中国药学杂志

Item	Mice	Guinea pigs
Mass/g	18~22	250~350
Number	Control group and experimental group with 5 each	Control group and experimental group with 2 each
Injection dose/mL	0.1 mL(0.015 mg α2-M)	1 mL(0.15 mg α2-M)
Administration frequency	Single administration	Single administration
Administration route	Intravenous injection	Intravenous injection
Criteria for determination	During the breeding observation period, all mice and guinea pigs in the control and experimental groups should be healthy and show no abnormal reactions. If the weight of each mouse and guinea pig increases, it is considered to meet the criteria after observing for 7 d

Item	Mice	Guinea pigs
Mass/g	18~22	250~350
Number	Control group and experimental group with 5 each	Control group and experimental group with 2 each
Injection dose/mL	0.1 mL(0.015 mg α2-M)	1 mL(0.15 mg α2-M)
Administration frequency	Single administration	Single administration
Administration route	Intravenous injection	Intravenous injection
Criteria for determination	During the breeding observation period, all mice and guinea pigs in the control and experimental groups should be healthy and show no abnormal reactions. If the weight of each mouse and guinea pig increases, it is considered to meet the criteria after observing for 7 d

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Observation time	Test tube 1	Test tube 2	Test tube 3	Test tube 4	Test tube 5	Test tube 6	Test tube 7
15 min	-	-	-	-	-	-	+
30 min	-	-	-	-	-	-	+
45 min	-	-	-	-	-	-	+
1 h	-	-	-	-	-	-	+
2 h	-	-	-	-	-	-	+
3 h	-	-	-	-	-	-	+
Determination result	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	Complete hemolysis

Observation time	Test tube 1	Test tube 2	Test tube 3	Test tube 4	Test tube 5	Test tube 6	Test tube 7
15 min	-	-	-	-	-	-	+
30 min	-	-	-	-	-	-	+
45 min	-	-	-	-	-	-	+
1 h	-	-	-	-	-	-	+
2 h	-	-	-	-	-	-	+
3 h	-	-	-	-	-	-	+
Determination result	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	Complete hemolysis

Item	Plasma/mL	Physiological saline/mL	Distilled water/mL
OD_{545 nm}	0.235±0.012	0.236±0.008	0.248±0.009	0.262±0.004	0.246±0.004	0.201±0.006	0.690±0.003
Hemolysis rate/%	6.9	7.1	9.6	12.5	9.2
Hemolysis result	+	+	+	+	+	-	-

Item	Plasma/mL	Physiological saline/mL	Distilled water/mL
OD_{545 nm}	0.235±0.012	0.236±0.008	0.248±0.009	0.262±0.004	0.246±0.004	0.201±0.006	0.690±0.003
Hemolysis rate/%	6.9	7.1	9.6	12.5	9.2
Hemolysis result	+	+	+	+	+	-	-

Parameters	After administration 3 d	After administration 14 d
WBC/×10⁹·L^-1	8.100 0(6.032 5-9.732 5)	9.600 0(6.970 0-10.315 0)	9.085 0(7.465 0-11.575 0)	11.215 0(10.107 5-12.230 0)
Neu/×10⁹·L^-1	1.185 0(0.960 0-1.392 5)	1.385 0(1.045-1.572 5)	1.390 0(1.197 5-1.660 0)	1.480 0(1.140 0-1.715 0)
Lym/×10⁹·L^-1	6.740 0(4.362 5-7.735 0)	7.585 0(5.500 0-8.087 5)	6.785 0(5.667 5-9.610 0)	8.885 0(7.660 0-10.112 5)
Mon/×10⁹·L^-1	0.380 0(0.275 0-0.492 5)	0.475 0(0.360 0-0.580 0)	0.535 0(0.455 0-0.687 5)	0.670 0(0.575 0-0.782 5)
Eos/×10⁹·L^-1	0.080 0(0.067 5-0.090 0)	0.085 0(0.065 0-0.115 0)	0.100 0(0.060 0-0.127 5)	0.115 0(0.110 0-0.125 0)
Bas/×10⁹·L^-1	0.010 0(0.010 0-0.027 5)	0.015 0(0.010 0-0.022 5)	0.010 0(0.007 5-0.015 0)	0.020 0(0.020 0-0.020 0)¹⁾
RBC/×10⁹·L^-1	6.440 0(6.032 5-6.880 0)	6.420 0(6.350 0-6.572 5)	6.665 0(6.480 0-6.910 0)	6.925 0(6.612 5-7.455 0)
HGB/g·L^-1	147.500 0(142.000 0-150.500 0)	147.000 0(143.750 0-157.000 0)	148.500 0(145.500 0-152.250 0)	151.000 0(149.250 0-156.250 0)
HCT/%	38.950 0(37.475 0-40.275 0)	38.850 0(38.775 0-41.225 0)	39.550 0(38.025 0-40.200 0)	39.350 0(38.300 0-41.925 0)
MCV/fL	59.350 0(58.075 0-63.375 0)	61.500 0(60.100 0-62.900 0)	59.050 0(57.500 0-60.175 0)	57.300 0(55.925 0-58.325 0)
MCH/pg	22.250 0(21.700 0-24.275 0)	22.950 0(22.675 0-23.725 0)	22.200 0(21.450 0-23.250 0)	22.050 0(21.375 0-22.550 0)
MCHC/g·L^-1	376.000 0(373.000 0-381.000 0)	377.000 0(369.750 0-379.750 0)	375.500 0(372.750 0-384.250 0)	382.500 0(377.000 0-390.250 0)
PLT/×10⁹·L^-1	896.500 0(793.250 0-990.500 0)	1036.500 0(951.750 0-1 160.500 0)	919.000 0(793.250 0-1 048.250 0)	858.000 0(729.500 0-950.750 0)
PDW	15.250 0(15.175 0-15.375 0)	15.450 0(15.375 0-15.500 0)	15.300 0(15.200 0-15.500 0)	15.300 0(15.225 0-15.400 0)
PCT/%	0.523 0(0.458 25-0.594 25)	0.621 5(0.581 5-0.705 5)¹⁾	0.544 0(0.474 25-0.614 5)	0.551 0(0.422 5-0.5707 5)

Parameters	After administration 3 d	After administration 14 d
WBC/×10⁹·L^-1	8.100 0(6.032 5-9.732 5)	9.600 0(6.970 0-10.315 0)	9.085 0(7.465 0-11.575 0)	11.215 0(10.107 5-12.230 0)
Neu/×10⁹·L^-1	1.185 0(0.960 0-1.392 5)	1.385 0(1.045-1.572 5)	1.390 0(1.197 5-1.660 0)	1.480 0(1.140 0-1.715 0)
Lym/×10⁹·L^-1	6.740 0(4.362 5-7.735 0)	7.585 0(5.500 0-8.087 5)	6.785 0(5.667 5-9.610 0)	8.885 0(7.660 0-10.112 5)
Mon/×10⁹·L^-1	0.380 0(0.275 0-0.492 5)	0.475 0(0.360 0-0.580 0)	0.535 0(0.455 0-0.687 5)	0.670 0(0.575 0-0.782 5)
Eos/×10⁹·L^-1	0.080 0(0.067 5-0.090 0)	0.085 0(0.065 0-0.115 0)	0.100 0(0.060 0-0.127 5)	0.115 0(0.110 0-0.125 0)
Bas/×10⁹·L^-1	0.010 0(0.010 0-0.027 5)	0.015 0(0.010 0-0.022 5)	0.010 0(0.007 5-0.015 0)	0.020 0(0.020 0-0.020 0)¹⁾
RBC/×10⁹·L^-1	6.440 0(6.032 5-6.880 0)	6.420 0(6.350 0-6.572 5)	6.665 0(6.480 0-6.910 0)	6.925 0(6.612 5-7.455 0)
HGB/g·L^-1	147.500 0(142.000 0-150.500 0)	147.000 0(143.750 0-157.000 0)	148.500 0(145.500 0-152.250 0)	151.000 0(149.250 0-156.250 0)
HCT/%	38.950 0(37.475 0-40.275 0)	38.850 0(38.775 0-41.225 0)	39.550 0(38.025 0-40.200 0)	39.350 0(38.300 0-41.925 0)
MCV/fL	59.350 0(58.075 0-63.375 0)	61.500 0(60.100 0-62.900 0)	59.050 0(57.500 0-60.175 0)	57.300 0(55.925 0-58.325 0)
MCH/pg	22.250 0(21.700 0-24.275 0)	22.950 0(22.675 0-23.725 0)	22.200 0(21.450 0-23.250 0)	22.050 0(21.375 0-22.550 0)
MCHC/g·L^-1	376.000 0(373.000 0-381.000 0)	377.000 0(369.750 0-379.750 0)	375.500 0(372.750 0-384.250 0)	382.500 0(377.000 0-390.250 0)
PLT/×10⁹·L^-1	896.500 0(793.250 0-990.500 0)	1036.500 0(951.750 0-1 160.500 0)	919.000 0(793.250 0-1 048.250 0)	858.000 0(729.500 0-950.750 0)
PDW	15.250 0(15.175 0-15.375 0)	15.450 0(15.375 0-15.500 0)	15.300 0(15.200 0-15.500 0)	15.300 0(15.225 0-15.400 0)
PCT/%	0.523 0(0.458 25-0.594 25)	0.621 5(0.581 5-0.705 5)¹⁾	0.544 0(0.474 25-0.614 5)	0.551 0(0.422 5-0.5707 5)

Parameters	After administration 3 d	After administration 14 d
ALB/g·L^-1	38.050 0(37.275 0-38.950 0)	38.000 0(36.925 0-39.000 0)	39.250 0(39.050 0-41.300 0)	39.500 0(38.575 0-41.225 0)
GLB/g·L^-1	11.950 0(9.900 0-13.200 0)	13.100 0(11.500 0-14.125 0)	14.800 0(14.025 0-17.250 0)	14.700 0(13.800 0-15.700 0)
TBIL/μmol·L^-1	4.530 0(3.460 0-4.705 0)	3.360 0(2.480 0-4.400 0)	3.675 0(2.965 0-4.477 5)	2.800 0(2.18 5-3.425 0)
ALT/U·L^-1	36.000 0(33.500 0-50.250 0)	43.000 0(38.750 0-46.500 0)	49.000 0(44.500 0-50.750 0)	44.500 0(41.500 0-47.000 0)
AST/U·L^-1	172.000 0(153.250 0-203.250 0)	215.000 0(197.500 0-261.000 0)	200.000 0(164.250 0-259.250 0)	166.000 0(142.000 0-204.750 0)
GGT/U·L^-1	0.800 0(0.475 0-1.325 0)	1.050 0(0.750 0-1.700 0)	1.200 0(0.875 0-1.700 0)	0.450 0(0.250 0-1.500 0)
BUN/mmol·L^-1	5.045 0(4.870 0-6.850 0)	5.090 0(4.775 0-5.405 0)	5.800 0(5.187 5-6.047 5)	5.040 0(4.115 0-5.400 0)
Cr/μmol·L^-1	43.000 0(40.750 0-46.500 0)	43.000 0(37.500 0-51.500 0)	53.500 0(50.750 0-59.750 0)	52.000 0(49.500 0-54.250 0)

Parameters	After administration 3 d	After administration 14 d
ALB/g·L^-1	38.050 0(37.275 0-38.950 0)	38.000 0(36.925 0-39.000 0)	39.250 0(39.050 0-41.300 0)	39.500 0(38.575 0-41.225 0)
GLB/g·L^-1	11.950 0(9.900 0-13.200 0)	13.100 0(11.500 0-14.125 0)	14.800 0(14.025 0-17.250 0)	14.700 0(13.800 0-15.700 0)
TBIL/μmol·L^-1	4.530 0(3.460 0-4.705 0)	3.360 0(2.480 0-4.400 0)	3.675 0(2.965 0-4.477 5)	2.800 0(2.18 5-3.425 0)
ALT/U·L^-1	36.000 0(33.500 0-50.250 0)	43.000 0(38.750 0-46.500 0)	49.000 0(44.500 0-50.750 0)	44.500 0(41.500 0-47.000 0)
AST/U·L^-1	172.000 0(153.250 0-203.250 0)	215.000 0(197.500 0-261.000 0)	200.000 0(164.250 0-259.250 0)	166.000 0(142.000 0-204.750 0)
GGT/U·L^-1	0.800 0(0.475 0-1.325 0)	1.050 0(0.750 0-1.700 0)	1.200 0(0.875 0-1.700 0)	0.450 0(0.250 0-1.500 0)
BUN/mmol·L^-1	5.045 0(4.870 0-6.850 0)	5.090 0(4.775 0-5.405 0)	5.800 0(5.187 5-6.047 5)	5.040 0(4.115 0-5.400 0)
Cr/μmol·L^-1	43.000 0(40.750 0-46.500 0)	43.000 0(37.500 0-51.500 0)	53.500 0(50.750 0-59.750 0)	52.000 0(49.500 0-54.250 0)

人血浆组分Ⅳ来源α2-巨球蛋白安全性的初步研究

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丰腾 ^1a^,² , 袁方 ² , 祁芳玲 ^1b^,² , 王少伟 ^1a , 贾俊婷 ² , 陈崇伟 ^1a^,^* , 马玉媛 ²^,^*

中国药学杂志 | 论著 2025,60(9): 941-648

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中国药学杂志 | 论著 2025, 60(9): 941-648

人血浆组分Ⅳ来源α2-巨球蛋白安全性的初步研究

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丰腾^1a^,², 袁方², 祁芳玲^1b^,², 王少伟^1a, 贾俊婷², 陈崇伟^1a^,^*, 马玉媛²^,^*

作者信息

¹ 山西医科大学, a.第二临床医学院, b.药学院, 太原 030001

² 军事医学研究院, 国家药品监督管理局血液制品质量控制重点实验室, 北京 100850

丰腾,男,硕士研究生研究方向:骨关节退行性疾病及损伤

通讯作者:

^*陈崇伟,男,博士,教授,硕士生导师研究方向:骨关节退行性疾病及损伤 Tel:(0351)3363158;

马玉媛,女,博士,副研究员,硕士生导师研究方向:生物制品及其病毒安全性 Tel:(010)66932222

Preliminary Study on the Safety of Human Plasma Fraction Ⅳ-Derived α2-Macroglobulin

Teng FENG^1a^,², Fang YUAN², Fangling QI^1b^,², Shaowei WANG^1a, Junting JIA², Chongwei CHEN^1a^,^*, Yuyuan MA²^,^*

Affiliations

¹ The Second Clinical Medical College, 1b.College of Pharmacy, Shanxi Medical University, Taiyuan 030001, China

² Academy of Military Medical Sciences, NMPA Key Laboratory for Quality Control of Blood Products, Beijing 100850, China

出版时间: 2025-05-01 doi: 10.11669/cpj.2025.09.006

文章导航

摘要

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目的评价关节腔内注射人血浆Cohn组分Ⅳ来源α2-巨球蛋白(α2-M)的安全性。方法以人血浆Cohn组分Ⅳ为原料制备α2-M并检测其纯度和活性;采用体外试管法进行兔红细胞溶血试验;通过向大鼠膝关节腔内注射α2-M评价局部刺激性作用;通过向小鼠与豚鼠腹腔内注射α2-M进行异常毒性试验。结果组分Ⅳ来源α2-M的纯度为95%,活性为(6.835±0.104)μg·mL^-1。在体外溶血试验中肉眼未见溶血或聚集现象,通过分光光度计检测观察到轻微溶血现象。刺激性试验结果显示,给药后大鼠注射局部未见明显异常,外观、行为活动、精神等一般状况良好,血常规与血生化指标除血小板压积(PCT)、嗜碱性粒细胞(Bas)计数外无异常变化。异常毒性试验中,小鼠和豚鼠经腹腔注射本品30 min内均无异常反应出现,观察期结束时,小鼠及豚鼠平均体质量均增加。结论人血浆Cohn组分Ⅳ来源α2-M体内安全性良好。

关键词

Cohn组分Ⅳ / α2-巨球蛋白 / 安全性 / 血液制品 / 关节腔注射

Abstract

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OBJECTIVE To evaluate the safety of intra-articular injection of α2-macroglobulin(α2-M) derived from human plasma Cohn fraction Ⅳ. METHODS α2-M was prepared from human plasma Cohn fraction Ⅳ and its purity and activity were tested. A rabbit red blood cell hemolysis test was conducted using an in vitro test tube method. The local irritancy was evaluated by injecting α2-M into the knee joint cavity of rats. The abnormal toxicity test was conducted by injecting α2-M into the abdominal cavity of mice and guinea pigs. RESULTS The purity of fraction Ⅳ derived α2-M was 95%, with an activity of (6.835±0.104) μg·mL^-1. In the in vitro hemolysis test, no hemolysis or aggregation was observed macroscopically, but slight hemolysis was detected by spectrophotometry. The irritancy test showed no obvious abnormalities at the injection site of the rats after administration, with good general condition, appearance, behavior, and spirit, and no abnormal changes in routine blood and blood biochemistry indicators except for PCT and Bas. In the abnormal toxicity test, no abnormal reactions were observed within 30 min after intraperitoneal injection of the test article in mice and guinea pigs, and the average body weight of mice and guinea pigs increased by the end of the observation period. CONCLUSION α2-M derived from human plasma Cohn fraction Ⅳ is considered to be safe in vivo.

Key words

Cohn fraction Ⅳ / α2-macroglobulin / safety / blood product / intra-articular injection

引用本文

丰腾, 袁方, 祁芳玲, 王少伟, 贾俊婷, 陈崇伟, 马玉媛. 人血浆组分Ⅳ来源α2-巨球蛋白安全性的初步研究. 中国药学杂志, 2025 , 60 (9) : 941 -648 . DOI: 10.11669/cpj.2025.09.006

Teng FENG, Fang YUAN, Fangling QI, Shaowei WANG, Junting JIA, Chongwei CHEN, Yuyuan MA. Preliminary Study on the Safety of Human Plasma Fraction Ⅳ-Derived α2-Macroglobulin[J]. Chinese Pharmaceutical Journal, 2025 , 60 (9) : 941 -648 . DOI: 10.11669/cpj.2025.09.006

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骨性关节炎(osteoarthritis,OA)是一种慢性退行性疾病,病理特征包括软骨变性、滑膜增生、韧带周围附着点及软骨下骨的增生性变化,继而导致关节疼痛、畸形和功能障碍。目前临床上对于OA的药物治疗以对症支持治疗为主,无法从根本上治愈或延缓OA的进展。研究表明,α2-巨球蛋白(α2-macroglobulin,α2-M)对关节软骨具有显著的保护作用,有望成为一种新型的骨关节炎疾病改善型药物(disease-modifying OA drugs,DMOADs)^[1-2]。

α2-M是一个相对分子质量为720×10³的同源四聚体糖蛋白,作为人体血浆中最大的非免疫球蛋白,其在血液中质量浓度为2~4 mg·mL^-1^[3-4]。α2-M是一种广谱的蛋白酶抑制剂,其诱饵区可被蛋白酶水解,这一过程可触发α2-M发生构象变化,从而将蛋白酶捕获并包裹。随后,α2-M暴露出受体结合位点,使得细胞(包括肝细胞、成纤维细胞、巨噬细胞等)表面受体能够识别并促进内吞作用,从而迅速将α2-M-蛋白酶复合物从体内清除出去^[5-8]。此外,α2-M还扮演着细胞因子结合蛋白和载体蛋白的角色,对于维持内环境的稳态至关重要^[9]。研究发现,α2-M可通过阻断白细胞介素-1β(interleukin-1β,IL-1β)/核因子κB(nuclear factor kappa-B,NF-κB)通路来抑制软骨细胞炎症因子和蛋白酶的分泌^[10],如IL-1β、IL-6、IL-8、肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)、MMP-9、MMP-13、血小板反应蛋白解整合素金属肽酶-4(a disintegrin and metalloproteinase with thrombospondin motifs 4,ADAMTS-4)、ADAMTS-5、ADAMTS-7和ADAMTS-12等^[4,11-12]。α2-M还可通过诱导增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达来促进软骨细胞的增殖和细胞外基质的合成^[13]。在大鼠前交叉韧带切断术模型、小鼠胶原诱导关节炎模型^[10]和小型猪前交叉韧带重建模型中研究发现关节腔内补充外源性的α2-M有助于保护关节软骨和延缓骨关节炎病程的进展^[4,14-15]。

美国Cytonics公司开发了一种自体血小板浓缩技术(autologous platelet integrated concentration,APIC),通过该技术可制得富α2-M血浆,现已获得美国食品药品监督管理局(FDA)的批准用于轻至中度膝关节、髋关节和肩关节骨关节炎的治疗^[16]。尽管这种技术在临床应用中取得了一定的成功,但是富α2-M血浆成分复杂、作用机制不明确。此外,自体血浆回注技术具有一定的局限性,这也限制了其更广泛的临床应用和规模化生产。同时Cytonics公司还开发了100余种α2-M重组变体,研究发现α2-M重组变体108(CYT-108)在众多变体和天然型α2-M中,其在抑制金属蛋白酶、减少促炎细胞因子以及促进Ⅱ型胶原蛋白和蛋白多糖合成方面的能力最为突出。动物实验结果也表明,与天然型α2-M相比,CYT-108在保护软骨、延缓退变进程方面具有更显著的效果^[17]。CYT-108已准备进入Ⅰ期临床研究阶段,以评估其对膝部OA的疗效。

目前,全球市场上尚未有α2-M药品获得批准上市。实验室前期已建立以人血浆Cohn组分Ⅳ为原料制备高纯度α2-M的工艺^[18],虽然α2-M是来源于血液的血浆蛋白,但由于制备过程工艺流程复杂,尚无法完全确定该制品的安全性。因此,对从Cohn组分Ⅳ中纯化人血浆来源的α2-M制品开展了安全性的初步评价,以进一步评估α2-M在OA治疗中成药的可能性。

1 材料

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1.1 实验动物

10只雄性昆明小鼠[SPF级,北京维通利华实验动物技术有限公司,动物生产许可证号SCXK(京)2021-0021];4只雄性Hartley豚鼠[SPF级,北京维通利华实验动物技术有限公司,动物生产许可证号SCXK(京)2021-0021];24只雄性SD大鼠[SPF级,北京维通利华实验动物技术有限公司,动物生产许可证号SCXK(京)2021-0006];1只雄性新西兰兔[普通级,北京科宇动物养殖中心,动物生产许可证号SCXK(京)2023-0016]。本实验方案中涉及操作均符合本单位伦理委员会的要求和标准,动物伦理编号:IACUC-DWZX-2024-551。

1.2 材料与仪器

Cohn组分Ⅳ(华兰生物工程股份有限公司);2%兔红细胞混悬液(由兔全血经脱纤处理后获得);硫酸铵、二水合磷酸二氢钠、氯化锌、乙二胺四乙酸二钠二水合物(分析纯);胰蛋白酶(货号:T1426-100MG)、胰蛋白酶抑制剂(货号:T9003-100MG)、Nα-苯甲酰基-DL-精氨酸4-硝基苯胺盐酸盐(货号:B4875-1G)(美国Sigma-Aldrich公司);8%非变性预制胶(货号:GPG00810-N,金克隆生物技术有限公司);非变性非还原性蛋白上样缓冲液5X(货号:P0016N,上海碧云天生物技术股份有限公司);金属螯合层析介质(货号:11-0010-06,中科森辉微球技术有限公司)。

双层玻璃反应釜(型号:S212-3L,上海霍桐实验仪器有限公司);低温高速离心机(型号:CR 30NX,日本Himac公司);AKTA层析系统(型号:purifier explorer 100,美国General Electric公司);中空纤维柱(型号:UFP-300-C-6A,瑞典Cytiva公司);NanoDrop^TM 8000 分光光度计(型号:ND-8000-GL,美国Thermo Fisher Scientific公司);低速台式离心机(型号:TDL-80-2B,上海安亭科学仪器厂);兽用全自动血液细胞分析仪(型号:BC-500Vet,深圳迈瑞生物医疗电子股份有限公司);微纳芯全自动生化分析仪(型号:Pointcare V2,天津微纳芯科技股份有限公司);真空冷冻干燥机(型号:Epsilon 2-4 LSC plus,德国Martin Christ公司);电泳仪(型号:PowerPac-Basic,美国Bio-Rad公司);紧凑型凝胶成像仪(型号:GeLDoc Go,美国Bio-Rad公司);全自动多功能酶标仪(型号:FLUOstar Omega,德国BMG LABTECH公司)。

2 方法

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2.1 α2-M的制备

基于课题组前期建立的方法进行α2-M制备^[18]。所有操作均在室温下进行,取1 000 g的Cohn组分Ⅳ沉淀溶解于5 000 mL的0.9%生理盐水中,经自然沉淀后取上清液。在上清液中缓慢添加硫酸铵(0.313 g·mL^-1)并通过反应釜持续搅拌2 h,使其饱和度达到50%。使用高速离心机离心10 min,得到离心沉淀后再使用蒸馏水重悬。将重悬液使用中空纤维柱超滤除盐,并收集超滤截流液。

将金属螯合层析介质装于层析柱内并连接AKTA层析系统,用氯化锌缓冲液(0.2 mol·L^-1)对金属螯合层析介质进行螯合锌离子处理。先用磷酸二氢钠(NaH₂PO₄)缓冲液(0.02 mol·L^-1 NaH₂PO₄,0.5 mol·L^-1 NaCl,pH值6.0)平衡层析介质约5个柱体积,再将超滤截流液进行上样。上样结束后继续用NaH₂PO₄缓冲液(0.02 mol·L^-1 NaH₂PO₄,0.5 mol·L^-1 NaCl,pH值6.0)淋洗至基线。然后用NaH₂PO₄缓冲液(0.02 mol·L^-1 NaH₂PO₄,0.5 mol·L^-1 NaCl,pH值5.3)淋洗至基线。最后使用乙二胺四乙酸二钠(EDTA-2Na)缓冲液(0.1 mol·L^-1 EDTA-2Na,pH值6.7)洗脱。用中空纤维柱对EDTA-2Na洗脱液进行二次超滤除盐,再用0.22 μm滤膜对二次超滤截流液除菌过滤,过滤后的蛋白溶液分装于西林瓶中进行冻干。

2.2 非变性聚丙烯酰胺凝胶电泳鉴定

将α2-M与非变性非还原性蛋白上样缓冲液混匀,上样10 μg蛋白。连接电泳装置,起始电压设为80 V恒压,30 min后电压调整为120 V恒压,当指示剂电泳至底部结束电泳。将凝胶转移至考马斯亮蓝染色液中染色20~30 min,使用脱色液脱色3次后用凝胶成像仪拍照。最后使用Image Lab软件分析凝胶条带进行纯度鉴定。

2.3 蛋白酶抑制活性检测

首先用40 μL样品与40 μL胰蛋白酶(20 μg·mL^-1)在37 ℃下孵育10 min。孵育结束后,加入12 μL大豆胰蛋白酶抑制剂(100 μg·mL^-1),37 ℃孵育10 min;然后加入75 μL的Nα-苯甲酰基-DL-精氨酸4-硝基苯胺盐酸盐(1 mg·mL^-1),37 ℃孵育30 min;最后在冰浴5 min后,加入25 μL的体积分数30%冰乙酸终止反应,酶标仪读取410 nm处的吸光度。用胰蛋白酶建立相应吸光度的标准曲线,通过标准曲线计算α2-M捕获的胰蛋白酶量^[19]。

2.4 异常毒性试验

根据《中国药典》2020年版三部关于生物制品相关研究内容设计了针对小鼠和豚鼠的异常毒性试验,试验方案设计见表1。

2.5 体外溶血试验

采用体外试管法,将本品复溶后观察制品在体外是否引起兔红细胞溶血或凝聚,试验设计分组见表2。将溶液混匀后,在37 ℃恒温箱中孵育。分别于15、30、45 min、1、2、3 h观察溶血情况。如溶液呈透明红色,即表示溶血;如溶液中有棕红色或红棕色絮状沉淀,振摇后不分散,表示有红细胞凝聚作用。吸取3 h后各组样品的上清液200 μL于96孔板中,用酶标仪测试545 nm处的光密度值,每管样品3个复孔测定(公式1)。

公式(1)溶血率(%)= $\frac{O{D}_{t}-O{D}_{nc}}{O{D}_{pc}-O{D}_{nc}}$×100%

OD_t:试验组光密度,OD_nc:阴性对照组光密度,OD_pc:阳性对照组光密度。溶血率>5%表明出现了溶血。

2.6 刺激性试验

为考察α2-M制品对给药部位及全身产生的毒性作用,设计了单次给药的药物刺激性试验。取24只雄性SD大鼠,将大鼠分为2组:盐水注射组和α2-M注射组,每组12只,注射体积为20 μL,采用关节腔内给药,α2-M剂量为每只0.15 mg。将大鼠右膝关节周围备皮,取仰卧位固定,以右膝关节外侧膝眼为进针点,屈膝90°后用胰岛素注射器向关节腔内垂直进针,有明显落空感时给药。试验期间每天对大鼠进行注射部位局部观察(主要包括坏死、出血和感染等)和大鼠一般情况观察(外观、行为活动、精神状态)。分别于3和14 d将盐水注射组和α2-M注射组各取6只对膝关节组织进行大体观察。

2.7 血常规与血生化

对“2.6”项下24只雄性SD大鼠分别于3、14 d将盐水注射组和α2-M注射组的6只大鼠在取材前进行血常规检测、血液生化学指标检测。

2.8 统计学处理

采用SPSS 26.0统计学软件进行数据分析,连续性变量资料(血常规、血生化数据)用中位数和四分位间距表示,采用曼-惠特尼秩和检验比较两组数据之间的差异。P<0.05表示具有显著性差异。

3 结果

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3.1 α2-M的制备、纯度鉴定以及活性检测

人血浆Cohn组分Ⅳ经硫酸铵粗纯和锌离子亲和层析两步法分离提纯α2-M。层析图(图1A)可见4个主要的峰,a峰为蛋白上样峰,b峰为NaH₂PO₄缓冲液(0.02 mol·L^-1 NaH₂PO₄,0.5 mol·L^-1 NaCl,pH值6.0)的蛋白洗脱峰,c峰为NaH₂PO₄缓冲液(0.02 mol·L^-1 NaH₂PO₄,0.5 mol·L^-1 NaCl,pH值5.3)的蛋白洗脱峰,d峰为EDTA-2Na缓冲液(0.1 mol·L^-1 EDTA-2Na,pH值6.7)的α2-M洗脱峰。通过电泳进行鉴定(图1B),从泳道c、d中可以看到经过硫酸铵粗纯处理后,将组分Ⅳ溶液中电泳迁移率低于α2-M的部分杂蛋白予以去除。与α2-M标准品(泳道a)相比,泳道f、g、h同样显示出高纯度的α2-M特征,这证实了经过锌离子亲和层析步骤后收集的蛋白溶液是高纯度的α2-M溶液;泳道i为超滤后去除EDTA-2Na的α2-M制备品,纯度为95%。然后采用胰蛋白酶活性试验来评价人血α2-M制品的生物活性。通过课题组前期建立的α2-M活性检测方法,测定了α2-M的活性为(6.835±0.104)μg·mL^-1。

3.2 异常毒性试验

试验组小鼠、豚鼠经腹腔注射α2-M 30 min后,未见异常反应。饲养观察期间,试验组和对照组小鼠、豚鼠全部健存,且无异常反应。观察期结束时,小鼠、豚鼠体质量均增加。

3.3 体外溶血试验

肉眼观察法:在3 h的观察结束后,阴性对照组和5个供试品管均未观察到溶血和凝聚现象。经振摇后,供试品管内液体可均匀分散。阳性对照管可见溶血,且管底无红细胞残留,结果见表3。分光光度法:3 h后,从试管中吸取上清液,并在545 nm处测定OD值。可以观察到1~5号管均有轻微溶血现象。结果见表4。综合以上结果,可以判定试验中的人α2-M表现出轻微溶血,但未观察到红细胞凝聚现象。

3.4 刺激性试验

刺激性试验显示,生理盐水注射组和α2-M关节腔内注射组的大鼠在给药30 min后大鼠活动量较注射前减少,均出现右后肢跛行情况。在给药后第2天开始每日观察至第14天结束,生理盐水组、α2-M组大鼠外观、行为、活动均正常,未见跛行,体质量增加;膝关节注射部位均未见感染、红肿和坏死等情况。给药后3、14 d分别观察关节腔内的膝关节滑膜及关节软骨情况,生理盐水注射组和α2-M关节腔内组织均未见明显差异。

3.5 血常规与血生化

血常规检查和血生化结果,见表5~6。与盐水对照组比较,给药3 d可见大鼠α2-M注射组血小板压积(PCT)数值增加,差异有显著性意义(P<0.05);给药14 d可见大鼠α2-M注射组嗜碱性粒细胞(Bas)计数增加,差异有显著性意义(P<0.05),血常规及血液生化其余各检查指标均未见明显差异。

4 讨论

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α2-M在人体中主要以天然型存在,天然型α2-M是血液中的常见成分,当它与蛋白酶结合后会转变为结构更紧凑的变构型α2-M^[7-8]。有研究显示,天然型α2-M在经铵盐、冷冻、冻干等条件处理后也会发生构象改变^[20-21]。本实验室建立的α2-M制备方法涉及复杂的工艺过程,存在产生变构型α2-M的可能性。此外,在目前α2-M的制备流程中,EDTA-2Na作为洗脱剂用于最终纯化阶段,有可能为最终产品引入杂质。制备过程中产生变构体及引入杂质可能影响到制品的安全性,因此本实验中通过溶血性试验、刺激性试验、异常毒性试验和血液相关化验指标来评价α2-M制品的安全性。

制备完成后,首先采用蛋白电泳技术对α2-M进行了纯度鉴定,确保其达到了高纯度标准。随后通过活性检测来确认制备的α2-M是否具备捕获胰蛋白酶的经典功能。在溶血试验中,通过肉眼观察法并未发现明显的溶血和聚集现象。然而,通过分光光度法分析检测到轻微溶血的存在。有研究表明,同为血液制品的静脉注射用免疫球蛋白在大剂量治疗后可能会引起严重的溶血反应,这可能与其中含有的较高滴度抗-A和抗-B血凝素有关^[22]。虽然通过蛋白电泳并未检测到α2-M制品中有其他明显的杂蛋白出现,鉴于溶血试验的结果,未来有必要对制品中可能残留的抗-A或抗-B血凝素进行检测。血常规与血生化检验结果表明,与盐水对照组相比,大鼠在给药3 d后PCT表现出显著性增高(P<0.05);而在给药14 d后Bas计数也出现显著性增高(P<0.05)。尽管这些指标与对照组相比具有统计学上的显著性差异,但大鼠的PCT(正常范围0.2%~0.78%)、Bas(正常范围每升血液中含嗜碱性粒细胞数量0~0.2×10⁹·L^-1)均在各自的正常生理范围内,因此认为这些变化不具有明显的生物学意义。刺激性试验中单次注射人α2-M后,未观察到对大鼠关节腔及周围软组织的明显局部刺激性作用,且大鼠的一般状态良好。异常毒性试验中小鼠和豚鼠正常生长,体质量增加,表明该制剂对实验动物无毒性。

α2-M作为一种广谱蛋白酶抑制剂,除骨关节炎外,在阿尔兹海默症^[23]、慢性阻塞性肺疾病^[24]、心血管疾病^[25]等多种疾病中均显示出重要的诊疗价值。因此,未来人α2-M制品存在多种给药途径的可能性。EDTA-2Na在静脉制剂中过量使用时,可能与钙离子结合形成络合物,导致血钙水平下降^[26]。目前的制备工艺尚未对EDTA-2Na进行定量控制,在后续的研究中需建立EDTA-2Na残留量检测方法并对制品中EDTA-2Na的残留量进行检测,根据给药途径进一步评估其对药品安全性的影响。

变构型α2-M也是α2-M在体内的一种存在形式,其可与低密度脂蛋白受体相关蛋白1(low density lipoprotein receptor-related protein 1,LRP1)的补体型重复域(complement-type repeat clusters,CR)Ⅰ、CR Ⅱ、CR Ⅳ相结合,结合后引发LRP1β链的YxxL基序与含磷酸酪氨酸结合结构域的蛋白相互作用从而引发细胞骨架重排与网格蛋白介导内吞^[9,27-28]。变构型α2-M与LRP1的结合还可激活下游信号通路,包括丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)通路,NF-kB通路、磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)通路,以及信号转导子和转录激活子3(signal transducer and activator of transcription 3,STAT3)通路,以往研究已证明变构型α2-M在细胞迁移、增殖和炎症反应过程中扮演重要角色^[29-33]。本实验室的α2-M制备工艺中在粗纯步骤使用了硫酸铵,在后续的研究中需考察硫酸铵在粗纯过程中的作用时间及其含量变化对α2-M构象的潜在影响,以进一步提高产品的安全性。

综上所述,本实验室制备的人α2-M产品各项安全性指标均在合理范围内,据此可初步判定本品具有临床前安全性。在后期的研究工作中,需要系统地评估新药的安全性、有效性,并深入探究其药理作用机制。本实验室将从原料的筛选、给药方式的优化、给药部位的选择,以及针对特定疾病类型的治疗策略等多个维度进行研究,旨在提升疗效和减少不良反应,从而为新药的开发提供科学依据和实验支持。

基金

收起

北京市科技计划项目资助(Z221100007922031)
北京市自然科学基金项目资助(7244471)

参考文献

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参考文献引证文献

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2025年第60卷第9期

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doi: 10.11669/cpj.2025.09.006

接收时间：2024-12-17
首发时间：2025-11-11
出版时间：2025-05-01

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作者

出版历史

收稿日期：2024-12-17

基金

北京市科技计划项目资助(Z221100007922031)

北京市自然科学基金项目资助(7244471)

作者信息

¹ 山西医科大学, a.第二临床医学院, b.药学院, 太原 030001

² 军事医学研究院, 国家药品监督管理局血液制品质量控制重点实验室, 北京 100850

通讯作者:

^*陈崇伟,男,博士,教授,硕士生导师研究方向:骨关节退行性疾病及损伤 Tel:(0351)3363158;

马玉媛,女,博士,副研究员,硕士生导师研究方向:生物制品及其病毒安全性 Tel:(010)66932222

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Item	Mice	Guinea pigs
Mass/g	18~22	250~350
Number	Control group and experimental group with 5 each	Control group and experimental group with 2 each
Injection dose/mL	0.1 mL(0.015 mg α2-M)	1 mL(0.15 mg α2-M)
Administration frequency	Single administration	Single administration
Administration route	Intravenous injection	Intravenous injection
Criteria for determination	During the breeding observation period, all mice and guinea pigs in the control and experimental groups should be healthy and show no abnormal reactions. If the weight of each mouse and guinea pig increases, it is considered to meet the criteria after observing for 7 d

Item

Mice

Guinea pigs

Mass/g

18~22

250~350

Number

Control group and experimental group with 5 each

Control group and experimental group with 2 each

Injection dose/mL

0.1 mL(0.015 mg α2-M)

1 mL(0.15 mg α2-M)

Administration frequency

Single administration

Administration route

Intravenous injection

Criteria for determination

During the breeding observation period, all mice and guinea pigs in the control and experimental groups should be healthy and show no abnormal reactions. If the weight of each mouse and guinea pig increases, it is considered to meet the criteria after observing for 7 d

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number

2% Red blood cell suspension

0.9% Sodium chloride injection

Distilled water

α2-M solution

2.5

2.0

0.5

2.5

2.1

0.4

2.5

2.2

0.3

2.5

2.3

0.2

2.5

2.4

0.1

2.5

Observation time	Test tube 1	Test tube 2	Test tube 3	Test tube 4	Test tube 5	Test tube 6	Test tube 7
15 min	-	-	-	-	-	-	+
30 min	-	-	-	-	-	-	+
45 min	-	-	-	-	-	-	+
1 h	-	-	-	-	-	-	+
2 h	-	-	-	-	-	-	+
3 h	-	-	-	-	-	-	+
Determination result	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	No hemolysis No agglutination	Complete hemolysis

Observation time

Test tube 1

Test tube 2

Test tube 3

Test tube 4

Test tube 5

Test tube 6

Test tube 7

15 min

30 min

45 min

1 h

2 h

3 h

Determination result

No hemolysis
No agglutination

Complete hemolysis

Item	Plasma/mL	Physiological saline/mL	Distilled water/mL
OD_{545 nm}	0.235±0.012	0.236±0.008	0.248±0.009	0.262±0.004	0.246±0.004	0.201±0.006	0.690±0.003
Hemolysis rate/%	6.9	7.1	9.6	12.5	9.2
Hemolysis result	+	+	+	+	+	-	-

Item

Plasma/mL

Physiological saline/mL

Distilled water/mL

Test tube 1'

Test tube 2'

Test tube 3'

Test tube 4'

Test tube 5'

Test tube 6'

Test tube 7'

0.5

0.4

0.3

0.2

0.1

2.5

OD_{545 nm}

0.235±0.012

0.236±0.008

0.248±0.009

0.262±0.004

0.246±0.004

0.201±0.006

0.690±0.003

Hemolysis rate/%

6.9

7.1

9.6

12.5

9.2

Hemolysis result

Parameters	After administration 3 d	After administration 14 d
WBC/×10⁹·L^-1	8.100 0(6.032 5-9.732 5)	9.600 0(6.970 0-10.315 0)	9.085 0(7.465 0-11.575 0)	11.215 0(10.107 5-12.230 0)
Neu/×10⁹·L^-1	1.185 0(0.960 0-1.392 5)	1.385 0(1.045-1.572 5)	1.390 0(1.197 5-1.660 0)	1.480 0(1.140 0-1.715 0)
Lym/×10⁹·L^-1	6.740 0(4.362 5-7.735 0)	7.585 0(5.500 0-8.087 5)	6.785 0(5.667 5-9.610 0)	8.885 0(7.660 0-10.112 5)
Mon/×10⁹·L^-1	0.380 0(0.275 0-0.492 5)	0.475 0(0.360 0-0.580 0)	0.535 0(0.455 0-0.687 5)	0.670 0(0.575 0-0.782 5)
Eos/×10⁹·L^-1	0.080 0(0.067 5-0.090 0)	0.085 0(0.065 0-0.115 0)	0.100 0(0.060 0-0.127 5)	0.115 0(0.110 0-0.125 0)
Bas/×10⁹·L^-1	0.010 0(0.010 0-0.027 5)	0.015 0(0.010 0-0.022 5)	0.010 0(0.007 5-0.015 0)	0.020 0(0.020 0-0.020 0)¹⁾
RBC/×10⁹·L^-1	6.440 0(6.032 5-6.880 0)	6.420 0(6.350 0-6.572 5)	6.665 0(6.480 0-6.910 0)	6.925 0(6.612 5-7.455 0)
HGB/g·L^-1	147.500 0(142.000 0-150.500 0)	147.000 0(143.750 0-157.000 0)	148.500 0(145.500 0-152.250 0)	151.000 0(149.250 0-156.250 0)
HCT/%	38.950 0(37.475 0-40.275 0)	38.850 0(38.775 0-41.225 0)	39.550 0(38.025 0-40.200 0)	39.350 0(38.300 0-41.925 0)
MCV/fL	59.350 0(58.075 0-63.375 0)	61.500 0(60.100 0-62.900 0)	59.050 0(57.500 0-60.175 0)	57.300 0(55.925 0-58.325 0)
MCH/pg	22.250 0(21.700 0-24.275 0)	22.950 0(22.675 0-23.725 0)	22.200 0(21.450 0-23.250 0)	22.050 0(21.375 0-22.550 0)
MCHC/g·L^-1	376.000 0(373.000 0-381.000 0)	377.000 0(369.750 0-379.750 0)	375.500 0(372.750 0-384.250 0)	382.500 0(377.000 0-390.250 0)
PLT/×10⁹·L^-1	896.500 0(793.250 0-990.500 0)	1036.500 0(951.750 0-1 160.500 0)	919.000 0(793.250 0-1 048.250 0)	858.000 0(729.500 0-950.750 0)
PDW	15.250 0(15.175 0-15.375 0)	15.450 0(15.375 0-15.500 0)	15.300 0(15.200 0-15.500 0)	15.300 0(15.225 0-15.400 0)
PCT/%	0.523 0(0.458 25-0.594 25)	0.621 5(0.581 5-0.705 5)¹⁾	0.544 0(0.474 25-0.614 5)	0.551 0(0.422 5-0.5707 5)

Parameters

After administration 3 d

After administration 14 d

Saline group

α2-M group

Saline group

α2-M group

WBC/×10⁹·L^-1

8.100 0(6.032 5-9.732 5)

9.600 0(6.970 0-10.315 0)

9.085 0(7.465 0-11.575 0)

11.215 0(10.107 5-12.230 0)

Neu/×10⁹·L^-1

1.185 0(0.960 0-1.392 5)

1.385 0(1.045-1.572 5)

1.390 0(1.197 5-1.660 0)

1.480 0(1.140 0-1.715 0)

Lym/×10⁹·L^-1

6.740 0(4.362 5-7.735 0)

7.585 0(5.500 0-8.087 5)

6.785 0(5.667 5-9.610 0)

8.885 0(7.660 0-10.112 5)

Mon/×10⁹·L^-1

0.380 0(0.275 0-0.492 5)

0.475 0(0.360 0-0.580 0)

0.535 0(0.455 0-0.687 5)

0.670 0(0.575 0-0.782 5)

Eos/×10⁹·L^-1

0.080 0(0.067 5-0.090 0)

0.085 0(0.065 0-0.115 0)

0.100 0(0.060 0-0.127 5)

0.115 0(0.110 0-0.125 0)

Bas/×10⁹·L^-1

0.010 0(0.010 0-0.027 5)

0.015 0(0.010 0-0.022 5)

0.010 0(0.007 5-0.015 0)

0.020 0(0.020 0-0.020 0)¹⁾

RBC/×10⁹·L^-1

6.440 0(6.032 5-6.880 0)

6.420 0(6.350 0-6.572 5)

6.665 0(6.480 0-6.910 0)

6.925 0(6.612 5-7.455 0)

HGB/g·L^-1

147.500 0(142.000 0-150.500 0)

147.000 0(143.750 0-157.000 0)

148.500 0(145.500 0-152.250 0)

151.000 0(149.250 0-156.250 0)

HCT/%

38.950 0(37.475 0-40.275 0)

38.850 0(38.775 0-41.225 0)

39.550 0(38.025 0-40.200 0)

39.350 0(38.300 0-41.925 0)

MCV/fL

59.350 0(58.075 0-63.375 0)

61.500 0(60.100 0-62.900 0)

59.050 0(57.500 0-60.175 0)

57.300 0(55.925 0-58.325 0)

MCH/pg

22.250 0(21.700 0-24.275 0)

22.950 0(22.675 0-23.725 0)

22.200 0(21.450 0-23.250 0)

22.050 0(21.375 0-22.550 0)

MCHC/g·L^-1

376.000 0(373.000 0-381.000 0)

377.000 0(369.750 0-379.750 0)

375.500 0(372.750 0-384.250 0)

382.500 0(377.000 0-390.250 0)

PLT/×10⁹·L^-1

896.500 0(793.250 0-990.500 0)

1036.500 0(951.750 0-1 160.500 0)

919.000 0(793.250 0-1 048.250 0)

858.000 0(729.500 0-950.750 0)

PDW

15.250 0(15.175 0-15.375 0)

15.450 0(15.375 0-15.500 0)

15.300 0(15.200 0-15.500 0)

15.300 0(15.225 0-15.400 0)

PCT/%

0.523 0(0.458 25-0.594 25)

0.621 5(0.581 5-0.705 5)¹⁾

0.544 0(0.474 25-0.614 5)

0.551 0(0.422 5-0.5707 5)

Parameters	After administration 3 d	After administration 14 d
ALB/g·L^-1	38.050 0(37.275 0-38.950 0)	38.000 0(36.925 0-39.000 0)	39.250 0(39.050 0-41.300 0)	39.500 0(38.575 0-41.225 0)
GLB/g·L^-1	11.950 0(9.900 0-13.200 0)	13.100 0(11.500 0-14.125 0)	14.800 0(14.025 0-17.250 0)	14.700 0(13.800 0-15.700 0)
TBIL/μmol·L^-1	4.530 0(3.460 0-4.705 0)	3.360 0(2.480 0-4.400 0)	3.675 0(2.965 0-4.477 5)	2.800 0(2.18 5-3.425 0)
ALT/U·L^-1	36.000 0(33.500 0-50.250 0)	43.000 0(38.750 0-46.500 0)	49.000 0(44.500 0-50.750 0)	44.500 0(41.500 0-47.000 0)
AST/U·L^-1	172.000 0(153.250 0-203.250 0)	215.000 0(197.500 0-261.000 0)	200.000 0(164.250 0-259.250 0)	166.000 0(142.000 0-204.750 0)
GGT/U·L^-1	0.800 0(0.475 0-1.325 0)	1.050 0(0.750 0-1.700 0)	1.200 0(0.875 0-1.700 0)	0.450 0(0.250 0-1.500 0)
BUN/mmol·L^-1	5.045 0(4.870 0-6.850 0)	5.090 0(4.775 0-5.405 0)	5.800 0(5.187 5-6.047 5)	5.040 0(4.115 0-5.400 0)
Cr/μmol·L^-1	43.000 0(40.750 0-46.500 0)	43.000 0(37.500 0-51.500 0)	53.500 0(50.750 0-59.750 0)	52.000 0(49.500 0-54.250 0)

Parameters

After administration 3 d

After administration 14 d

Saline group

α2-M group

Saline group

α2-M group

ALB/g·L^-1

38.050 0(37.275 0-38.950 0)

38.000 0(36.925 0-39.000 0)

39.250 0(39.050 0-41.300 0)

39.500 0(38.575 0-41.225 0)

GLB/g·L^-1

11.950 0(9.900 0-13.200 0)

13.100 0(11.500 0-14.125 0)

14.800 0(14.025 0-17.250 0)

14.700 0(13.800 0-15.700 0)

TBIL/μmol·L^-1

4.530 0(3.460 0-4.705 0)

3.360 0(2.480 0-4.400 0)

3.675 0(2.965 0-4.477 5)

2.800 0(2.18 5-3.425 0)

ALT/U·L^-1

36.000 0(33.500 0-50.250 0)

43.000 0(38.750 0-46.500 0)

49.000 0(44.500 0-50.750 0)

44.500 0(41.500 0-47.000 0)

AST/U·L^-1

172.000 0(153.250 0-203.250 0)

215.000 0(197.500 0-261.000 0)

200.000 0(164.250 0-259.250 0)

166.000 0(142.000 0-204.750 0)

GGT/U·L^-1

0.800 0(0.475 0-1.325 0)

1.050 0(0.750 0-1.700 0)

1.200 0(0.875 0-1.700 0)

0.450 0(0.250 0-1.500 0)

BUN/mmol·L^-1

5.045 0(4.870 0-6.850 0)

5.090 0(4.775 0-5.405 0)

5.800 0(5.187 5-6.047 5)

5.040 0(4.115 0-5.400 0)

Cr/μmol·L^-1

43.000 0(40.750 0-46.500 0)

43.000 0(37.500 0-51.500 0)

53.500 0(50.750 0-59.750 0)

52.000 0(49.500 0-54.250 0)

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-

Test tube number	2% Red blood cell suspension	0.9% Sodium chloride injection	Distilled water	α2-M solution
1	2.5	2.0	-	0.5
2	2.5	2.1	-	0.4
3	2.5	2.2	-	0.3
4	2.5	2.3	-	0.2
5	2.5	2.4	-	0.1
6	2.5	2.5	-	-
7	2.5	-	2.5	-