS of equal volume was injected in control group. All mice of the 4 groups were injected consecutively 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, 15. The difference between the rMuMig groups and the control group on peripheral white blood cells was only observed on day 5 between the 15μg/kg group and the control. For the bone marrow cells, the two groups of 1.5 and 15μg/kg had lower cell counts at day 5 and recovered at day 10 and 15. Histology of 15μg/kg group showed more sinus and larger diameters of vessels than that of the control group. The results were similar to that of mice after plasmid transfection.
We further studied the effect of rMuMig on the 5-Fu treated mice. Two ways of administration were taken: administration of rMuMig before and after the 5-Fu treatment. PBS was injected as control. After treated with 250mg/kg 5-Fu the mice in the control group had a survival rate of only 10%, while administration of rMuMig before 5-Fu could protect more than 50% of mice from dying. But if rMuMig was administrated after 5-Fu treatment, no mice survived. The peripheral white blood and bone marrow cells were counted at day 0, 3, 7, 11, and 14 after 5-Fu treatment. At ay 7, 11 and 14, rMuMig group mice had more cells than control. Especially at day 14, the bone marrow of rMuMig group mice had already recovered to near the normal level, while that of control group was only 1/10 of the normal level. Histology revealed that bone marrow of rMuMig group was suppressed less severe at day 7. At day 11 and 14, more hematopoietic cells and regular blood vessels were observed in the rMuMig treated mice than that of control group.
The anti-Mig antibody was used to neutralize elevated expression of endogenous Mig after 5-Fu treatment. The anti-rMuMig polyclonal antibody raised in rat was administrated to mice after 5-Fu treatment consecutively for 10 days once a day. At day 0, 3, 7, 11, and 14 mice were sacrificed to count the WBC and BM cells, and histology femur tissue sections were examined. The result was similar to that of administration of rMuMig before 5-Fu treatment. The survival rate was up-regulated to 60% compared with 10% of the control preimmune serum group. And the mice injected with polyclonal antibody serum had a better and more rapid BM recovery than the control.
From the experiments of rMuMig and its antibody we conclude that: prior administration of rMuMig before 5-Fu protects mouse bone marrow form the chemotherapy damage; using anti-Mig antibody after 5-Fu enhance the bone marrow recovery.
To further study the mechanism of the protection effect of rMuMig, we administrated rMuMig to mice during the BM regeneration phase after 5-Fu treatment. We found that rMuMig inhibited bone marrow cells transition to S-phase of cell cycle, suggesting that the protection role of rMuMig is mediated by slowing down the BM cell cycle rendering the cells less vulnerable to cell-cycle specific chemotherapy drugs, like 5-Fu.
Human Mig is 82% identical to mouse Mig. We propose that human Mig may play similar roles as mouse Mig. We expressed and purified the recombinant human Mig (rHuMig). The prokaryotic expressing plasmid pET28-h was constructed, which contained the coding sequence of mature human Mig protein. When the bacteria culture reached 0.8 at OD600, the protein expression was induced using 1mM IPTG. Protein purification process was carried out similar to rMuMig. SDS-PAGE showed the purity was above 95%.
In summary, this work purified the recombinant murine Mig, studied the function of Mig in regeneration of bone marrow after chemotherapy, which suggest that Mig may be an important BM protector against chemotherapy induced BM side effect. Moreover, we also expressed and purified recombinant human Mig, which should facilitate the studies of human Mig as a BM protector in new protein drug development.
引文
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