WO2022228223A1 - 工程化线粒体及其制备方法 - Google Patents

工程化线粒体及其制备方法 Download PDF

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WO2022228223A1
WO2022228223A1 PCT/CN2022/087837 CN2022087837W WO2022228223A1 WO 2022228223 A1 WO2022228223 A1 WO 2022228223A1 CN 2022087837 W CN2022087837 W CN 2022087837W WO 2022228223 A1 WO2022228223 A1 WO 2022228223A1
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mitochondria
exogenous
engineered
cells
cell
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周兴
林治华
沈燕
李雪梅
张晗奕
张清
娄杰
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重庆理工大学
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  • the present invention relates to the technical field of mitochondria, in particular, to engineered mitochondria and a preparation method thereof.
  • Mitochondria are organelles that provide energy in cells, provide 90% of the ATP of human cells, and regulate apoptosis. Mitochondrial dysfunction will cause ATP synthesis obstacles, so that the energy source of human cells is insufficient, causing a series of diseases.
  • biologically active free mitochondria are isolated and extracted from cells or body tissues and administered intravenously or locally, so that exogenous mitochondria can be targeted to reach the site of disease and replace the damaged mitochondria.
  • exogenous mitochondria can be targeted to reach the site of disease and replace the damaged mitochondria.
  • mitochondrial dysfunction diseases To exert normal mitochondrial function for the treatment of mitochondrial dysfunction diseases.
  • the first object of the present invention is to provide a method for preparing engineered mitochondria, which can prepare engineered mitochondria with higher biological activity and better therapeutic effect on mitochondrial dysfunction diseases.
  • the second object of the present invention is to provide engineered mitochondria, the engineered mitochondria obtained by the preparation method have higher biological activity and have better therapeutic effect on mitochondrial dysfunction diseases.
  • the engineered mitochondria are bound from the exogenous cell membrane to the outer membrane of the exogenous mitochondria.
  • Normal mitochondria exist in the matrix of cells and are adapted to live in a membrane-coated environment; the present invention combines the exogenous cell membrane with the outer membrane of the exogenous mitochondria, providing a similar membrane for the naked exogenous mitochondria. coated environment, thereby making the biological activity of exogenous mitochondria more stable.
  • the exogenous cell membrane is extracted from any one of neutrophils, monocytes, lymphocytes or tumor cells.
  • exogenous mitochondria are isolated from cells or body tissues.
  • the body tissue is selected from any one of myocardial tissue, liver tissue, brain tissue, muscle tissue, blood or tissue fluid.
  • the preparation method of engineered mitochondria includes the following steps:
  • the body tissue is used to extract the cells through the kit, and then the cells are disrupted by a mechanical method, and the exogenous cell membrane is prepared after freeze-drying.
  • step S2 cells or body tissues are used to separate and extract exogenous mitochondria using a cell mitochondria separation kit.
  • the exogenous mitochondria and the exogenous cell membrane are mixed at a mass ratio of 1:1 to 1:4 of protein.
  • the exogenous cell membrane can be sufficiently and effectively combined with the outer membrane of the exogenous mitochondria, so that a membrane-like environment can be formed outside the exogenous mitochondria, so as to improve the biological activity.
  • the exogenous mitochondria and the exogenous cell membrane are mixed in an appropriate amount of 0.01M PBS solution in proportion, ultrasonicated in a water bath at 4°C for 2-5 minutes, centrifuged at 3500g for 10-15 minutes, discard the supernatant, and use 0.01M PBS.
  • the solution was washed and precipitated 2-3 times to remove unbound exogenous cell membranes; and then centrifuged at 3500g at 4°C for 10-15min to obtain engineered mitochondria.
  • steps S1 and S2 C57BL/6J mice were used to extract exogenous cell membranes and exogenous mitochondria.
  • the engineered mitochondria are prepared by combining the outer membrane of the biologically active exogenous mitochondria with the outer membrane of the isolated and extracted exogenous mitochondria, so that the engineered mitochondria have higher biological activity than the bare exogenous mitochondria, and the mitochondrial dysfunction is improved. Disease treatment is better.
  • Fig. 1 is a graph of the results provided by Experimental Example 1, wherein Fig. 1A is the zete potential, Fig. 1B is the particle size, and Fig. 1C is a TEM image;
  • Fig. 2 is the result graph provided by Experimental Example 2, wherein Fig. 2A is the ATP level, and Fig. 2B is the mitochondrial membrane potential (MMP) level;
  • Fig. 3 is the result graph that Experimental Example 3 provides, wherein, Fig. 3A is ALT level, Fig. 3B is AST level;
  • Fig. 4 is the result graph provided by Experimental Example 3, wherein Fig. 4A is the ATP level, Fig. 4B is the ROS level, and Fig. 4D is the MMP level;
  • Fig. 5 is the result graph that Experimental Example 4 provides, wherein, Fig. 5A is ALT level, Fig. 5B is AST level;
  • Fig. 6 is the result graph provided by Experimental Example 4, wherein Fig. 6A is the level of IL-10, Fig. 6B is the level of IL-12, and Fig. 6C is the level of TNF- ⁇ ;
  • Fig. 7 is the result graph provided by Experimental Example 4, wherein Fig. 7A is the ATP level, and Fig. 7B is the ROS level;
  • Figure 8 shows the results provided in Experimental Example 4, wherein Figure 8a is the liver tissue of the blank group mouse, Figure 8b is the liver tissue of the model mouse, Figure 8c is the liver tissue of the Mito control group mouse, and Figure 8d is the NEM-Mito experiment Group mouse liver tissue.
  • This example provides engineered mitochondria that are bound from exogenous cell membranes to the outer membrane of exogenous mitochondria.
  • This embodiment provides a method for preparing engineered mitochondria, comprising the following steps:
  • This example provides engineered mitochondria that are bound from exogenous cell membranes to the outer membrane of exogenous mitochondria.
  • This embodiment provides a method for preparing engineered mitochondria, comprising the following steps:
  • S1 Use C57BL/6J mouse liver tissue to separate and extract monocytes by using Solarbio's mouse organ tissue mononuclear cell separation solution kit, and then crush monocytes by shaking method, and freeze-dry to prepare exogenous monocyte membranes fragments;
  • S2 Use C57BL/6J mouse liver tissue to isolate and extract exogenous mitochondria using Solarbio's mitochondrial isolation kit;
  • This example provides engineered mitochondria that are bound from exogenous cell membranes to the outer membrane of exogenous mitochondria.
  • This embodiment provides a method for preparing engineered mitochondria, comprising the following steps:
  • S1 Use C57BL/6J mouse spleen tissue to separate and extract lymphocytes using Solarbio's mouse spleen lymphocyte separation liquid kit, and then disrupt the lymphocytes by probe ultrasonic method, and freeze-dried to prepare exogenous lymphocyte membrane fragments;
  • S2 Use C57BL/6J mouse brain tissue to isolate and extract exogenous mitochondria by Solarbio's mitochondrial isolation kit;
  • Example 1 The free neutrophil membrane fragments (NEM), isolated and extracted exogenous mitochondria (Mito) and finally prepared engineered mitochondria (NEM-Mito) obtained in Example 1 were measured for Zeta potential and particle size, respectively, And irradiated transmission electron microscope, the results are shown in Figure 1.
  • the particle size of the NEM-Mito prepared in Example 1 of the present invention is: 1104.55 ⁇ 227.97nm; the Zeta potential is: -38 ⁇ 0.26mV.
  • the engineered mitochondria (NEM-Mito) prepared in Example 1 and the isolated exogenous mitochondria (Mito) were detected by Biyuntian's enhanced ATP detection kit, respectively, and their ATP levels were detected by Biyuntian's mitochondrial membrane potential.
  • the kit (JC-1) detects the mitochondrial membrane potential (MMP), and the results are shown in Figure 2.
  • Experimental L02 cells cultured in a culture flask with 1640 medium containing 10% serum, and subcultured in a sterile constant temperature incubator at 37°C and 5% CO 2 .
  • APAP solution After fully dissolving APAP powder in 1640 culture medium containing 0.125% DMSO and containing 1% serum, it was prepared into a certain concentration of APAP solution.
  • the exogenous mitochondria (Mito) and engineered mitochondria ( NEM-Mito), 2 ml each, and 3 duplicate wells were set for each concentration gradient; the blank group was set to add 2 ml of 1640 culture medium containing 1% serum to each well of normal L02 cells on a six-well plate; Incubate for 24h in a sterile constant temperature incubator with 5% CO2 .
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ROS reactive oxygen species
  • both the isolated Mito and the NEM-Mito prepared by the present invention can inhibit the release of ALT and AST in the L02 cell model of mitochondrial dysfunction, but the NEM-Mito prepared by the present invention is stronger than the isolated NEM-Mito.
  • the source Mito was more capable of inhibiting ALT and AST release.
  • both the isolated and extracted exogenous Mito and the NEM-Mito prepared by the present invention can inhibit the release of ROS in the L02 cell model of mitochondrial dysfunction and increase the levels of ATP and MMP, but the NEM-Mito prepared by the present invention can inhibit the release of ROS in the L02 cell model of mitochondrial dysfunction.
  • Mito was more capable of inhibiting the release of ROS than isolated exogenous Mito, resulting in a higher increase in ATP and MMP levels.
  • mice Kunming mice, random male and female, 4-5 weeks old, weighing 18-22 g, fed ad libitum.
  • acetaminophen (APAP) solution After fully mixing the APAP powder with physiological saline, add an equal volume of PEG400 to mix and dissolve to prepare a 400 mg/kg APAP solution.
  • mice were injected intraperitoneally with 400 mg/kg APAP solution at a dose of 10 ml/kg, and the modeling time was 24 hours to induce serum AST and ALT in mice. Elevated, mitochondrial dysfunction in liver cells, hepatocyte rupture and apoptosis;
  • Example 1 The exogenous Mito and NEM-Mito obtained in Example 1 were formulated into 100 ⁇ g/ml using normal saline and PEG400 with a volume ratio of 1:1, and were injected into the mouse model of liver cell mitochondrial dysfunction by tail vein injection, and the dosage was 10ml/kg; the blank group was set as healthy Kunming mice by tail vein injection of a mixed solution of physiological saline and PEG400 with a volume ratio of 1:1, and the dose was 10ml/kg; 7 parallel groups were set in each group.
  • mice were sacrificed 24 hours after administration, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the mice were detected by a biochemical analyzer.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the mouse IL-12 ELISA kit from Hangzhou Lianke Biotechnology was used for detection, and the results are shown in Figure 6; and the levels of ATP and ROS in mitochondria of mouse liver tissue were detected.
  • the detection method of ATP and ROS was the same as that of Experimental Example 2. , the results are shown in Figure 7.
  • the engineered mitochondria (NEM-Mito) prepared in the present application have high biological activity and have a good therapeutic effect on mitochondrial dysfunction.

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Abstract

一种工程化线粒体及其制备方法，所述工程化线粒体，由外源细胞膜结合至外源线粒体的外膜。其制备方法包括以下步骤：S1：从细胞提取制备外源细胞膜；S2：从细胞或机体组织分离提取外源线粒体；S3：将分离提取的外源线粒体与外源细胞膜，按比例混合，将外源细胞膜结合至外源线粒体的外膜得到工程化线粒体。可以制得具有较高的生物活性，对线粒体功能障碍性疾病具有较好的治疗效果的工程化线粒体。

Description

工程化线粒体及其制备方法 技术领域
本发明涉及线粒体技术领域，具体而言，涉及工程化线粒体及其制备方法。
背景技术
线粒体是细胞内提供能量的细胞器，提供人体细胞90％的ATP，并调控细胞凋亡。线粒体功能障碍会造成ATP合成障碍，使人体细胞能量来源不足，引发一系列疾病。
目前，现有从细胞或机体组织分离提取出有生物活性的游离线粒体通过静脉给药或局部给药的方式，使外源线粒体靶向的到达疾病发生部位，替换功能受损的线粒体，在体内发挥正常的线粒体功能，以对线粒体功能障碍性疾病进行治疗。
但是，从细胞或机体组织分离提取出有生物活性的游离线粒体极其不稳定，很快便会失去其正常的生物活性，且对病灶组织无靶向作用，对线粒体功能障碍性疾病的治疗效果不佳。
发明内容
本发明的第一个目的在于提供工程化线粒体的制备方法，可以制备得 到具有较高的生物活性，对线粒体功能障碍性疾病具有较好的治疗效果的工程化线粒体。
本发明的第二个目的在于提供工程化线粒体，通过制备方法制得的具有较高的生物活性，对线粒体功能障碍性疾病具有较好的治疗效果的工程化线粒体。
本发明的实施例通过以下技术方案实现：
工程化线粒体，由外源细胞膜结合至外源线粒体的外膜。
正常的线粒体是存在于细胞的基质中的，其适应生活于有膜包被的环境中；本发明将外源细胞膜结合至外源线粒体的外膜，为裸露的外源线粒体提供了一个类似膜包被的环境，从而使外源线粒体的生物活性更稳定。
进一步地，所述外源细胞膜提取制备自中性粒细胞、单核细胞、淋巴细胞或肿瘤细胞中的任一种。
进一步地，所述外源线粒体分离自细胞或机体组织。
进一步地，所述机体组织选自心肌组织、肝脏组织、脑组织、肌肉组织、血液或组织液中的任一种。
工程化线粒体的制备方法，包括以下步骤：
S1：从细胞提取制备外源细胞膜(NEM)；
S2：从细胞或机体组织分离提取外源线粒体(Mito)；
S3：将分离提取的外源线粒体与外源细胞膜，按比例混合，将外源细 胞膜结合至外源线粒体的外膜得到工程化线粒体(NEM-Mito)。
进一步地，所述步骤S1中使用机体组织通过试剂盒提取细胞，再使用机械法进行细胞破碎，冷冻干燥后制备得到外源细胞膜。
进一步地，所述步骤S2中使用细胞或机体组织通过细胞线粒体分离试剂盒分离提取外源线粒体。
进一步地，所述步骤S3中外源线粒体与外源细胞膜按蛋白质的质量比1:1～1:4混合。
使外源细胞膜可以充分有效的结合至外源线粒体的外膜上，使外源线粒体外部充分形成类似膜包被的环境，以提高生物活性。
进一步地，所述步骤S3中外源线粒体与外源细胞膜按比例混合于适量的0.01M PBS溶液，于4℃水浴超声2～5min，3500g离心10～15min，弃掉上清液，用0.01M PBS溶液洗涤沉淀2～3次，去掉未结合的外源细胞膜；再于4℃，3500g离心10～15min即制得到工程化线粒体。
进一步地，所述步骤S1和S2中均采用C57BL/6J小鼠进行提取外源细胞膜和外源线粒体。
本发明实施例的技术方案至少具有如下优点和有益效果：
本发明通过在分离提取出有生物活性的外源线粒体的外膜上结合外源细胞膜制得工程化线粒体，使工程化线粒体比裸露的外源线粒体具有更高的生物活性，对线粒体功能障碍性疾病的治疗效果更好。
附图说明
图1为实验例1提供的结果图，其中，图1A为zete电位，图1B为粒径大小，图1C为射透射电镜图；
图2为实验例2提供的结果图，其中，图2A为ATP水平，图2B为线粒体的膜电位(MMP)水平；
图3为实验例3提供的结果图，其中，图3A为ALT水平，图3B为AST水平；
图4为实验例3提供的结果图，其中，图4A为ATP水平，图4B为ROS水平，图4D为MMP水平；
图5为实验例4提供的结果图，其中，图5A为ALT水平，图5B为AST水平；
图6为实验例4提供的结果图，其中，图6A为IL-10水平，图6B为IL-12水平，图6C为TNF-α水平；
图7为实验例4提供的结果图，其中，图7A为ATP水平，图7B为ROS水平；
图8为实验例4提供的结果图，其中，图8a为空白组小鼠肝脏组织，图8b为模型小鼠肝脏组织，图8c为Mito对照组小鼠肝脏组织，图8d为NEM-Mito实验组小鼠肝脏组织。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本发明实施例提供的工程化线粒体及其制备方法进行具体说明。
实施例1
本实施例提供了工程化线粒体，由外源细胞膜结合至外源线粒体的外膜。
本实施例提供了工程化线粒体的制备方法，包括以下步骤：
S1：采用C57BL/6J小鼠骨髓通过Solarbio的小鼠骨髓中性粒细胞分离液试剂盒分离提取中性粒细胞，再通过探头超声法破碎中性粒细胞，冷冻干燥后制备得到外源中性粒细胞膜碎片；
S2：采用C57BL/6J小鼠心肌组织通过碧云天的细胞线粒体分离试剂盒分离提取外源线粒体；
S3：将分离的外源线粒体与外源中性粒细胞膜碎片，按蛋白质的质量比1:1混合于适量的0.01M PBS溶液，于4℃水浴超声2min，3500g离心10min，弃掉上清液，用0.01M PBS溶液洗涤沉淀2次，去掉未结合的外源中性粒细胞膜碎片；再于4℃，3500g离心10min即制得工程化线粒体。
实施例2
本实施例提供了工程化线粒体，由外源细胞膜结合至外源线粒体的外膜。
本实施例提供了工程化线粒体的制备方法，包括以下步骤：
S1：采用C57BL/6J小鼠肝脏组织通过Solarbio的小鼠脏器组织单核细胞分离液试剂盒分离提取单核细胞，再通过振荡法破碎单核细胞，冷冻干燥后制备得到外源单核细胞膜碎片；
S2：采用C57BL/6J小鼠肝脏组织通过Solarbio的线粒体分离试剂盒分离提取外源线粒体；
S3：将分离的外源线粒体与外源单核细胞膜碎片，按蛋白质的质量比1:2混合于适量的0.01M PBS溶液，于4℃水浴超声4min，3500g离心15min，弃掉上清液，用0.01M PBS溶液洗涤沉淀3次，去掉未结合的外源单核细胞膜碎片；再于4℃，3500g离心15min即制得工程化线粒体。
实施例3
本实施例提供了工程化线粒体，由外源细胞膜结合至外源线粒体的外膜。
本实施例提供了工程化线粒体的制备方法，包括以下步骤：
S1：采用C57BL/6J小鼠脾脏组织通过Solarbio的小鼠脾脏淋巴细胞分离液试剂盒分离提取淋巴细胞，再通过探头超声法破碎淋巴细胞，冷冻干燥后制备得到外源淋巴细胞膜碎片；
S2：采用C57BL/6J小鼠脑组织通过Solarbio的线粒体分离试剂盒分离提取外源线粒体；
S3：将分离的外源线粒体与外源淋巴细胞膜碎片，按蛋白质的质量比1:4混合于适量的0.01M PBS溶液，于4℃水浴超声5min，3500g离心15min，弃掉上清液，用0.01M PBS溶液洗涤沉淀3次，去掉未结合的外源淋巴细胞膜碎片；再于4℃，3500g离心15min即制得工程化线粒体。
实验例1
将实施例1分离制得的游离中性粒细胞膜碎片(NEM)、分离提取的外源线粒体(Mito)和最终制得的工程化线粒体(NEM-Mito)分别进行测定Zeta电位和粒径大小，并照射透射电镜图，结果如图1所示。
由图1可以看出，本发明实施例1制得的NEM-Mito的粒径大小为：1104.55±227.97nm；Zeta电位为：-38±0.26mV。
实验例2
将实施例1制得的工程化线粒体(NEM-Mito)和分离提取的外源线粒体(Mito)分别使用碧云天的增强型ATP检测试剂盒检测ATP水平，并分别使用碧云天的线粒体膜电位检测试剂盒(JC-1)检测线粒体的膜电位(MMP)，结果如图2所示。
由图2可以看出，本发明制得的NEM-Mito的ATP和MMP水平与分离提取的Mito相比较显著升高，说明本发明制得的NEM-Mito比Mito具有更好的生物活性。
实验例3
1.建立线粒体功能障碍的L02细胞模型
(1)实验L02细胞：在培养瓶中，使用含10％血清的1640培养液培养，于37℃、5％CO 2的无菌恒温培养箱中传代培养。
(2)对乙酰氨基酚(APAP)溶液配制：使用含0.125％DMSO的含1％血清的1640培养液将APAP粉末充分溶解后，配制为一定浓度的APAP溶液。
(3)线粒体功能障碍的L02细胞模型的建立：取对数生长期的L02细胞悬液进行消化后，使用六孔板上每孔接种2ml，调节L02细胞密度为5×10 3个/孔，于37℃、5％CO 2的无菌恒温培养箱中培育24h，至细胞单层铺满六孔板孔底，将上层培养液吸出，在板孔中加入APAP溶液，使培养液中APAP的终浓度为10mM，于37℃、5％CO 2的无菌恒温培养箱中培养24h，诱使L02细胞线粒体功能障碍，ALT、AST和ROS释放量增多，ATP和MMP水平降低。
2.体外细胞实验
于六孔板上每孔线粒体功能障碍的L02细胞模型中分别加入浓度梯度为6.25μg/ml、12.5μg/ml和25μg/ml的实施例1得到的外源线粒体(Mito)和工程化线粒体(NEM-Mito)各2ml，每个浓度梯度均设置3个复孔；空白组设置为于六孔板上每孔正常的L02细胞中加入含1％血清的1640培养液2ml；均于37℃、5％CO 2的无菌恒温培养箱中孵育24h。
孵育24h后再通过生化分析仪检测细胞上清液中的谷丙转氨酶(ALT)和谷草转氨酶(AST)水平，结果如图3所示；孵育24h后再检测细胞线粒体的ATP、MMP和活性氧(ROS)水平，ATP和MMP的检测方法与实验例2的检测方法相同，活性氧(ROS)水平采用DCFH-DA探针进行检测，结果如图4所示。
由图3可以看出，分离提取的Mito和本发明制得的NEM-Mito均可以抑制线粒体功能障碍的L02细胞模型的ALT和AST释放，但是本发明制得的NEM-Mito比分离提取的外源Mito对ALT和AST释放的抑制能力更强。
由图4可以看出，分离提取的外源Mito和本发明制得的NEM-Mito均可以抑制线粒体功能障碍的L02细胞模型的ROS释放，提高ATP和MMP水平，但是本发明制得的NEM-Mito比分离提取的外源Mito对ROS的释放抑制能力更强，使ATP和MMP水平增长更高。
实验例4
1.建立肝脏细胞线粒体功能障碍的小鼠模型
(1)实验小鼠：昆明系小鼠，雌雄随机，4～5周龄、体重18～22g，自由采食。
(2)对乙酰氨基酚(APAP)溶液配制：使用生理盐水将APAP粉末充分混匀后，再加入等体积的PEG400混合溶解，配制为400mg/kg的APAP溶液。
(3)肝脏细胞线粒体功能障碍的小鼠模型的建立：对小鼠一次性腹腔 注射400mg/kg的APAP溶液，给药剂量为10ml/kg，建模时间24h，诱发小鼠血清中AST、ALT升高，肝脏细胞内线粒体功能障碍，肝细胞破裂与凋亡；
2.小鼠体内实验
将实施例1得到的外源Mito和NEM-Mito使用体积比为1:1的生理盐水和PEG400配制为100μg/ml，尾静脉注射于肝脏细胞线粒体功能障碍的小鼠模型体内，给药剂量为10ml/kg；空白组设置为健康的昆明小鼠尾静脉注射体积比为1:1的生理盐水和PEG400的混合溶液，给药剂量为10ml/kg；每组分别设置7个平行组。
给药24h后处死小鼠，通过生化分析仪检测小鼠血清中的谷丙转氨酶(ALT)和谷草转氨酶(AST)水平，结果如图5所示；同时检测小鼠血清中的炎症因子TNF-α、IL-10和IL-12的水平，TNF-α使用欣博盛生物的小鼠TNF-αELISA试剂盒检测，IL-10使用杭州联科生物的小鼠IL-10ELISA试剂盒检测，IL-12使用杭州联科生物的小鼠IL-12ELISA试剂盒检测，结果如图6所示；并检测小鼠肝脏组织线粒体的ATP和ROS水平，ATP和ROS的检测方法与实验例2的检测方法相同，结果如图7所示。
单独取一组的实验小鼠取肝脏组织制作病理切片，结果如图8所示。
由图5可以看出，本发明制得的NEM-Mito治疗肝脏细胞线粒体功能障碍的小鼠后，显著降低了小鼠血清中AST和ALT的水平；与分离提取的外源Mito治疗肝脏细胞线粒体功能障碍的小鼠组对比，AST和ALT的水平降低得更显著。
由图6可以看出，本发明制得的NEM-Mito治疗肝脏细胞线粒体功能障碍的小鼠后，显著降低了小鼠血清中炎症因子TNF-α、IL-10和IL-12的水平，抑制了小鼠血清中炎症因子的聚集；与分离提取的外源Mito治疗肝脏细胞线粒体功能障碍的小鼠组对比，其抑制效果更显著。
由图7可以看出，本发明制得的NEM-Mito治疗肝脏细胞线粒体功能障碍的小鼠后，显著提高了小鼠肝脏组织线粒体的ATP水平，显著抑制了小鼠肝脏组织线粒体ROS的释放；与分离提取的Mito治疗肝脏细胞线粒体功能障碍的小鼠组对比，其治疗效果更显著。
由图8可以看出，本发明制得的NEM-Mito治疗肝脏细胞线粒体功能障碍的小鼠后，小鼠损伤的肝组织得到了较大程度的修复，与分离提取的外源Mito治疗肝脏细胞线粒体功能障碍的小鼠组对比，治疗后的肝小叶结构更加完整，肝细胞结构恢复正常，大部分细胞核结构完整。
综上，本申请制得的工程化线粒体(NEM-Mito)，具有较高的生物活性，对于线粒体功能障碍具有较好的治疗效果。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 工程化线粒体，其特征在于，由外源细胞膜结合至外源线粒体的外膜。
  2. 根据权利要求1所述的工程化线粒体，其特征在于，所述外源细胞膜提取制备自中性粒细胞、单核细胞、淋巴细胞或肿瘤细胞中的任一种。
  3. 根据权利要求1所述的工程化线粒体，其特征在于，所述外源线粒体分离自细胞或机体组织。
  4. 根据权利要求3所述的工程化线粒体，其特征在于，所述机体组织选自心肌组织、肝脏组织、脑组织、肌肉组织、血液或组织液中的任一种。
  5. 一种根据权利要求1～4任一项所述的工程化线粒体的制备方法，其特征在于，包括以下步骤：
    S1：从细胞提取制备外源细胞膜；
    S2：从细胞或机体组织分离提取外源线粒体；
    S3：将分离提取的外源线粒体与外源细胞膜，按比例混合，将外源细胞膜结合至外源线粒体的外膜得到工程化线粒体。
  6. 根据权利要求5所述的工程化线粒体的制备方法，其特征在于，所述步骤S1中使用机体组织提取细胞，再使用机械法进行细胞破碎，冷冻干燥后制备得到外源细胞膜。
  7. 根据权利要求5所述的工程化线粒体的制备方法，其特征在于，所述 步骤S2中使用细胞或机体组织通过细胞线粒体分离试剂盒分离提取外源线粒体。
  8. 根据权利要求5所述的工程化线粒体的制备方法，其特征在于，所述步骤S3中外源线粒体与外源细胞膜按蛋白质的质量比1:1～1:4混合。
  9. 根据权利要求5所述的工程化线粒体的制备方法，其特征在于，所述步骤S3中外源线粒体与外源细胞膜按比例混合后离心并洗涤沉淀，去掉未结合的外源细胞膜，即制得工程化线粒体。
  10. 根据权利要求5所述的工程化线粒体的制备方法，其特征在于，所述步骤S1和S2中均采用C57BL/6J小鼠进行提取外源细胞膜和外源线粒体。
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CN112020553A (zh) * 2018-04-26 2020-12-01 白雁生物技术公司 修饰的线粒体及其应用
WO2021004477A1 (zh) * 2019-07-09 2021-01-14 唐凌峰 一种基于线粒体的药物递送系统及其用途
CN110627875A (zh) * 2019-10-18 2019-12-31 合肥修合生物科技有限公司 一种线粒体靶向透膜环肽及其制备方法和用途
CN110790783A (zh) * 2019-11-19 2020-02-14 湖北科技学院 一种线粒体靶向抗肿瘤药物的制备方法及其应用

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CN112941025A (zh) * 2021-03-25 2021-06-11 四川大学华西医院 血液中中性粒细胞的分离方法和分离试剂盒
CN112941025B (zh) * 2021-03-25 2023-05-02 四川大学华西医院 血液中中性粒细胞的分离方法和分离试剂盒

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