WO2021204061A1 - 胰腺细胞的扩增和分化方法以及应用 - Google Patents

胰腺细胞的扩增和分化方法以及应用 Download PDF

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WO2021204061A1
WO2021204061A1 PCT/CN2021/085019 CN2021085019W WO2021204061A1 WO 2021204061 A1 WO2021204061 A1 WO 2021204061A1 CN 2021085019 W CN2021085019 W CN 2021085019W WO 2021204061 A1 WO2021204061 A1 WO 2021204061A1
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cells
pancreatic
medium
content
density
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French (fr)
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张洪丹
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上海赛立维生物科技有限公司
上海慧存医疗科技有限公司
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Priority to EP21785369.6A priority Critical patent/EP4063487A4/en
Priority to US17/776,594 priority patent/US20220389387A1/en
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Definitions

  • the present invention relates to the field of biotechnology, in particular to methods and applications for the expansion and differentiation of pancreatic cells.
  • Diabetes mellitus is a chronic metabolic disorder characterized by insufficient insulin secretion or hyperglycemia caused by insulin resistance in surrounding tissues. The late stage can cause damage to the whole body tissues and organs, especially the eyes, kidneys, cardiovascular and nervous systems, and their dysfunction. Organ failure. Drug therapy and insulin supplementation are currently the most common treatments for diabetes, but they cannot completely cure diabetes, and it is difficult to prevent the occurrence and development of long-term complications.
  • Human ⁇ -cells can secrete insulin and lower blood sugar to regulate fat and protein metabolism.
  • Human embryonic stem cells have infinite proliferation capacity and theoretically can generate unlimited human ⁇ -cells, so they have attracted much attention in recent years.
  • embryonic stem cells are gradually induced to obtain cells with insulin secretion function, but the induction process is complicated, the cycle is long, and the cost is high; in addition, in the cell mass induced by embryonic stem cells, cells with insulin secretion function The proportion generally does not exceed 30%, and the later purification process is likely to contaminate the cell mass, so that the resulting cells with insulin secretion function have a potential carcinogenic risk. Therefore, it is urgent to find effective replacement cells for pancreatic islet cells and obtain a sufficient number of seed cells that can be used for cell replacement therapy.
  • the purpose of the present invention is to provide a method for expansion, differentiation, and application of pancreatic cells to provide a good alternative to pancreatic islet cells and avoid the ethical limitations and possible consequences of embryonic stem cell-derived therapies in the prior art. Carcinogenic risk, as well as non-specific and off-target deletion risks that are easily caused by the use of gene editing methods to change the gene sequence.
  • the method for expanding pancreatic cells of the present invention includes the following steps:
  • pancreatic tissue which is derived from a pancreatic duct of a mammal
  • S3 Amplify the primary cells with an expansion medium to induce reprogramming of the primary cells to obtain pancreatic precursor-like cells;
  • the amplification medium includes reprogramming substances composed of several small molecule compounds, and the reprogramming substances include growth factors, Rock signaling pathway inhibitors, WNT signaling pathway agonists, and TGF- ⁇ signaling pathway inhibitors.
  • the beneficial effect of the amplification method of the present invention is that a mammalian pancreatic duct is used as the source of the pancreatic precursor-like cells, and after the discontinuous density gradient centrifugation process is used, the obtained cell cluster is removed.
  • the pancreatic islet cells and acinar cells ensure that most of the cells in the primary cells are pancreatic ductal epithelial cells, which improves the yield of the pancreatic precursor-like cells and avoids the use of embryonic stem cells.
  • the amplification medium of step S3 includes a reprogramming substance composed of several small molecule compounds, and the reprogramming substance includes growth factors and Rock signaling pathway Inhibitors, WNT signaling pathway agonists, and TGF- ⁇ signaling pathway inhibitors can obtain pancreatic precursor-like cells by inducing the reprogramming of the primary cells, avoiding the risk of changing gene sequences using gene editing methods Risks such as non-specific and off-target deletion.
  • the pancreatic tissue is derived from a human pancreatic duct.
  • the growth factor includes at least one of epidermal cell growth factor, hepatocyte growth factor and fibroblast growth factor.
  • the epidermal cell growth factor and the hepatocyte growth factor The content of any one of the fibroblast growth factor and the fibroblast growth factor is 5-100 ng/ml.
  • the beneficial effect is that it is beneficial to promote the transformation of the primary cells into pancreatic precursor-like cells.
  • the content of the Rock signaling pathway inhibitor is 2-15 micromol/l
  • the content of the WNT signaling pathway inhibitor is 2-10 micromol/l
  • the TGF The content of - ⁇ signaling pathway inhibitor is 0.5-20 micromol/l.
  • the beneficial effect is that it is beneficial to promote the proliferation of the primary cells.
  • the Rock signaling pathway inhibitor is at least one of Y27632, Fasudil, Thiazovivin and SB-772077-B
  • the WNT signaling pathway agonist is recombinant WNT protein, recombinant R-spondin protein and glycogen synthesis
  • the glycogen synthase kinase 3 ⁇ inhibitor is at least one of BIO, CHIR99021 and TWS119
  • the TGF- ⁇ signaling pathway inhibitor is A83-01, RepSox and SB431542 At least one of them.
  • the expansion medium further includes a basal medium, serum and a serum-free neuron supplement, and in the expansion medium, the total volume percentage of the serum and the serum-free neuron supplement is not high At 15%, the volume percentage of the serum is 2-10%.
  • the reprogramming substance further includes a lipid signal substance, and the content of the lipid signal substance in the amplification medium is 0.5-10 ⁇ mol/L.
  • the lipid signal substance is at least one of sphingosine 1-phosphate and lysophosphatidic acid.
  • the basal medium is at least one of MEM, DMEM, BME, DMEM/F12, RPMI1640, CMRL1066, WilliamE, Neurobasal and Fischers medium
  • the serum is fetal bovine serum
  • the meta additive is at least one of N2 additive and B27 additive.
  • the adjacent layering agents are a low-density layering agent and a high-density layering agent respectively
  • the density of the low-density layering agent is reduced by 0.1-3% relative to the density of the high-density layering agent.
  • the density of the layering agent with the highest density is not higher than 1.1 g/ml, and the density of the layering agent with the lowest density is not low At 1.03 g/ml.
  • the differentiation method of the pancreatic precursor-like cells of the present invention includes: seeding the pancreatic precursor-like cells in a culture container, and then inducing the pancreatic precursor-like cells to differentiate into islet-like cells through a differentiation medium.
  • the beneficial effect of the differentiation method of the present invention is that since the pancreatic precursor-like cells are obtained by the expansion method of the present invention, it is beneficial to obtain good replacement cells for pancreatic islet cells.
  • the differentiation medium includes several small molecule inducers, and the several small molecule inducers include heparin, deacetylase inhibitors, ALK5 inhibitors, T3 and GLP-1 receptor agonists.
  • the beneficial effect is that it is beneficial to induce the differentiation of the pancreatic precursor-like cells into the islet-like cells with good pancreatic islet function.
  • the deacetylase inhibitor is at least one of nicotinamide and nitrastatin A
  • the GLP-1 receptor agonist is glucagon-like peptide-1 and Exendin- At least one of 4.
  • the differentiation medium further includes a basal medium and a serum-free neuron supplement
  • the basal medium is MEM, DMEM, BME, DMEM/F12, RPMI1640, CMRL1066, WilliamE, Neurobasal and Fischers medium
  • At least one of the serum-free neuron additives is at least one of N2 additives and B27 additives.
  • the content of the heparin is 5-15 ⁇ g/ml
  • the content of the deacetylase inhibitor is 5-15 mmol/L
  • the content of the ALK5 inhibitor The content of the T3 is 5-15 micromol/L
  • the content of the T3 is 0.5-2 micromol/L
  • the content of the GLP-1 receptor agonist is 10-50 nanomol/L.
  • the present invention also provides the application of the islet-like cells obtained by the differentiation method, including: the islet-like cells used in the in vitro high glucose reagent to secrete insulin, and 8 weeks of applying the islet-like cells to an animal model Afterwards, the body weight change rate of the animal model is -5%-+5%, and the blood glucose value is 10-20 mmol/L.
  • Figure 1 is a photo of the proliferation of primary cells on the first day after adding expansion medium in an embodiment of the present invention
  • Figure 2 is a photo of the proliferation of the primary cells shown in Figure 1 on the 7th day after adding the expansion medium;
  • Figure 3 is a photo of the proliferation of primary cells of the comparative example of the present invention on the first day after adding the expansion medium;
  • Figure 4 is a photo of the proliferation of the primary cells shown in Figure 3 on the 7th day after adding the expansion medium;
  • Fig. 5 is a schematic diagram of the comparison of the proliferation performance of three pancreatic precursor-like cells according to an embodiment of the present invention.
  • Figure 6 is a photo of the proliferation state of a pancreatic precursor-like cell proliferated to the 10th generation according to an embodiment of the present invention
  • Fig. 7 is a microscopic morphology diagram of pancreatic islet-like cells according to an embodiment of the present invention at a magnification of 100 times;
  • Fig. 8 is a graph showing changes in blood glucose values of different NSG severely immunodeficient mice of the present invention and the comparative example over time after the transplantation experiment is completed;
  • Fig. 9 is a curve of weight change over time of different NSG severe immunodeficiency mice of the embodiment of the present invention and the comparative example after the transplantation experiment is completed.
  • embryonic stem cells can be used to obtain single hormone-secreting cells with similar morphology and function to human pancreatic ⁇ -cells, they express mature ⁇ -cell markers, such as PDX1, MAFA, Insulin, etc., when transplanted into immunodeficient diabetic mice The latter partly reversed hyperglycemia, but its function and transcription level still lag behind those of real ⁇ cells. Potential tumorigenicity and ethical issues also limit its application in humans.
  • Other non-pancreatic islet cells such as gastrointestinal epithelial cells, hepatocytes, and other ⁇ -like cells transformed from ⁇ -like cells are quite different from human ⁇ -cells in molecular and functional properties, and have an epigenetic unstable state.
  • the embodiments of the present invention provide a method for expanding pancreatic cells and a method for differentiation of pancreatic precursor-like cells obtained by the expanding method.
  • Type V collagenase, Ficoll cell separation medium, Percoll cell separation medium, streptozotocin, dithizone, heparin, niacinamide, fetal bovine serum and T3 are from Sigma; HBSS buffer and Hanks are from Biyuntian Biotechnology Co., Ltd.; DMEM/F12 is from Shanghai Yuanpei Biotechnology Co., Ltd.; N2 additives, B27 additives and Y27632 are from Thermo Fisher Scientific; epidermal cell growth factor, hepatocyte growth factor and human fibroblast growth factor are derived from PeproTech company; Thiazovivin, SB431542, BIO, CHIR99021, sphingosine 1-phosphate, lysophosphatidic acid, A83-01, ALK5inhibitorII and GLP-1 are from Tao Plastic Biochemical Co., Ltd.; transferrin, insulin, and Accutase cell digestion are from Shanghai Yisheng Biological Technology Co., Ltd.
  • the amplification method includes the following steps:
  • pancreatic tissue which is derived from a pancreatic duct of a mammal
  • S3 Amplify the primary cells with an expansion medium to induce reprogramming of the primary cells to obtain pancreatic precursor-like cells.
  • the pancreas is responsible for the endocrine and exocrine functions of the human body.
  • the exocrine glands of the pancreas are composed of acini and ducts, which are responsible for secreting and expelling pancreatic juice to assist digestion;
  • the endocrine glands are composed of islets, which are responsible for regulating the body's stable level of glucose, and the endocrine in the islets
  • Cells are usually continuously differentiated from pancreatic ductal stem cells, and studies have shown that pancreatic ducts are the source of new pancreatic islets under certain pathological conditions.
  • the pancreatic tissue is derived from a pancreatic duct of a mammal, which is beneficial to obtain cells with good pancreatic islet function through the expansion method and the differentiation method.
  • the pancreatic duct of the mammal has no pathological changes.
  • the mammal is a human
  • the pancreatic tissue is derived from a human pancreatic duct.
  • the human pancreatic duct has no pathological changes.
  • the digestion reagent used for the digestion treatment includes type V collagenase.
  • the digestion reagent is HBSS buffer containing type V collagenase, and every 100 ml of digestion reagent contains 1 gram of type V collagenase.
  • pancreatic tissue is repeatedly washed with physiological saline containing 10% bi-antibody to remove blood stains, it is cut into pieces of pancreatic tissue about 1 cubic millimeter, and 5 times the volume of the digestion reagent is added, and then placed in Digestion is performed for 30 minutes in a constant temperature shaking water bath at 37° C. at a rate of 120 revolutions per minute; then, the digestion is terminated with HBSS buffer containing fetal bovine serum to complete the digestion treatment.
  • the mass concentration of fetal bovine serum is 10%
  • the temperature of the HBSS buffer is 4°C.
  • Hanks liquid containing fetal bovine serum is used to terminate the digestion, the mass concentration of fetal bovine serum is 10%, and the temperature of Hanks liquid is 4°C.
  • the digestion reagent contains DNase, type V collagenase and type IV collagenase.
  • the enzyme activity unit of the DNase is 15
  • the volume of the type IV collagenase is equal to the volume of the type V collagenase
  • the concentration is both 1 mg/ml.
  • the digested product obtained after the digestion treatment is filtered using a sieve with a pore size of 150 microns, and the precipitate is collected after centrifugation at a centrifugal force of 1000 g for 3 minutes. Resuspend the pellet with a layering agent to obtain a cell suspension.
  • step S2 of the embodiment of the present invention in order to determine that most of the obtained primary cells are pancreatic ductal epithelial cells, so as to increase the yield of the pancreatic precursor-like cells, it is necessary to collect various density gradients.
  • the cell cluster at the junction is then removed from the islet cells and acinar cells in the cell cluster.
  • At least three layering agents with different densities are used for the discontinuous density gradient centrifugation treatment, and the adjacent layering agents are low-density layering agents and high-density layering agents.
  • a layering agent, the density of the low-density layering agent is reduced by 0.1-3% relative to the density of the high-density layering agent, which is conducive to making most of the pancreatic duct epithelial cells exist at the junction between adjacent density gradients Place.
  • the density of the layering agent with the highest density is not higher than 1.1 g/ml, and the density of the layering agent with the lowest density is not lower than 1.03 g/ml.
  • the layering agent is Percoll cell separation solution.
  • Example 1 of the present invention three kinds of Percoll cell separation liquids with densities of 1.075g/ml, 1.045g/ml and 1.035g/ml were arranged, and they were added to the centrifuge tube in sequence, and then put into the resuspended liquid.
  • the cell suspension obtained after the treatment was centrifuged to obtain the suspension; the centrifuged suspension was centrifuged at 4° C., 2500 g centrifugal force for 15 min, and the acceleration was controlled to be 3 m/s 2 and the deceleration was 0 to perform the process.
  • the discontinuous density gradient centrifugation treatment was performed.
  • the volume of the Percoll cell separation solution of each density is the same, and is twice the volume of the suspension to be centrifuged.
  • Example 1 of the present invention the volume of the Percoll cell separation solution of each density is 10 ml, and the volume of the suspension to be centrifuged is 5 ml.
  • the layering agent is Ficoll cell separation solution.
  • Example 2 of the present invention four Ficoll cell separation solutions with densities of 1.088g/ml, 1.076g/ml, 1.066g/ml and 1.040g/ml were arranged, and they were added to the centrifuge tube in sequence, and then put The suspension to be centrifuged; Centrifuge the suspension to be centrifuged at 4° C., 3000g centrifugal force for 20 minutes, and control the acceleration to 3m/s 2 and the deceleration to 0 to perform the discontinuous density gradient centrifugation process .
  • Example 3 of the present invention four Ficoll cell separation solutions with densities of 1.1 g/ml, 1.080 g/ml, 1.060 g/ml, and 1.040 g/ml were configured.
  • the volume of the Ficoll cell separation solution of each density is 10 ml, and the volume of the suspension to be centrifuged is 5 ml.
  • the cell clusters existing at the junction between adjacent density gradients include ductal epithelial cells, pancreatic islet cells, acinar cells and other types of ductal cells, and the cell clusters need to be further removed In order to ensure that most of the obtained primary cells are pancreatic ductal epithelial cells.
  • the red or scarlet islet cells and the acinar cells that look like grape bunches are manually removed under a microscope.
  • DTZ Dithizone
  • the amplification medium includes a reprogramming substance composed of several small molecule compounds, and the reprogramming substance includes growth factors, Rock signaling pathway inhibitors, WNT signaling pathway agonists, and
  • the TGF- ⁇ signaling pathway inhibitor can obtain pancreatic precursor-like cells by inducing the reprogramming of the primary cells, avoiding the risks of non-specific and off-target deletion that are easily caused by the use of gene editing methods to change the gene sequence.
  • the primary cells are seeded in a petri dish pre-coated with Matrigel and cultured for 48 hours, and then the expansion medium is used to continue the expansion deal with.
  • the expansion medium is composed of a basal medium, serum, serum-free neuron supplements, and the reprogramming substance.
  • the total volume percentage of the serum and the serum-free neuron additive is not higher than 15%.
  • the volume percentage of the serum in the amplification medium is 2-10%.
  • the basic medium is at least one of MEM, DMEM, BME, DMEM/F12, RPMI1640, CMRL1066, WilliamE, Neurobasal or Fischers medium.
  • the serum is fetal bovine serum
  • the serum-free neuron additive is at least one of N2 additive and B27 additive.
  • the growth factors include epidermal cell growth factor, hepatocyte growth factor and fibroblast growth factor to facilitate the development of the primary cells.
  • the contents of the epidermal cell growth factor, the human hepatocyte growth factor and the human fibroblast growth factor are all 5-100 ng/ml.
  • the epidermal cell growth factor is human epidermal cell growth factor
  • the hepatocyte growth factor is human hepatocyte growth factor
  • the fibroblast growth factor is human fibroblast growth factor.
  • the content of the Rock signaling pathway inhibitor is 2-15 micromol/L.
  • the Rock signaling pathway inhibitor is at least one of Y27632, Fasudil, Thiazovivin and SB-772077-B.
  • the content of the WNT signaling pathway agonist is 2-10 ⁇ mol/L.
  • the WNT signaling pathway inhibitor is at least one of recombinant Wnt protein, recombinant R-spondin protein and glycogen synthase kinase 3 ⁇ inhibitor.
  • the glycogen synthase kinase 3 ⁇ inhibitor is at least one of BIO, CHIR99021 and TWS119.
  • the content of the TGF- ⁇ signaling pathway inhibitor is 0.5-20 ⁇ mol/L.
  • the TGF ⁇ signaling pathway inhibitor is at least one of A83-01, RepSox and SB431542.
  • the amplification medium further includes a lipid signal substance, and the content of the lipid signal substance is 0.5-10 micromol/L.
  • the lipid signal substance is at least one of sphingosine 1-phosphate and lysophosphatidic acid.
  • the expansion medium is composed of DMEM/F12, fetal bovine serum, N2 additive, B27 additive, human epidermal growth factor, human hepatocyte growth factor, human fibroblast growth factor, Y27632, CHIR99021. , A83-01, sphingosine 1-phosphate and lysophosphatidic acid.
  • the content of human epidermal growth factor is 20 ng/ml
  • the content of human hepatocyte growth factor is 20 ng/ml
  • the content of human fibroblast growth factor is 20 ng/ml.
  • G/ml the content of Y27632 is 10 micromol/l
  • the content of CHIR99021 is 3 micromol/l
  • the content of sphingosine 1-phosphate is 1 micromol/l
  • the content of lysophosphatidic acid is 5 micromol/l
  • the content of A83-01 is 1 micromol/L
  • the volume percentage of the fetal bovine serum is 2%
  • the volume percentage of the N2 additive is 0.5%
  • the volume percentage of the B27 additive is 1%.
  • the amplification medium of Example 2 of the present invention is composed of DMEM/F12, fetal bovine serum, N2 additive, B27 additive, human epidermal growth factor, human fibroblast growth factor, human hepatocyte growth factor, Thiazovivin, BIO and A83- 01 composition.
  • the content of human epidermal growth factor is 20 ng/ml
  • the content of human fibroblast growth factor is 20 ng/ml
  • the content of Thiazovivin is 2 micron.
  • Mol/L the content of BIO is 10 micromol/liter
  • the content of A83-01 is 1 micromol/liter
  • the volume percentage of fetal bovine serum is 5%
  • the volume percentage of N2 additive is 0.5%
  • the volume percentage of B27 additive is Is 1%.
  • the amplification medium of Example 3 of the present invention is composed of DMEM/F12, fetal bovine serum, N2 additive, B27 additive, human epidermal growth factor, human fibroblast growth factor, human hepatocyte growth factor, Y27632, CHIR99021 and SB431542 .
  • the content of human epidermal growth factor is 20 ng/ml
  • the content of human hepatocyte growth factor is 20 ng/ml
  • the content of Y27632 is 10 micromolar. /L
  • the content of CHIR99021 is 3 micromoles/liter
  • the content of SB431542 is 10 micromoles/liter
  • the volume percentage of fetal bovine serum is 5%
  • the volume percentage of N2 additive is 0.5%
  • the volume percentage of B27 additive is 1% .
  • the comparative expansion medium is composed of DMEM/F12, epidermal cell growth factor, fetal bovine serum, transferrin and insulin, the volume percentage of fetal bovine serum is 10%, and epidermal growth factor
  • the content is 20 ng/ml
  • the transferrin content is 5 ⁇ g/ml
  • the insulin content is 10 ⁇ g/ml.
  • Example 1 of the present invention and Comparative Example 1 the amplification treatment time is at least 72 hours, and the amplification medium is replaced every 48 hours after the cells adhere to the wall, except that the composition of the amplification medium is different ,
  • the other operation controls of the steps S1 to S3 are the same.
  • Example 1 of the present invention the degree of removal of pancreatic islet cells and acinar cells in the primary cells was investigated.
  • the primary cells were statically cultured for 3 days using the amplification medium, and it was found that most of the cells adhered to the wall and spread into a single cell layer.
  • the RT-PCR technology was used to detect the expression of related genes on these cells.
  • For the specific detection method please refer to CN108330099A, which will not be repeated here.
  • the primary cells significantly express HNF1B, CK19, and FOXA2, and the expression levels can reach 3000, 300,000, and 10,000, respectively.
  • HNF1B, CK19, and FOXA2 are expressed.
  • the expression levels are all lower than 100 and are almost negligible, indicating that the islet cells and acinar cells in the primary cells are almost negligible.
  • Figure 1 and Figure 2 are photos of the proliferation of the primary cells of Example 1 of the present invention on the 1st and 7th day after adding the expansion medium, respectively.
  • Figures 3 and 4 are the primary cells of Comparative Example 1 of the present invention. Photographs of proliferation on the 1st and 7th day after adding the expansion medium.
  • Example 1 of the present invention showed significant proliferation on the first day after adding the expansion medium, and showed significant expansion on the seventh day.
  • the proliferation of the primary cells of Comparative Example 1 was not significant on the first day after adding the expansion medium, and there was no significant expansion until the seventh day.
  • the expansion medium can endow the primary cells with a strong expansion ability, which is conducive to solving the problem of low yield of replacement cells for pancreatic islet cells.
  • Examples 1-3 of the present invention respectively investigated the proliferation performance of the pancreatic precursor-like cells, and further investigated the proliferation state of the pancreatic precursor-like cells proliferated to the 10th generation through the Edu infiltration experiment.
  • 5000 precursor cells to be investigated were seeded in a six-well plate pre-coated with Matrigel and allowed to stand for 24 hours, and then the cells were digested and counted using a countstar to calculate the doubling time. Please refer to http://www.doubling-time.com/compute.php for specific technical methods and calculation methods of doubling time.
  • BeyoClickTM EdU-488 cell proliferation detection kit from Biyuntian Biotechnology Co., Ltd. was used to stain the 10th generation cells, and the nuclei were calibrated by Hoechst 33342, and then photographed with a Nikon Ts2-FL fluorescent inverted microscope.
  • Fig. 5 is a schematic diagram of comparison of the proliferation performance of pancreatic precursor-like cells in Examples 1-3 of the present invention.
  • the proliferation performance of the pancreatic precursor-like cells of Examples 1-3 shown in Figure 5 shows that the doubling times of the pancreatic precursor-like cells of Examples 1, 2 and 3 are 14 hours, 15 hours and 15 hours, respectively. 12 hours. It can be seen that the three kinds of pancreatic precursor-like cells have good proliferation ability.
  • Figure 6 is a photo of the proliferation state of the pancreatic precursor-like cells of Example 1 of the present invention proliferating to the 10th generation. It can be seen that the pancreatic precursor-like cells of Example 1 of the present invention have proliferated to at least the 10th generation and can still remain active Proliferation state.
  • the number of passages of each type of pancreatic precursor-like cells in Examples 1-3 of the present invention were 15, 16, and 22, respectively.
  • the 10th generation cells formed by the proliferation of each pancreatic precursor-like cell were incubated with 100 ng/ml colchicine at 37°C for 40 minutes, and then the resulting culture was washed, and Use Accutase cell digestion solution to dissociate it into single cells for karyotype analysis and count the chromosomes of at least 40 metaphase arrest cells.
  • the karyotype analysis was performed in the karyotype analysis department of Hangzhou Polar Gene Co., Ltd. Among them, the proliferation of each type of pancreatic precursor-like cells to the 10th generation is still in the exponential growth phase. The result of the karyotype analysis found that the cells can still maintain a stable diploid karyotype without mutation.
  • Examples 1-3 of the present invention the expression levels of stem cell-related markers of each pancreatic precursor-like cell obtained on the 5th and 9th days after adding the expansion medium were investigated by flow cytometry.
  • EpCAM, Pdx1 and Krt19 were all significantly expressed on the 5th and 9th days after adding the expansion medium, indicating that the pancreatic precursor-like cells obtained in the examples of the present invention obtained part of the precursor cells characteristic.
  • the specific operation method of the flow cytometry please refer to CN108330099A, which will not be repeated here.
  • the proportion of positive cells expressing stem cell related markers EpCAM, Pdx1 and Krt19 was counted by flow cytometry on the 5th day after adding the expansion medium as an example, which were 99.2 ⁇ 0.7% and 74.8 ⁇ respectively. 2.3% and 92.0 ⁇ 4.7%.
  • Table 1 shows the average expression levels of pancreatic precursor-like cells of Examples 1-3 of the present invention that express acinar-related markers, endocrine-related markers, and duct or precursor-related markers compared with pancreatic tissue
  • the specific statistical methods are conventional technical means of those skilled in the art.
  • the expression levels of acinar-related markers in pancreatic tissue are extremely low, which proves that the present invention
  • the step S2 of the embodiment effectively removed acinar cells.
  • pancreatic precursor-like cells of the embodiment of the present invention are extremely low, It is proved that the endocrine function of the pancreatic islet-like cells in the examples of the present invention is indeed derived from the differentiated pancreatic precursor-like cells.
  • pancreatic precursor-like cells obtained in the embodiment of the present invention Compared with the expression levels of ducts or precursor-related markers of pancreatic tissue, specifically Pdx1, Nkx6.1, Foxa2, Hnf1b, EpCAM and Krt19, the aforementioned ducts or precursors in the pancreatic precursor-like cells obtained in the embodiment of the present invention
  • the expression levels of related markers are not much different, which proves that the pancreatic precursor-like cells obtained in the examples of the present invention have the potential to differentiate into islet-like cells with endocrine function.
  • the differentiation method of the pancreatic precursor-like cells in the embodiment of the present invention includes: seeding the pancreatic precursor-like cells in a culture container, and then inducing the pancreatic precursor-like cells to differentiate into islet-like cells through a differentiation medium.
  • the differentiation medium includes several small molecule inducers, and the several small molecule inducers include heparin, deacetylase inhibitors, ALK5 inhibitors, T3 and GLP-1 receptor agonists, In order to facilitate the targeted induction of the pancreatic precursor-like cells to differentiate into the islet-like cells with good pancreatic islet function.
  • the content of the heparin is 5-15 ⁇ g/ml
  • the content of the deacetylase inhibitor is 5-15 mmol/L
  • the ALK5 inhibitor The content of T3 is 5-15 micromol/L
  • the content of T3 is 0.5-2 micromol/L
  • the content of GLP-1 receptor agonist is 10-50 nanomol/L.
  • the differentiation medium is composed of a basal medium, a serum-free neuron supplement, and the several small molecule inducers.
  • the deacetylase inhibitor is at least one of niacinamide and nitrastatin A.
  • the GLP-1 receptor agonist is at least one of GLP-1 and Exendin-4.
  • the basal medium in the differentiation medium is at least one of MEM, DMEM, BME, DMEM/F12, RPMI1640, CMRL1066, WilliamE, Neurobasal, and Fischers medium.
  • the serum-free neuron additive in the differentiation medium is at least one of N2 additive and B27 additive.
  • the differentiation medium is composed of DMEM/F12 medium, N2 additive, B27 additive, nicotinamide, heparin, ALK5 inhibitor II, T3 and GLP-1.
  • the volume percentages of N2 additive and B27 additive are both 5%
  • the content of nicotinamide is 10 mmol/L
  • the content of heparin is 10 ⁇ g/ml
  • the content of ALK5inhibitor II is 10 mmol. /L
  • the content of T3 is 1 micromole/liter
  • the content of GLP-1 is 30 nanomole/liter.
  • the differentiation medium in Examples 2 and 3 of the present invention has the same material composition as the differentiation medium in Example 1.
  • the volume percentages of N2 additive and B27 additive are both 3%
  • the content of nicotinamide is 5 mmol/L
  • the content of heparin is 5 ⁇ g/ml
  • the content of ALK5inhibitor II is 5 milliliter.
  • Moles/liter the content of T3 is 0.5 micromol/liter
  • the content of GLP-1 is 10 nanomol/liter.
  • the volume percentages of N2 additive and B27 additive are both 10%, the content of nicotinamide is 15 mmol/L, the content of heparin is 15 ⁇ g/ml, and the content of ALK5inhibitor II is 15 milliliter. Moles/liter, the content of T3 is 2 micromoles/liter, and the content of GLP-1 is 50 nanomoles/liter.
  • the differentiation culture time is 7-10 days, and the differentiation medium is replaced every 48 hours.
  • Fig. 7 is a microscopic morphology diagram of the islet-like cells obtained in Example 1 of the present invention at a magnification of 100 times.
  • the pancreatic islet-like cells present a clear outline, and the outline shape is similar to a polygon.
  • GAPDH was used as the internal reference gene to detect CK19, SOX9, NKX2-2, PDX1, INS, GCG, CK19, SOX9, NKX2-2, PDX1, INS, GCG, and CK19, SOX9, NKX2-2, PDX1, INS, GCG, etc. of each pancreatic precursor-like cell and the corresponding islet-like cell using GAPDH as the internal reference gene. SST and MAFA gene level.
  • pancreatic islet-like cells On the one hand, at the mRNA level, compared with undifferentiated pancreatic precursor-like cells, the expression levels of the precursor-related genes CK19 and SOX9 of pancreatic islet-like cells have decreased.
  • Example 1 the expression level of CK19 decreased by 80%, and the expression level of SOX9 decreased by 30%.
  • the expression levels of mature islet-related genes are significantly up-regulated.
  • Example 1 the expression level of PDX1 is up-regulated by 110%, the expression level of NKX2-2 is up-regulated by 300%, the expression level of MAFA is up-regulated by 200%, the expression level of INS is up-regulated by 350%, and the expression level of GCG is up-regulated by 220%, SST The expression level was increased by 900%.
  • Example 1-3 were subjected to an in vitro insulin stimulation secretion experiment to investigate the islet function of the islet-like cells.
  • the Krb buffer has 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl 2 , 1.2 mM MgCl 2 , 1 mM Na 2 HPO 4 , 1.2 mM KH 2 PO 4 , 5 mM NaHCO 3 , 10 mM HEPES and 0.1% BSA.
  • the concentration of glucose is 2 mmol/L; in the high-sugar Krb buffer solution, the concentration of glucose is 20 mmol/L. In the Krb excitation solution, the content of glucose is 2 mmol/L, and the concentration of KCl is 30 mmol/L.
  • the in vitro insulin stimulation and secretion experiment showed that for the islet-like cells of Example 1, every 103 islet-like cells after the first challenge, the second challenge, and the third challenge
  • the insulin content obtained was 0.6 ⁇ U/ml, 1 ⁇ U/ml and 1.5 ⁇ U/ml, respectively.
  • the insulin content of every 10 3 islet-like cells after the first challenge, the second challenge, and the third challenge is 0.5 ⁇ U/ml, respectively , 1.3 ⁇ U/ml and 1.5 ⁇ U/ml.
  • the insulin content of every 103 islet-like cells after the first challenge, the second challenge, and the third challenge is 0.9 ⁇ U/ml, respectively , 0.7 ⁇ U/ml and 2.2 ⁇ U/ml.
  • Example 1 the stimulation index calculated from the ratio of insulin secreted by high sugar to insulin secreted by low sugar was 2.2 ⁇ 0.4.
  • the islet-like cells applied to the in vitro high glucose reagent can effectively secrete insulin, and the islet-like cells have the potential to differentiate into islet cells.
  • pancreatic islet-like cells were applied to animal models to investigate the rate of change in body weight and blood glucose of experimental animals.
  • the citric acid buffer solution was dissolved with 1% concentration of streptozocin (Streptozocin, STZ) to form an injection solution.
  • streptozocin Streptozocin, STZ
  • the injection was injected intraperitoneally at 130 mg/kg.
  • the blood glucose level of NSG severely immunodeficient mice is ⁇ 20mM, it is considered that the NSG type I diabetes model has been established.
  • 1 ⁇ 10 6 of the pancreatic islet-like cells were mixed with appropriate amount of Matrigel and transplanted under the renal capsule of successfully modeled NSG severe immunodeficiency mice, and then the NSG severe immunodeficiency mice were recovered for one week to complete the transplantation experiment . After the transplantation experiment was completed, the blood glucose level and body weight of the NSG severely immunodeficient mice were monitored.
  • the embodiment of the present invention also provides Comparative Example 2. After the injection is injected into the NSG severe immunodeficiency mice at 130 mg/kg intraperitoneally, Matrigel is transplanted under the renal capsule of the successfully modeled NSG severe immunodeficiency mice. The rest of the process applied to the animal model is the same as in Example 1-3.
  • Figures 8 and 9 are the blood glucose level and body weight of the NSG severe immunodeficiency mice of Examples 1-3 and Comparative Example 2 at the second, fourth, sixth, and eighth weeks after the completion of the transplantation experiment, respectively. Curve over time. It can be seen that within 8 weeks after the completion of the transplantation experiment, the body weight change rate of the animal model of Examples 1-3 is -5%-+5%, and the blood glucose value is 10-20 mmol/L, indicating the present invention The islet-like cells of the examples can effectively help control blood sugar and body weight.

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Abstract

提供了一种胰腺细胞的扩增方法，该方法包括对胰腺组织依次进行消化处理、重悬处理、不连续密度梯度离心处理和扩增处理。该方法使用哺乳动物的胰腺导管作为胰腺前体样细胞的来源，并在不连续密度梯度离心处理后去除得到的细胞团中的胰岛细胞和腺泡细胞。使用的扩增培养基包括由若干小分子化合物组成的重编程物质。还提供了经该扩增方法得到的胰腺前体样细胞的分化方法和应用。

Description

胰腺细胞的扩增和分化方法以及应用 技术领域
本发明涉及生物技术领域，尤其涉及胰腺细胞的扩增和分化方法以及应用。
背景技术
糖尿病是一种以胰岛素分泌不足或周围组织胰岛素抵抗导致的高血糖为特征的慢性代谢紊乱疾病，晚期可导致全身组织器官，特别是眼、肾、心血管及神经系统的损害及其功能障碍和器官衰竭。药物治疗和胰岛素补充是糖尿病目前最常见的治疗方法，但并不能完全治愈糖尿病，同时难以阻止远期并发症的发生发展。
人类β细胞能够分泌胰岛素，降低血糖，以调节脂肪和蛋白质代谢，而人胚胎干细胞由于具有无限的增殖能力，理论上可以从其产生无限量的人类β细胞，因而近年来备受关注。现有技术中对胚胎干细胞进行逐步诱导能够得到具有胰岛素分泌功能的细胞，但诱导的过程程序复杂，周期长且成本高；另外，由胚胎干细胞诱导得到的细胞团中，具有胰岛素分泌功能的细胞占比一般不会超过30％，后期的纯化过程容易污染细胞团，从而使得到的具有胰岛素分泌功能的细胞具有潜在的致癌风险。因此，找到切实有效的胰岛细胞的替代细胞并获得足够数量的可用于细胞替代治疗的种子细胞刻不容缓。
因此，有必要开发一种新型的胰腺细胞的扩增和分化方法，以解决现有技术中存在的上述问题。
发明内容
本发明的目的在于提供一种胰腺细胞的扩增方法、分化方法以及应用，以提 供良好的胰岛细胞的替代选择，避免现有技术中的胚胎干细胞衍生疗法的伦理方面的限制和可能带来的致癌风险，以及由于使用基因编辑方法改变基因序列而容易造成的非特异性及脱靶缺失等风险。
为实现上述目的，本发明的所述胰腺细胞的扩增方法，包括以下步骤：
S1：获取胰腺组织，所述胰腺组织来源于哺乳动物的胰腺导管；
S2：对所述胰腺组织依次进行消化处理、重悬处理和不连续密度梯度离心处理后，收集各密度梯度交界处的细胞团，然后去除所述细胞团中的胰岛细胞和腺泡细胞，得到原代细胞；
S3：采用扩增培养基对所述原代细胞进行扩增处理，以诱导所述原代细胞重编程，得到胰腺前体样细胞；
所述扩增培养基包括由若干小分子化合物组成的重编程物质，所述重编程物质包括生长因子、Rock信号通路抑制剂、WNT信号通路激动剂和TGF-β信号通路抑制剂。
本发明的所述扩增方法的有益效果在于：使用哺乳动物的胰腺导管作为所述胰腺前体样细胞的来源，并采用所述不连续密度梯度离心处理后，去除得到的所述细胞团中的胰岛细胞和腺泡细胞，保证了所述原代细胞中绝大部分细胞为胰腺导管上皮细胞，提高了所述胰腺前体样细胞的得率，同时避免了由于使用胚胎干细胞而带来的伦理方面的限制和可能带来的致癌风险；另外，所述步骤S3的所述扩增培养基包括由若干小分子化合物组成的重编程物质，且所述重编程物质包括生长因子、Rock信号通路抑制剂、WNT信号通路激动剂和TGF-β信号通路抑制剂，能够通过诱导所述原代细胞重编程的方式得到胰腺前体样细胞，避免了由于使用基因编辑方法改变基因序列而容易造成的非特异性及脱靶缺失等风险。
优选的，所述胰腺组织来源于人胰腺导管。
优选的，所述生长因子包括表皮细胞生长因子、肝细胞生长因子和成纤维细胞生长因子中的至少一种，所述扩增培养基中，所述表皮细胞生长因子、所述肝细胞生长因子和所述成纤维细胞生长因子中的任意一种的含量为5-100纳克/毫升。其有益效果在于：有利于促进所述原代细胞向胰腺前体样细胞转化。
优选的，所述扩增培养基中，所述Rock信号通路抑制剂的含量为2-15微摩尔/升，所述WNT信号通路抑制剂的含量为2-10微摩尔/升，所述TGF-β信号通路抑制剂的含量为0.5-20微摩尔/升。其有益效果在于：有利于促进所述原代细胞的增殖。
进一步优选的，所述Rock信号通路抑制剂为Y27632、Fasudil、Thiazovivin和SB-772077-B中的至少一种，所述WNT信号通路激动剂为重组WNT蛋白、重组R-spondin蛋白和糖原合成酶激酶3β抑制剂中的至少一种，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种，所述TGF-β信号通路抑制剂为A83-01、RepSox和SB431542中的至少一种。
优选的，所述扩增培养基还包括基础培养基、血清和无血清神经元添加物，所述扩增培养基中，所述血清和所述无血清神经元添加物的总体积百分比不高于15％，所述血清的体积百分比为2-10％。
进一步优选的，所述重编程物质还包括脂类信号物质，所述扩增培养基中，所述脂类信号物质的含量为0.5-10微摩尔/升。
进一步优选的，所述脂类信号物质为1-磷酸鞘氨醇和溶血磷脂酸中的至少一种。
进一步优选的，所述基础培养基为MEM、DMEM、BME、DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal和Fischers培养基中的至少一种， 所述血清为胎牛血清，所述无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
优选的，所述步骤S2中，使用至少三种具有不同密度的分层剂进行所述不连续密度梯度离心处理，相邻所述分层剂分别为低密度分层剂和高密度分层剂，所述低密度分层剂的密度相对所述高密度分层剂的密度下降0.1-3％。其有益效果在于：有利于使绝大部分胰腺导管上皮细胞存在于相邻的密度梯度之间的交界处。
进一步优选的，所述步骤S2中，所述至少三种具有不同密度的分层剂中，密度最高的分层剂的密度不高于1.1克/毫升，密度最低的分层剂的密度不低于1.03克/毫升。
本发明的所述胰腺前体样细胞的分化方法包括：将所述胰腺前体样细胞接种于培养容器中，然后通过分化培养基诱导所述胰腺前体样细胞分化为胰岛样细胞。
本发明的所述分化方法的有益效果在于：由于所述胰腺前体样细胞由本发明的所述扩增方法得到，有利于得到胰岛细胞的良好替代细胞。
优选的，所述分化培养基包括若干小分子诱导剂，所述若干小分子诱导剂包括肝素、去乙酰化酶抑制剂、ALK5抑制剂、T3和GLP-1受体激动剂。其有益效果在于：有利于定向诱导所述胰腺前体样细胞分化为具有良好胰岛功能的所述胰岛样细胞。
进一步优选的，所述去乙酰化酶抑制剂为尼克酰胺和尼曲古抑菌素A中的至少一种，所述GLP-1受体激动剂为胰高血糖素样肽-1和Exendin-4中的至少一种。
进一步优选的，所述分化培养基还包括基础培养基和无血清神经元添加物， 所述基础培养基为MEM、DMEM、BME、DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal和Fischers培养基中的至少一种，所述无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
进一步优选的，所述分化培养基中，所述肝素的含量为5-15微克/毫升，所述去乙酰化酶抑制剂的含量为5-15毫摩尔/升，所述ALK5抑制剂的含量为5-15微摩尔/升，所述T3的含量为0.5-2微摩尔/升，所述GLP-1受体激动剂的含量为10-50纳摩尔/升。
本发明还提供给了由所述分化方法得到的胰岛样细胞的应用，包括：应用于体外高糖试剂的所述胰岛样细胞分泌胰岛素，以及将所述胰岛样细胞应用于动物模型的8周后，所述动物模型的体重变化率为-5％-+5％，血糖值为10-20毫摩尔/升。
附图说明
图1为本发明实施例的原代细胞加入扩增培养基后第1天的增殖情况照片；
图2为图1所示的原代细胞加入扩增培养基后第7天的增殖情况照片；
图3为本发明对比例的原代细胞加入扩增培养基后第1天的增殖情况照片；
图4为图3所示的原代细胞加入扩增培养基后第7天的增殖情况照片；
图5为本发明实施例的三种胰腺前体样细胞的增殖性能对比示意图；
图6为本发明实施例的一种胰腺前体样细胞增殖至第10代的增殖状态照片；
图7为本发明实施例的胰岛样细胞在100倍的放大倍数下的微观形貌图；
图8为本发明实施例和对比例的不同NSG重度免疫缺陷小鼠在移植实验完毕后的血糖值随时间的变化曲线；
图9为本发明实施例和对比例的不同NSG重度免疫缺陷小鼠在移植实验完毕后的体重随时间的变化曲线。
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，下面结合附图对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
现有技术中，尽管使用胚胎干细胞能够得到与人胰岛β细胞形态功能相似的单激素分泌细胞，其表达成熟β细胞标志物，例如PDX1，MAFA，Insulin等在移植入免疫缺陷的糖尿病小鼠体内后部分逆转了高血糖，但其功能及转录水平仍与真正的β细胞存在差距，潜在的致瘤性及伦理问题也限制了其在人体的应用。其他的非胰岛细胞如胃肠道上皮细胞，肝细胞等转化而来的β样细胞其分子和功能特性均与人β细胞差异较大，并存在表观遗传的不稳定状态。
针对现有技术存在的问题，本发明实施例提供了一种胰腺细胞的扩增方法，以及对经所述扩增方法得到的胰腺前体样细胞进行的分化方法。
本发明实施例主要试剂来源如下：
V型胶原酶、Ficoll细胞分离液、Percoll细胞分离液、链脲佐菌素、双硫腙、肝素、尼克酰胺、胎牛血清和T3来源于Sigma公司；HBSS缓冲液和Hanks液来源于碧云天生物科技有限公司；DMEM/F12来源于上海源培生物科技有限公司；N2添加剂、B27添加剂和Y27632来源于Thermo Fisher Scientific公司；表 皮细胞生长因子、肝细胞生长因子和人成纤维细胞生长因子来源于PeproTech公司；Thiazovivin、SB431542、BIO、CHIR99021、1-磷酸鞘氨醇、溶血磷脂酸、A83-01、ALK5inhibitorⅡ和GLP-1来源于陶塑生化有限公司；转铁蛋白、胰岛素、Accutase细胞消化液来源于上海翊圣生物科技有限公司。
本发明实施例的所有统计分析均使用GraphPad Prism 7进行。为了比较两个平均值，使用two-tailed unpaired t-test来计算统计显着性。为了比较多个值，将one-wayANOVA与Dunnett校正一起使用进行多个值与单个值比较，将多个值相互比较时，进行Tukey校正。P值小于0.05被认为具有统计学意义。
所述扩增方法包括以下步骤：
S1：获取胰腺组织，所述胰腺组织来源于哺乳动物的胰腺导管；
S2：对所述胰腺组织依次进行消化处理、重悬处理和不连续密度梯度离心处理后，收集各密度梯度交界处的细胞团，然后去除所述细胞团中的胰岛细胞和腺泡细胞，得到原代细胞；
S3：采用扩增培养基对所述原代细胞进行扩增处理，以诱导所述原代细胞重编程，得到胰腺前体样细胞。
胰腺负责人体的内分泌和外分泌功能，胰腺的外分泌腺由腺泡和腺管组成，负责分泌并排出胰液，以辅助消化；内分泌腺由胰岛组成，负责调节机体的葡萄糖稳定水平，而胰岛中的内分泌细胞通常都是从胰腺导管干细胞连续分化而来，且有研究证明在某些病理条件下，胰腺导管是新生胰岛的来源。本发明实施例的所述步骤S1中，所述胰腺组织来源于哺乳动物的胰腺导管，有利于通过所述扩增方法和所述分化方法得到具有良好胰岛功能的细胞。
本发明实施例中，所述哺乳动物的胰腺导管不存在病变。
本发明实施例1-3中，所述哺乳动物为人，所述胰腺组织来源于人胰腺导管。
具体的，所述人胰腺导管不存在病变。
本发明一些实施例的所述步骤S2中，进行所述消化处理使用的消化试剂包含V型胶原酶。
本发明实施例1-3中，所述消化试剂为含有V型胶原酶的HBSS缓冲液，每100毫升消化试剂中含有1克V型胶原酶。
具体的，将所述胰腺组织用含有10％双抗的生理盐水反复冲洗以去除血污后，剪碎为1立方毫米左右的胰腺组织碎块并加入5倍体积的所述消化试剂，然后置于37℃的恒温振荡水浴中以120转/分钟的速率进行30分钟的消化；然后使用含胎牛血清的HBSS缓冲液终止所述消化，以完成所述消化处理。胎牛血清的质量浓度为10％，HBSS缓冲液的温度为4℃。
本发明一些实施例中，使用含胎牛血清的Hanks液终止所述消化，胎牛血清的质量浓度为10％，Hanks液的温度为4℃。
本发明一些具体的实施例中，所述消化试剂含有DNA酶、V型胶原酶和IV型胶原酶。
具体的，所述DNA酶的酶活力单位为15，所述IV型胶原酶的体积与所述V型胶原酶的体积相等，浓度均为1毫克/毫升。
本发明实施例1-3中，所述消化处理结束后，使用孔径为150微米的筛网对经所述消化处理后得到的消化产物过滤后，在1000g的离心力下离心3分钟后取沉淀，使用分层剂对所述沉淀进行重悬，得到细胞悬液。
本发明实施例的所述步骤S2中，为确定得到的所述原代细胞中的绝大部分细胞为胰腺导管上皮细胞，以提高所述胰腺前体样细胞的得率，需要收集各密度梯度交界处的细胞团，然后去除所述细胞团中的胰岛细胞和腺泡细胞。
本发明一些实施例的所述步骤S2中，使用至少三种具有不同密度的分层剂 进行所述不连续密度梯度离心处理，相邻所述分层剂分别为低密度分层剂和高密度分层剂，所述低密度分层剂的密度相对所述高密度分层剂的密度下降0.1-3％，有利于使绝大部分胰腺导管上皮细胞存在于相邻的密度梯度之间的交界处。
进一步的，所述具有不同密度的分层剂中，密度最高的分层剂的密度不高于1.1克/毫升，密度最低的分层剂的密度不低于1.03克/毫升。
本发明一些实施例中，所述分层剂为Percoll细胞分离液。
本发明实施例1中，配置密度分别为1.075g/ml、1.045g/ml以及1.035g/ml的三种Percoll细胞分离液，并顺次加入到离心管中，然后放入经所述重悬处理后得到的细胞悬液，得到待离心悬液；对所述待离心悬液在4℃，2500g的离心力下离心15min，并控制注意加速度为3m/s 2，减速度为0，以进行所述不连续密度梯度离心处理。
本发明一些具体的实施例中，每种密度的Percoll细胞分离液的体积相同，且为所述待离心悬液体积的2倍。
本发明实施例1中，每种密度的Percoll细胞分离液的体积为10毫升，所述待离心悬液的体积为5毫升。
本发明一些实施例中，所述分层剂为Ficoll细胞分离液。
本发明实施例2中，配置密度分别为1.088g/ml、1.076g/ml、1.066g/ml和1.040g/ml的四种Ficoll细胞分离液，并顺次加入到离心管中，然后放入所述待离心悬液；对所述待离心悬液在4℃，3000g的离心力下离心20min，并控制注意加速度为3m/s 2，减速度为0，以进行所述不连续密度梯度离心处理。
本发明实施例3中，配置密度分别为1.1g/ml、1.080g/ml、1.060g/ml和1.040g/ml的四种Ficoll细胞分离液。
本发明实施例2和实施例3中，每种密度的Ficoll细胞分离液的体积为10毫升，所述待离心悬液的体积为5毫升。
本发明实施例的所述步骤S2中，存在于相邻的密度梯度之间的交界处细胞团包括导管上皮细胞、胰岛细胞、腺泡细胞以及其他类型的导管细胞，需要进一步去除所述细胞团中的胰岛细胞和腺泡细胞，以保证得到的所述原代细胞中的绝大部分细胞为胰腺导管上皮细胞。
本发明一些实施例中，对所述细胞团进行双硫腙(Dithizone，DTZ)染色后，在显微镜下手动去除呈现红色或猩红色的胰岛细胞，以及呈葡萄串珠样的腺泡细胞。具体的染色和去除方法为本领域技术人员的常规技术手段，在此不做赘述。
本发明实施例的所述步骤S3中，所述扩增培养基包括由若干小分子化合物组成的重编程物质，所述重编程物质包括生长因子、Rock信号通路抑制剂、WNT信号通路激动剂和TGF-β信号通路抑制剂，能够通过诱导所述原代细胞重编程的方式得到胰腺前体样细胞，避免了由于使用基因编辑方法改变基因序列而容易造成的非特异性及脱靶缺失等风险。
本发明一些实施例的所述步骤S3中，将所述原代细胞接种于预先包被有基质胶的培养皿中静置培养48小时后，然后使用所述扩增培养基继续所述扩增处理。
本发明一些实施例中，所述扩增培养基由基础培养基、血清、无血清神经元添加物以及所述重编程物质组成。所述扩增培养基中，所述血清和所述无血清神经元添加物的总体积百分比不高于15％。
本发明一些实施例中，所述扩增培养基中，所述血清的体积百分比为2-10％。
本发明一些具体的实施例中，所述基础培养基为MEM、DMEM、BME、 DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal或Fischers培养基中的至少一种。
本发明一些具体的实施例中，所述血清为胎牛血清，所述无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
本发明一些实施例中，所述生长因子包括表皮细胞生长因子、肝细胞生长因子和成纤维细胞生长因子，以有利于促进所述原代细胞的发育。所述扩增培养基中，所述表皮细胞生长因子、所述人肝细胞生长因子和所述人成纤维细胞生长因子的含量均为5-100纳克/毫升。
本发明一些实施例中，所述表皮细胞生长因子为人表皮细胞生长因子，所述肝细胞生长因子为人肝细胞生长因子，所述成纤维细胞生长因子为人成纤维细胞生长因子。
本发明一些实施例的所述扩增培养基中，所述Rock信号通路抑制剂的含量为2-15微摩尔/升。
具体的，所述Rock信号通路抑制剂为Y27632，Fasudil，Thiazovivin和SB-772077-B中的至少一种。
本发明一些实施例的所述扩增培养基中，所述WNT信号通路激动剂的含量为2-10微摩尔/升。
具体的，所述WNT信号通路抑制剂为重组Wnt蛋白，重组R-spondin蛋白和糖原合成酶激酶3β抑制剂中的至少一种。所述糖原合成酶激酶3β抑制剂为BIO，CHIR99021和TWS119中的至少一种。
本发明一些实施例的所述扩增培养基中，所述TGF-β信号通路抑制剂的含量为0.5-20微摩尔/升。
具体的，所述TGFβ信号通路抑制剂为A83-01，RepSox和SB431542中至少 一种。
本发明一些实施例中，所述扩增培养基还包括脂类信号物质，所述脂类信号物质的含量为0.5-10微摩尔/升。
具体的，所述脂类信号物质为1-磷酸鞘氨醇和溶血磷脂酸中的至少一种。
本发明实施例1中，所述扩增培养基由DMEM/F12、胎牛血清、N2添加剂、B27添加剂、人表皮细胞生长因子、人肝细胞生长因子、人成纤维细胞生长因子、Y27632、CHIR99021、A83-01、1-磷酸鞘氨醇和溶血磷脂酸组成。
具体的，所述扩增培养基中，人表皮细胞生长因子的含量为20纳克/毫升，人肝细胞生长因子的含量为20纳克/毫升，人成纤维细胞生长因子的含量为20纳克/毫升、Y27632的含量为10微摩尔/升，CHIR99021的含量为3微摩尔/升，1-磷酸鞘氨醇的含量为1微摩尔/升，溶血磷脂酸的含量为5微摩尔/升，A83-01的含量为1微摩尔/升，所述胎牛血清的体积百分比为2％，N2添加剂的体积百分比为0.5％，B27添加剂的体积百分比为1％。
本发明实施例2的扩增培养基由DMEM/F12、胎牛血清、N2添加剂、B27添加剂、人表皮细胞生长因子、人成纤维细胞生长因子、人肝细胞生长因子、Thiazovivin、BIO和A83-01组成。
具体的，本发明实施例2的扩增培养基中，人表皮细胞生长因子的含量为20纳克/毫升，人成纤维细胞生长因子的含量为20纳克/毫升、Thiazovivin的含量为2微摩尔/升，BIO的含量为10微摩尔/升，A83-01的含量为1微摩尔/升，胎牛血清的体积百分比为5％、N2添加剂的体积百分比为0.5％，B27添加剂的体积百分比为1％。
本发明实施例3的扩增培养基由DMEM/F12、胎牛血清、N2添加剂、B27添加剂、人表皮细胞生长因子、人成纤维细胞生长因子、人肝细胞生长因子、 Y27632、CHIR99021和SB431542组成。
具体的，本发明实施例3的扩增培养基中，人表皮细胞生长因子的含量为20纳克/毫升，人肝细胞生长因子的含量为20纳克/毫升，Y27632的含量为10微摩尔/升，CHIR99021的含量是3微摩尔/升，SB431542的含量为10微摩尔/升，胎牛血清的体积百分比为5％、N2添加剂的体积百分比为0.5％、B27添加剂的体积百分比为1％。
本发明实施例的对比例1中，对比扩增培养基由DMEM/F12、表皮细胞生长因子、胎牛血清、转铁蛋白和胰岛素组成，胎牛血清的体积百分比为10％，表皮细胞生长因子的含量为20纳克/毫升，转铁蛋白的含量为5微克/毫升，胰岛素的含量为10微克/毫升。
本发明实施例1和对比例1中，所述扩增处理的时间至少为72小时，待细胞贴壁后每48小时更换所述扩增培养基，除所述扩增培养基的组成不同外，所述步骤S1至S3的其他操作控制均相同。
本发明实施例1考察了所述原代细胞中的胰岛细胞和腺泡细胞的去除程度。使用所述扩增培养基对所述原代细胞进行3天的静置培养，发现大部分细胞贴壁并平铺为单细胞层，利用RT-PCR技术对这些细胞进行相关基因表达的检测。具体的检测方法请参见CN108330099A，在此不做赘述。
具体的，所述原代细胞显著表达HNF1B、CK19以及FOXA2，表达量分别可达3000、300000和10000，而在与腺泡细胞和胰岛细胞相关的基因中，仅HNF1A和INS有表达的现象，但表达量均低于100，几乎可以忽略不计，说明所述原代细胞中的胰岛细胞和腺泡细胞几乎可以忽略不计。
图1和图2分别为本发明实施例1的原代细胞加入扩增培养基后第1天和第7天的增殖情况照片，图3和图4分别为本发明对比例1的原代细胞加入扩增培养基后第1天和第7天的增殖情况照片。
参照图1和图2，本发明实施例1的原代细胞在加入扩增培养基后第1天就表现出了显著的增殖，第7天表现出了明显的扩增。
参照图3和图4，对比例1的原代细胞在加入扩增培养基后第1天的增殖情况不显著，直至第7天也没有出现明显的扩增，可见本发明实施例的所述扩增培养基能够赋予所述原代细胞强大的扩增能力，有利于解决胰岛细胞的替代细胞得率低的问题。
本发明实施例1-3分别考察了所述胰腺前体样细胞的增殖性能，并通过Edu渗入实验进一步考察了增殖至第10代的胰腺前体样细胞的增殖状态。
具体的，将5000个待考察前体细胞接种于预包被有基质胶的六孔板中静置24小时，然后消化细胞并使用countstar计数，计算倍增时间。具体的技术方法以及倍增时间的计算方法请参见http://www.doubling-time.com/compute.php。
所述Edu渗入实验使用碧云天生物科技有限公司的BeyoClickTMEdU-488细胞增值检测试剂盒将第10代细胞染色，由Hoechst33342标定细胞核，然后用NikonTs2-FL荧光倒置显微镜拍照。
图5为本发明实施例1-3的胰腺前体样细胞的增殖性能对比示意图。
参照图5，由图5所示的实施例1-3的胰腺前体样细胞的增殖性能可以得到实施例1、2和3的胰腺前体样细胞的倍增时间分别为14小时、15小时和12小时。可见，三种胰腺前体样细胞具有良好的增殖能力。
图6为本发明实施例1的胰腺前体样细胞增殖至第10代的增殖状态照片，可见本发明实施例1的所述胰腺前体样细胞至少增殖至第10代，仍能保持活跃的增殖状态。
本发明实施例1-3中的每种胰腺前体样细胞的传代次数分别为15、16和22。
本发明实施例1-3中，将每种胰腺前体样细胞增殖形成的第10代细胞与100 纳克/毫升的秋水仙碱在37℃下孵育40分钟，然后洗涤得到的培养物，并使用Accutase细胞消化液将其解离为单细胞，以进行核型分析并计数至少40个中期停滞细胞的染色体。所述核型分析在杭州极地基因有限公司核型分析部门进行。其中，每种胰腺前体样细胞增殖至第10代仍处于指数增长期。所述核型分析的结果发现细胞仍能保持稳定的二倍体核型而不会发生变异。
本发明实施例1-3中，通过流式细胞术考察加入所述扩增培养基后的第5天和第9天得到的每种胰腺前体样细胞的干细胞相关标志物的表达水平。
一方面，EpCAM、Pdx1以及Krt19在加入所述扩增培养基后的第5天和第9天后均显著表达，表明本发明实施例得到的所述胰腺前体样细胞获得了部分的前体细胞特性。所述流式细胞术的具体操作方法请参见CN108330099A，在此不做赘述。
具体的，以加入所述扩增培养基后的第5天通过流式细胞术统计表达干细胞相关标志物EpCAM、Pdx1以及Krt19的阳性细胞数占比为例，分别为99.2±0.7％，74.8±2.3％和92.0±4.7％。
另一方面，表1统计了和胰腺组织相比，本发明实施例1-3的胰腺前体样细胞表达腺泡相关标志物、内分泌相关标志物以及导管或前体相关标志物的平均表达水平，具体的统计方法为本领域技术人员的常规技术手段。
表1
Figure PCTCN2021085019-appb-000001
Figure PCTCN2021085019-appb-000002
从表2中可以看到：
相较于胰腺组织的腺泡相关标志物，具体为Ptf1a、Gata4和Cpa1的表达水平，本发明实施例的胰腺前体样细胞中上述腺泡相关标志物的表达水平极低， 证明通过本发明实施例的所述步骤S2有效去除了腺泡细胞。
相较于胰腺组织的内分泌相关标志物，具体为Nkx2.2、Mafb、Gcg、Sst和Ins的表达水平，本发明实施例的胰腺前体样细胞中上述内分泌相关标志物的表达水平极低，证明本发明实施例的胰岛样细胞的内分泌功能确实来源于分化后的胰腺前体样细胞。
相较于胰腺组织的导管或前体相关标志物，具体为Pdx1、Nkx6.1、Foxa2、Hnf1b、EpCAM和Krt19的表达水平，本发明实施例获得的胰腺前体样细胞中上述导管或前体相关标志物的表达水平相差并不大，证明本发明实施例获得的胰腺前体样细胞具有分化为具有内分泌功能的胰岛样细胞的潜力。
本发明实施例的所述胰腺前体样细胞的分化方法包括：将所述胰腺前体样细胞接种于培养容器中，然后通过分化培养基诱导所述胰腺前体样细胞分化为胰岛样细胞。
本发明一些实施例中，所述分化培养基包括若干小分子诱导剂，所述若干小分子诱导剂包括肝素、去乙酰化酶抑制剂、ALK5抑制剂、T3和GLP-1受体激动剂，以有利于定向诱导所述胰腺前体样细胞分化为具有良好胰岛功能的所述胰岛样细胞。
本发明一些实施例的所述分化培养基中，所述肝素的含量为5-15微克/毫升，所述去乙酰化酶抑制剂的含量为5-15毫摩尔/升，所述ALK5抑制剂的含量为5-15微摩尔/升，所述T3的含量为0.5-2微摩尔/升，所述GLP-1受体激动剂的含量为10-50纳摩尔/升。
本发明一些实施例中，所述分化培养基由基础培养基、无血清神经元添加物和所述若干小分子诱导剂组成。
本发明一些实施例中，所述去乙酰化酶抑制剂为尼克酰胺和尼曲古抑菌素A 中的至少一种。
本发明一些实施例中，所述GLP-1受体激动剂为GLP-1和Exendin-4中的至少一种。
本发明一些实施例中，所述分化培养基中的基础培养基为MEM、DMEM、BME、DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal和Fischers培养基中的至少一种。
本发明一些具体的实施例中，所述分化培养基中的无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
本发明实施例1中，所述分化培养基由DMEM/F12培养基、N2添加剂、B27添加剂、尼克酰胺、肝素、ALK5inhibitorⅡ、T3和GLP-1组成。
具体的，所述分化培养基中，N2添加剂和B27添加剂的体积百分比均为5％，尼克酰胺的含量为10毫摩尔/升，肝素的含量为10微克/毫升，ALK5inhibitorⅡ的含量为10毫摩尔/升，T3的含量为1微摩尔/升，GLP-1的含量为30纳摩尔/升。
本发明实施例2和3中的分化培养基具有与实施例1的分化培养基相同的物质组成。
本发明实施例2的分化培养基中，N2添加剂和B27添加剂的体积百分比均为3％，尼克酰胺的含量为5毫摩尔/升，肝素的含量为5微克/毫升，ALK5inhibitorⅡ的含量为5毫摩尔/升，T3的含量为0.5微摩尔/升，GLP-1的含量为10纳摩尔/升。
本发明实施例3的分化培养基中，N2添加剂和B27添加剂的体积百分比均为10％，尼克酰胺的含量为15毫摩尔/升，肝素的含量为15微克/毫升，ALK5inhibitorⅡ的含量为15毫摩尔/升，T3的含量为2微摩尔/升，GLP-1的含 量为50纳摩尔/升。
本发明实施例1-3中，所述分化培养的时间为7-10天，每48小时更换所述分化培养基。
图7为本发明实施例1得到的胰岛样细胞在100倍的放大倍数下的微观形貌图。参照图7，所述胰岛样细胞呈现出明显的轮廓，轮廓形状类似多边形。
本发明实施例1-3中，以GAPDH为内参照基因，使用Q-PCR方法检测每种胰腺前体样细胞和对应的胰岛样细胞的CK19、SOX9、NKX2-2、PDX1、INS、GCG、SST以及MAFA的基因水平。
一方面，在mRNA水平上，与未分化的胰腺前体样细胞相比，胰岛样细胞的前体相关基因CK19和SOX9的表达水平有所下降。
以实施例1为例，CK19的表达水平下降80％，SOX9的表达水平下降30％。
另一方面，在mRNA水平上，与未分化的胰腺前体样细胞相比，成熟胰岛相关基因，诸如PDX1、NKX2-2、MAFA、INS、GCG以及SST的表达水平有显著上调。
以实施例1为例，PDX1的表达水平上调110％，NKX2-2的表达水平上调300％，MAFA的表达水平上调200％，INS的表达水平上调350％，GCG的表达水平上调220％，SST的表达水平上调900％。
进一步的，对实施例1-3的胰岛样细胞进行体外胰岛素激发分泌实验，以考察胰岛样细胞的胰岛功能。
首先通过Krb缓冲液配置低糖Krb缓冲液、高糖Krb缓冲液和Krb激发溶液；取1×10 6个胰岛样细胞，用所述Krb缓冲液洗涤两次后在所述低糖Krb缓冲液中孵育1小时以平衡细胞；然后使用所述Krb缓冲液洗涤后再放置于所述低糖Krb缓冲液中温育30min，以完成第一次激发；去除上清然后使用所述Krb缓冲液洗 涤洗涤两次后在所述高糖Krb缓冲液中温育30min，以完成第二次激发，再次去除上清并使用所述Krb缓冲液洗涤两次，最后将得到的细胞放在所述Krb激发溶液中温育30min，最后去除上清，以完成第三次激发，对每次激发得到的细胞用TrypLE消化并使用countstar计数。
具体的，所述Krb缓冲液具有128mM NaCl、5mM KCl、2.7mM CaCl 2、1.2mM MgCl 2、1mM Na 2HPO 4、1.2mM KH 2PO 4、5mM NaHCO 3、10mM HEPES和0.1％BSA。
所述低糖Krb缓冲液中，葡萄糖的浓度为2毫摩尔/升；所述高糖Krb缓冲液缓冲液中，葡萄糖的浓度为20毫摩尔/升。所述Krb激发溶液中，葡萄糖的含量为2毫摩尔/升，KCl的浓度为30毫摩尔/升。
所述体外胰岛素激发分泌实验的表明，对于实施例1的胰岛样细胞来说，每10 3个胰岛样细胞经所述第一次激发、所述第二次激发和所述第三次激发后得到的胰岛素含量分别为0.6μU/ml、1μU/ml以及1.5μU/ml。
对于实施例2的胰岛样细胞来说，每10 3个胰岛样细胞经所述第一次激发、所述第二次激发和所述第三次激发后得到的胰岛素含量分别为0.5μU/ml、1.3μU/ml以及1.5μU/ml。
对于实施例3的胰岛样细胞来说，每10 3个胰岛样细胞经所述第一次激发、所述第二次激发和所述第三次激发后得到的胰岛素含量分别为0.9μU/ml、0.7μU/ml以及2.2μU/ml。
另外，以实施例1为例，由高糖分泌的胰岛素与低糖分泌的胰岛素之比计算得出的刺激指数为2.2±0.4。
综合所述体外胰岛素激发分泌实验的实验结果可知，应用于体外高糖试剂的所述胰岛样细胞能够有效分泌胰岛素，所述胰岛样细胞具有分化为胰岛细胞的 潜力。
本发明实施例1-3将所述胰岛样细胞应用于动物模型，考察实验动物的体重变化率和血糖值。
具体的，将柠檬酸缓冲液以1％的浓度溶解链脲佐菌素(Streptozocin，STZ)，形成注射液。对NSG重度免疫缺陷小鼠称重后以130mg/kg腹腔注射所述注射液。注射完毕的一周后，NSG重度免疫缺陷小鼠血糖值≥20mM即视为建立了NSGⅠ型糖尿病模型。将1×10 6个所述胰岛样细胞分别与适量基质胶混合后移植入造模成功的NSG重度免疫缺陷小鼠的肾被膜下，然后使NSG重度免疫缺陷小鼠恢复一周，以完成移植实验。所述移植实验完毕后，监测NSG重度免疫缺陷小鼠的血糖值和体重。
本发明实施例还提供了对比例2，对NSG重度免疫缺陷小鼠按130mg/kg腹腔注射所述注射液后，将基质胶移植入造模成功的NSG重度免疫缺陷小鼠的肾被膜下，其余应用于动物模型的过程与实施例1-3相同。
图8和图9分别为实施例1-3以及对比例2的NSG重度免疫缺陷小鼠在所述移植实验完毕后的第2、4、6和8周的血糖值随时间的变化曲线和体重随时间的变化曲线。可见，所述移植实验完毕后的8周内，实施例1-3的所述动物模型的体重变化率为-5％-+5％，血糖值为10-20毫摩尔/升，表明本发明实施例的所述胰岛样细胞能有效协助控制血糖和体重。
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。

Claims (18)

  1. 一种胰腺细胞的扩增方法，其特征在于，包括以下步骤：
    S1：获取胰腺组织，所述胰腺组织来源于哺乳动物的胰腺导管；
    S2：对所述胰腺组织依次进行消化处理、重悬处理和不连续密度梯度离心处理后，收集各密度梯度交界处的细胞团，然后去除所述细胞团中的胰岛细胞和腺泡细胞，得到原代细胞；
    S3：采用扩增培养基对所述原代细胞进行扩增处理，以诱导所述原代细胞重编程，得到胰腺前体样细胞；
    所述扩增培养基包括由若干小分子化合物组成的重编程物质，所述重编程物质包括生长因子、Rock信号通路抑制剂、WNT信号通路激动剂和TGF-β信号通路抑制剂。
  2. 根据权利要求1所述的扩增方法，其特征在于，所述胰腺组织来源于人胰腺导管。
  3. 根据权利要求1所述的扩增方法，其特征在于，所述生长因子包括表皮细胞生长因子、肝细胞生长因子和成纤维细胞生长因子中的至少一种，所述扩增培养基中，所述表皮细胞生长因子、所述肝细胞生长因子和所述成纤维细胞生长因子中的任意一种的含量为5-100纳克/毫升。
  4. 根据权利要求1所述的扩增方法，其特征在于，所述扩增培养基中，所述Rock信号通路抑制剂的含量为2-15微摩尔/升，所述WNT信号通路抑制剂的含量为2-10微摩尔/升，所述TGF-β信号通路抑制剂的含量为0.5-20微摩尔/升。
  5. 根据权利要求4所述的扩增方法，其特征在于，所述Rock信号通路抑制剂为Y27632、Fasudil、Thiazovivin和SB-772077-B中的至少一种，所述WNT信 号通路激动剂为重组WNT蛋白、重组R-spondin蛋白和糖原合成酶激酶3β抑制剂中的至少一种，所述糖原合成酶激酶3β抑制剂为BIO、CHIR99021和TWS119中的至少一种，所述TGF-β信号通路抑制剂为A83-01、RepSox和SB431542中的至少一种。
  6. 根据权利要求1所述的扩增方法，其特征在于，所述扩增培养基还包括基础培养基、血清和无血清神经元添加物，所述扩增培养基中，所述血清和所述无血清神经元添加物的总体积百分比不高于15％，所述血清的体积百分比为2-10％。
  7. 根据权利要求6所述的扩增方法，其特征在于，所述重编程物质还包括脂类信号物质，所述扩增培养基中，所述脂类信号物质的含量为0.5-10微摩尔/升。
  8. 根据权利要求7所述的扩增方法，其特征在于，所述脂类信号物质为1-磷酸鞘氨醇和溶血磷脂酸中的至少一种。
  9. 根据权利要求1所述的扩增方法，其特征在于，所述基础培养基为MEM、DMEM、BME、DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal和Fischers培养基中的至少一种，所述血清为胎牛血清，所述无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
  10. 根据权利要求1所述的扩增方法，其特征在于，所述步骤S2中，使用至少三种具有不同密度的分层剂进行所述不连续密度梯度离心处理，相邻所述分层剂分别为低密度分层剂和高密度分层剂，所述低密度分层剂的密度相对所述高密度分层剂的密度下降0.1-3％。
  11. 根据权利要求10所述的扩增方法，其特征在于，所述步骤S2中，所述至少三种具有不同密度的分层剂中，密度最高的分层剂的密度不高于1.1克/毫升，密度最低的分层剂的密度不低于1.03克/毫升。
  12. 一种胰腺前体样细胞的分化方法，其特征在于，所述胰腺前体样细胞由权利要求1-11中任一项所述的扩增方法得到，将所述胰腺前体样细胞接种于培养容器中，然后通过分化培养基诱导所述胰腺前体样细胞分化为胰岛样细胞。
  13. 根据权利要求12所述的分化方法，其特征在于，所述分化培养基包括若干小分子诱导剂，所述若干小分子诱导剂包括肝素、去乙酰化酶抑制剂、ALK5抑制剂、T3和GLP-1受体激动剂。
  14. 根据权利要求13所述的分化方法，其特征在于，所述去乙酰化酶抑制剂为尼克酰胺和尼曲古抑菌素A中的至少一种，所述GLP-1受体激动剂为胰高血糖素样肽-1和Exendin-4中的至少一种。
  15. 根据权利要求13所述的分化方法，其特征在于，所述分化培养基还包括基础培养基和无血清神经元添加物，所述基础培养基为MEM、DMEM、BME、DMEM/F12、RPMI1640、CMRL1066、WilliamE、Neurobasal和Fischers培养基中的至少一种，所述无血清神经元添加物为N2添加剂和B27添加剂中的至少一种。
  16. 根据权利要求13所述的分化方法，其特征在于，所述分化培养基中，所述肝素的含量为5-15微克/毫升，所述去乙酰化酶抑制剂的含量为5-15毫摩尔/升，所述ALK5抑制剂的含量为5-15微摩尔/升，所述T3的含量为0.5-2微摩尔/升，所述GLP受体激动剂的含量为10-50纳摩尔/升。
  17. 如权利要求12-16中任一项所述的分化方法得到的胰岛样细胞的应用，其特征在于，应用于体外高糖试剂的所述胰岛样细胞分泌胰岛素。
  18. 如权利要求17所述的应用，其特征在于，将所述胰岛样细胞应用于哺乳动物模型的8周后，所述哺乳动物模型的体重变化率为-5％-+5％，血糖值为10-20毫摩尔/升。
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