Establishment method of naturally immortalized pilose antler regeneration bud cell line and engineering preparation method of functional factors of naturally immortalized pilose antler regeneration bud cell line
By constructing a naturally immortalized deer antler regenerated bud-based cell line and purifying cell secretion factors by engineering preparation methods, the problem of poor stability of the deer antler regenerated bud-based cell line in vitro is solved, and the stability of the cell line and the efficient production of functional factors is achieved, and the safety and ethical risks of clinical applications are reduced.
Patent Information
- Application Number
- CN202411741392.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-03-07
AI Technical Summary
The regenerated bud-based cell lines of deer antlers have poor stability in culture and expansion in vitro, and cannot achieve large-scale culture and application. At the same time, as xenogeneic animal cells, there are ethical and safety restrictions on direct application in clinical drugs.
By obtaining the regenerated bud base cells of the deer antler, a spontaneous immortalized cell line was constructed using the preferred adherent and starved cell selection method, and the cell secretion factor was collected and purified by suspended culture through a cell culture bioreactor.
The natural immortalization of the deer antler regenerated bud-based cell line was achieved, which improved the stability and proliferation activity of cells, solved the problem of poor amplification stability in in vitro culture, and achieved efficient purification and mass production of functional factors through engineering preparation methods, reducing safety and ethical risks.
Smart Images
Figure HDA0005162677280000011 
Figure HDA0005162677280000012 
Figure HDA0005162677280000021
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for establishing a naturally immortalized antler regeneration blastema cell line and an engineering preparation method for its functional factors. Background Art
[0002] Deer antler is a precious traditional Chinese medicine with the effects of "strengthening kidney yang, benefiting essence and blood, strengthening tendons and bones, regulating Chong and Ren meridians, and supporting sores and toxins". The discovery of these therapeutic effects of deer antler is not only the result of traditional application practice of Chinese medicine, but also the result of modern scientific research.
[0003] Deer antler is the only mammalian organ in nature that can fully regenerate periodically. Its fastest growth rate can reach 2.73 cm / day (Landete-Castillejos et al, Bone 2019), so it is rich in active factors, proteins and peptides, phospholipids and other nutrients necessary for tissue regeneration, repair, anti-aging and development. The regeneration of deer antler is a process that depends on stem cells, which are located in the periosteum tissue at the end of the horn stalk and are called antler horn stalk periosteal stem cells (Li et al, Cell Tissue Res 2007). The rapid growth of deer antler also depends on stem cells, which are located in the interstitial tissue under the skin at the top of the antler and are called antler mesenchymal stem cells (Li et al, Anat Rec 2002).
[0004] Recently, our research team found that when the antler starts to regenerate, the antler periosteal stem cells first divide and differentiate into a very small cone-shaped tissue (maximum diameter length is 0.5 cm), which is the antler regeneration blastema tissue; the blastema tissue contains regeneration blastema cells (Antler Blastema Progenitor Cells, ABPC), which gradually differentiate into antler mesenchymal stem cells within 5 days, and regenerate and develop the entire antler (the relevant results "A population of stem cells with strong regenerative potential discovered in deer antlers" was published in the international top journal "Science" in 2023). It is worth noting that compared with the other two types of antler stem cells, ABPC has stronger proliferation activity, multidirectional differentiation potential, and the potential and efficacy of secretion of regeneration and repair factors, so it is inferred that it has stronger development value.
[0005] Although this research team identified and isolated ABPC for the first time, its in vitro culture and expansion stability are poor, making it impossible to achieve large-scale culture and application; at the same time, as xenogeneic animal cells, there are ethical and safety limitations for direct use in clinical drugs. Summary of the invention
[0006] The purpose of the present invention is to provide a method for establishing a naturally immortalized antler regeneration blastema cell line without gene editing and an engineered preparation method of its functional factors, so as to overcome the technical defects of the prior art that ABPC in vitro culture and amplification have poor stability, cannot be cultured and applied on a large scale, and are difficult to be directly applied in clinical drugs as xenogeneic animal cells.
[0007] The objective of the present invention is achieved through the following technical solutions:
[0008] A method for establishing a naturally immortalized antler regeneration blastema cell line comprises the following steps:
[0009] S1 Obtaining antler regeneration blastema cells:
[0010] After the male sika deer antler disc falls off 2-5 days (regeneration starts), the antler apex tissue is cut after disinfection with iodine and alcohol gradient, and the antler regeneration bud base tissue is cut under a sterile environment and cut into small pieces, which are then washed with PBS and digested with collagenase in sequence, and then the tissue pieces are transferred and dispersed to the bottom of the culture bottle for primary cell culture, and the obtained cells are identified with antler regeneration bud base cell markers (TNN and TNC);
[0011] S2 Natural Immortality Process:
[0012] The superior cell strain of antler regeneration blastema cells was selected, and the preferential adherent cell and starvation cell selection methods were used to obtain a cell strain with fast proliferation and strong monoclonal ability for subsequent subculture to construct a spontaneously immortalized cell line.
[0013] Preferably, the collagenase used in the digestion in step S1 is type I collagenase; the digestion temperature is 37° C.; and the digestion time is 30 minutes.
[0014] Preferably, the specific steps of the preferential adherent cell and starving cell selection method are as follows:
[0015] The cells were inoculated into a culture flask and cultured with a medium containing 10% FBS. After 3 hours, the cells that were not attached to the wall were removed and replaced with a medium containing 3% FBS for starvation culture. After 12 hours, the size of the cell colony was observed and the cell colony with a large colony was selected for digestion and passage.
[0016] The above treatment was repeated 5 times.
[0017] In addition, to achieve the above purpose, the present invention also provides a method for engineering the preparation of secretory factors of antler regeneration blastema cells, comprising the following steps:
[0018] S1: Inoculate an appropriate amount of established antler regeneration blastema cell lines into a cell culture bioreactor (PBS Biotech) for suspension culture, use a serum replacement medium, and collect and add medium at regular intervals;
[0019] S2 performs density gradient centrifugation on the collected culture medium to remove cells, dead cells, apoptotic bodies and exosomes respectively, and then uses nuclease to remove residual nucleic acids, freeze-drying the final culture medium, and finally obtaining the secretory factors of antler regeneration blastema cells.
[0020] Preferably, in step S1, the culture medium is collected and added every 48 hours.
[0021] Preferably, in step S1, the steps of collecting and adding culture medium are: after stopping stirring for 10 minutes, 3 / 4 of the culture medium is extracted, and then new culture medium of the same volume is added.
[0022] Preferably, in step S2, the specific steps of density gradient centrifugation are: 300g centrifugation for 10 minutes → 2000g centrifugation for 10 minutes → 10000g centrifugation for 10 minutes → 100000g centrifugation for 60 minutes.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] 1. The present invention provides for the first time a method for establishing a naturally immortalized antler regeneration blastema cell line without gene editing, which can obtain the most effective naturally immortalized antler regeneration blastema cell line in antler without gene editing. The immortalized cells have the characteristics of good stability, which solves the problems of poor amplification stability and low viability encountered in in vitro culture and amplification applications; the preferential adherent cell and starvation cell selection method is adopted to select a antler regeneration blastema cell line with high survival rate, fast proliferation rate, and at least 200 passages, thereby realizing the natural immortalization of cells. At the same time, the biological characteristics of immortalized antler regeneration blastema cells are identified by cell morphology identification, cell immunofluorescence and other methods.
[0025] 2. The present invention provides a method for engineering preparation and purification of secretory factors of antler regeneration blastema cells, which solves the safety and ethical constraints of directly using cells for human body, and realizes the low-cost batch production of secretory factors; the cells can be used to engineer functional factors, which can be directly applied to various forms of medicines, medical device products, foods, Health products and cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a flow chart of the construction of the natural immortalized antler regeneration blastema cell line of the present invention;
[0027] Figure 2This is a comparison diagram of the morphology of primary cells and naturally immortalized cells of the present invention;
[0028] Figure 3 This is a diagram of the identification of primary cells and naturally immortalized cells (expression of blastema cell markers TNN and TNC) of the present invention;
[0029] Figure 4 This is a graph showing the proliferation activity of primary cells and naturally immortalized cells of the present invention; *p<0.05, ns, no significance;
[0030] Figure 5 This is a diagram verifying the activity of the secretion factor of the engineered antler regeneration blastema cells of the present invention;
[0031] Control: PBS treatment; 2D-factor: secretory factor prepared by 2D cultured antler regeneration blastema cells (50 μg / ml); 3D-factor: secretory factor prepared by engineered antler regeneration blastema cells (50 μg / ml). BMSC: bone marrow mesenchymal stem cells; HUVEC: vascular endothelial cells; NIH3T3, mouse embryonic fibroblasts. **p<0.01, ****p<0.0001, ns not significant.
[0032] Figure 6 This is a comparison chart of the activity of the antler regeneration blastema cell secretion factor of the present invention and the other two antler stem cell secretion factors;
[0033] Control: PBS treatment; PPC-factor: secretory factor prepared from periosteal stem cells of antler horn handle (50 μg / ml); RMC-factor: secretory factor prepared from mesenchymal stem cells of antler horn (50 μg / ml); ABPC-factor: secretory factor prepared from antler regeneration blastema. BMSC: bone marrow mesenchymal stem cells; HUVEC: vascular endothelial cells; NIH3T3, mouse embryonic fibroblasts. **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION
[0034] Example 1: Obtaining antler regeneration blastema cells
[0035] In April and May, 3-4 year old male sika deer were selected. After the antler discs fell off for 2-5 days, professional veterinarians anesthetized the deer. The hair on the horn handle was shaved off with a scalpel, and then it was gradiently disinfected with iodine and alcohol. The skin of the horn handle was circumcised (the distal 1 / 2), and then the distal horn handle tissue was sawed off, placed in a ziplock bag (ice temperature) and brought back to the laboratory. In the laboratory, the tissue was gradiently disinfected again, and the skin was cut open in a biosafety cabinet to expose the antler regeneration bud base tissue. The bud base tissue was then cut and cut into small pieces (about 0.2 mm). After washing with PBS 3 times, it was placed in a refrigerator. Digest with type I mixed collagenase (200 U / ml) for about 30 minutes (until rough edges appear around the tissue); then, transfer the tissue pieces to the bottom of the culture bottle for primary cell culture (a small amount of culture medium). After the cells crawl out, add sufficient culture medium ( Figure 1 When the cells grew to 90% density, photos were taken under a microscope ( Figure 2 ), harvest the cells. Then, immunofluorescence staining was used to detect the expression of antler regeneration blastema cell markers (TNN and TNC) on the obtained cells, so as to identify whether the obtained cells are antler regeneration blastema cells ( Figure 3 The results showed that the obtained cells had uniform morphology, were long spindle-shaped, and expressed TNN and TNC at high abundance, which could be confirmed as antler regeneration blastema cells.
[0036] Example 2: Establishment of a naturally immortalized antler regeneration blastema cell line
[0037] First, select the dominant growth cell line of antler regeneration blastema cells, and use the preferential adherent cell and starvation cell selection method. Specifically, the cells are inoculated into a culture bottle, and at this time, a 10% FBS culture medium is used for culture (Note: 1 / 5 of the normal cell inoculation amount, and the inoculation must be particularly uniform). After 3 hours, the suspended cells that are not attached to the wall are removed, and the culture medium containing 3% FBS is replaced for starvation culture. After 12 hours, the size of the cell colony is observed, and the 5 largest cell colonies are circled on the back of the culture bottle with a marker, and then digested and passaged separately. Repeat the above treatment 5 times to obtain the cell line with the fastest proliferation and the strongest monoclonal ability for subsequent subculture to construct a spontaneously immortalized cell line ( Figure 1 ). Take photos under a microscope at specified times ( Figure 2 ) and tested whether it retained its blastema cell properties ( Figure 3 and Figure 4 The results showed that the cells at the 50th, 100th and 200th generations all showed a uniform long spindle shape, without obvious enlargement or deformation, and were able to express TNN and TNC ( Figure 3 ), and the cell proliferation activity did not decrease significantly (a slight decrease at 200 generations, Figure 4 ). Therefore, it can be determined that this cell did not lose its blastema cell properties during the subculture process.
[0038] Example 3: Engineering preparation of secretory factors from antler regeneration blastema cells
[0039] Inoculate an appropriate amount of antler regeneration blastema cell line into a cell culture bioreactor (PBS Biotech), and perform cell suspension culture according to the method in the manual, using a serum replacement medium. Check the sugar content in the culture medium regularly every day (blood sugar test strips). After every 48 hours, stop stirring for 10 minutes, and then collect 3 / 4 of the culture medium in the supernatant; then add a new culture medium of the same volume and start stirring. Repeat this step continuously to collect the conditioned culture medium. Then perform gradient centrifugation on the collected culture medium: 300g (10 minutes) → 2000g (10 minutes) → 10000g (10 minutes) →
[0040] 100000g (60 minutes) to remove cells, dead cells, apoptotic bodies and exosomes respectively, and then use nucleases (DNase and RNase) to remove residual nucleic acids, and then inactivate nucleases. The final culture medium is freeze-dried to finally obtain the secretory factors of antler regeneration blastema cells.
[0041] In order to identify the activity of the obtained factors, bone marrow mesenchymal stem cells (BMSC), human vascular endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH3T3) were selected to detect the effects of the obtained factors on the proliferation of the three types of cells; at the same time, the factors obtained by 2D plate culture ( Figure 5 ), as well as secretory factors of antler horn stalk periosteal stem cells and antler mesenchymal stem cells ( Figure 6 ) as a control, the results showed that the ability of the engineered factors to promote cell proliferation was not significantly different from that of the factors obtained by 2D plate culture ( Figure 5 ), which proves that the engineered active factors have reached the standard of factors obtained by 2D plate culture; but are significantly stronger than the secretory factors of the other two antler stem cells ( Figure 6 ), which proves that the secretory factor of antler regeneration blastema cells may be the factor with the strongest efficacy and development value among all the secretory factors of antler stem cells.
Claims
1. A method for establishing a naturally immortalized antler regeneration blastema cell line, characterized in that: The steps include: S1 Obtaining antler regeneration blastema cells: After the male sika deer antler disc falls off for 2-5 days, the top tissue of the horn handle is cut, and the antler regeneration bud base tissue is cut under a sterile environment and cut into small pieces, which are then washed with PBS and digested with collagenase in sequence, and then the tissue pieces are transferred and dispersed to the bottom of the culture bottle for primary cell culture, and the obtained cells are identified with antler regeneration bud base cell markers; S2 Natural Immortality Process: The superior cell strain of antler regeneration blastema cells was selected, and the preferential adherent cell and starvation cell selection methods were used to obtain the cell strain with fast proliferation and strong monoclonal ability for subsequent subculture to construct a naturally immortalized cell line.
2. A method for establishing a naturally immortalized antler regeneration blastema cell line according to claim 1, characterized in that: The collagenase used in the digestion in step S1 is type I collagenase; the digestion temperature is The digestion time was 30 minutes.
3. A method for establishing a naturally immortalized antler regeneration blastema cell line according to claim 1, characterized in that: The specific steps of the preferential adherent cell and starving cell selection method are as follows: The cells were inoculated into a culture flask and a medium containing 10% FBS was used. After 3 hours, the cells that did not adhere to the wall were removed and replaced with a medium containing 3% FBS for starvation culture. After 12 hours, the size of the cell colony was observed and the cell colony with a large colony was selected for digestion and passage. The above treatment was repeated 5 times.
4. A method for engineering the preparation of secretory factors of antler regeneration blastema cells, characterized in that: The steps include: S1. Inoculate an appropriate amount of the antler regeneration blastema cell line established in claims 1-3 into a cell culture bioreactor for suspension culture, use a serum replacement medium, and collect and add the medium at regular intervals; S2 performs density gradient centrifugation on the collected culture medium to remove cells, dead cells, apoptotic bodies and exosomes respectively, and then uses nuclease to remove residual nucleic acids, freeze-drying the final culture medium, and finally obtaining the secretory factors of antler regeneration blastema cells.
5. The method for engineering preparation of secretory factors of antler regeneration blastema cells according to claim 4, characterized in that: In step S1, the culture medium is collected and added every 48 hours.
6. The method for engineering preparation of secretory factors of antler regeneration blastema cells according to claim 4, characterized in that: In step S1, the steps of collecting and adding culture medium are as follows: after stopping stirring for 10 minutes, 3 / 4 of the culture medium is extracted, and then new culture medium of the same volume is added.
7. The method for engineering preparation of secretory factors of antler regeneration blastema cells according to claim 4, characterized in that: In step S2, the specific steps of density gradient centrifugation are: 300g centrifugation for 10 minutes → 2000g centrifugation for 10 minutes → 10000g centrifugation for 10 minutes → 100000g centrifugation for 60 minutes.