WO2010099643A1 - 一种促进体细胞增殖的方法 - Google Patents

一种促进体细胞增殖的方法 Download PDF

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WO2010099643A1
WO2010099643A1 PCT/CN2009/001453 CN2009001453W WO2010099643A1 WO 2010099643 A1 WO2010099643 A1 WO 2010099643A1 CN 2009001453 W CN2009001453 W CN 2009001453W WO 2010099643 A1 WO2010099643 A1 WO 2010099643A1
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cells
culture
cell
supernatant
somatic
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王智崇
陈冬
刘颖
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中山大学中山眼科中心
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells

Definitions

  • the present invention is in the field of cell engineering and tissue engineering, and in particular relates to a method of promoting somatic cell proliferation.
  • the proliferative capacity of the cells gradually weakens and eventually stops proliferating. This is the aging process of cells from stem cells through transient expansion of cells to terminal cells. This process is affected by many factors, and some factors can be slowed or even reversed. This aging process allows the cells to regain the ability to differentiate and proliferate.
  • the main strategies for re-acquiring differentiation and proliferative ability of terminally differentiated mature cells in vitro include nuclear transplantation (Wilmut I, et al. Viable offspring derived from fetal and adult mammalian cells. Nature, 1997, 385: 810-813), Cell fusion (Tada M 5 et al. Embryonic germ cell induce epigenetic reprogramming of somatic nucleus in hybrids cells. EMBO J, 1997, 16: 6510-6520; Tada M, et al. Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells. Dev Dyn, 2003, 227:504-510; Tada M, et al.
  • telomere transfection Robottson, DM, et al. Characterization of growth and differentiation telomerase-immortalized Human corneal epithelial cell line.
  • Nuclear transfer is the most extreme example.
  • the somatic cells of adult animals can be re-emerged in the development process by integrating with mature eggs, somatic cell unclear transplantation (SCNT). This process relies on the participation of the egg, so there is an ethical controversy; although many types of cloned animals have been obtained, their efficiency is very low, and the individuals produced by nuclear transfer may develop abnormalities during development, thus limiting the method. Applications.
  • Terminal-differentiated rat myogenic-derived lineage myoblast C2C12 can be reprogrammed to mesenchymal state under the induction of 2, 6-substituted guanidine or additional expression of Msxl gene. Somatic cells, which then proliferate and re-differentiate into bone and fat cells. Oligodindrocyte Precursor Cells (OPCs) can be treated with fetal bovine serum serum or Bone morphogenic proteins (BMPs) and basic fibroblast growth factor (bFGF). Programmatically form pluripotent CNS stem cells. But these compounds or The application of factors is limited to a specific type of cell and has poor versatility.
  • the combination of transcription factors induces the reprogramming of fibroblasts or skin cells, which is one of the most popular research fields.
  • the commonly used transcription factor combinations mainly include Oct-4, Sox-2, c-myc and Klf ⁇ , and the main way to introduce genes is through adenovirus or lentiviral vectors.
  • the cells obtained by this method are similar in characteristics to ES cells and are therefore called induced pluripotent stem cells (iPS).
  • Totipotent cell lysates pluripotent cells - Embryonic Carcinoma (EC) and ES cell nucleus and cytosol extract (ES cell or EC cell lysate (10 mM HEPES, pH 8.2, After treatment with 50 mM NaCl, 5 mM MgCl 2 , 1 mM dithiothreitol, protease inhibitors, repeated freeze-thaw, ultrasonic lysis, centrifugation or filtration to remove insoluble cellular components, since they do not contain intact Cells, in some literature, these extracts are also called cell free extracts, but the nature of this extract is the soluble product extracted after cell lysis, and is essentially different from the culture supernatant of the present invention.
  • the culture supernatant of the present invention is completely cell-free, that is, a component such as a growth factor secreted by a cell, and can reprogram human fibroblasts to express partial characteristics and functions of ES or EC cells.
  • this method has certain limitations: Proteins entering the cells require reversible perforation on the cell membrane using streptococcal hemolysin (SLO), which is toxic to cells and requires a large amount of protein (20-30 mg/ mL), the operation process is complicated, and the efficiency is low; the cells can only be induced into ES-like cells, have poor controllability, are tumorigenic, and are inefficient to differentiate into specific terminally differentiated cells.
  • SLO streptococcal hemolysin
  • telomerase transfection Cells can be immortalized by transfection of SV40 and L. sarcoma virus genes or telomerase catalytic subunits (hTERT). However, after the cells are transfected with oncogenes, the phenotype of some cells is easy to change, and a few cell lines are even potentially tumorigenic. Some cells have virus particles released during the culture. Morphology, genotype, karyotype and molecular level have changed after long-term growth of telomerase-transfected cells in vitro.
  • the most basic requirement for tissue engineering for the required seed cells is rapid amplification and high efficiency.
  • the first five methods listed above have one thing in common. These treatments, while enhancing cell activity, alter the characteristics of the original cells, and are inefficient, requiring re-induction into the desired target cells before they can be applied. And get The ES-like cells or iPS may be tumorigenic and limit their clinical application.
  • the cells obtained by the transfection method can proliferate, since the transfection process uses mostly viral vectors, or the virus itself is transfected, there are potential safety problems, and the transgenic operation itself may The foreign gene is transferred into the genome of the receptor and interferes with the sequence of its original DNA. Therefore, how to achieve rapid and efficient amplification of seed cells on the basis of maintaining the original cell characteristics has become a major issue to be solved in tissue engineering. Summary of the invention
  • the object of the present invention is to provide a method for promoting somatic cell proliferation, which can effectively enhance the ability of somatic cell proliferation and delay somatic cell aging while maintaining the original characteristics of the cell.
  • the present invention adopts the following technical solutions:
  • a method for promoting cell proliferation by culturing a somatic cell or stem cells cultured with embryonic stem cells cultured from a human or animal derived from a commercial product and co-cultured with a somatic cell.
  • the embryonic stem cells are homologous and/or xenogeneic stem cells, and the supernatant of the embryonic stem cell culture is a stock solution, a diluent, a concentrate or a dry powder.
  • the somatic cells include, but are not limited to, corneal epithelial cells, conjunctival cells, corneal epithelial cells, skin cells, hepatocytes, and cardiomyocytes of humans and animals, which are difficult to expand in vitro.
  • the co-cultures are homologous and/or xenogeneic embryonic stem cells cultured in direct contact with cells or in vitro and/or xenogeneic embryonic stem cells are in indirect contact with cells via liquid exchange medium.
  • the above method of directly contacting the somatic cells with the embryonic stem cells for co-culture includes mixing the two cells and then culturing one cell and then inoculating the other.
  • One of the stem cells and somatic cells in the co-culture is a cell labeled with a fluorescent dye.
  • the above-mentioned co-cultured cells can be isolated by the passage process (the cells whose proliferation is inhibited gradually disappear with passage), and can also be separated by utilizing the characteristics of the fluorescent dye (separating the fluorescent-positive and fluorescent-negative cells), and also depending on the cells. Separated by different characteristics.
  • Embryonic stem cells in co-culture are cells that have been inhibited from proliferation by physical and/or chemical means.
  • the method for inhibiting stem cell proliferation is treatment with 2 mM mitomycin C for 0.5-5 hours, or with gamma irradiation for 0.5-5 hours, or with 1% paraformaldehyde for 0.5-5 hours.
  • the invention utilizes the microenvironment of embryonic stem cells to delay the process of somatic cell senescence and promote somatic cell proliferation Methods.
  • the former researchers either co-cultured somatic cells and stem cells to induce stem cell differentiation (Wang, Z, C, et al. Preliminary experimental study on commitment differentiation of embryonic stem cells induced by corneal limbal stoma in vitro Eye Science 1999, 15(4), 195-198; Wang, Z. C, et al Differentiation of embryonic stem cells into corneal epithelium. Science In China Ser. C Life Sciences 2005, 48(5), 471-480.
  • the greatest innovation of the present invention is that the rapid and efficient amplification of cells is realized on the basis of maintaining the original biological characteristics of the cells, which makes it possible to amplify cells from small pieces of tissue, and will provide sufficient seed cells for tissue engineering research.
  • the method for promoting somatic cell proliferation of the invention is simple and practical, high in efficiency and high in purity.
  • the treated somatic cells maintain the original somatic characteristics while increasing the proliferative activity, and there is no heterologous rejection when used for in vivo transplantation or in vitro tissue engineered organ construction.
  • Figure 1 is a partial biological characteristic of human corneal epithelial cells after continuous culture of supernatant after stem cell culture
  • Figure 2 is a partial biological characteristic of rabbit corneal epithelial cells after continuous culture of supernatant after stem cell culture
  • Figure 3 is a partial biological characteristic of rabbit conjunctival epithelial cells after continuous culture of supernatant after stem cell culture
  • Fig. 4 is a view showing the morphology of cat corneal endothelial cells after continuous culture of the supernatant after stem cell culture
  • Fig. 5 is a view showing the morphology of rabbit skin cells after continuous culture of the supernatant after stem cell culture
  • Figure 6 is the effect of direct contact culture with stem cells on the proliferation of human corneal epithelial cells
  • Figure 7 is the effect of indirect contact culture with stem cells on the proliferation of human corneal epithelial cells
  • Figure 8 is a diagram showing the functional recovery of cells.
  • the application method of the supernatant after the embryonic stem cell culture of the invention comprises adding to the culture solution in vitro to promote somatic cell growth, proliferation, rapid expansion, establishment, and providing seed cells for tissue engineering organ construction; After the drug is injected into the damaged part of the body, it is added to the damaged part of the body surface and made into cosmetics to delay and resist skin aging.
  • the manner in which embryonic stem cells are co-cultured with somatic cells includes direct contact culture or culture through a medium in which liquid exchange can occur.
  • the invention systematically studies the biological characteristics and safety of somatic cells, and carries out experiments for promoting the aging process of human and rabbit corneal epithelial cells, rabbit conjunctival epithelial cells and rabbit endothelial cells, and preparation of eye drops treatment. Dry eye experiment. The results all show that the method of the invention can delay the aging process of somatic cells and promote somatic cell proliferation.
  • Mouse E14 embryonic stem cells (American Type Culture Collection Center, ATCCCRL-1821), GFP-labeled mouse embryonic stem cells (OriCdlTM C57BL/6 mouse embryonic cells/GFP, Cyagen Biosciences), human embryonic stem cells (OriCdlTM human) Embryonic stem cells, Cyagen Biosciences), human GFP-labeled embryonic stem cells (OriCellTM human embryonic stem cells/GFP, Cyagen Biosciences).
  • the ES cell culture medium used in the following examples was high glucose Knockout DMEM medium (Gibco BRL) or high glucose DMEM medium (GibcoBRL), and added 5-15% (by volume) fetal bovine serum (Hyclone), 100 U/mL. Streptomycin (Sigma), 1% (by volume) glutamine substitute (Glutamax-I, Invitrogen), 100 mol/L 2-mercaptoethanol (Sigma), leukemia inhibitory factor (LIF) l l0 6 IU/L (Millepore), 1% by volume of non-essential amino acids (GibcoBRL)
  • the method for preparing the supernatant after stem cell culture used in the following examples: Inoculation of mouse E14 embryonic stem cells (American Type Culture Collection Center, ATCC CRL-1821) at 0.1% at a cell density of 1 ⁇ 10 4 -1 ⁇ 10 5 /mL In a gelatin (Sigma) coated flask (BD), culture in a 5% CO 2 , 37 ° C incubator, half daily change, and pass through for 2-3 days. After each 24h culture supernatant, 1500r / mi n after centrifugation 0.22 ⁇ filter membrane sterilization, i.e. an embryonic stem cell culture supernatant.
  • Inoculation of mouse E14 embryonic stem cells American Type Culture Collection Center, ATCC CRL-1821
  • BD gelatin coated flask
  • Example 1 Using supernatant from embryonic stem cell culture to promote corneal epithelial cell proliferation and using corneal epithelial cell culture medium (high glucose DMEM basal medium (GibcoBRL), adding 10-15% by weight (volume ratio) fetal bovine serum, 5 g/mL insulin (Gibco BRL), 5 g/mL transfer iron Protein (Simga), l ( ⁇ g/mL epidermal growth factor (EGF) (Gibco BRL), lOOU/mL penicillin (Simga), 1% (by volume) Glutamax-I, 400 g/mL hydrogenated Gibco BRL, 1% by volume of non-essential amino acids (Gibco BRL) Culture human and rabbit corneal epithelial cells, adding 10-50% (by volume) of mice to corneal epithelial cultures starting from P1 generation cells The supernatant (stock solution) after the embryonic stem cell culture was continuously cultured.
  • corneal epithelial cell culture medium high glucose DM
  • A Morphology of human corneal epithelial cells cultured in conventional corneal epithelial culture medium under inverted phase contrast microscope (Pl, culture for 10 days);
  • CK3/CK12 corneal epithelial cells cultured in the supernatant after stem cell culture were positively expressed by CK3/CK12 (corneal specific cell surface marker);
  • E Human corneal epithelial cells cultured in the supernatant after stem cell culture were positively expressed by CK19 (proliferation-associated cell surface marker);
  • FIG. 2 is a partial biological characteristic of rabbit corneal epithelial cells after continuous culture of supernatant after stem cell culture.
  • the control group was a cell cultured in a conventional corneal culture medium, and the treatment group was a cell in which the supernatant after the stem cell culture was continuously cultured.
  • AI Morphology of rabbit corneal epithelial cells cultured under inverted phase contrast microscope, AF is P1 cells, GI was treated with P10, P20, and P40 cells, respectively, and % was the supernatant concentration after the addition of stem cells.
  • Example 2 Using supernatant from embryonic stem cell culture to promote conjunctival epithelial cell proliferation
  • EGF epidermal growth factor
  • Simga streptomycin
  • Figure 3 is a partial biological characteristic of rabbit conjunctival epithelial cells after continuous culture of supernatant after stem cell culture, wherein
  • Example 3 Using supernatant from stem cell culture to promote corneal endothelial cell proliferation
  • the supernatant of the collected mouse embryonic stem cell culture was dispensed and completely lyophilized using a vacuum freeze-drying machine, and stored at -20 Torr for use.
  • the cells treated with the supernatant after stem cell culture were typically polygonal and the cell morphology was more regular (Fig. 4).
  • the supernatant treated cells after stem cell culture were expanded approximately 100-fold, whereas the conventionally cultured cells were only expanded approximately 30-fold.
  • Fig. 4 shows the morphology of cat corneal endothelial cells after continuous culture of supernatant after stem cell culture, wherein A: morphology of P3 endothelial cells cultured in conventional corneal endothelial culture medium
  • Example 4 Using supernatant from stem cell culture to promote skin cell proliferation
  • the supernatant of the collected mouse embryonic stem cell culture was dispensed and frozen by a vacuum freeze-drying machine for 1-2 times, and stored at -20 Torr for use.
  • the rabbit skin cells (cells with little proliferative activity in vitro) were cultured with keratinocyte culture medium K-SFM (Gibco), and 2.5%-25% (by volume) of mice were added to K-SFM from P1 generation cells. The supernatant (concentration) after the embryonic stem cell culture is continuously cultured.
  • Figure 5 shows the morphology of rabbit skin cells after continuous culture of supernatant after stem cell culture, wherein: A: morphology of P2 skin cells cultured in K-SFM culture medium;
  • Example 5 Proliferation of corneal epithelial cells is promoted by direct contact with stem cells (Note: The embryonic stem cells used in this embodiment may be homologous and/or xenogeneic stem cells, and the somatic cells may be any algebra, and the effect is the same as below)
  • Figure 6 shows the effect of direct contact culture with stem cells on the proliferation of human corneal epithelial cells, wherein A: the morphology of human P3 corneal epithelial cells directly exposed to green fluorescent protein-labeled stem cells observed under an inverted phase contrast microscope;
  • Example 6 Promoting corneal epithelial cell proliferation by a method of inhibiting the co-culture of proliferating stem cells by physical and/or chemical means (Note: The embryonic stem cells used in the present embodiment may be homologous and/or xenogeneic stem cells, and the somatic cells may be For any algebra, the effect is the same below)
  • Example 7 Promoting corneal epithelial cell proliferation by indirect contact culture with stem cells 1. Obtain and culture human corneal epithelial cells according to conventional methods;
  • Plant mouse ES cells on the bottom or lower surface of a six-well plate lay 1-5 layers of extracellular matrix components (including but not limited to type I collagen, type IV collagen, laminin, silk fibronectin) P1 corneal epithelial cells were implanted on Transwell membrane (BD) (using corneal epithelial complete medium), Transwell membrane was placed in a six-well plate, and culture was continued for 2-6 days.
  • ES cells are planted at the bottom of the plate, and corneal epithelial cells are planted on the Transwell membrane. Since the ES cells and the corneal epithelial cells are separated by a Transwell membrane, the two cells cannot directly contact each other and avoid cell fusion, but can be secreted. The growth factors interact with each other.
  • the medium between the two cells includes, but is not limited to, a Transwell membrane, and a medium that can exchange liquids such as a heat sensitive membrane or an amnion membrane can also be used.
  • Figure 7 is the effect of indirect contact culture with stem cells on the proliferation of human corneal epithelial cells, wherein
  • A Morphology of human corneal epithelium cultured in indirect contact with stem cells. Human corneal epithelial cells were seeded on Transwell membranes, and ES cells were seeded on 6-well plates at 100 Torr.
  • ⁇ The morphology of normal cultured human corneal epithelial cells, 100 ⁇ .
  • the rabbit corneal epithelial cells treated by the present invention can form a stratified epithelium under the stimulation of the gas-liquid interface in vitro, and are injected into Balb/c nude mice without tumor formation. It was confirmed that the present invention not only maintains the normal function of the cells, but also has safety.
  • the P20 rabbit corneal epithelial cells obtained by the method of Example 1 were inoculated onto the acellular porcine corneal stroma at a density of 3.5 ⁇ 10 5 /ml, and cultured in a cell culture incubator. After the cells were fused, after 3 weeks of culture using the gas-liquid interface, HE staining showed that the cells could form a 2-3 layer stratification, the lower cells were round, and the upper cells gradually became flat. Scanning electron microscopy showed that the cells were superimposed in a multi-layered structure with abundant microvilli and pseudopods on the surface (Fig. 8).
  • FIG. 8 shows the functional recovery of cells
  • the P20 generation rabbit corneal epithelial cells obtained by the method of Example 1 were inoculated subcutaneously into the groin of Balb/c nude mice at a density of 5 ⁇ 10 6 /ml. After the inoculation, no obvious adverse reactions were observed. After 80 days of word cultivation, no tumor growth was observed in the subcutaneous tissue of the neck and back. After the nude mice were sacrificed, there were no nodules and masses in the subcutaneous tissue of the inoculated site after gross dissection. No obvious pathological changes were found in the liver, lung, kidney and spleen.

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Description

一种促进体细胞增殖的方法 技术领域
本发明属于细胞工程和组织工程领域，具体地是涉及一种促进体细胞增殖的 方法。
背景技术
随着分裂次数的不断增多， 细胞的增殖能力逐渐减弱， 最终停止增殖， 这是 细胞从干细胞经过短暂扩增细胞到终末细胞的老化过程， 此过程受诸多因素影 响， 有些因素可以减缓甚至逆转这一老化过程， 使细胞重新获得分化、增殖的能 力。
目前， 在体外使终末分化的成熟细胞重新获得分化、增殖能力主要策略包括核 移植 (Wilmut I, et al. Viable offspring derived from fetal and adult mammalian cells. Nature, 1997, 385:810-813)、 细胞融合 (Tada M5 et al. Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrids cells. EMBO J, 1997, 16: 6510-6520； Tada M, et al. Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells. Dev Dyn, 2003, 227:504-510； Tada M, et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol, 2001, 11 :1553-1558)、 利用细胞裂解液（Taranger C K， et al. Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extract of carcinoma and embryonic stem cells. Mol Bio Cell, 2005, 16: 5719-35)或者特殊成分的培养液 (Chen S, et al. Dedifferentiated of lineage-committed cells by a small molecule. J Am Chem Soc， 2004, 126: 410-411； Shannon J， et al. Dedifferentiation of mammalian myotubes induced by Msxl. Cell, 2000,103: 1099-1109 ; Kondo T & Raff M. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science, 2000, 289: 1754-1757)诱导、 多个转录因子组合转染 (Takahashi K & Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126:663-676； Okita K, et al. Generation of germline-competent
- l - induced pluripotent stem cells. Nature, 2007, 448:313-7； Wernig M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 2007, 448:318-24； Maherali N, et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 2007, 1 :55-70)，病毒或端粒酶转染 ( Robertson, D. M.,et al. Characterization of growth and differentiation telomerase-immortalized human corneal epithelial cell line. Investig Ophthalmol Vis Sci,2005,46: 470-478; Araki-Sasaki, K.， et al. An SV40 immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci. 1995,36: 614-21; ahn, C.R., et al. Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies. Invest Ophthalmol Vis Sci. 1993,34: 3429-41. ) 等。
(1) 核移植： 核移植是最极端的例子， 成年动物的体细胞能够通过与成熟卵子融 合即体细胞核移植 (Somatic cell unclear transplantation, SCNT)的方法实现发育过 程的重演。该过程依赖于卵子的参与， 因此存在伦理上的争议； 虽然目前已经获 得了许多种类的克隆动物，但是其效率非常低，而且核移植产生的个体在发育中 可能出现异常， 因此限制了该方法的应用。
(2) 细胞融合： 不同细胞间的融合是研究分化细胞可塑性的常用手段， 在大多数 "杂交 "细胞中， 参与融合的分化程度较低的细胞处于支配地位， 而分化程度高的 细胞失去了原有的细胞特性转而表达分化程度较低的细胞的特性，如将体细胞与 胚胎配子细胞 (Embryonic germ, EG)或者胚胎干细胞 (Embryonic Stem Cells, ES) 以一定的比例混合培养后筛选出融合的细胞，这些融合产生的"杂交"细胞的表型 呈现 EG或者 ES细胞的特征。 同样， 利用细胞融合进行分化细胞的效率非常低， 大约为万分之一， 而且在这个过程中会产生异倍体。
(3)特殊成分培养液诱导： 终末分化的鼠肌源性定向谱系成肌细胞 C2C12可以在 2, 6位取代嘌呤的诱导下或者额外表达 Msxl基因的条件下再程序化为间充质前 体细胞， 随后增殖再分化为骨和脂肪细胞。 少突前体细胞 (Oligodendrocyte Precursor Cells， OPCs)依次经胎牛血清血清或者骨形态发生蛋白（Bone morphogenic proteins , BMPs)禾口碱性成纤维生长因子 (basic fibroblast growth factor, bFGF)处理后能够再程序化形成多能的 CNS干细胞。但是这些化合物或者 因子的应用只局限于特定种类的细胞， 通用性差。
(4)转录因子组合： 转录因子组合诱导成纤维细胞或者皮肤细胞发生再程序化是 目前最热门的研究领域之一， 常用的转录因子组合主要包括 Oct-4、 Sox-2, c-myc 和 Klf^， 而导入基因的主要方式则是通过腺病毒或者慢病毒载体。 经过这种方 法获得的细胞的特性与 ES细胞类似因此被称为诱导的全能性干细胞 (induced pluripotent stem cells, iPS)。 但是， 对这些杂核后代的研究发现， 这些细胞具有致 瘤性， 可能是由于利用逆转录病毒作为载体以及转入原癌基因 c-myc带来的潜在 危险， 除此之外制备 iPS的效率非常低， 不是所有的 iPS都具有发育的全能性； iPS 向特定细胞诱导分化的效率低， 分化的细胞中含有不等量的杂细胞。
(5)全能性细胞细胞裂解液：全能性细胞—胚胎癌细胞 (Embryonic Carcinoma, EC) 和 ES细胞的细胞核和胞浆提取液 (ES细胞或 EC细胞用裂解液 （10mM HEPES, pH8.2, 50mM NaCl, 5 mM MgCl2, ImM二硫苏糖醇,蛋白酶抑制剂）处理后经反复 冻融， 超声波裂解后， 离心或过滤后除去不溶性的细胞成分后使用， 因其中己经 不含有完整的细胞，在有些文献中这些提取液也被称为 cell free extracts,但是这 种提取液的本质还是将细胞裂解后提取的可溶性的产物，和本发明所述培养培养 上清是有本质区别的， 本发明所述培养培养上清是完全不含细胞的， 就是细胞分 泌的生长因子等成分 ）可以使人成纤维细胞发生再程序化， 而表达 ES或 EC细胞 的部分特性和功能。但是该方法具有一定局限性： 蛋白进入细胞内需要使用链球 菌溶血素 (SLO)在细胞膜上可逆性的打孔， 对细胞有一定的毒性， 并且需要的蛋 白量也比较大 (20-30mg/mL)， 操作过程复杂， 效率低； 细胞只能被诱导成类 ES 细胞， 可控性差， 具有致瘤性， 向特定终末分化细胞分化的效率低。
(6)病毒或端粒酶转染： 细胞转染 SV40及劳氏肉瘤病毒基因或者端粒酶催化亚 单位 （hTERT) 后可以实现"永生化"。 但是细胞在转染癌基因之后， 部分细胞 的表型容易发生改变， 少数细胞系甚至有潜在的致瘤性， 培养过程中部分细胞有 病毒颗粒的释放。 而端粒酶转染后的细胞在体外长期生长后， 形态学， 基因型， 核型和分子水平都发生了改变
组织工程对所需的种子细胞的最基本要求就是扩增快， 效率高。上述列出的 前五种方法存在一个共同点，即这些处理在增强细胞活性的同时却改变了原有细 胞的特性， 而且效率低， 需要重新诱导为需要的靶细胞， 才能够应用。 并且获得 的类 ES细胞或者 iPS可能存在致瘤性而限制了其临床应用。 而通过转染方法获 得的细胞， 虽然可以增殖， 但是由于转染过程使用的多是病毒载体， 或者本身转 染的就是病毒， 也存在潜在的安全性问题， 而本身这种转基因操作可能会将外源 基因转入受体的基因组而干扰其原有 DNA的序列。 因此如何在保持原有细胞特 性的基础上实现种子细胞的快速高效扩增已经成为组织工程亟待解决的重大课 题。 发明内容
本发明的目的在于提供一种促进体细胞增殖的方法，该方法在保持细胞原有 特性的基础上能有效地增强体细胞增殖能力， 延缓体细胞衰老。
为实现上述目的， 本发明采用了以下技术方案：
一种促进细胞增殖的方法，所述方法为利用来自商品的人或动物的胚胎干细 胞培养后的上清液培养体细胞或干细胞与体细胞共培养。
所述胚胎干细胞为同种和 /或异种干细胞， 所述胚胎干细胞培养后的上清液 为原液、 稀释液、 浓缩液或干粉。
所述的体细胞包括但不仅限于人、 动物的角膜上皮细胞、 结膜细胞、 角膜内 皮细胞、 皮肤细胞、 肝细胞及心肌细胞等在体外扩增困难的细胞。
所述共培养为同种和 /或异种的胚胎干细胞与细胞直接接触培养或同种和 /或 异种的胚胎干细胞与细胞通过液体交换的介质间接接触培养。
上述将体细胞与胚胎干细胞直接接触共培养的方法包括将两种细胞混和后培 养， 以及将一种细胞种植后再接种另一种细胞。
共培养中的干细胞和体细胞中的一种是经过荧光染料标记的细胞。 上述共培 养的细胞可以通过传代过程而进行分离 （增殖受到抑制的细胞随传代而逐渐消 失）， 也可以通过利用荧光染料的特性进行分离 （将荧光阳性和荧光阴性细胞分 离）， 还可以根据细胞特性不同而得到分离。
共培养中的胚胎干细胞是经过物理和 /或化学方法抑制增殖的细胞。 所述抑 制干细胞增殖的方法为用 2 mM丝裂霉素 C处理 0.5-5小时，或用 γ射线照射 0.5-5 小时， 或用 1%的多聚甲醛处理 0.5-5小时。
本发明是一种利用胚胎干细胞微环境延缓体细胞衰老过程、促进体细胞增殖 的方法。在实际应用中， 以往研究者或者是将体细胞和干细胞共培养以诱导干细 胞发生定向分化 (Wang, Z, C, et al. Preliminary experimental study on commitment differentiation of embryonic stem cells induced by corneal limbal stoma in vitro. Eye Science 1999,15(4), 195-198; Wang, Z. C, et al Differentiation of embryonic stem Cells into corneal epithelium. Science In China Ser. C Life Sciences 2005, 48(5), 471-480. ), 或者是在细胞可塑性的研究中， 利用细胞间自然发生的融合作用， 使 分化程度高的细胞获得分化程度低的细胞（一般为干细胞， 原癌细胞， 配子细胞 等） 的特性（Tada M，et al.l997; 2001； 2003 )， 却并未有人报道在不改变原有细 胞特性的基础上利用胚胎干细胞微环境来促进细胞的增殖，这也是本发明的最大 创新。本发明的最大创新在于在保持细胞原有生物学特性的基础上实现了细胞的 快速， 高效扩增， 使从小块组织扩增细胞成为可能， 将为组织工程研究提供充足 的种子细胞。 本发明促进体细胞增殖的方法简单实用， 效率高， 纯度高。 经过处 理的体细胞，在增殖活性提高的同时保持了原有的体细胞特性， 用于体内移植或 者体外组织工程化器官的构建时没有异源排斥问题。 附图说明
图 1是干细胞培养后的上清液连续培养后的人角膜上皮细胞的部分生物学特 性；
图 2是干细胞培养后的上清液连续培养后的兔角膜上皮细胞的部分生物学特 性；
图 3是干细胞培养后的上清液连续培养后的兔结膜上皮细胞的部分生物学特 性；
图 4是干细胞培养后的上清液连续培养后的猫角膜内皮细胞的形态； 图 5是干细胞培养后的上清液连续培养后的兔皮肤细胞的形态；
图 6是与干细胞直接接触培养对人角膜上皮细胞增殖的影响；
图 7是与干细胞间接接触培养对人角膜上皮细胞增殖的影响；
图 8是细胞的功能恢复情况。 具体实施方式 本发明胚胎干细胞培养后的上清液的应用方式包括在体外添加到培养液中 促进体细胞生长、 增殖、 快速扩增、 建系、 为组织工程器官构建提供种子细胞； 以适当的浓度制成药品后注射到体内细胞受损部位，滴加到身体表面细胞受损部 位及将其制成化妆品延缓及抵抗皮肤衰老。
胚胎干细胞与体细胞共培养的方式包括直接接触培养，或者通过可以发生液 体交换的介质进行培养。
本发明从体细胞的生物学特性、安全性等方面进行了系统研究， 进行了促进 人和兔的角膜上皮细胞、兔结膜上皮细胞、兔内皮细胞衰老过程的实验， 以及制 成滴眼液治疗干眼的实验。结果都显示：本发明方法能够延缓体细胞的衰老过程， 促进体细胞增殖。
以下通过具体实施例来进一步阐述本发明。
小鼠 E14胚胎干细胞（美国模式菌种收集中心， ATCCCRL-1821), GFP标 记的小鼠胚胎干细胞 （ OriCdl™ C57BL/6 小鼠胚胎千细胞 /GFP， Cyagen Biosciences ) ， 人胚胎干细胞 （ OriCdl™ 人胚胎干细胞 ， Cyagen Biosciences), 人 GFP标记的胚胎干细胞（OriCell™人胚胎干细胞 /GFP， Cyagen Biosciences )。
以下实施例中所用的 ES细胞培养液为高糖 Knockout DMEM培养基（Gibco BRL) 或者高糖 DMEM培养基 （GibcoBRL), 添加 5-15% (体积比） 胎牛血清 (Hyclone) , 100U/mL 青链霉素（Sigma)、 1% (体积比） 谷氨酰胺替代物 (Glutamax-I, Invitrogen)、 100 mol/L2-巯基乙醇（Sigma)、白血病抑制因子（LIF) l l06IU/L (Millepore)、 1% (体积比） 非必需氨基酸 （GibcoBRL)
以下实施例中所用的干细胞培养后的上清液的制备方法： 以 1χ104-1χ105个 /mL 细胞密度接种小鼠 E14 胚胎干细胞 （美国模式菌种收集中心， ATCC CRL-1821) 于 0.1%明胶 (Sigma) 包被的培养瓶 （BD) 中， 置 5%C02、 37°C 培养箱内培养， 每日半量换液， 2-3天传代。 培养每 24h后取上清， 1500r/min离 心后用 0.22μιη微孔滤膜过滤除菌， 即为胚胎干细胞培养上清。
实施例 1: 利用胚胎干细胞培养后的上清液促进角膜上皮细胞增殖 取材并用角膜上皮细胞培养液（高糖 DMEM基础培养基（GibcoBRL), 添 加 10-15%% (体积比) 胎牛血清、 5 g/mL胰岛素 (Gibco BRL), 5 g/mL转铁 蛋白 （Simga)、 l(^g/mL表皮生长因子（EGF) (Gibco BRL)、 lOOU/mL青链霉 素 （Simga)、 1% (体积比） Glutamax-I、 400 g/mL氢化可的松 （Gibco BRL)、 1% (体积比） 非必需氨基酸 （Gibco BRL) 培养人和兔角膜上皮细胞， 从 P1代 细胞开始在角膜上皮培养液中添加 10-50% (体积比)的小鼠胚胎干细胞培养后的 上清液 (原液)， 进行连续培养。
测定利用胚胎干细胞培养后的上清液连续培养后的角膜上皮细胞的生物学 特性。 结果显示： （1 ) 在倒置相差显微镜下， 干细胞培养后的上清液培养的 P1 人角膜上皮细胞增殖更快， 形态更典型， 细胞仍然表达角膜特异性标记物 CK3/CK12, 处于 S期的细胞更多 （图 1 )。 (2) 研究结果显示： 经过 ES上清液 处理的细胞增殖能力明显增强， 在体外可以连续传代， 形态规则， 大小均一， 并 表达特异性的标记物 （图 2)。 图 1 为胚胎干细胞培养后的上清液连续培养后的人角膜上皮细胞的部分生 物学特性。
A: 倒置相差显微镜下常规角膜上皮培养液培养的人角膜上皮细胞的形态 (Pl， 培养 10天）；
B:倒置相差显微镜下干细胞培养后的上清液连续培养后的人角膜上皮细胞 的形态 （Pl， 培养 10天）；
C: MTT法测定的 P1细胞的生长曲线；
D: 干细胞培养后的上清液培养的人角膜上皮细胞 CK3/CK12 (角膜特异性 细胞表面标记物） 呈阳性表达；
E: 干细胞培养后的上清液培养的人角膜上皮细胞 CK19 (增殖相关的细胞 表面标记物） 呈阳性表达；
F: 常规角膜上皮培养液培养的人角膜上皮细胞 （P5 ) 的细胞周期分析； G: 干细胞培养后的上清液培养的人角膜上皮细胞 （P5 ) 的细胞周期分析。 图 2为干细胞培养后的上清液连续培养后的兔角膜上皮细胞的部分生物学 特性。对照组为常规角膜培养液培养的细胞， 处理组为干细胞培养后的上清液连 续培养后的细胞。
A-I: 倒置相差显微镜下培养的兔角膜上皮细胞的形态， A-F为 P1细胞， G-I分别为处理组 P10、 P20、 P40细胞，％为添加的干细胞培养后的上清液浓度。
J: P1细胞表面标记物（CK3， CK12, P63 , ABCG2) mRNA表达的 RT-PCR 分析， 持家基因 GAPDH作为阳性对照
K: MTT法测定的细胞的生长曲线
L: 细胞的克隆形成能力分析。
M: 克隆的形态
N: 细胞表面标记物 （CK3/CK12, P63 , ABCG2 ) 表达的免疫荧光检测结 果。 实施例 2: 利用胚胎干细胞培养后的上清液促进结膜上皮细胞增殖
取材并利用常规结膜上皮细胞培养液 (高糖 DMEM: F12=3 : 1 基础培养基 (Gibco),添加 5- 15% (体积比）胎牛血清、 l( g/mL表皮生长因子（EGF) (Gibco BRL)、 lOOU/mL青链霉素 （Simga), 从 PI代细胞开始添加用常规结膜上皮细 胞培养液稀释的小鼠胚胎干细胞培养后的上清液 （稀释为 10%-50%， 体积比）， 进行连续培养。
结果显示： 干细胞培养后的上清液能够促进结膜上皮增殖， 在形态上， ES 上清液处理的细胞呈典型的多边形， 细胞形态规则 （图 3 )。 图 3 为干细胞培养后的上清液连续培养后的兔结膜上皮细胞的部分生物学 特性， 其中，
A: 常规结膜培养液培养的 P2结膜细胞的形态
B: 干细胞培养后的上清液连续培养后的 P2结膜细胞的形态
C: MTT法测定的细胞的生长曲线 （P2)
D: 结膜细胞表面标记物的表达的免疫荧光检测结果 实施例 3: 利用干细胞培养后的上清液促进角膜内皮细胞增殖
将收集的小鼠胚胎干细胞培养后的上清液分装后利用真空冷冻冻干机完全 冻干， -20Ό保存备用。
取材并用常规内皮细胞培养液：高糖 DMEM: F12=3 : 1基础培养基（Gibco) , 添加 5-15% (体积比） FBS, 硫酸软骨素 (lug/ml)， bFGF(lng/ml), EGF(l(^g/mL)， NGF(5ng/ml), lOOU/mL青链霉素 （Simga)、 1% (体积比） Glutamax-I、 1% (体 积比） 非必需氨基酸 （Gibco BRL) 培养猫角膜内皮细胞 （在体外几乎没有增殖 活性的细胞）， 从 P1代细胞开始在 lOOmL常规内皮细胞培养液中添加 l-100mg 冻干的小鼠胚胎干细胞培养后的上清液干粉， 进行连续培养 (6代)。
结果显示： 干细胞培养后的上清液能够促进猫内皮细胞增殖， 在形态上， 干 细胞培养后的上清液处理的细胞呈典型的多边形， 细胞形态更规则 （图 4)。 经 过 6代体外培养， 干细胞培养后的上清液处理的细胞扩增了约 100倍， 而常规培 养的细胞仅扩增了大约 30倍。
图 4为干细胞培养后的上清液连续培养后的猫角膜内皮细胞的形态， 其中， A: 常规角膜内皮培养液培养的 P3内皮细胞的形态
B: 干细胞培养后的上清液连续培养后的 P3内皮细胞的形态。
实施例 4: 利用干细胞培养后的上清液促进皮肤细胞增殖
将收集的小鼠胚胎干细胞培养后的上清液分装后利用真空冷冻冻干机冻千 1-2倍， -20Ό保存备用。
取材并用角质细胞培养液 K-SFM (Gibco)培养兔皮肤细胞 （在体外几乎没 有增殖活性的细胞）， 从 P1代细胞开始在 K-SFM中添加 2.5%-25% (体积比)的小 鼠胚胎干细胞培养后的上清液 (浓缩液)， 进行连续培养。
结果显示： 干细胞培养后的上清液能够促进兔皮肤细胞增殖， 在形态上， 干 细胞培养后的上清液处理的细胞形态更规则， 更典型 （图 5 )。
图 5干细胞培养后的上清液连续培养后的兔皮肤细胞的形态， 其中， A: K-SFM培养液培养的 P2皮肤细胞的形态；
B: 干细胞培养后的上清液连续培养后的 P2皮肤细胞的形态。
实施例 5: 利用与干细胞直接接触培养的方法促进角膜上皮细胞增殖 （注： 本实施例所用的胚胎干细胞可以是同种和 /或异种干细胞， 体细胞可以为任何代 数， 效果与以下相同）
取材并培养人角膜上皮细胞， 将人角膜上皮细胞与绿色荧光蛋白 (GFP)标记 的 ES细胞 (GFP-ES)直接接接触培养， 培养 1代后， 利用流式细胞仪分离荧光阴 性细胞。 结果显示： 与胚胎干细胞直接接触共培养的人角膜上皮细胞体积变小， 核 浆比变大， 形态也变得比较规则， 更接近原代从角膜缘萌出的细胞。 流式细胞仪 分选前荧光阴性率为 4.2%， 分选后阴性率为 92.2% (图 6)。
图 6为与干细胞直接接触培养对人角膜上皮细胞增殖的影响， 其中， A: 倒置相差显微镜下观察的人 P3角膜上皮细胞与绿色荧光蛋白标记的干细胞 直接接触后的形态；
B: 荧光显微镜下干细胞克隆；
C: 流式细胞仪分选前荧光阴性细胞比例 （4.2%);
D: 流式细胞仪分选后荧光阴性细胞比例 （92.2%)。 实施例 6:利用与经过物理和 /或化学方法抑制增殖后的干细胞共培养的方法促进 角膜上皮细胞增殖 （注： 本实施例所用的胚胎干细胞可以是同种和 /或异种干细 胞， 体细胞可以为任何代数， 效果以下相同）
1. 按常规方法取材并培养兔角膜上皮细胞；
2. 按常规方法培养小鼠 ES细胞达到 80%融合后， 用 2mM丝裂霉素 C抑 制细胞增殖 0.5-5小时后， 用无菌 PBS充分洗涤 5 _6次； （也可以用 γ 射线照射 0.5-5小时后； 或者用 1 %多聚甲醛抑制细胞增殖 0.5-5小时 后）， 用 PBS充分洗涤；
3. 将角膜上皮细胞种植在经过处理的 ES 细胞上继续培养至 80%细胞融 合.
4. 根据细胞扩增情况和生长状态决定是否继续将角膜上皮细胞种植在经 过处理的 ES细胞上；
5. 由于经过处理的 ES细胞失去了继续增殖能力， 最终可以通过连续传代 的方法将 ES细胞逐渐除掉。
虽然 ES细胞经过处理后失去了连续增殖能力， 但是仍然能够其分泌的生长 因子影响与
其共培养的细胞， 而不会发生细胞融合。 实施例 7: 利用与干细胞间接接触培养的方法促进角膜上皮细胞增殖 1. 按常规方法取材并培养人角膜上皮细胞；
2. 将小鼠 ES细胞种植在六孔板底部或者下表面； 铺上 1-5层细胞外基质 成分（包括但不仅限于 I型胶原， IV型胶原，层粘联蛋白，丝粘联蛋白）， 将 P1代角膜上皮细胞种植在 Transwell膜上 （BD) (使用角膜上皮完全 培养液)， 将 Transwell膜置于六孔板中， 继续培养 2-6天。 ES细胞种植 于培养板底部， 而角膜上皮细胞种植在 Transwell膜上， 由于 ES细胞和 角膜上皮细胞之间隔了一层 Transwell膜， 两种细胞不能相互直接接触 而避免了细胞融合， 但可以通过分泌的生长因子相互影响。 而两种细胞 之间的介质包括但不仅仅限于 Transwell膜， 也可以使用热敏膜， 羊膜 等可以发生液体交换的介质。
结果发现:与干细胞间接接触培养的人角膜上皮细胞增殖速度更快， 形态也更典 型 （图 7)。 图 7为与干细胞间接接触培养对人角膜上皮细胞增殖的影响， 其中，
A: 与干细胞间接接触培养的人角膜上皮的形态， 人角膜上皮细胞种植于 Transwell膜上， ES细胞种植于 6孔板上， 100χ。
Β:正常培养的人角膜上皮细胞的形态， 100χ。
实施例 8: 细胞的功能恢复和安全性检验
通过本发明所处理的兔角膜上皮细胞， 在体外气一液界面刺激下可以形成 复层上皮，注射到 Balb/c裸鼠皮下不致瘤。证实本发明不但可以维持细胞正常的 功能， 还具有安全性。
功能恢复试验：
以实施例 1方法获得的 P20代兔角膜上皮细胞， 以 3.5χ 105个 /ml的密度接 种到脱细胞猪角膜基质上， 细胞培养箱中静置培养。 当细胞融合后， 利用气-液 界面培养 3周后， HE染色可见细胞能够形成 2-3层的复层， 下层细胞比较圆， 上层细胞逐渐变得扁平。扫描电镜下可见细胞呈重叠的多层结构， 细胞表面有丰 富的微绒毛和伪足 （图 8)。
图 8 为细胞的功能恢复情况， 其中
A: 接种于 Transwell培养池 48h后角膜上皮细胞形成单层。 标尺： lOOum B: 气 -液界面培养 10天后角膜上皮细胞的形态。 标尺： lOOum
C: 复层上皮表面的扫描电镜分析。 标尺： lOumD: 接种于猪脱细胞角膜基 质上的角膜上皮细胞形成的复层上皮的 HE染色结果
E: 形成的复层上皮呈 CK3/CK12阳性表达。 标尺： 20um
F: 形成的复层上皮呈 P63阴性表达。 标尺： 20um
G: 形成的复层上皮呈 ABCG2阴性表达。 标尺： 20um。
安全性检验：
以实施例 1 方法获得的 P20代兔角膜上皮细胞， 以 5χ 106个 /ml 的密度接种到 Balb/c裸鼠腹股沟皮下。 接种后， 未见有明显不良反应。 词养 80天后， 颈背部 皮下组织未见有肿瘤生长。处死裸鼠后， 经大体解剖未见接种部位皮下组织有结 节和肿块， 肝脏， 肺脏， 肾脏和脾脏未见明显病理性改变。

Claims

权 利 要 求 一种促进体细胞增殖的方法， 其特征在于： 所述方法为将来自商品的人和 / 或动物的胚胎干细胞培养的上清液培养体细胞； 或所述方法为不改变原有细 胞的特性的情况下将来自商品的人和 /或动物的胚胎干细胞与体细胞共培养。 根据权利要求 I所述的促进体细胞增殖的方法， 其特征在于： 所述胚胎干细 胞为同种和 /或异种胚胎干细胞，所述干细胞培养后的上清液为原液、稀释液、 浓缩液或干粉。 根据权利要求 1所述的促进体细胞增殖的方法， 其特征在于： 共培养中的胚 胎干细胞和体细胞中的一种是经过荧光染料标记的细胞。 根据权利要求 1所述的促进体细胞增殖的方法， 其特征在于： 共培养中的胚 胎干细胞是经过物理和 /或化学方法抑制增殖的细胞。 根据权利要求 4所述的促进体细胞增殖的方法， 其特征在于： 所述抑制胚胎 干细胞增殖的方法为用丝裂霉素 C处理 0.5-5小时， 或用 γ射线照射 0.5-5 小时， 或用多聚甲醛处理 0.5-5小时。 根据权利要求 1所述的促进体细胞增殖的方法， 其特征在于： 所述体细胞选 自但不限于角膜细胞、 结膜细胞、 内皮细胞、皮肤细胞、肝细胞或心肌细胞。
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