WO2023217121A1 - 含肾前体样细胞的生物制剂及其制备方法和应用 - Google Patents

含肾前体样细胞的生物制剂及其制备方法和应用 Download PDF

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WO2023217121A1
WO2023217121A1 PCT/CN2023/092954 CN2023092954W WO2023217121A1 WO 2023217121 A1 WO2023217121 A1 WO 2023217121A1 CN 2023092954 W CN2023092954 W CN 2023092954W WO 2023217121 A1 WO2023217121 A1 WO 2023217121A1
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cells
renal
precursor
hla
preparation
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张琴
周伸奥
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上海赛立维生物科技有限公司
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    • C12N5/0602—Vertebrate cells
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    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
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    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to biological preparations containing renal precursor-like cells and preparation methods and applications thereof.
  • Traditional drug treatment and dialysis treatment are difficult to reverse the damage to kidney structure and the continued decline of kidney function.
  • Type 1 diabetic nephropathy, glomerulonephropathy, chronic nephritis, etc. The process causes damage to the kidneys, resulting in a continued decline in the glomerular filtration rate.
  • the accumulation of scars in the renal parenchyma leads to renal fibrosis.
  • the precipitation of fibrous matrix disrupts the organ structure and reduces blood supply. Fibrosis will reduce the ability of tissue repair.
  • Renal progenitors are distributed in renal tubules and renal capsules.
  • kidney precursor-like cells It is difficult to isolate kidney precursor-like cells using embryonic kidney, and the ability of continuous cell passage is limited.
  • the method of isolating and screening renal precursor cells from urine has the problem of introducing contamination from exogenous microorganisms, and the precursor cells obtained by this method cannot achieve colonization after being reinfused through the blood system. Renal precursor-like cells induced by differentiation from embryonic stem cells or iPS cells are at risk of developing teratomas.
  • kidney precursor-like cells It is difficult to isolate kidney precursor-like cells using embryonic kidney, and the ability of continuous cell passage is limited.
  • the method of isolating and screening renal precursor cells from urine has the problem of introducing contamination from exogenous microorganisms, and the precursor cells obtained by this method cannot achieve colonization after being reinfused through the blood system. Renal precursor-like cells induced by differentiation from embryonic stem cells or iPS cells are at risk of developing teratomas.
  • the purpose of the present invention is to provide novel biological agents containing renal precursor-like cells and their preparation methods and applications, so as to facilitate the regeneration or repair of damaged renal tissue.
  • the biological preparation of the present invention includes renal precursor-like cells that positively express CD133, CD24 and CD44, have strong continuous passage ability, can inhibit the fibrosis process of renal tissue, and promote renal tissue regeneration or repair.
  • the renal precursor-like cells are epithelial tissue-derived precursor-like cells.
  • the renal precursor-like cells have characteristics of epithelial precursor cells.
  • epithelial tissue is coated epithelium.
  • epithelial tissue-derived precursor-like cells are derived from renal cortical tissue.
  • the renal precursor-like cells also positively express at least one of CD73 and SOX9.
  • the renal precursor-like cells positively express CD133, CD24, CD44, CD73 and SOX9.
  • the renal precursor-like cells also negatively express at least one MHC class II molecule.
  • the renal precursor-like cells also negatively express at least one of HLA-DR, HLA-DP and HLA-DQ.
  • the renal precursor-like cells negatively express HLA-DR, HLA-DP, HLA-DQ, CD34 and CD45.
  • the preparation method of the biological preparation includes: using an amplification transformation medium to culture primary renal cells in vitro to obtain renal precursor-like cells that positively express CD133, CD24 and CD44.
  • the amplification transformation medium includes a basic medium and the following components based on the volume content of the basic medium: point:
  • the supplement ITS is insulin-transferrin.
  • the basal culture medium consists of DMEM/F12 culture medium and MCDB culture medium with a volume ratio of 3:1-1:1.
  • the MCDB medium is MCDB-131 medium.
  • the ROCK kinase inhibitor is any one of Fasudil, Y-27632, Thiazovivin and SB-772077-B.
  • the TGF- ⁇ signal inhibitor is any one of RepSox, SB431542 and A83-01.
  • the glycogen synthase kinase 3 ⁇ inhibitor is at least one of BIO, CHIR99021 and TWS119.
  • the present invention also provides the application of the biological preparation in the preparation of drugs for treating kidney disease.
  • the biological agent of the present invention includes renal precursor-like cells that positively express CD133, CD24 and CD44, has strong continuous passage ability, can inhibit the fibrosis process of renal tissue, and promote renal tissue regeneration or repair.
  • Figure 2 is a schematic diagram of the expression of HLA-DR, HLA-DP, HLA-DQ, CD34 and CD45 in renal precursor cells according to an embodiment of the present invention
  • Figure 3 is an optical microscope photograph of renal precursor-like cells according to an embodiment of the present invention.
  • Figure 4 is a comparison chart of the proliferation properties of renal precursor-like cells and cells obtained through control culture according to the embodiment of the present invention.
  • Figure 5 is a comparison diagram of photos of the left kidneys of the experimental group, the normal saline control group and the blank control group according to the embodiment of the present invention
  • Figure 6 is a Masson stained photo of the experimental group, normal saline control group and blank control group in the embodiment of the present invention
  • Figure 7 is a picture of Sirius red staining of the experimental group, normal saline control group and blank control group in the embodiment of the present invention.
  • Figure 8 is a comparison chart of Urea levels in the normal control group, the model control group, the test substance group A and the test substance group B according to the embodiment of the present invention.
  • Figure 9 is a comparison chart of Cr levels in the normal control group, the model control group, the test substance group A and the test substance group B in the embodiment of the present invention.
  • This embodiment provides a method for obtaining renal primary cells.
  • kidney tissue was obtained, which was derived from patient surgical samples provided by Renji Hospital. The patient had no infectious viral infection after medical examination, and the patient had not used steroids within 6 months before surgery. The patient was fully informed about the purpose of obtaining surgical samples before surgery and signed an informed consent form.
  • the cortex was cut away from the medulla, and the medulla was removed.
  • a total of 3 ml of cell digestion solution consisting of type I collagenase, sterile PBS buffer and trypsin digestion solution was used at 37 degrees Celsius. Digest the tissue for 90 minutes to obtain a primary cell suspension. Among them, sterile PBS buffer and trypsin digestion solution have the same volume, and the volume percentage of type I collagenase in the modified buffer is 1%.
  • the primary cell suspension was screen sorted with the assistance of sterile PBS buffer using a 70-micron sterile mesh, and the filtrate was collected and mucus and undigested tissue were removed to complete the screen sorting .
  • the filtrate was collected and mucus and undigested tissue were removed to complete the screen sorting .
  • add red blood cell lysis balance solution to the obtained precipitate and resuspend it, centrifuge again and repeat the above process until it is resuspended.
  • red blood cells are not observed in the cell pellet to complete the lysis and removal of red blood cells.
  • the centrifugation rate for each time was 1000g, and the centrifugation time was 3 minutes.
  • the resulting cell pellet contains renal primary cells.
  • This example provides a method for amplifying, transforming, and culturing the renal primary cells obtained in Example 1.
  • the basal culture medium used in this example consisted of DMEM/F12 culture medium and MCDB131 culture medium with a volume ratio of 3:2.
  • the DMEM/F12 culture medium and MCDB131 used in this example are from Shanghai Yuanpei Biotechnology Co., Ltd.; EGF and bFGF are from Nearshore Biotechnology; Y-27632, CHIR99021 and A83-01 are from Taoshu Biotechnology; N2 additive and B27
  • the additives are from Invitrogen, with product numbers 17502001 and 17504044 respectively; ITS is from Merck, with product number I1884-1VL; dexamethasone is from Selleck, product number is S1322, platelet-derived growth factor is from Merck, product number is GF142, and FBS is from Corning, item number 35081-CV.
  • the specific method of amplifying, transforming, and culturing the renal primary cells obtained in Example 1 to obtain renal precursor cells is as follows: seeding the cell pellets obtained in Example 1 into a 6-well plate at a seeding area of 104 cells/cm2, Add 2 ml of amplification transformation medium to the wells and culture for 5-7 days until the cell confluence is no less than 80%. Add 0.5 ml of trypsin digestion solution to each well and digest for 1-5 minutes until the proteins connecting the cells and culture medium are completely digested. Then add 2 ml of amplification and transformation medium to each well to terminate digestion.
  • Terminate the digestion in the amplification and transformation medium collect the cell suspension, centrifuge at 200g for 5 minutes, collect the cell pellet, resuspend the cell pellet in the amplification and transformation medium, and inoculate an appropriate number of T75 cells at an inoculation area of 10 4 cells/cm2. Complete one subculture in the culture bottle. Repeat the above subculture process until the tenth generation is reached to complete the subculture.
  • FIG. 1 The schematic diagram of the expression of CD133, CD24, CD44, CD73 and SOX9 in renal precursor cells shown in Figure 1 and the expression of HLA-DR, HLA-DP, HLA-DQ, CD34 and CD45 in renal precursor cells shown in Figure 2 were obtained.
  • Schematic diagram It can be seen from Figure 1 and Figure 2 that the cells obtained by amplification, transformation and culture in this example positively expressed CD133, CD24, CD44, CD73 and SOX9, and the expression rates were 79.5%, 73.0%, 85.2%, 92.6% and 99.9%, negative expression of HLA-DR, HLA-DP, HLA-DQ, CD34 and CD45, the expression rate is less than 1%.
  • CD133 and CD24 are markers characteristic of renal progenitor cells. The double-positive expression of the two indicates that the renal precursor-like cells obtained in this example have the characteristics of renal progenitor cells.
  • CD44, CD73 and SOX9 are markers characteristic of stem cells, among which CD133 is the characteristic marker in this field.
  • CD34 and CD45 are well-known specific markers for epithelial cell dedifferentiation in the process of renal injury repair.
  • CD34 and CD45 are hematopoietic stem cell and leukocyte markers.
  • the positive expression of CD133 and the negative expression of CD34 and CD45 indicate that the renal precursor-like cells obtained in this example of epithelial origin.
  • the negative expression of HLA-DR, HLA-DP, and HLA-DQ indicates that the renal precursor obtained in this example Such cells are non-immunogenic for human or mammalian allogeneic transplantation.
  • the morphological characteristics of the cells in the cell clusters obtained after subculture to the tenth generation were also observed using an optical microscope, and the optical microscope photos shown in Figure 3 were obtained.
  • the cells are polygonal, grow in clusters, and exhibit the characteristics of epithelial precursor cells.
  • This example also uses a control medium to conduct control culture on the renal primary cells obtained in Example 1.
  • the difference between the specific culture method and the method of amplifying, transforming and culturing the renal primary cells obtained in Example 1 in Example 1 is only that: using a volume ratio of 3:2 and containing FBS (final volume concentration 5%) DMEM/F12 medium and MCDB131 medium replace amplification and transformation medium.
  • the cell cluster obtained by comparative culture is the comparative cell cluster.
  • cells of different generations (P0-P10, P0 is the primary cell) were counted during amplification, transformation, and control culture respectively, and the cell proliferation performance was examined, and the proliferation performance comparison chart shown in Figure 4 was obtained.
  • P0-P10 the primary cell
  • This embodiment provides the application of the renal precursor cells obtained in Example 2 in the preparation of drugs for treating kidney disease, specifically, drugs for treating chronic kidney disease.
  • the modeling method of the unilateral ureteral ligation renal fibrosis model in this embodiment is as follows: select several male SD rats aged 9-10 weeks and weighing 250-300g, anesthetize each rat with isoflurane, and shave the abdomen. Mao, fixed on the operating board in a supine position and under continuous anesthesia. The abdomen was disinfected, a surgical incision was made along the midline of the abdomen, the skin and muscles were incised in sequence, the kidneys and ureters were freed, the middle part of the left ureter was held up with tissue forceps, and the two ends were ligated with sutures, and the ureter was severed in the middle.
  • the silicone tube is inserted into the renal artery through the femoral artery, and the injection line is left in place. Then the muscles and skin were sutured, wiped and disinfected with iodophor, and placed on a heating pad to rewarm. After they regained consciousness, they were placed back in the cage and raised.
  • a part of the precipitate obtained after centrifuging the digestion product containing cells in Example 2 to collect the precipitate was added to a 50 mL sterile centrifuge tube and centrifuged for 5 minutes under the action of 200 g centrifugal force. The supernatant was discarded and the cells were diluted to 2 ⁇ 10 with physiological saline. 7 /mL.
  • Each rat was administered at a dose of 6.6 ⁇ 10 6 /kg via the left renal artery to serve as the experimental group.
  • the normal saline control group was injected with normal saline at the same dose. Specific administration methods are routine technical means for those skilled in the art.
  • each rat was euthanized, and the left kidney was dissected and photographed.
  • the comparison of the photographs of the left kidney of the experimental group, the normal saline control group, and the blank control group was obtained as shown in Figure 5.
  • the kidneys in the normal saline control group showed significant edema, while the kidneys in the experimental group basically maintained a normal shape.
  • the left kidney of each rat was cut along the coronal plane and half of it was fixed in 10% formalin solution, and each part was stained with Masson and Sirius red to obtain the experimental group, normal saline control group and blank shown in Figure 6 Masson staining photos of the control group, and Sirius red staining photos of the experimental group, saline control group and blank control group shown in Figure 7.
  • Masson stained photos compared with the normal saline control group, the degree of renal fibrosis in the experimental group was significantly reduced, indicating that renal precursor-like cells have a positive effect on alleviating renal fibrosis.
  • Sirius red stained photos compared with the normal saline control group, the degree of renal fibrosis in the experimental group was significantly reduced, indicating that injection of renal precursor-like cells can alleviate renal fibrosis.
  • This embodiment provides the application of the renal precursor cells obtained in Example 2 in the preparation of drugs for treating kidney disease, specifically drugs for treating acute kidney disease.
  • the cortical tissue of the kidneys of healthy male SD rats aged 9-10 weeks and weighing 250-300g was also used for amplification, transformation and culture to obtain mouse-derived kidney precursor-like cells.
  • the difference between the specific culture process and the culture process of human-derived renal precursor-like cells in Example 2 is only that the source of the renal cortical tissue is different.
  • the modeling method of the glycerol-induced acute kidney injury model is as follows: several healthy male SD rats aged 9-10 weeks and weighing 250-300g are adaptively raised for 3 days and deprived of water for 24 hours before being weighed. weight, intramuscularly inject 50% glycerol at 10 mL/kg.
  • Example 2 A part of the precipitate obtained after centrifuging the digestion product containing human renal precursor-like cells in Example 2 was added to a 50 mL sterile centrifuge tube and centrifuged for 5 minutes under a centrifugal force of 200 g. The supernatant was discarded and washed with physiological saline. The cells were diluted to 2 ⁇ 10 7 cells/mL and administered to the left renal artery of 15 glycerol-induced acute kidney injury model rats at a dose of 6.6 ⁇ 10 6 cells/kg at 24 hours after glycerol injection as the test substance. Group A.
  • a part of the precipitate obtained after centrifugation of the digestion product containing mouse-derived renal precursor-like cells obtained in this example was added to a 50 mL sterile centrifuge tube and centrifuged for 5 minutes under the action of 200 g centrifugal force. The supernatant was discarded and washed with physiological saline. The cells were diluted to 2 ⁇ 10 7 cells/mL, and 15 glycerol-induced acute kidney injury model rats were administered to the left renal artery at a dose of 6.6 ⁇ 10 6 cells/kg starting from the 24th hour after glycerol injection as subjects.
  • Object group B The cells were diluted to 2 ⁇ 10 7 cells/mL, and 15 glycerol-induced acute kidney injury model rats were administered to the left renal artery at a dose of 6.6 ⁇ 10 6 cells/kg starting from the 24th hour after glycerol injection as subjects.
  • glycerol-induced acute kidney injury model rats were selected as the model control group.
  • the rats in each group were taken 6 hours and 72 hours after glycerin injection and anesthetized with isoflurane.
  • 1 mL of blood was taken from the orbital venous plexus and placed in a procoagulant tube. The blood was centrifuged at 3000 rpm for 10 minutes. The serum was taken to detect Urea and Cr. The figure was obtained.
  • the Urea level comparison chart of the normal control group, model control group, test substance group A and test substance group B shown in 8 in the several bar graphs corresponding to each time point, from left to right are the normal control group, model control group, test substance group A and test substance group B
  • the Cr level comparison chart of the normal control group, model control group, test substance group A and test substance group B shown in Figure 9 Among the several bar graphs corresponding to each time point, from left to right are the normal control group, the model control group, the test substance group A and the test substance group B).
  • test substance group A and test substance group B were injected with glycerol, Urea and Cr in the serum were significantly increased compared with the normal control group (P ⁇ 0.01).
  • test substance group A and test substance group B used human-derived renal precursor-like cells and mouse-derived renal precursor-like cells for therapeutic intervention respectively, and 48 hours after treatment (i.e. 72 hours after glycerol injection) , compared with the model control group, the serum Urea and Cr levels of both test substance group A and test substance group B were significantly reduced, indicating that the mouse-derived and human-derived renal precursor-like cells of this example and Example 2 It has the function of repairing acute kidney injury.
  • test substance group A and test substance group B each showed similar trends in changes in Urea and Cr levels compared with the model control group, indicating that human-derived renal precursor-like cells exhibit similar effects to mouse-derived renal precursors. Such cells have considerable therapeutic effect and will not cause xenogeneic rejection.
  • cell culture was performed in a cell culture incubator at 37 degrees Celsius and with a carbon dioxide concentration of 5%.
  • the culture medium used for cell culture and various reagents used to process cells, such as buffers, are sterilized and filtered with a 0.22 micron filter to remove impurities before use.
  • Each embodiment of the present invention involves statistical analysis of data.
  • Each set of experiments is repeated at least three times, and the experimental result data is analyzed using GraphPad Prism 8.0 software.
  • the biological preparations of the embodiments of the present invention include renal precursor-like cells that positively express CD133, CD24, and CD44. They have strong continuous passage ability, can inhibit the fibrosis process of renal tissue, and promote renal tissue regeneration or repair.
  • the renal precursor-like cells are epithelial tissue-derived precursor-like cells.
  • the renal precursor-like cells have characteristics of epithelial precursor cells.
  • the "epithelial tissue” is coated epithelium.
  • the epithelial tissue-derived precursor-like cells are derived from renal cortical tissue.
  • the renal precursor-like cells also positively express at least one of CD73 and SOX9.
  • the renal precursor-like cells positively express CD133, CD24, CD44, CD73 and SOX9.
  • the renal precursor-like cells also negatively express at least one MHC class II molecule. Specifically, the renal precursor-like cells also negatively express at least one of HLA-DR, HLA-DP and HLA-DQ.
  • the renal precursor-like cells also negatively express at least one of CD34 and CD45.
  • the renal precursor-like cells negatively express HLA-DR, HLA-DP, HLA-DQ, CD34, and CD45.
  • the preparation method of the biological preparation includes: using an amplification transformation medium to culture primary renal cells in vitro to obtain renal precursor-like cells that positively express CD133, CD24 and CD44.
  • the amplification transformation medium includes a basal medium, and the following components based on the volume of the basal medium: 15-25 ng/ml of epidermal cell growth factor EGF, 40-60 nm g/ml basic fibroblast growth factor bFGF, volume percentage 0.5%-1.5% N2 additive, volume percentage 0.5%-1.5% B27 additive, 5-15 micromol/L ROCK kinase inhibitor, 2-4 ⁇ mol/L glycogen synthase kinase 3 ⁇ inhibitor, 0.5-1.5 ⁇ mol/L TGF- ⁇ signaling inhibitor, 0.5%-1.5% by volume supplement ITS, not to exceed 1 nanomolar /L dexamethasone, 5-15 ng/mL platelet-derived growth factor PDGF, 1-10% serum by volume.
  • the supplement ITS is insulin-transferrin.
  • the basal culture medium consists of DMEM/F12 culture medium and MCDB culture medium with a volume ratio of 3:1-1:1.
  • the MCDB medium is MCDB-131 medium.
  • the ROCK kinase inhibitor is any one of Fasudil, Y-27632, Thiazovivin and SB-772077-B.
  • the TGF- ⁇ signaling inhibitor is any one of RepSox, SB431542 and A83-01.
  • the glycogen synthase kinase 3 ⁇ inhibitor is at least one of BIO, CHIR99021, and TWS119.
  • the embodiments of the present invention also provide the application of the biological preparation in the preparation of drugs for treating kidney disease.

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Abstract

本发明提供了一种含肾前体样细胞的生物制剂及其制备方法和应用。所述生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。

Description

含肾前体样细胞的生物制剂及其制备方法和应用
本申请要求申请日为2022年05月10日，申请号为2022105036786，发明名称为“生物制剂、细胞衍生物及制备方法和应用”的中国专利申请的优先权。上述申请的内容以引用方式被包含于此。
技术领域
本发明涉及生物技术领域，尤其涉及含肾前体样细胞的生物制剂及其制备方法和应用。
背景技术
急慢性肾脏疾病导致的肾功能衰竭严重威胁人类健康，传统的药物治疗和透析治疗难以逆转肾脏结构的损伤和肾功能的持续下降，Ⅰ型糖尿病肾病、肾小球肾病、慢性肾炎等在疾病进展过程对肾脏造成损伤，造成肾小球滤过率持续下降，肾实质内瘢痕的堆积成肾纤维化，纤维基质沉淀扰乱器官结构，减少血液供应，纤维化会降低组织修复能力，最后不可避免的会进展为终末期肾病。肾前体细胞(Renal progenitors)分布在肾小管和肾小囊中，能够分化成为肾小管细胞和肾足细胞，恢复肾功能，逆转肾损伤，是肾组织修复最重要的细胞。体外构建肾前体细胞培养体系，研究肾前体细胞对肾脏疾病模型的治疗作用，对于研究慢性和急性肾脏疾病的意义重大。
利用胚肾分离得到肾前体样细胞的难度大，细胞连续传代能力有限。从尿液中分离筛选取得肾前体细胞的方法存在引入外源微生物污染的问题，且该种方法得到的前体细胞通过血液系统回输后无法实现定植。由胚胎干细胞或iPS细胞诱导分化形成的肾前体样细胞有发展成畸胎瘤的风险。
因此，有必要开发新型的含肾前体样细胞的生物制剂。
技术问题
利用胚肾分离得到肾前体样细胞的难度大，细胞连续传代能力有限。从尿液中分离筛选取得肾前体细胞的方法存在引入外源微生物污染的问题，且该种方法得到的前体细胞通过血液系统回输后无法实现定植。由胚胎干细胞或iPS细胞诱导分化形成的肾前体样细胞有发展成畸胎瘤的风险。
技术解决方案
本发明的目的在于提供新型的含肾前体样细胞的生物制剂及其制备方法和应用，以利于受损肾组织的再生或修复。
为实现上述目的，本发明的生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。
优选的，所述肾前体样细胞为上皮组织来源前体样细胞。具体的，所述肾前体样细胞具有上皮前体细胞特征。
所述的“上皮组织”为被覆上皮。具体的，所述上皮组织来源前体样细胞来源于肾皮质组织。
优选的，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种。
优选的，所述肾前体样细胞阳性表达CD133、CD24、CD44、CD73和SOX9。
优选的，所述肾前体样细胞还阴性表达至少一种MHC二类分子。具体的，所述肾前体样细胞还阴性表达HLA-DR、HLA-DP和HLA-DQ的至少一种。
优选的，所述肾前体样细胞还阴性表达CD34和CD45中的至少一种。
优选的，所述肾前体样细胞阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45。
所述生物制剂的制备方法包括：使用扩增转化培养基对肾原代细胞进行体外培养，得到阳性表达CD133、CD24和CD44的肾前体样细胞。
所述扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成 分：
15-25纳克/毫升的表皮细胞生长因子EGF，40-60纳克/毫升的碱性成纤维细胞生长因子bFGF，体积百分比为0.5％-1.5％的N2添加剂，体积百分比为0.5％-1.5％的B27添加剂，5-15微摩尔/升的ROCK激酶抑制剂，2-4微摩尔/升的糖原合成酶激酶3β抑制剂，0.5-1.5微摩尔/升的TGF-β信号抑制剂，体积百分比为0.5％-1.5％的补充剂ITS，不超过1纳摩尔/升的地塞米松，5-15纳克/毫升的血小板源生长因子PDGF，体积百分比为1-10％的血清。其中，补充剂ITS为胰岛素-转铁蛋白。
优选的，所述基础培养基由体积比为3:1-1:1的DMEM/F12培养基和MCDB培养基组成。
优选的，MCDB培养基为MCDB-131培养基。
优选的，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的任意一种。
优选的，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的任意一种。
优选的，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种。
本发明还提供了所述生物制剂在肾病治疗药物制备方面的应用。
有益效果
本发明的生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。
附图说明
图1为本发明实施例的肾前体细胞CD133、CD24、CD44、CD73和SOX9表达情况示意图；
图2为本发明实施例的肾前体细胞HLA-DR、HLA-DP、HLA-DQ、CD34和CD45的表达情况示意图；
图3为本发明实施例的肾前体样细胞的光学显微镜照片；
图4为本发明实施例的肾前体样细胞和经对照培养得到的细胞两者的增殖性能对比图；
图5为本发明实施例的实验组、生理盐水对照组以及空白对照组各左侧肾脏照片的对比图；
图6为本发明实施例的实验组、生理盐水对照组和空白对照组的Masson染色照片；
图7为本发明实施例的实验组、生理盐水对照组和空白对照组的天狼星红染色照片；
图8为本发明实施例的正常对照组、模型对照组、受试物组A和受试物组B的Urea水平对比图；
图9为本发明实施例的正常对照组、模型对照组、受试物组A和受试物组B的Cr水平对比图。
本发明的最佳实施方式
实施例1：
本实施例提供了肾原代细胞的获取方法。
首先，获取正常无病变的肾组织，该肾组织来源于仁济医院提供的患者手术样本。患者经医学检查无传染性病毒感染，患者在术前6个月内未使用过类固醇激素药物。患者在术前对手术样本的获取目的充分知情，并签署了知情同意书。
去除前述肾组织的外部组织、脂肪和囊状物后，将皮质从髓质上切下，去除髓质。使用无菌PBS缓冲液对2g的得到的皮质组织进行清洗和灭菌处理后，再使用由Ⅰ型胶原酶、无菌PBS缓冲液和胰酶消化液组成的共3毫升细胞消化液在37摄氏度下对组织消化90分钟得到原代细胞悬液。其中，无菌PBS缓冲液和胰酶消化液具有相同的体积，Ⅰ型胶原酶占改良缓冲液的体积百分比为1％。
然后，使用70微米的无菌筛网对所述原代细胞悬液在无菌PBS缓冲液的辅助下进行筛网分选，收集滤液并去除粘液和未消化的组织，以完成筛网分选。将得到的滤液离心并去除上清后，向得到的沉淀物加入红细胞溶解平衡液进行重悬后再次离心并重复上述过程直至再 次离心后在细胞沉淀中观察不到红细胞为止，以完成红细胞裂解去除。具体的，每次离心的速率为1000g，离心时间为3分钟。得到的细胞沉淀中包含肾原代细胞。
实施例2：
本实施例提供了对实施例1得到的肾原代细胞进行扩增转化培养的方法。
本实施例进行所述扩增转化培养所使用的扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：
20纳克/毫升的EGF，50纳克/毫升的bFGF，体积百分比为1％的N2添加剂，体积百分比为1％的B27添加剂，10微摩尔/升的Y-27632，3微摩尔/升的CHIR99021，1微摩尔/升的A83-01，体积百分比为1％的ITS，1纳摩尔/升的地塞米松，10纳克/毫升的血小板源生长因子，体积百分比为5％的FBS。
本实施例所用的基础培养基由体积比为3:2的DMEM/F12培养基和MCDB131培养基组成。
本实施例使用的DMEM/F12培养基和MCDB131来源于上海源培生物科技有限公司；EGF和bFGF来源于近岸生物；Y-27632、CHIR99021和A83-01来源于陶术生物；N2添加剂和B27添加剂来源于Invitrogen，货号分别为17502001和17504044；ITS来源于Merck，货号为I1884-1VL；，地塞米松来源于Selleck，货号为S1322，血小板源生长因子来源于Merck，货号为GF142，FBS来源于Corning，货号为35081-CV。
对实施例1得到的肾原代细胞进行扩增转化培养得到肾前体细胞的方法具体为：将实施例1得到的细胞沉淀按104个/平方厘米的接种面积接种于6孔板中，每孔加2毫升的扩增转化培养基培养5-7天直至细胞融合度不低于80％。向每孔加入0.5毫升胰酶消化液进行1-5分钟的消化直至连接细胞及培养基的蛋白质消化完全，再向每孔加入2毫升的扩增转化培养基终止消化，收集细胞悬液并对得到的细胞悬液在不超过20摄氏度的室温以及200g离心力下离心5分钟以收集细胞沉淀。用扩增转化培养基继续对得到的细胞沉淀进行传代培养，具体传代培养如下：使用扩增转化培养基重悬前述离心得到的细胞沉淀，按照104个/平方厘米的接种面积接种于T75细胞培养瓶中继续进行培养4-5天后，向每个细胞培养瓶加入2.0毫升胰酶消化液进行1-5分钟的消化直至连接细胞及培养基的蛋白质消化完全，再向每孔加入5.0毫升的扩增转化培养基终止消化，收集细胞悬液，200g离心5分钟，收集细胞沉淀，并用扩增转化培养基重悬细胞沉淀，按照按104个/平方厘米的接种面积接种于合适数量的T75细胞培养瓶中完成一次传代培养。重复上述传代培养过程直至传代到第十代以完成传代培养。
传代培养结束后，提取每孔中的细胞团后用无菌PBS缓冲液进行润洗后，用胰酶消化液消化连接细胞蛋白质，然后对得到的包含细胞的消化产物离心收集沉淀。向部分沉淀中加入100微升染色缓冲液重悬细胞至流式管中，再向流式管中加入5微升待测流式抗体孵育20分钟，然后向每管加入400微升的染色缓冲液重悬后用于表面抗体流式检测。得到图1所示的肾前体细胞CD133、CD24、CD44、CD73和SOX9表达情况示意图以及图2所示的肾前体细胞HLA-DR、HLA-DP、HLA-DQ、CD34和CD45的表达情况示意图。从图1和图2中可以看到，本实施例经扩增转化培养得到的细胞阳性表达CD133、CD24、CD44、CD73和SOX9，表达率分别为79.5％，73.0％，85.2％，92.6％和99.9％，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45，表达率都低于1％。
CD133和CD24是肾祖细胞特征标志物，两者的双阳性表达说明本实施例得到的肾前体样细胞具有肾祖细胞特征，CD44、CD73和SOX9是干细胞特征标志物，其中CD133是本领域公知的肾损伤修复过程中上皮细胞去分化的特异性标志物，CD34和CD45是造血干细胞和白细胞标志物，CD133的阳性表达以及CD34和CD45的阴性表达说明本实施例得到的肾前体样细胞为上皮来源。HLA-DR、HLA-DP、HLA-DQ的阴性表达说明本实施例得到的肾前体 样细胞对于人或哺乳动物的异体移植而言就有非免疫原性。
本实施例还通过光学显微镜观察传代培养至第十代后得到的细胞团中细胞的形态特征，得到图3所示的光学显微镜照片。从图3可以看到，细胞呈多边形，聚集生长，表现出了上皮前体细胞特征。
本实施例还使用对照培养基对实施例1得到的肾原代细胞进行对照培养。具体的培养方法与本实施例1的对实施例1得到的肾原代细胞进行扩增转化培养的方法区别仅在于：使用体积比为3：2，并含FBS(体积终浓度5％)的DMEM/F12培养基和MCDB131培养基替代扩增转化培养基。对比培养得到的细胞团为对比细胞团。
本实施例分别对扩增转化培养以及对照培养过程中不同代次(P0-P10，P0为原代细胞)细胞进行计数，考察细胞增殖性能，得到图4所示的增殖性能对比图。从图4可以看到，对照培养得到的细胞不具备增殖性能，而经扩增培养得到的细胞，即使传到第十代，仍具有稳定的增殖性能。
实施例3：
本实施例提供了实施例2得到的肾前体细胞在肾病治疗药物，具体为慢性肾病治疗药物制备方面的应用。
本实施例的单侧输尿管结扎肾纤维化模型的造模方法如下：选择周龄9-10周体重在250-300g的若干雄性SD大鼠，将各大鼠用异氟烷麻醉后，腹部剃毛，仰卧位固定于手术板上并持续麻醉。腹部消毒，沿腹中线作手术切口，依次切开皮肤和肌肉，游离肾脏和输尿管，用组织钳托起左侧输尿管中段部位，在两端用缝合线结扎后，于中间离断输尿管。造模成功后，将硅胶管经股动脉插管入肾动脉，并留置注射管路。然后缝合肌肉和皮肤，用碘伏擦拭消毒后，放置于加热垫复温，待其苏醒后放回笼盒饲养。
将实施例2的包含细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL。通过各大鼠左肾动脉按照6.6×106个/公斤的剂量给药，作为实验组。生理盐水对照组按同等剂量注射生理盐水。具体给药操作方法为本领域技术人员的常规技术手段。
给药结束后的第28天将各大鼠安乐死，解剖取左侧肾脏拍照，得到图5所示的实验组、生理盐水对照组以及空白对照组各左侧肾脏照片的对比图。从图中可以看到，生理盐水对照组的肾脏出现了明显的水肿增大现象，而实验组的肾脏基本维持正常的形态。
将各大鼠左肾沿冠状面切开后取一半固定于10％福尔马林溶液，并分别取部分进行Masson和天狼星红染色，得到图6所示的实验组、生理盐水对照组和空白对照组的Masson染色照片，和图7所示的实验组、生理盐水对照组和空白对照组的天狼星红染色照片。在Masson染色照片中可以看到，与生理盐水对照组相比，实验组的肾脏纤维化程度显著降低，说明肾前体样细胞对缓解肾纤维化具有积极作用。在天狼星红染色照片中，与生理盐水对照组相比，实验组的肾脏纤维化程度显著降低，说明肾前体样细胞注射后有缓解肾纤维化的作用。
实施例4
本实施例提供了实施例2得到的肾前体细胞在肾病治疗药物，具体为急性肾病治疗药物制备方面的应用。
本实施例还取健康的周龄9-10周，体重在250-300g的雄性SD大鼠肾脏的皮质组织进行扩增转化培养得到含鼠源肾前体样细胞。具体培养过程与实施例2的人源的肾前体样细胞培养过程的区别仅在于肾皮质组织的来源不同。
本实施例中，甘油诱导急性肾损伤模型的建模方法如下：选择健康的周龄9-10周，体重在250-300g的若干只雄性SD大鼠适应性饲养3天并禁水24h后称重，按10mL/kg肌肉注射50％的甘油。
将实施例2的包含人源的肾前体样细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL，对15只甘油诱导急性肾损伤模型大鼠左肾动脉按照6.6×106个/公斤的剂量在注射甘油后的第24小时给药作为受试物组A。
将本实施例得到包含鼠源的肾前体样细胞的消化产物离心收集沉淀后得到的一部分沉淀加入到50mL无菌离心管后在200g离心力的作用下离心5分钟，弃上清，用生理盐水将细胞稀释至2×107个/mL，对15只甘油诱导急性肾损伤模型大鼠左肾动脉按照6.6×106个/公斤的剂量在注射甘油后的第24小时开始给药作为受试物组B。
取15只健康的周龄9-10周，体重在250-300g的雄性SD大鼠作为正常对照组。
取15只甘油诱导急性肾损伤模型大鼠作为模型对照组。各组大鼠分别于注射甘油后6h和72h后取大鼠用异氟烷麻醉，通过眼眶静脉丛取血1mL置于促凝管中，3000rpm离心10分钟，取血清检测Urea、Cr，得到图8所示的正常对照组、模型对照组、受试物组A和受试物组B的Urea水平对比图(每个时间点所对应的若干柱形图中，从左至右依次为正常对照组、模型对照组、受试物组A和受试物组B)，以及图9所示的正常对照组、模型对照组、受试物组A和受试物组B的Cr水平对比图(每个时间点所对应的若干柱形图中，从左至右依次为正常对照组、模型对照组、受试物组A和受试物组B)。参图可知，模型对照组、受试物组A和受试物组B的各大鼠注射甘油后6h，血清中Urea和Cr相较于正常对照组都显著升高(P<0.01)。注射后的24h，受试物组A和受试物组B分别使用人源的肾前体样细胞和鼠源的肾前体样细胞进行了治疗干预，治疗后48h(即注射甘油后72h)，相较于模型对照组，无论是受试物组A还是受试物组B的血清Urea和Cr水平显著降低，说明本实施例和实施例2的鼠源以及人源的肾前体样细胞有修复急性肾损伤的作用。而且，受试物组A和受试物组B各自相较模型对照组而言表现出的Urea和Cr水平变化趋势相当，说明人源的肾前体样细胞表现出与鼠源的肾前体样细胞相当的治疗效果，且不会引起异种排斥。
虽然在上文中详细说明了本发明的实施方式，但是对于本领域的技术人员来说显而易见的是，能够对这些实施方式进行各种修改和变化。但是，应理解，这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且，在此说明的本发明可有其它的实施方式，并且可通过多种方式实施或实现。
本发明的实施方式
本发明各实施例中，如无特别说明，细胞培养均在37摄氏度环境下且二氧化碳浓度为5％的细胞培养箱中进行。细胞培养使用的培养基以及处理细胞所使用的各类试剂，例如缓冲液使用前均经无菌化处理以及0.22微米滤器过滤以去除杂质。
本发明各实施例中涉及统计学分析的数据，每组实验至少重复3次，实验结果数据利用GraphPad Prism 8.0软件进行分析。
本发明实施例的生物制剂包括阳性表达CD133、CD24和CD44的肾前体样细胞，具有强的连续传代能力，能够抑制肾脏组织成纤维化进程，促进肾组织再生或修复。
一些实施例中，所述肾前体样细胞为上皮组织来源前体样细胞。具体的，所述肾前体样细胞具有上皮前体细胞特征。
一些实施例中，所述的“上皮组织”为被覆上皮。具体的，所述上皮组织来源前体样细胞来源于肾皮质组织。
一些实施例中，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种。
一些实施例中，所述肾前体样细胞阳性表达CD133、CD24、CD44、CD73和SOX9。
一些实施例中，所述肾前体样细胞还阴性表达至少一种MHC二类分子。具体的，所述肾前体样细胞还阴性表达HLA-DR、HLA-DP和HLA-DQ的至少一种。
一些实施例中，所述肾前体样细胞还阴性表达CD34和CD45中的至少一种。
一些实施例中，所述肾前体样细胞阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45。
本发明实施例中，所述生物制剂的制备方法包括：使用扩增转化培养基对肾原代细胞进行体外培养，得到阳性表达CD133、CD24和CD44的肾前体样细胞。
一些实施例中所述扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：15-25纳克/毫升的表皮细胞生长因子EGF，40-60纳克/毫升的碱性成纤维细胞生长因子bFGF，体积百分比为0.5％-1.5％的N2添加剂，体积百分比为0.5％-1.5％的B27添加剂，5-15微摩尔/升的ROCK激酶抑制剂，2-4微摩尔/升的糖原合成酶激酶3β抑制剂，0.5-1.5微摩尔/升的TGF-β信号抑制剂，体积百分比为0.5％-1.5％的补充剂ITS，不超过1纳摩尔/升的地塞米松，5-15纳克/毫升的血小板源生长因子PDGF，体积百分比为1-10％的血清。其中，补充剂ITS为胰岛素-转铁蛋白。
一些实施例中，所述基础培养基由体积比为3:1-1:1的DMEM/F12培养基和MCDB培养基组成。
一些实施例中，MCDB培养基为MCDB-131培养基。
一些实施例中，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的任意一种。
一些实施例中，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的任意一种。
一些实施例中，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种。
本发明实施例还提供了所述生物制剂在肾病治疗药物制备方面的应用。

Claims (11)

  1. 一种生物制剂，其特征在于，包括肾前体样细胞，所述肾前体样细胞阳性表达CD133、CD24和CD44。
  2. 根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种。
  3. 根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞还阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
  4. 根据权利要求1所述的生物制剂，其特征在于，所述肾前体样细胞为上皮组织来源前体样细胞。
  5. 根据权利要求4所述的生物制剂，其特征在于，所述上皮组织来源前体样细胞来源于肾皮质组织。
  6. 一种生物制剂的制备方法，其特征在于，包括：
    使用扩增转化培养基对肾原代细胞进行体外培养，得到阳性表达CD133、CD24和CD44的肾前体样细胞；
    所述扩增转化培养基包含基础培养基，以及以占所述基础培养基的体积含量计的以下成分：
    15-25纳克/毫升的EGF，40-60纳克/毫升的bFGF，体积百分比为0.5％-1.5％的N2添加剂，体积百分比为0.5％-1.5％的B27添加剂，5-15微摩尔/升的ROCK激酶抑制剂，2-4微摩尔/升的糖原合成酶激酶3β抑制剂，0.5-1.5微摩尔/升的TGF-β信号抑制剂，体积百分比为0.5％-1.5％的补充剂ITS，不超过1纳摩尔/升的地塞米松，5-15纳克/毫升的血小板源生长因子，体积百分比为1-10％的血清。
  7. 根据权利要求6所述的生物制剂的制备方法，其特征在于，所述基础培养基由体积比为3:1-1:1的DMEM/F12培养基和MCDB培养基组成。
  8. 根据权利要求6所述的生物制剂的制备方法，其特征在于，所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的任意一种，所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的任意一种，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种。
  9. 根据权利要求6所述的生物制剂的制备方法，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
  10. 一种生物制剂在治疗肾病药物的制备方面的应用，其特征在于，所述生物制剂包含阳性表达CD133、CD24和CD44的肾前体样细胞。
  11. 根据权利要求10所述的生物制剂在治疗肾病药物的制备方面的应用，其特征在于，所述肾前体样细胞还阳性表达CD73和SOX9中的至少一种，阴性表达HLA-DR、HLA-DP、HLA-DQ、CD34和CD45中的至少一种。
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