WO2021185341A1 - 线粒体萃取物用于治疗或/及预防肾脏损伤相关疾病的用途 - Google Patents

线粒体萃取物用于治疗或/及预防肾脏损伤相关疾病的用途 Download PDF

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WO2021185341A1
WO2021185341A1 PCT/CN2021/081686 CN2021081686W WO2021185341A1 WO 2021185341 A1 WO2021185341 A1 WO 2021185341A1 CN 2021081686 W CN2021081686 W CN 2021081686W WO 2021185341 A1 WO2021185341 A1 WO 2021185341A1
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kidney
renal
mitochondrial
cells
mitochondria
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PCT/CN2021/081686
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English (en)
French (fr)
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郑汉中
许智凯
曾惠卿
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台湾粒线体应用技术股份有限公司
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Priority to CN202180014670.7A priority Critical patent/CN115135328B/zh
Priority to US17/912,394 priority patent/US20230137870A1/en
Priority to CN202311570784.7A priority patent/CN117357559A/zh
Priority to JP2022556517A priority patent/JP7563782B2/ja
Priority to CN202311621466.9A priority patent/CN117379458A/zh
Publication of WO2021185341A1 publication Critical patent/WO2021185341A1/zh
Priority to JP2024112807A priority patent/JP2024133717A/ja

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Definitions

  • the present invention relates to the second use of mitochondrial extracts, and particularly refers to the use of mitochondrial extracts to treat or/and prevent kidney injury-related diseases.
  • Mitochondria are organelles that exist in human cells to provide ATP needed for normal cell operation. Recent studies have pointed out that the increase and activation of mitochondria in cells will provide energy for stem cell differentiation and contribute to the success of stem cells. Differentiation. In other words, mitochondria play a very important role in human energy metabolism. For example, if mitochondria are defective, it will cause degenerative or aging-related diseases, such as brain degeneration, muscle weakness, muscle disease, etc.; and many current studies have pointed out If patients with Parkinson’s disease or Alzheimer’s disease caused by oxidative damage can maintain normal mitochondrial function or improve the body’s antioxidant capacity, it will contribute to the deterioration of neurodegenerative diseases.
  • kidney disease When the kidney tissue is damaged for more than 3 months, so that the structure or function of the kidney cannot recover its original function, it is called chronic kidney disease.
  • clinical treatment is mostly based on drug treatment, supplemented by diet and lifestyle habits.
  • chronic kidney disease gradually worsens with the course of the disease, and the patient faces renal fibrosis and gradually loses kidney function, the patient needs to maintain life through hemodialysis or kidney transplantation. This is not only a very uncomfortable process for the patient, but also for the national medical treatment. The cost is also a high burden.
  • an effective treatment for chronic kidney disease and kidney disease is provided.
  • the composition or method of fibrosis has become a top priority in clinical medicine.
  • the main purpose of the present invention is to provide a second use of mitochondrial extract, which can effectively improve or prevent kidney damage-related diseases, thereby achieving the effect of treating kidney disease or slowing down the deterioration of kidney disease.
  • the present invention discloses the use of mitochondria for preparing a composition for preventing or/and treating kidney injury-related diseases, specifically, by administering a certain amount of mitochondrial extract to a kidney affected Damage to individuals with related diseases can improve the condition of kidney cell damage, thereby achieving the effect of treating or preventing deterioration of kidney damage-related diseases.
  • the kidney injury-related disease is renal fibrosis, kidney inflammation, chronic kidney disease, acute kidney disease, renal tubular injury, renal failure, prerenal injury, nephrogenic injury, and post-renal injury , Glomerulitis, pyelonephritis, renal disease syndrome, uremia.
  • the kidney injury-related disease has mitochondrial damage and at least one of the following symptoms: proteinuria, edema, oliguria, high urea nitrogen, high creatinine, abnormal uric acid, stones, glomeruli
  • the filtering rate is abnormal.
  • the mitochondria are isolated from a stem cell, such as adipose stem cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, bone follower stem cells, umbilical cord stem cells, amniotic stem cells, amniotic fluid stem cells, placental stem cells, iPS, neural stem cells.
  • a stem cell such as adipose stem cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, bone follower stem cells, umbilical cord stem cells, amniotic stem cells, amniotic fluid stem cells, placental stem cells, iPS, neural stem cells.
  • the dosage of mitochondria in the composition is 5 to 80 ⁇ g, and the dosage of mitochondria in the composition is preferably 40 ⁇ g or more.
  • administering the mitochondrial extract provided by the present invention or a composition containing the same can effectively improve the damage of kidney cell mitochondria, and then It can improve or treat kidney cell damage or related diseases.
  • Figure 1A is the result of statistical analysis of the survival rate of renal epithelial cells treated with different concentrations of hydrogen peroxide for 24 hours.
  • Figure 1B is a statistical analysis of the survival rate of kidney epithelial cells treated with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial sediment.
  • Figure 2A shows the results of the detection and analysis of the collagen secretion of the renal epithelial cells after the renal epithelial cells were treated with different concentrations of AGEs-BSA for different times.
  • Figure 2B shows the results of the detection and analysis of the collagen secretion of the renal epithelial cells after the renal epithelial cells were treated with different concentrations of AGEs-BSA and then administered with different doses of mitochondrial sediment.
  • Figure 2C shows the results of the detection and analysis of the collagen secretion of the renal epithelial cells after the renal epithelial cells were treated with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial sediment.
  • Figure 3 shows the results of the detection and analysis of renal epithelial cell mitochondrial damage after the renal epithelial cells were treated with hydrogen oxide or AGEs-BSA, and then administered with different doses of mitochondrial sediment.
  • the present invention provides a second use of mitochondrial extract, which means that by administering a certain amount of mitochondrial extract or a composition containing it to an individual suffering from kidney injury-related diseases, it can effectively improve kidney injury-related diseases, and It is necessary to prevent the deterioration of kidney damage-related diseases.
  • the dosage of mitochondria provided by the present invention to an individual is 5-80 ⁇ g, such as 5, 10, 15, 20, 25, 30, 40, 50, 60, 65, 70, 80 ⁇ g, etc., wherein The dosage is preferably 15-40 ⁇ g; and, in order to achieve a better effect of treating or improving kidney disease, the mitochondria provided by the present invention can be combined with other components to prepare a composite, and the combined components are again It is better to have growth factors, such as blood products containing growth factors, platelet-rich plasma (PRP), plasma, serum, platelet-rich fibrin and so on.
  • PRP platelet-rich plasma
  • mitochondria extract refers to the mitochondria isolated from a cell, and the separation technology or method used should be able to maintain the integrity of the mitochondrial structure and function. According to those skilled in the art, separation The technique or method can be physical or chemical.
  • Cells refer to cells with mitochondria, such as adipose stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, blood cells, etc.
  • composition can be pharmaceutical compositions, foods, functional foods, nutritional supplements, etc., and can be matched with different components according to different types, and have different dosage forms and different administration methods.
  • kidney injury-related diseases are diseases caused by damage to kidney cells and have symptoms of mitochondrial damage, such as renal fibrosis, kidney inflammation, kidney disease, acute kidney disease, renal tubular injury, and renal failure , Prerenal injury, nephrogenic injury, post-renal injury, glomerulitis, pyelonephritis, renal disease syndrome, uremia, etc.
  • the mitochondria used in the following examples are taken from human adipose-derived stem cells, but without limitation, the mitochondria of the present invention can only be derived from human adipose-derived stem cells, which means that the mitochondria of the present invention are taken from any cell of the human body.
  • the dose of mitochondria used in the following examples is only an example.
  • the dose of mitochondria is 15 ⁇ g as a low dose and 40 ⁇ g as a high dose. It is not intended to limit the technical features of the present invention, which means that the mitochondrial dose provided by the present invention is 5 ⁇ 80 ⁇ g can achieve the desired effect of the invention.
  • the kidney epithelial cell line was cultured in a cell culture medium containing MEM- ⁇ Earl's salt and 5% fetal bovine serum, and placed at 37°C (containing 5% carbon dioxide) for culture.
  • transfer Remove the cell culture medium and add phosphate buffer to wash the cells.
  • phosphate buffer After removing the phosphate buffer, add 0.25% trypsin/2.21mM EDTA.
  • MEM- ⁇ containing 5% fetal bovine serum to neutralize Trypsin, collect suspended cells, centrifuge, then count the cells, and then dilute with MEM- ⁇ containing 5% fetal bovine serum to a final concentration of 5x10 4 cells per milliliter for subsequent subculture Or for analysis purposes.
  • Example 1 The kidney epithelial cells cultured in Example 1 were subcultured in 96-well plates, where the concentration of each well was 5 ⁇ 10 4 cells/200 ⁇ L. After 8 hours of culture, the supernatant was removed and washed with phosphate buffer. Add 200 ⁇ L of cell culture medium without 5% fetal bovine serum to each well for 8 hours of culture, and then give different concentrations of hydrogen peroxide (0.3, 0.5, 1, 3, 5, 10 mM) for treatment after culture. After culturing with different concentrations of hydrogen peroxide for 24 hours, remove the supernatant from each well, and then add cell culture medium (100 ⁇ L/well) containing 10% Alamar blue. After culturing for 3-4 hours , Perform fluorescence signal measurement (Excitation/Emission:560/590nm), and the result is shown in Figure 1A.
  • the renal epithelial cells will begin to be damaged after 24 hours of treatment with different concentrations of hydrogen peroxide.
  • the hydrogen peroxide concentration is more than 1 mM
  • the renal epithelial cells will be damaged due to damage.
  • the number of deaths increased significantly.
  • the survival rate of renal epithelial cells was 81.4% after treatment with hydrogen peroxide at a concentration of 1mM for 24 hours; the survival rate of renal epithelial cells was 81.4% when treated with hydrogen peroxide at a concentration of 5mM.
  • the survival rate of renal epithelial cells was 31.3%; after treating the renal epithelial cells with hydrogen peroxide at a concentration of 10 mM for 24 hours, the survival rate of renal epithelial cells was 24.2%.
  • the results show that the treatment of renal epithelial cells with hydrogen peroxide can indeed construct a pattern of renal epithelial cell damage and death. Moreover, as the concentration of hydrogen peroxide increases, the damage of renal epithelial cells worsens. The number of renal epithelial cell death also increased.
  • Example 2 The kidney epithelial cells cultured in Example 1 were subcultured in a 96-well plate, where the concentration of each well was 1 ⁇ 10 4 cells/200 ⁇ L. After culturing for 8 hours, the supernatant was removed and washed with phosphate buffer. Add a volume of 200 ⁇ L of cell culture medium without 5% fetal bovine serum to each well and incubate for 8 hours, then give 1mM and 3mM hydrogen peroxide treatments for 4 hours, and then give cells treated with different concentrations of hydrogen peroxide. Different doses (15 ⁇ g and 40 ⁇ g) of mitochondrial sediments (prepared in Example 2) were cultured for 24 hours.
  • the mitochondrial extract provided by the present invention can indeed protect kidney epithelial cells, avoid oxidative or inflammatory reactions that lead to damage to kidney epithelial cells, and can repair damaged kidney epithelial cells, effectively avoiding kidney epithelial cells. Cell death.
  • the mitochondrial extract or the composition containing the mitochondrial extract provided by the present invention indeed has the effect of improving or/and preventing kidney damage or kidney disease caused by oxidative stress.
  • the kidney epithelial cells cultured in Example 1 were cultured in a 6-well plate with a culture medium containing 5% fetal bovine serum at a concentration of 1 ⁇ 10 5 cells/2ml per well. After 24 hours of culture, the supernatant was removed, and then phosphate was added. After washing with buffer, add 1ml of cell culture medium without 5% fetal bovine serum to each well for 8 hours, and then give different concentrations (100 ⁇ g/ml and 400 ⁇ g/ml) of AGEs-BSA for 4 hours.
  • the process of this example is basically the same as that of Example 5. The difference is that after the cell culture medium containing AGEs-BSA is removed, the cell culture medium that does not contain 5% fetal bovine serum and different doses (15 ⁇ g) are added to each well. And 40 ⁇ g) of the mitochondrial sediment (prepared in Example 2), cultured for 24 hours, after the completion of the culture, the supernatant was collected separately, and the water-soluble collagen assay kit was used to detect collagen secretion. The results are shown in Figure 2B Shown.
  • the procedure of this embodiment is substantially the same as that of Embodiment 6, except that the stimulant that induces renal epithelial cell fibrosis is changed from AGEs-BSA to hydrogen peroxide; the detection result is shown in Figure 2C.
  • the kidney epithelial cells cultured in Example 1 were subcultured in a 96-well plate with a cell culture medium containing 5% fetal bovine serum at a concentration of 5x10 4 cells/200 ⁇ L per well. After 24 hours of culture, the supernatant was removed After washing with phosphate buffer, add 1ml of cell culture medium without 5% fetal bovine serum to each well for 8 hours. After incubation, 3mM hydrogen peroxide and 100 ⁇ g/ml AGEs-BSA are given and incubated for 4 hours.
  • I /R kidney injury mode ischemia-reperfusion, hereinafter referred to as I /R kidney injury mode
  • the steps are as follows: first, 150mg/Kg of phenobarbital (phenobarbital) is injected into the abdomen of the mouse by intraperitoneal injection. Move the left kidney from the incision to the outside, and then use a vascular clip to block the renal artery flowing into the kidney. After 30 minutes of blocking, remove the vascular clip to allow blood to pass through again, and complete the I/R kidney injury model
  • mice treated with the I/R kidney injury model were injected with different doses of mitochondria (15 ⁇ g and 40 ⁇ g) into the kidney by renal artery vascular injection, namely the mitochondrial high-dose group and the mitochondrial low-dose group; the control group (I/R) Group) were injected with phosphate buffer. After the mice in each group completed their treatment, the kidneys were moved back into the body and the wound was sutured; blood samples were collected on the first day (D1) and the second day (D2) after each group of mice, and the serum creatinine was measured.
  • mice Creatinine analysis kit brand: Crystal Chem; model: 80350
  • serum urea nitrogen is tested with urea analysis kit (brand: abcam; model: ab83362) for analysis.
  • D7 7th day after transplantation, the mice in each group were sacrificed, the left kidney of each mouse was perfused and fixed with formalin, paraffin embedding and tissue sectioning were performed, and then H&E staining was performed.
  • the staining results were based on Histological study of the morphological changes caused by ischemic injury, using Jablonski semi-quantitative standard to score the degree of renal injury: 0 is divided into normal tissue; 1 is divided into renal tubule damage area less than 5%; 2 is divided into renal tubular damage area of 5% or more To less than 25%; 3 is divided into renal tubular damage area greater than 25% to 75% or less; 4 is divided into renal tubular damage area greater than 75%.
  • the urea in the serum of the mice in the mitochondrial group The content of nitrogen and creatinine was significantly reduced, showing that the administration of mitochondria can effectively improve the kidney damage, and the results of the kidney injury score show that the administration of mitochondria can restore the damaged kidney cells and improve the condition of renal tubular damage, and with the administration of mitochondria
  • the increase in the dose increases the effect of improving kidney damage.
  • Table 1 Analysis results of serum and kidney tissue sections of mice in each group after different treatments

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Abstract

一种线粒体萃取物用于治疗或预防肾脏损伤相关疾病的用途，该线粒体萃取物能够有效改善肾脏损伤相关疾病，并预防肾脏损伤相关疾病的恶化。

Description

线粒体萃取物用于治疗或/及预防肾脏损伤相关疾病的用途 技术领域
本发明有关于线粒体萃取物的第二用途，特别指线粒体萃取物用于治疗或/及预防肾脏损伤相关疾病的用途。
背景技术
线粒体为存在人体细胞内的一个胞器，用以提供细胞正常运作所需要的ATP，并且，近期研究指出，细胞内线粒体数量增加及活化，将能够提供干细胞分化所需能量，有助于干细胞成功分化。换言之，线粒体在人体能量代谢上扮演着十分重要的角色，举例来说，若线粒体产生缺陷，将会造成退化性或老化相关疾病，例如脑退化、肌无力、肌肉病变等；而目前许多研究指出，氧化损伤造成的巴金森氏症或是阿兹海默症患者若能维持线粒体正常功能或是提升体内抗氧化能力，将有助于神经退化性疾病恶化。
肾脏组织受损超过3个月，以至于肾脏结构或功能无法恢复原有功能时，就被称为慢性肾脏疾病，目前临床治疗上多以药物治疗为主，饮食及生活习惯控制为辅，然而当慢性肾脏疾病随着病程逐渐恶化，患者面临肾脏纤维化而逐渐丧失肾脏功能时，患者需要通过血液透析或是肾脏移植来维持生命，这不仅对于患者来说是十分难受的过程，对于国家医疗成本也是高额的负担。换言之，由于目前对于慢性肾脏疾病的致病机制与治疗方法皆未有明确的认识，导致临床上对于慢性肾脏疾病并无法提供有效的治疗方式，是以，提供一种有效治疗慢性肾脏疾病及肾脏纤维化的组合物或是方法成为临床医疗上的当务之急。
发明内容
本发明的主要目的在于提供一种线粒体萃取物的第二用途，其能够有效地改善或预防肾脏受损相关疾病，进而达到治疗肾脏病或减缓肾脏病恶化的功效。
于是，为能达成上述目的，本发明揭露一种线粒体用于制备预防或/及治疗肾脏损伤相关疾病的组合物的用途，具体来说，通过投予一定量的线粒体萃取物至一罹患肾脏受损相关疾病的个体时，能够改善肾脏细胞受损的情形，进而达到肾脏受损相关疾病的治疗或预防恶化的功效。
在本发明的实施例中，该肾脏损伤相关疾病为肾脏纤维化、肾脏发炎、慢性肾脏病、急性肾脏病、肾小管损伤、肾衰竭、肾前性损伤、肾因性损伤、 肾后性损伤、肾小球炎、肾盂肾炎、肾病症候群、尿毒症。
在本发明的一实施例中，该肾脏损伤相关疾病具有线粒体受损及下列至少一病征：蛋白尿、水肿、少尿、尿素氮过高、肌酸酐过高、尿酸异常、结石、肾丝球过滤率异常。
在本发明的另一实施例中，该线粒体分离自一干细胞，如脂肪干细胞、CD34+造血干细胞、间质干细胞、骨随干细胞、脐带干细胞、羊膜干细胞、羊水干细胞、胎盘干细胞、iPS、神经干细胞。
在本发明的实施例中，线粒体在组合物中的剂量为5～80μg，又以线粒体在组合物中的剂量为40μg以上为佳。
本发明的有益效果在于：
当肾脏细胞因纤维化、氧化压力或是发炎环境造成线粒体受损情形发生时，投予本发明所提供线粒体萃取物或含有其的组合物，能够有效地改善肾脏细胞线粒体受损的情形，进而能够达到改善或治疗肾脏细胞受损或与之相关疾病的功效。
附图说明
图1A为统计分析肾脏上皮细胞经不同浓度过氧化氢处理24小时后的存活率的结果。
图1B为统计分析肾脏上皮细胞经不同浓度过氧化氢处理后，再投予不同剂量的线粒体沉淀物后的存活率的结果。
图2A为肾脏上皮细胞经不同浓度糖化终产物(AGEs-BSA)分别处理不同时间后，检测分析肾脏上皮细胞胶原蛋白分泌量的结果。
图2B为肾脏上皮细胞经不同浓度AGEs-BSA分别处理并再投予不同剂量的线粒体沉淀物后，检测分析肾脏上皮细胞胶原蛋白分泌量的结果。
图2C为肾脏上皮细胞经不同浓度的过氧化氢分别处理并再投予不同剂量的线粒体沉淀物后，检测分析肾脏上皮细胞胶原蛋白分泌量的结果。
图3为肾脏上皮细胞经过氧化氢或AGEs-BSA分别处理，并再投予不同剂量的线粒体沉淀物后，检测分析肾脏上皮细胞线粒体受损情形的结果。
具体实施方式
本发明提供一种线粒体萃取物的第二用途，意即通过投予一定量的线粒体萃取物或含有其的组合物至一罹患肾脏损伤相关疾病的个体，能够有效地改善肾脏损伤相关疾病，并得预防肾脏损伤相关疾病的恶化。
一般来说，本发明所提供线粒体投予至个体的剂量为5～80μg，如5、10、15、20、25、30、40、50、60、65、70、80μg等，其中，又以投予剂量为15～40 μg为佳；并且，为能达到较佳的治疗或改善肾脏疾病的功效，本发明所提供线粒体能搭配其他组成份制备为一组合物，而所搭配的组成份又以具有生长因子为佳，例如含有生长因子的血液制品、富含血小板的血浆(PRP)、血浆、血清、富含血小板纤维蛋白(Platelet-Rich Fibrin)等。
本发明所指“线粒体萃取物”，指分离自一细胞中的线粒体，而所使用的分离技术或方法应要能维持线粒体结构及功能的完整性，依据本发明本领域技术人员来说，分离技术或方法可为物理性或化学性。
本发明所指“细胞”，指具有线粒体的细胞，如脂肪干细胞、间质干细胞、骨骼肌细胞、肝脏细胞、肾脏细胞、纤维母细胞、神经细胞、皮肤细胞、血球细胞等。
本发明所指“组合物”，得为医药组合物、食品、机能性食品、营养补充品等，并依据种类不同得与不同组成份搭配而成，而具有不同剂型及不同投予方法。
本发明所指“肾脏损伤相关疾病”，为肾脏细胞受损而引发的疾病，并且具有线粒体受损的病征，如肾脏纤维化、肾脏发炎、肾脏病、急性肾脏病、肾小管损伤、肾衰竭、肾前性损伤、肾因性损伤、肾后性损伤、肾小球炎、肾盂肾炎、肾病症候群、尿毒症等。
以下，为能证实本发明所提供线粒体萃取物的功效，将举若干实施例并搭配附图做详细说明如后。
以下实施例中所使用的线粒体取自人体脂肪干细胞(adipose-derived stem cell)，但非限制本发明的线粒体仅能来自人体脂肪干细胞，意即本发明的线粒体取自人体任何细胞。
以下实施例中所使用的线粒体剂量仅为例示，以线粒体剂量为15μg作为低剂量代表，40μg作为高剂量代表，并非用于限制本发明的技术特征，意即本发明所提供线粒体在剂量为5～80μg皆能达成本发明所欲达成的功效。
实施例一：培养肾脏上皮细胞株
将肾脏上皮细胞株培养于含有MEM-αEarl’s salt与5％胎牛血清的细胞培养基中，并置于37℃(含有5％二氧化碳)下进行培养，当细胞培养达到8成满度时，移除细胞培养基并加入磷酸盐缓冲液清洗细胞，再移除磷酸盐缓冲液后，加入0.25％胰蛋白酶/2.21mM EDTA，反应20分钟后，加入含有5％胎牛血清的MEM-α中和胰蛋白酶，收集悬浮的细胞，进行离心，而后对细胞进行计数，再以含5％胎牛血清的MEM-α进行稀释至终浓度为每毫升中5x10 4个细胞，用以做后续继代培养或分析之用。
实施例二：线粒体萃取
将人体脂肪干细胞培养至细胞数量为1.5x10 8个细胞，以杜氏缓冲液(DPBS)冲洗细胞后移除杜氏缓冲液，再加入胰蛋白酶反应3分钟后，加入干细胞培养液(Keratinocyte SFM(1X)液体、bovine pituitary extract、10wt％胎牛血清)终止反应，而后，收集细胞后进行离心(600g、10分钟)，移除上清液，加入80毫升的IBC-1缓冲液(缓冲液(225mM甘露醇、75mM蔗糖、0.1mM EDTA、30mMTris-HCl pH 7.4)至细胞中，进行均质后离心，得到的沉淀物即为线粒体(下称线粒体沉淀物)。将线粒体沉淀物中加入1.5毫升IBC-1缓冲液及蛋白质分解酶抑制剂，并置于4℃，供以下实施例使用。
实施例三：肾脏上皮细胞损伤试验(一)
将实施例一所培养的肾脏上皮细胞在96孔盘中进行继代培养，其中，每孔浓度为5x10 4cells/200μL，培养8小时后移除上清液，以磷酸盐缓冲液清洗后，在每孔加入200μL未含有5％胎牛血清的细胞培养液进行培养8小时，培养后分别给予不同浓度的过氧化氢(0.3、0.5、1、3、5、10mM)进行处理。以不同浓度的过氧化氢培养24小时后，分别移除每孔的上清液，再加入含有10％阿尔玛蓝(Alamar blue)的细胞培养液(100μL/孔)，培养3-4小时后，进行荧光信号测量(Excitation/Emission:560/590nm)，结果如图1A所示。
由图1A的结果可知，肾脏上皮细胞通过不同浓度的过氧化氢处理24小时后，肾脏上皮细胞会开始有损伤的情形发生，其中，当过氧化氢浓度为1mM以上时，肾脏上皮细胞因损伤而导致死亡的情形大幅增加，具体来说，以浓度为1mM的过氧化氢处理肾脏上皮细胞24小时后，肾脏上皮细胞的存活率为81.4％；以浓度为5mM的过氧化氢处理肾脏上皮细胞24小时后，肾脏上皮细胞的存活率为31.3％；以浓度为10mM的过氧化氢处理肾脏上皮细胞24小时后，肾脏上皮细胞的存活率为24.2％。由此结果显示，以过氧化氢处理肾脏上皮细胞确实能够建构出肾脏上皮细胞受损及死亡的模式，并且，随着添加过氧化氢的浓度增加，肾脏上皮细胞受损情形随的恶化，并肾脏上皮细胞死亡数量亦随的增加。
实施例四：肾脏上皮细胞损伤试验(二)
将实施例一所培养的肾脏上皮细胞在96孔盘中进行继代培养，其中，每孔浓度为1x10 4cells/200μL，培养8小时后移除上清液，通过磷酸盐缓冲液清洗后，在每孔加入体积为200μL未含有5％胎牛血清的细胞培养液培养8小时，而后分别给予浓度为1mM及3mM的过氧化氢处理4小时，再分别给予经不同浓度过氧化氢处理的细胞不同剂量(15μg及40μg)的线粒体沉淀 物(实施例二所制备)后，培养24小时，培养完后移除上清液，再加入含有10％阿尔玛蓝的细胞培养液(100μL/孔)，在37℃环境下培养3-4小时后，在培养结束时进行荧光信号测量(Excitation/Emission:560/590nm)，结果如图1B所示。
由图1B的结果可知，经过氧化氢处理的肾脏上皮细胞会受到损伤而导致细胞存活率下降，而当肾脏上皮细胞已经被过氧化氢诱导受损后，再投予一定量的线粒体沉淀物后，能够明显提升肾脏上皮细胞的存活率，并且，随着投予剂量的增加，肾脏上皮细胞的存活率亦随的增加。
由此结果可知，本发明所提供线粒体萃取物确实能够保护肾脏上皮细胞，避免氧化或发炎反应而导致肾脏上皮细胞受损的情形发生，并且能够修复受损的肾脏上皮细胞，有效地避免肾脏上皮细胞死亡。换言之，本发明所提供线粒体萃取物或含其的组合物确实具有能够改善或/及预防因氧化压力而导致的肾脏受损或肾脏疾病的功效。
实施例五：肾脏上皮细胞纤维化试验(一)
将实施例一所培养的肾脏上皮细胞以含有5％胎牛血清的培养液培养于6孔盘，每孔浓度为1x10 5cells/2ml，培养24小时后移除上清液，再以磷酸盐缓冲液清洗后，在每孔加入1ml未含有5％胎牛血清的细胞培养液培养8小时，再给予不同浓度(100μg/ml及400μg/ml)的糖化终产物(AGEs-BSA)培养4小时，培养完成后，移除含有AGEs-BSA的细胞培养基并以磷酸盐缓冲液进行清洗，而后在每孔加入1ml未含有5％胎牛血清的细胞培养液分别培养24及48小时，培养结束后分别收集上清液，再以水溶性胶原蛋白(soluble collagen)测定试剂盒(Sircol TM Soluble Collagen Assay Kit)进行胶原蛋白分泌检测，结果如图2A所示。
由图2A的结果显示，不论以高浓度(400μg/ml)或低浓度(100μg/ml)的AGEs-BSA处理肾脏上皮细胞，皆会增加肾脏上皮细胞胶原蛋白的分泌表现量，并且胶原蛋白的分泌表现量会随着处理时间增加而提升，显示AGEs-BSA确实会诱导肾脏上皮细胞病变且产生纤维化的情形，其即为慢性肾脏病的前期，且若胶原蛋白的分泌表现量持续增加，则会导致慢性肾脏病的发生。
实施例六：肾脏上皮细胞纤维化试验(二)
本实施例的流程大体上等同于实施例五，不同在于，在移除含有AGEs-BSA的细胞培养基后，在每孔中加入未含有5％胎牛血清的细胞培养液及不同剂量(15μg及40μg)的线粒体沉淀物(实施例二所制备)后，分别 培养24小时，培养完成后，分别收集上清液，并以水溶性胶原蛋白测定试剂盒进行胶原蛋白分泌检测，结果如图2B所示。
由图2B的结果可知，投予线粒体沉淀物后能够降低肾脏上皮细胞内因AGEs-BSA诱导所产生的胶原蛋白分泌量，显示本发明所提供线粒体或含有其的组合物能够达到有效地改善或/及预防肾脏纤维化或与的相关肾脏疾病的功效。
实施例七：肾脏上皮细胞纤维化试验(三)
本实施例的流程大体上等同实施例六，不同在于，将诱导肾脏上皮细胞纤维化的刺激剂由AGEs-BSA改为过氧化氢；检测结果如图2C所示。
由图2C的结果可知，以过氧化氢处理肾脏上皮细胞会诱导胶原蛋白分泌量增加，意即透过过氧化氢处理肾脏上皮细胞确实能够建构出肾脏细胞纤维化的模式；而给予经过氧化氢处理后的肾脏上皮细胞线粒体沉淀物后，能够明显降低肾脏上皮细胞内的胶原蛋白分泌量，显示本发明所提供线粒体或含有其的组合物能够达到有效地改善或/及预防肾脏纤维化或与之相关肾脏疾病的功效。
实施例八：肾脏上皮细胞内线粒体功能损伤试验
将实施例一所培养的肾脏上皮细胞在96孔盘中以含有5％胎牛血清的细胞培养液进行继代培养，每孔浓度为5x10 4cells/200μL，培养24小时后移除上清液，以磷酸盐缓冲液清洗后，在每孔加入1ml未含有5％胎牛血清的细胞培养液进行培养8小时，培养后分别给予3mM过氧化氢及100μg/ml的AGEs-BSA培养4小时后，移除含有过氧化氢或AGEs-BSA的细胞培养液，并以磷酸盐缓冲液进行清洗，而后在每孔加入1ml未含有5％胎牛血清的细胞培养液及不同剂量(15μg及40μg)的线粒体沉淀物(实施例二所制备)，再分别进行培养24小时，培养完成后，以磷酸盐缓冲液进行清洗，再加入含有10μM的JC-1染色试剂的缓冲液，在37℃下反应10分钟，经清洗后，进行荧光信号测量(Excitation/Emission:488/530nm)，结果如图3所示。
由图3的结果可知，仅有以过氧化氢或AGEs-BSA处理的肾脏上皮细胞，其内JC-1单体(monomer)的表现增加，显示肾脏上皮细胞内线粒体被过氧化氢或AGEs-BSA所造成的发炎环境而导致损伤；而先以过氧化氢或AGEs-BSA处理，再以不同剂量线粒体沉淀物处理的肾脏上皮细胞，其内JC-1单体的表现明显下降，并且随着线粒体沉淀物的剂量增加，肾脏上皮细胞中JC-1单体的表现随之下降。
实施例九：动物试验
取10周大的C57BL/6小鼠，饲养于恒温和恒湿度下及12:12小时的明暗循环环境；而小鼠肾脏损伤模式将以缺血再灌流的模式(ischemia-reperfusion，下称I/R肾脏损伤模式)进行，步骤如下：先以腹腔注射将150mg/Kg的苯巴比妥(phenobarbital)注射至小鼠腹部，待小鼠昏迷后在其小鼠左侧肾脏位置进行手术，将左侧肾脏自切口处移至外面，接着以血管夹将肾动脉流入肾脏的血管阻断，阻断30分钟后移除血管夹使血流再度通过，及完成I/R肾脏损伤模式
经I/R肾脏损伤模式处理后的小鼠，以肾动脉血管注射将不同剂量的线粒体(15μg及40μg)送入肾脏，即为线粒体高剂量组及线粒体低剂量组；对照组(I/R组)则注射磷酸盐缓冲液。各组小鼠完成其处理后，分别将肾脏移回体内并进行伤口缝合；并在各组小鼠进行后的第1天(D1)及第2天(D2)收集血液样品，测量血清肌酸酐(Creatinine，Cr)和血液尿素氮(BUN)；接着将所抽取到的血液利进行离心，分离且收集血清，分析血清中的尿素氮及肌酸酐的含量；其中，血清肌酸酐检测以小鼠肌酸酐分析套组(厂牌:Crystal Chem；型号:80350)进行分析；血清尿素氮检测以尿素分析套组(厂牌:abcam；型号:ab83362)进行分析。在各组小鼠移植后第7天(D7)牺牲后，对各小鼠左侧肾脏进行灌流并用福尔马林固定后，进行石蜡包埋及组织切片，再进行H&E染色，对染色结果根据组织学研究缺血性损伤引起的形态变化，采用Jablonski半定量标准评分肾损伤程度：0分为正常组织；1分为肾小管损伤面积小于5％；2分为肾小管损伤面积为5％以上至25％以下；3分为肾小管损伤面积为大于25％至75％以下；4分为肾小管损伤面积大于75％。
上述结果如下表1所示。由表1的结果可知，相较于控制组来说，I/R组血清中的尿素氮与肌酸酐的表现量会有明显的增加，显示I/R肾脏损伤模式确实会造成肾脏损伤，并且，由肾脏损伤评分的结果可得知I/R组的分数为3-4分，代表肾小管损伤严重；而相较于I/R组来说，给予线粒体的组别的小鼠血清中尿素氮与肌酸酐的含量明显降低，显示给予线粒体能够有效地改善肾脏损伤，并由肾脏损伤评分的结果可知投予线粒体能够使受损肾脏细胞恢复，改善肾小管损伤的状况，且随着给予线粒体剂量的增加，改善肾脏损伤的效果增加。
表1：各组小鼠经不同处理后的血清及肾脏组织切片的分析结果
Figure PCTCN2021081686-appb-000001
Figure PCTCN2021081686-appb-000002
由上述结果显示，当肾脏细胞因纤维化、氧化压力或是发炎环境造成线粒体受损情形发生时，投予本发明所提供线粒体萃取物或含有其的组合物，能够有效地改善肾脏细胞线粒体受损的情形，进而能够达到改善或治疗肾脏细胞受损或与之相关疾病的功效。

Claims (9)

  1. 一种线粒体作为制备预防或/及治疗肾脏损伤相关疾病的组合物的用途。
  2. 如权利要求1所述用途，其特征在于，该肾脏损伤相关疾病为肾脏纤维化。
  3. 如权利要求1所述用途，其特征在于，该肾脏损伤相关疾病为肾脏发炎。
  4. 如权利要求1所述用途，其特征在于，该肾脏损伤相关疾病具有线粒体受损的病征。
  5. 如权利要求1所述用途，其特征在于，该肾脏损伤相关疾病为慢性肾脏病。
  6. 如权利要求1所述用途，其特征在于，该肾脏损伤相关疾病为肾小管损伤。
  7. 如权利要求6所述用途，其特征在于，该肾小管损伤的病征为损伤面积大于25％。
  8. 如权利要求1所述用途，其特征在于，该组合物中的该线粒体的剂量为5μg以上。
  9. 如权利要求1所述用途，其特征在于，该组合物中的该线粒体的剂量为40μg以上。
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