WO2015032233A1 - 重组灵芝免疫调节蛋白在制备治疗局灶性脑缺血药物中的应用 - Google Patents

重组灵芝免疫调节蛋白在制备治疗局灶性脑缺血药物中的应用 Download PDF

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WO2015032233A1
WO2015032233A1 PCT/CN2014/079816 CN2014079816W WO2015032233A1 WO 2015032233 A1 WO2015032233 A1 WO 2015032233A1 CN 2014079816 W CN2014079816 W CN 2014079816W WO 2015032233 A1 WO2015032233 A1 WO 2015032233A1
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rlz
focal cerebral
recombinant
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张喜田
孙非
梁重阳
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Zhang Xitian
Sun Fei
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Priority to US14/912,914 priority Critical patent/US20160199439A1/en
Priority to EP14843115.8A priority patent/EP3028712B1/en
Publication of WO2015032233A1 publication Critical patent/WO2015032233A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • the present invention belongs to the field of oyster medicinal, and relates to the use of recombinant 3 ⁇ 4 ⁇ immunomodulatory protein (rLZ 8) expressed by Pichia ia pas tori s in the preparation of a medicament for treating focal cerebral deficiency.
  • rLZ 8 recombinant 3 ⁇ 4 ⁇ immunomodulatory protein
  • Ganoderma lucidum is a precious medicinal fungus in traditional Chinese medicine, belonging to the genus Basidiomycetes i3 ⁇ 4 Ganoderma lucidum.
  • Ganoderma lucidum rabbit regulatory protein was first isolated from the extract of Ganoderma lucidum fruit extract by Kino et al., consisting of 1 10 3 ⁇ 4 base acids, N-terminal silk acid acetylation, protein molecular mass is i.2 , 4kD, and named LZ 8.
  • LZ 8 protein molecular mass is i.2 , 4kD, and named LZ 8.
  • Cerebral ischemia is a kind of accident of brain M tube. Cerebrovascular accident, also known as stroke and stroke, is a common and frequently-occurring disease in middle-aged and elderly people. It seriously threatens the well-being and life of human beings. With the continuous development of science and technology, researchers have conducted in-depth research on the pathogenesis of cerebral ischemia and the cascade of brain damage in brain tissue, and found that it can inhibit cell apoptosis, reduce inflammation and free radicals. Relieve brain damage
  • the recombinant Antrodia camphorata immunomodulating egg of the invention not only can improve the behavioral dysfunction caused by cerebral ischemic injury, but also can alleviate brain tissue damage by reducing inflammatory reaction and inhibiting apoptosis, and regulating egg by applying recombinant t art rabbit disease
  • the development of drugs for the treatment of cerebral ischemic injury has a positive effect on the "Description"
  • the present invention relates to the use of recombinant Ganoderma lucidum immunoregulatory protein rLZ 8 in the preparation of the treatment of focal cerebral dysfunction 1 & drug, through a series of experimental means and knots show that TLZ 8 has significant significance in peripheral focal cerebral ischemia in rats Therapeutic effect, specific invention content ⁇ ;
  • the present invention takes rats as a research object, and adopts a modified Zea Longa method to prepare a rat model of focal cerebral ischemia.
  • the model is designed to include a negative control group (sham operation group) and a positive drug control group (single sialic acid hexose Ganglioside sodium injection (GM-i), model group, rLZ-8 high dose group (70 ⁇ g . kg' s ), rLZ 8 medium dose group ( 35 ⁇ g « kg .. s ), rLZ 8 low-dose group ( ⁇ .
  • the dead area is larger: the brain tissue of the high-dose group of ⁇ 8 has different degrees of improvement compared with the model group, which is characterized by nucleus pyknosis, mild pathological changes such as dissolution, and small necrotic area.
  • PV 6000 two-way immunohistochemical detection reagent to detect the expression of ED i positive cells in experimental rats the results showed that compared with the model group, the number of ED 1 positive cells in the high dose group of rLZ 8 was significantly reduced, which could reduce brain deficiency. An inflammatory reaction caused by blood damage.
  • the TUNEL kit was used to detect apoptosis, and the test was carried out according to the instructions of the kit. The results showed that the number of apoptotic cells in the rLZ dose group was significantly lower than that in the model group, and the number of apoptosis gradually increased with the increase of rLZ 8 concentration. Reduction:. Further explanation, the therapeutic effect of rLZ 8 on focal cerebral ischemia
  • S is the sham operation group
  • M is the model group
  • m-l is the positive drug control group
  • H is the high dose group
  • Z is the rLZ medium dose group
  • L is! 8 low dose group
  • ⁇ is a sham operation group
  • B is a model group
  • C is a GM i group
  • ⁇ ) is a rLZ 8 3 ⁇ 4 dose group
  • E is a rLZ 8 medium dose group
  • A is the sham operation group
  • B is the model group
  • C is 1 group
  • D is rLZ 8 high dose group
  • E is rLZ 8 medium dose group
  • F is rLZ «low dose group
  • Circle 4 modeled apoptotic cells (40 X) after 8 days
  • A is a sham operation group
  • B is a model group
  • C is GM
  • D is r!.2TM8 high dose group
  • E is r!.2TM8 medium dose group
  • F is rLZ 8 low dose group
  • Example 1 Treatment of nerve functional spasm with rLZ 8
  • the model of focal cerebral ischemia was prepared by modified Zea Longa method. After the rats were fully awake, they were scored daily according to the modified SS score (16-point system).
  • Positive control drug is monosialic acid Hexose ganglioside sodium injection (GM i )
  • Table 1 shows that the animals in each drug-administered group and model group showed different degrees of neurological dysfunction after modeling (on the same day, postoperatively), and the model group had the most ffi, rLZ ⁇ S avian, low-dose group score. Significant improvement (/ tt indicates that rLZ 8 W reduces neurological function scores and improves neurological function.
  • Model ⁇ rats were weighed, and the model of focal cerebral ischemia was prepared by modified 3 ⁇ 4a Longa method. Weighed on the first day, after 7 days of continuous administration, weighed
  • Table 2 and Figure i show that there was no significant difference in body weight between the groups in the model group. After the model, the body weight of the rats in each group was significantly reduced. After 7 days of administration, the weight of the rats in the high dose group of ⁇ -8 increased. It is shown to reduce the damage caused by cerebral ischemia.
  • Perfusion fixation rats were fixed in supine position after anaesthesia with hydrated gas aldehyde, open and expose the heart, insert the perfusion needle into the ascending aorta through the tip of the left ventricle, first) 3 ⁇ 4 rapid perfusion of normal saline, while cutting the right atrial appendage, hemostasis clamp Abdominal aorta, when the effluent does not see blood color, rapid perfusion fixative": stop the perfusion when the body becomes hard, and remove the brain tissue from the broken head. Coronary brain slices were prepared using a rat brain slice mold, formalin solid, and the horizontal cross-horizon brain slices (3rd slice) were selected, and the H scale was embedded and sliced.
  • Fig. 2 It can be seen from Fig. 2 that the number and morphology of neurons and glial cells in the sham operation group are normal, the cell membrane is intact, the cytoplasm is uniform, the nucleus is located in the center, and the chromatin distribution is relatively uniform: the brain tissue of the model group is sparse, and a large number of necrotic nerves are visible. The cell structure disappeared, the nucleus condensed and stained deeply, the cell necrosis collapsed, the cell gap increased, and the necrotic area was larger. The brain tissue of the rLZ 83 ⁇ 4 dose group showed different degrees of improvement compared with the model group, which showed the nucleus shrinkage. Dissolution, etc., a light change, less necrotic area
  • rLZ-8 is set to high, medium, and low: ⁇ one dose group, with concentration of raw spotted saline with m concentration of 70 ⁇ s? * l ⁇ , 35 ⁇ g ⁇ , 5 ⁇ g ⁇ ! : , EIH antibody (Wuhan De Bio: I Cheng Co., Ltd., PV 6000 two-way immunohistochemical detection reagent (Beijing Zhongshang Jinqiao Biotechnology Co., Ltd.).
  • the experimental group was set in the operation group, the model group, the drug control group, the rLZ 8 high dose group (70 ⁇ g »kg " ! ) vrLZ-S medium dose group ( 35 ⁇ g * kg , rLZ-8 low dose group ( 1 5 ⁇ g ⁇ kg ) honor
  • Example 5 TU EL kit detection of apoptosis rLZ-S was set high, medium, Low, three dose groups, prepared with physiological saline at a concentration of 7 (g * ml 35 ⁇ g ⁇ ⁇ , 17.5 ⁇ g * ml" TUNEL kit

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Abstract

本发明公开了毕赤酵母（Pichiapastoris）所表达的重组灵芝免疫调节蛋白（rLZ_8）在制备治疗局灶性脑缺血药物中的应用。rLZ-8能够治疗神经功能损伤，降低脑损伤引起的神经功能评分，减少ED-1阳性细胞数，从而降低炎性反应，并可抑制细胞凋亡。

Description

技术领域
[0001 ] 本发明属于生狻制药领域，涉及使用毕赤酵母（Pich ia pas tori s)表达的重组 ¾ 芝免疫调节蛋白（rLZ 8)在制备治疗局灶性脑缺 fi药物中的应用。 背景技术
[0002] 灵芝是中医药学中一种珍贵的药用真菌，属担子菌纲多孔菌 i¾灵芝科。 灵芝兔 疫调节蛋白首次由 Kino等从灵芝子实体提取物中分离纯化得到的真菌免疫调节蛋 ，由 1 10个 ¾基酸所组成， N端丝戴酸乙酰化,蛋白分子质量为 i.2, 4kD，并命名为 LZ 8。 很多研 究表明， 具有促迸细胞有 分裂和兔疫调节的作用等多种活性。 重组灵芝免疫调节 ( Recombinant limnunoregii ί a tory Protein of (kmoderma L.uc'idi碰， rLZ 8 ')是经由 rLZ~8真核表达载体 PiChiaPinkra菌株，筛选出稳定高效分泌表达的工程瞎株中提取，并验 证 rLZ 8具有与 LZ 8相同的兔疫学活性与生物活性„
[0003] 脑缺血是脑 M管意外的 -种，脑血管意外又称脑卒中、中风，是中老年人的常见 病、多发病，严重烕胁着人类的徤康与生活《近些年来,随着科学技术的不断发展，研究学者 对脑缺血病发病机制、脑組织细胞损伤级联反应等的深入研究，发现可通过抑制细胞凋亡、 降低炎症反应与自由基等多种途径来缓解脑组织损伤
[0004] 本发明的重组炅芝免疫调节蛋 不仅可改善脑缺血损伤引起的行为功能障碍，而 且可通过降低炎症反应、抑制细胞凋亡缓解脑组织损伤,对应用重组t艺兔疫调节蛋 研 制治疗脑缺血损伤药物有 ······定的积极作用《 发明内容
[0005] 本发明涉及重组灵芝免疫调节蛋白 rLZ 8在制备治疗局灶性脑缺 1&药物中的应 用，通过一系列的实验手段和结梁表明 TLZ 8对大鼠周灶性脑缺血具有显著的治疗作用， 具体发明内容 ^下；
本发明以大鼠作为研究对象，釆用改良的 Zea Longa法制备大鼠局灶性脑缺血.模型， 设计了包括阴性对照组 (假手术组)，阳性药对照组 (单唾液酸已 糖神经节昔脂钠注射液 ( GM-i ))，模型组， rLZ-8高剂量组 ( 70 μ g . kg's)、rLZ 8中剂量组 ( 35 μ g « kg ..s )、 rLZ 8低 剂量组（Π. 5 μ g · k 在内的 6个实验組,。 在给药程序方面设计为：腹麼注射，每天给药 2次，假手术组与模型组注射等体积生理盐水，连续给药 7天 首先,考察了 rLZ 8对神经 功能损伤的治疗作用，按改良的 SS评分标准对其进行评分（1.6分制)，各给药组及模型组 动物在造模后（¾日，术后约 5h )均出现不同程度神经功能障碍， rLZ~8高、低剂量组评分效 果显著 (/ 0. 01 )，说明 rLZ 8可降低神经功能评分，改善神经功能。 有力说明了， rLZ 8对 局灶性脑缺血的治疗作用；通过 HE染色实验,假手术组脑组织神经元及胶质细胞数量、形 态分布正常，细胞膜完整，胞浆均勾，细胞核位于中央，染色质分布较均勻；模型组脑组织稀 疏，可见大量坏死神经元,细胞结构消失，胞核 [11缩深染，细胞坏死崩解，细胞间隙增大，坏 死面积较大：Γϋί···8高剂量组动物脑组织较模型組有不同程度的改善，表现为胞核固缩,溶 解等病理改变较轻，坏死面积较小。 釆用 PV 6000二歩法免疫组化检測试剂对实验大鼠进 行 ED i阳性细胞表达的检溯，结果表明与模型组比较, rLZ 8高剂量组 ED 1阳性细胞数明 显减少，可降低脑缺血损伤引起的炎性反应。衆用 TUNEL试剂盒检測细胞凋亡,按照试剂盒 说明书指导歩骤进行试验,实验结果表明 rLZ 剂量組凋亡细胞数明显低于模型組，并且 随着 rLZ 8浓度的增加，细胞凋亡数逐渐减少:. 进一歩说明了， rLZ 8对局灶性脑缺血的治 疗作用
[0006] 经过上述重组灵芝兔疫调节蛋白 rLZ 8在制备治疗局灶性脑缺血药物中的应用 的一系列实验结果表明， rLZ 8能够治疗和缓解实验大鼠的神经损伤和生存状态，抑制脑 组织细胞蠲亡而治疗脑缺血损伤
[0007] 说明书 |¾图说明
图 i各组大鼠体重变化
其中， S为假手术组 , M为模型组， m-l为阳性药对照組， H为 高剂量組， Z为 rLZ 中剂量组， L为！ 8低剂量组
閱 2各组大鼠脑组织形态学观察（ffi染色, 0 X )
其中， Λ为假手术组， B为糢型组， C为 GM i组， ί)为 rLZ 8 ¾剂量组， E为 rLZ 8中剂 量组， 为 rLZ 8低剂量组
图 3造模 8天后各组 ED 1阳性细胞（40 X )
其中， A为假手术組， B为模型组， C为 1組， D为 rLZ 8高剂量组， E为 rLZ 8中剂 量组， F为 rLZ «低剂量组
圈 4造模 8天后各组凋亡细胞（40 X )
其中， A为假手术组， B为模型组， C为 GM ,组， D为 r!.2™8高剂量組， E为 r!.2™8中剂 量组， F为 rLZ 8低剂量组 实施例 1： rLZ 8对神经功能掼伤的治疗^用
1. 实验材料
大默，体重 250 300g,雌雄各半,由吉林大学实验动物中心提供。 单唾液酸已己糖神经 节普脂钠注射液（GM i )，北京四环制药有 »公司，手术器械。
[0008] 2, 实验方法
釆用改良的 Zea Longa法制备大鼠局灶性脑缺血模型，术后待大鼠完全清醒后，每天 按改良的 SS评分标准对其迸行评分（16分制)。 实验设假手术组,模型组，阳性药对照组， rS.2-8高剂量組（ 70 μ g « kg"! )、 rLZ 8中剂量组 ( 35 ,μ g -kg 1 )、.r:LZ .8低細量组 ( 17. 5 μ g « kg L 腹腔注射，每天给药 2次，假手术组¾模型组注射等体积生理盐水，连续给药 7天。 阳 性对照药为单唾液酸已己糖神经节普脂钠注射液 ( GM i )
[0009] 3. 实验结果
表 1显示，各给药组及模型组动物在造模后（当日，术后约 )均出现不同程度神经功 能障碍，以模型组神经功能评分最 ffi， rLZ~S禽、低剂量组评分可明显改善 (/ tt 说明 rLZ 8 W降低神经功能评分，改善神经功能。
[0010] 表 1 各组大鼠 NSS评分结果（X 士 s , f^i 2)
Figure imgf000005_0001
注：各给药组及模型组与假手术组比较有显著性幾异 / 01
ρ<ϊλ (》5， " ρ<(). 01；
实施例 2 :ri.. H对大鼠体重：的影响
造模 ίίί,对大鼠迸行称重，采用改良的 ¾a Longa法制备大鼠局灶性脑缺血模型，术后 第 1.天称重，连续给药 7天后，称重
[0011] 2. 实验结果
表 2和图 i显示，造模離，各组大鼠体重无明显差异,遗模后，各组大鼠体重明显降低， 给药 7天后， ΠΖ-8高剂量组大鼠体重有所增加，表明 可降低脑缺血引起的损伤。
[0012]
表 2各组大鼠体重变化（X土 s)
Figure imgf000006_0001
注：与假手术組比较 < ΰδ, »*p<a
实施例 3 :HE染色实验
i. 实验方法
灌注固定：大鼠以水合氣醛麻醉后仰卧位固定，开與暴露心脏，将灌注针经左心室尖部 插入升主动脉，先) ¾生理盐水快速灌注，同时剪开右心耳，止血钳夹腹主动脉，当流出液不 见血色时，快速灌注固定液》：身体变硬时停止灌注，断头取出脑组织。利用大鼠脑切片模具 , 制备冠状脑切片，福尔马林固 ，选择视交叉水平脑切片（第 3片）,常规; H鳞包埋，切片。
[0013] 染色：（1)二甲苯 ί、二甲苯 II脱蜡，各 15mi.n :2)无水乙醇 Ϊ、无水乙醇 Π 95%乙醇、 90%乙醇識乙醇水化，各 ¾βίη，蒸馏水， mii ； (3)苏木萦染色， δΊθ η ;(4)流 水吸取苏木素， in ;(5)]%盐酸-乙醇分化， Γ¾ί ;(6)返蓝，流水洗 Γ— 3 i.n ;(7)伊红染 色, Γ ； (8)蒸馏水洗，广;¾ ;(9)脱水、透明,中性树胶封片；（1.0)光学显微镜下初歩 观察病理切片《
[0014] 2. 实验结果
由图 2可见，假手术组脑组织神经元及胶质细胞数量、形态分布正常，细胞膜完整，胞 浆均匀，细胞核位于中央，染色质分布较均匀：模型组脑组织稀疏，可见大量坏死神经 ¾，细 胞结构消失，胞核固缩深染，细胞坏死崩解,细胞间隙增大，坏死面积较大； rLZ 8¾剂量组 动物脑组织较模型組有不同程度的改善，表现为胞核画缩、溶解等病 a改变较轻，坏死面积 较小
[0015] 实施劍 4 iri2~B可降低炎性反应
I. 实验试剂
rLZ-8分别设高、中、低： Ξ个剂量组，用生斑盐水配 m成浓度为 70 μ s? * l}, 35 μ g Π, 5 μ g ιΓ! :, EIH抗体（武汉博士德生物: I程有限公司）， PV 6000二歩法免 疫组化检测试剂 (北京中杉金桥生物技术有限公司）。
[0016] 2. 实验分组
实验设煆手术组，模型组， l 性药对照組, rLZ 8高剂量组（70 μ g »kg "!)vrLZ-S中剂量 组（ 35 μ g * kg 、 rLZ-8低剂量组（ 1 5 μ g · kg )„
[0017] 3. 实验方法
常规石蜡包埋、切片后，釆用免疫组织化学染色的方法检測 ED»i阳性细胞的衰达 (1) 烤片：脑組织石鳞切片置于 6ΰΌ烤箱中 Γ60 η，自然浪； (2)脱蜡；脑组织切片于二甲苯 ί >二甲苯 ίί各 lomin : (3)水化；脑组织切片干 水乙醇 I、无水乙醇 Ϊ1 ,90%乙醇、 70%乙 醇、蒸馏水中各 2ffiin ;(4)预处理：根据所应用的一抗的特殊要求，对组织切片进行预处理。 0. (UM枸橼酸盐缓冲液（pH6.0)热修复抗原；（5) 3%¾¾去离子水婦育 iOmi 以阻断内源 性过氧化物酶， PBS清洗 3次，每次 in ;(6) '滴加 》 1抗体， 过夜， PBS清洗 3次，每 次 δίϊίϊη ;(7)滴加通用型 igG抗体 HRP多聚体，室温鮮育 1.5mi PBS淸洗 3次，每次 5mw； (8)应用 DAB溶液显色；（9)蒸馏水冲洗、苏木精复染、胶水、透明，中性树胶封片；（ίθ)光学 S徵镜下观察。
[0018] 4. 实验结果
型组比较，; riLZ 8高剂量组 1¾> 1阳性细胞数明显减少，可降低
Figure imgf000007_0001
[0019] 表 3各组造模 S天后 ED— 1阳性细胞数 (X 土 s)
Figure imgf000007_0002
与假手术比较 05 , <0.01；与模型组比较 Κ¾ 05 ；与 G -1组比较 05.β [0020] 实施例 5： TU EL试剂盒检測细胞凋亡 rLZ-S分别设高.、中、低,：三个剂量组，用生理盐水配制成浓度为 7( g * ml 35 μ g · ΓηΓ、 17.5μ g * ml" TUNEL试剂盒
[0021] 2, 实验分组
实验设假手术組，模型组，阳性药对照组， rLZ 8高剂量组（70 μ g · i ^M^中剂量 组（35μ g♦ βίΓ' ΐΙΖ--·^低剂量组（Π.5μ g « atl"!);:
[0022] 3, 实验方法
常规石蜡包埋、切片，按 TUMiL试剂盒说明书操作。 （1)脑组织 ^蜡切片常规脱蜡、水 化；（2) 胰 S白酶处理， 3 t，暗湿盒中 l^ i ;(3) PBS灘 2次，每次 ftrain ;(4)加 50 μ I TONEL反应混合液（阴性对照组尽加 50 n 1荧光素标记的 dUTP液)于标本上，在 暗湿盒中反应， Ih； (δ) PBS漂洗 3次，每次 5rain； (6)加 50 μ 1 Converter POD 于标本」::， 在 暗湿盒中反应， ； ( 7)PBS漂洗 3次，每次 5mi n； (8)在组织处加 5 "ί00 μ Ϊ DAB 溶液显色；（9)蒸馏水冲洗、苏木精复染、脱水、透明，中性树胶封片；（10)光学显微镜下观 察,：
[0023] 4. 实验结果
由表 4和图 4可见， rLZ»8剂量组 «亡细艇数明显低于模型组，并且随着 TLZ 8浓度的 增加，细胞凋亡数遂渐减少。
[00243 表 4各组造模 8天后凋亡细胞数（X 土 s)
Figure imgf000008_0001
GM ί组比较 - 01„

Claims

ΨΪ 要 氷 书
1. 重组灵芝免疫调节蛋白在制备治疗局灶性脑缺血药物中的应用。
2. 如权利要求 1所述的治疗局灶性脑缺血換伤的应用,其特征在于重组灵芝兔疫调节 蛋 !ίΐ可降低 ώ脑缺血损伤引起的神经功能评分
3. 如权利要求 1所述的治疗局灶性脑缺血损伤的应用，其特征在 : 重组灵芝免疫调节 蛋 e可减少 阳性细胞数，降低炎性反应，治疗脑缺血损伤。
4. 如权利要求 ί所述的治疗局灶性脑缺血损伤的应用，其特征在于重組 ¾芝免疫调节 蛋1¾可降低凋亡细胞数，神制脑组织细胞凋亡，
5. 如权利耍求 1所述的制备治疗局灶性脑缺血药物，其特従在干该药物制剂核心成分 是 权利要求 .所述的童组灵芝免疫调节蛋白（rLZ 8 )和任逸的药学可接受的辅剂组成。
6. 如权利要求 ί所述的药物制剂给药途径为口服和 Φ肠道给药，其中，口服包括口服 液，片剂，丸剂和胶囊；非肠道给药包括钋用药和注射剂。
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