Zhang Yaozhou, Chen Jian, Lv Zhengbing, Nie Zuoming, Zhang Xiaoyan, Wu Xiangfu
Institute of Biochemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Eur J Pharm Sci. 2006 Jun;28(3):212-23. doi: 10.1016/j.ejps.2006.02.014. Epub 2006 Apr 17.
In order to study the effect of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as active cytokine through orally administration, we expressed hGM-CSF within silkworm pupae bioreactor. The purified rhGM-CSF named as BmrhGM-CSF is characterized as 29kDa glycoprotein, and its biological activity was measured both in vitro and in vivo. We found out BmrhGM-CSF could stimulate the colony formation of human bone marrow cells in a dose-dependent manner whether which were treated with or without gamma-ray 24h before. The ability of colony formation induced by BmrhGM-CSF is negatively correlated with gamma-ray intensity. As soon as 15min post oral administration with BmrhGM-CSF labeled with (125)I, an approximately 20kDa protein fragment was detected within mice blood by SDS-PAGE followed by autoradiography. In blood sample of test mice, a protein was also recognized by anti-hGM-CSF antibody using ELISA. The immunohistochemical analysis showed that BmrhGM-CSF was detected within intestinal histiocyte. This indicated it might be absorbed into blood via intestinal microvillus. Pharmacokinetics analysis after orally administered BmrhGM-CSF in animal model of leucopenia including mice, Beagle dogs and macaques showed that: (1) BmrhGM-CSF promoted the CFU-S formation in mice spleen and the synthesis of DNA in bone marrow cells of mice; (2) BmrhGM-CSF induced bone marrow karyocyta granulocyte growth significantly in both macaques and Beagle dogs compared to the negative control group. On the 9th day of orally administration, the animal WBC significantly increased in a dose-dependant manner, in which neutrophilic granulocyte was predominant. The WBC level of dogs in high dose group was about 1.5x10(9)cells/L more than that in the negative control. And the bone marrow smear revealed that the percents of both myloblast and progranulocyte in WBC in the hGM-CSF group were obviously higher than those in the negative control. These results proved that BmrhGM-CSF, a 29kDa glycoprotein expressed by Silkworm pupae bioreactor, could bring into the effect as active cytokine through oral administration.
为研究人粒细胞巨噬细胞集落刺激因子（hGM-CSF）作为活性细胞因子口服给药的效果，我们在蚕蛹生物反应器中表达了hGM-CSF。纯化后的重组人粒细胞巨噬细胞集落刺激因子（BmrhGM-CSF）为29kDa糖蛋白，并对其体外和体内生物学活性进行了检测。我们发现，无论人骨髓细胞在24小时前是否接受γ射线照射，BmrhGM-CSF均能以剂量依赖的方式刺激其集落形成。BmrhGM-CSF诱导集落形成的能力与γ射线强度呈负相关。口服用（125）I标记的BmrhGM-CSF后15分钟，通过SDS-PAGE和放射自显影在小鼠血液中检测到一个约20kDa的蛋白片段。在受试小鼠的血液样本中，用ELISA法也能检测到一种可被抗hGM-CSF抗体识别的蛋白。免疫组化分析显示，在肠道组织细胞中检测到了BmrhGM-CSF。这表明它可能通过肠道微绒毛吸收进入血液。在包括小鼠、比格犬和猕猴在内的白细胞减少症动物模型中口服BmrhGM-CSF后的药代动力学分析表明：（1）BmrhGM-CSF促进了小鼠脾脏中CFU-S的形成以及小鼠骨髓细胞中DNA的合成；（2）与阴性对照组相比，BmrhGM-CSF在猕猴和比格犬中均显著诱导了骨髓有核细胞粒细胞生长。口服给药第9天，动物白细胞以剂量依赖的方式显著增加，其中以中性粒细胞为主。高剂量组犬的白细胞水平比阴性对照组高约1.5×10⁹个细胞/L。骨髓涂片显示，hGM-CSF组白细胞中早幼粒细胞和原粒细胞的百分比明显高于阴性对照组。这些结果证明，由蚕蛹生物反应器表达的29kDa糖蛋白BmrhGM-CSF口服给药后可作为活性细胞因子发挥作用。
