CN113025661A - Construction method of immortalized musk glandular epithelial cells - Google Patents
Construction method of immortalized musk glandular epithelial cells Download PDFInfo
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Abstract
The invention provides a construction method of an immortalized forest musk gland epithelial cell. It comprises the following steps: (1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period; (2) separating and purifying the musk forest gland epithelial cells and fibroblasts of the musk forest gland tissues to obtain purified musk forest gland epithelial cells; (3) transfecting the purified musk forest glandular epithelial cells with lentiviruses to obtain transfected musk forest glandular epithelial cells; (4) screening the transfected forest musk glandular epithelial cells through puromycin to obtain successfully transfected forest musk glandular epithelial cells; (5) and performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells. The construction method of the immortalized forest musk gland epithelial cells provided by the invention adopts a lentivirus transfection mode to immortalize the musk gland epithelial cells, can completely maintain the characteristics of the epithelial cells after 12 generations of culture, and overcomes the defect of epithelial cell apoptosis.
Description
Technical Field
The invention relates to the technical field of animal tissue culture, in particular to a construction method of immortalized forest musk gland epithelial cells.
Background
Musk is an important component of traditional Chinese medicine. As recorded in the '2020 edition of Chinese pharmacopoeia', musk has the functions of inducing resuscitation, refreshing mind, promoting blood circulation, dredging channels, relieving swelling and pain. Modern pharmaceutical works all refer to musk as a treasure of medicinal materials. In the practical application of Chinese patent medicines and patent medicines, musk often accounts for the first place of rare animal medicines such as bezoar, rhinoceros horn, bear gall and the like. In 2621 prescriptions recorded in the national Chinese patent medicine prescription Collection, 295 prescriptions containing musk are recorded. Because the yield of the natural musk is extremely low, each male forest musk deer can only produce about 15 g per year, the musk is extremely precious, the market price is more than 3 times of gold, the musk gland epithelial cells are functional cells playing a role in secretion in the musk glands, the forest musk glands begin to swell before and after 5 months of each year, and the primary fragrant liquid secreted by the musk gland cells flows into the sachet along a conduit. The results of these studies lead people to have a more comprehensive understanding of the macrostructure and ultrastructure of the musk forest glands.
In order to meet the demand of the market for musk, in recent years, researchers are actively seeking to obtain musk through in vitro cell biological culture. The previous people also tried for the purpose, but the problems of difficult collection of musk forest gland tissues, slow growth of musk forest gland epithelial cells during culture and apoptosis of musk forest gland epithelial cells cannot be solved.
The applicant has found that the prior art has at least the following technical problems:
1. because no effective and safe forest musk deer anesthesia method exists at present, the existing method for anesthetizing the forest musk deer to collect the musk gland tissue causes damage to the forest musk deer, and the cell amount is insufficient;
2. in the prior art, when the musk forest gland cells are cultured, epithelial cells grow slowly;
3. in the prior art, when the musk forest gland cells are cultured, the cells die after epithelial cells are cultured to the 5 th generation, and the requirement of producing musk by in vitro cell culture cannot be met.
Disclosure of Invention
The invention aims to provide a construction method of immortalized forest musk gland epithelial cells, which aims to solve the technical problem that the forest musk gland cells are apoptotic after the epithelial cells are cultured to the 5 th generation in the prior art. The technical effects that can be produced by the preferred technical scheme in the technical schemes provided by the invention are described in detail in the following.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a construction method of immortalized forest musk glandular epithelial cells, which comprises the following steps:
(1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period;
(2) separating and purifying the forest musk gland epithelial cells and the fibroblasts of the forest musk gland tissues collected in the step (1) to obtain purified forest musk gland epithelial cells;
(3) transfecting the purified lentivirus of the musk forest glandular epithelial cells obtained in the step (2) to obtain transfected musk forest glandular epithelial cells;
(4) screening the transfected forest musk glandular epithelial cells obtained in the step (3) through puromycin to obtain successfully transfected forest musk glandular epithelial cells;
(5) and performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells.
Further, in the step (1), the forest musk gland tissue is transferred to PBS containing streptomycin immediately after being collected.
Further, the streptomycin qing is 100U/ml.
Further, in the step (2), the method for separating and purifying the musk forest glandular epithelial cells and the fibroblasts comprises the following steps:
firstly, cutting the musk forest gland tissue collected in the step (1) to 1-2mm3(ii) a Then digesting the collagen IV at 36-38 ℃ for 3-5 hours by 0.05-0.3 percent of collagenase IV; then using complete culture medium to perform suspension washing for 2-4 times; centrifuging at 2000r/m for 2-3min at 1300-;
secondly, inoculating the supernatant collected in the first step into a complete culture medium, wherein the content of serum in the complete culture medium is 10%; culturing at 37 deg.C under 5% carbon dioxide and saturated humidity, and changing the culture solution every 24-36 h for 1 time;
thirdly, after culturing for 3 to 5 days, when the fibroblasts adhere to the wall and the epithelial cells do not adhere to the wall, sucking the supernatant and inoculating the supernatant into another cell bottle;
fourthly, after the culture is continued for 2 to 3 days, epithelial cells start to grow adherent to the wall, the complete culture medium is replaced, and the serum content in the replaced complete culture medium is 2 to 5 percent; if the fibroblasts are attached to the wall, scraping fibroblast agglomerates by using an aseptic inoculating loop;
fifthly, continuously culturing for 7-10 days, replacing the complete culture medium when the growth of the epithelial cells of the single layer is accelerated, and continuously culturing until the serum content in the replaced complete culture medium is 8-10%; and culturing to obtain purified musk forest glandular epithelial cells.
Further, the complete culture medium used in the first step and the second step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 10%, and the content of the EPC factors is 10-15 ng/ml;
the complete culture medium used in the step (c) is a basal culture medium which is DMEM and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 2-5%, and the content of the EPC factors is 10-15 ng/ml;
the complete culture medium used in the fifth step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 8-10%, and the content of the EPC factors is 10-15 ng/ml;
furthermore, in the fifth step, when the monolayer cells begin to be combined, 0.25-0.50mg/ml trypsin is used for digestion.
Further, in the step (3), the lentivirus transfection is SV40 virus transfection, EB virus transfection or human papilloma virus transfection.
Further, in the step (3), the lentivirus transfection is SV40 virus transfection, specifically: inoculating purified forest musk glandular epithelial cells obtained in the step (2) into 6-hole plates, wherein the number of the cells in each hole is about 1 multiplied by 105A plurality of; the next day, after the cells adhere to the wall, changing the culture solution, adding 1mL of complete culture medium, adding 20 μ L of SV40 overexpression lentivirus, mixing uniformly, continuing culturing, observing the cell state after 12h, changing the culture medium into a fresh culture medium, after the cells grow to the bottom, passing the cells into a T25 culture bottle, and culturing to obtain the immortalized forest musk deer epithelial cells.
Further, in the step (3), the complete culture medium used is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factor, wherein the content of the double antibody is 1%, the content of the serum is 10%, and the content of the EPC factor is 10-15 ng/ml.
Further, in the step (4), the step of screening the transfected forest musk glandular epithelial cells for successfully transfected forest musk glandular epithelial cells by puromycin comprises the following specific steps:
firstly, determining the use concentration of puromycin to be 2ug/mL, and the action time to be 2 d;
secondly, screening the transfected forest musk glandular epithelial cells by the puromycin use concentration and action time determined in the step I.
Further, in the step (i), the specific steps for determining the use concentration and the action time of puromycin are as follows:
a. laying untransfected musk forest gland epithelial cells into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2-5%;
b. after 24-26h, removing the culture medium in the 24-well plate in the step a;
c. respectively adding fresh culture media containing puromycin with different concentrations into a 24-pore plate paved with cells, correspondingly replacing the fresh culture media once in 48 hours, and observing the survival ratio of the cells every day; the concentration of the puromycin is 1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL and 7ug/mL respectively; the culture medium is epithelial cell complete culture medium, and the serum concentration is 2-5%;
d. the minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from puromycin screening; as a result, it was confirmed that puromycin was used at a concentration of 2ug/mL for an action time of 2 days.
Further, in the second step, the screening of the successful transfection forest musk glandular epithelial cells comprises the following specific steps:
a. paving the transfected forest musk gland epithelial cells in the step (1) into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2-5%;
b. after 24-26h, removing the culture medium in the 24-well plate in the step a,
c. adding a screening culture medium, incubating, wherein the screening culture medium is an epithelial cell complete culture medium, the serum concentration is 2-5%, and the concentration of the added puromycin is 2 ug/mL; replacing the fresh screening culture medium once in 48 hours, and observing the survival rate of the cells every day;
d. the cells which survive at the same time point are the forest musk glandular epithelial cells which are successfully transfected.
Based on the technical scheme, the embodiment of the invention can at least produce the following technical effects:
the construction method of the immortalized forest musk deer glandular epithelial cells collects the massive musk deer glandular tissues of forest musk deer died due to illness in the secretion period, ensures enough cell quantity and does not cause damage to living forest musk deer; during cell culture, the fibroblast and the epithelial cell are separated and purified by adopting a differential adherence method and a physical scraping method, so that the influence of the fibroblast on the epithelial cell is reduced, the growth speed of the epithelial cell is ensured, after the cell is purified, the tonka epithelial cell is immortalized by adopting a lentivirus transfection mode, the characteristics of the epithelial cell can be completely maintained after 12 generations of culture, and the defect of epithelial cell apoptosis is overcome.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a diagram of a culture of primary cells of forest musk glands in example 1 of the present invention (wherein the paving stone cells are epithelial cells, and the fluorescence inverted microscope is 200 times);
FIG. 2 is a drawing showing the culture of primary cells of forest musk glands in example 1 of the present invention (the paving stone cells are epithelial cells, 400 times fluorescence inverted microscope)
FIG. 3 shows the culture of the forest musk gland P2 cells in example 1 of the present invention (after purification, the paving stone cells are epithelial cells, 400 times fluorescence inverted microscope)
FIG. 4 is a photograph showing the culture of forest musk gland cells before immortalization in example 1 of the present invention (the paving stone cells are epithelial cells, 400 times fluorescence inverted microscope)
FIG. 5 is a drawing showing the culture of forest musk gland cells after immortalized transfection in example 1 of the present invention (the paving stone cells are epithelial cells, 800 times fluorescence inverted microscope)
FIG. 6 shows the immortalization of the forest musk gland cell (expanded culture P12) in example 1 of the present invention (the paving stone cells are epithelial cells, 400 times fluorescence inverted microscope);
FIG. 7 is a photograph (100X) of the immunofluorescence assay of cells of the epithelial cells of the immortalized forest musk gland obtained in example 1 of the present invention;
FIG. 8 is a photograph (200X) of the immunofluorescence assay of cells of the epithelial cells of the immortalized forest musk gland obtained in example 1 of the present invention;
FIG. 9 is a map of SV40 overexpression lentiviral vectors used in the examples of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
In the following examples 1-3:
the complete epithelial cell culture medium used was PriMed-iCell-001 Sapococcus (Shanghai) Biotech Co., Ltd;
the SV40 overexpression lentivirus used (as shown in FIG. 9) was purchased from Hemmy's organism;
the sequence information of the vector is as follows:
gtggttcaaagtttttttcttccatttcaggtgtcgtgaggatctatttccggtgaattc(atggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgcctgggggaatattcctctgatgagaaaggcatatttaaaaaaatgcaaggagtttcatcctgataaaggaggagatgaagaaaaaatgaagaaaatgaatactctgtacaagaaaatggaagatggagtaaaatatgctcatcaacctgactttggaggcttctgggatgcaactgagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatccattttctgttattgaggaaagtttgccaggtgggttaaaggagcatgattttaatccagaagaagcagaggaaactaaacaagtgtcctggaagcttgtaacagagtatgcaatggaaacaaaatgtgatgatgtgttgttattgcttgggatgtacttggaatttcagtacagttttgaaatgtgtttaaaatgtattaaaaaagaacagcccagccactataagtaccatgaaaagcattatgcaaatgctgctatatttgctgacagcaaaaaccaaaaaaccatatgccaacaggctgttgatactgttttagctaaaaagcgggttgatagcctacaattaactagagaacaaatgttaacaaacagatttaatgatcttttggataggatggatataatgtttggttctacaggctctgctgacatagaagaatggatggctggagttgcttggctacactgtttgttgcccaaaatggattcagtggtgtatgactttttaaaatgcatggtgtacaacattcctaaaaaaagatactggctgtttaaaggaccaattgatagtggtaaaactacattagcagctgctttgcttgaattatgtggggggaaagctttaaatgttaatttgcccttggacaggctgaactttgagctaggagtagctattgaccagtttttagtagtttttgaggatgtaaagggcactggaggggagtccagagatttgccttcaggtcagggaattaataacctggacaatttaagggattatttggatggcagtgttaaggtaaacttagaaaagaaacacctaaataaaagaactcaaatatttccccctggaatagtcaccatgaatgagtacagtgtgcctaaaacactgcaggccagatttgtaaaacaaatagattttaggcccaaagattatttaaagcattgcctggaacgcagtgagtttttgttagaaaagagaataattcaaagtggcattgctttgcttcttatgttaatttggtacagacctgtggctgagtttgctcaaagtattcagagcagaattgtggagtggaaagagagattggacaaagagtttagtttgtcagtgtatcaaaaaatgaagtttaatgtggctatgggaattggagttttagattggctaagaaacagtgatgatgatgatgaagacagccaggaaaatgctgataaaaatgaagatggtggggagaagaacatggaagactcagggcatgaaacaggcattgattcacagtcccaaggctcatttcaggcccctcagtcctcacagtctgttcatgatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacagagcaaaagctcatttctgaagaggacttgtaa) tctagacacagtgcagcactctcaacgttcaaggacactacgcgtctggaacaatcaacc, respectively; the sequence region of interest is in parentheses.
First, an embodiment
Example 1:
a construction method of immortalized musk forest glandular epithelial cells comprises the following steps:
(1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period; immediately transferring the collected solution to PBS containing streptomycin, wherein the streptomycin is 100U/ml;
(2) separating and purifying the musk forest gland epithelial cells and fibroblasts of the musk forest gland tissues (shown in figures 1 and 2) collected in the step (1) to obtain purified musk forest gland epithelial cells; the method specifically comprises the following steps:
firstly, cutting the musk forest gland tissue collected in the step (1) to 1-2mm3(ii) a Then digested for 4 hours at 37 ℃ with 0.2% collagenase type IV; then using complete culture medium to perform suspension washing for 3 times; centrifuging at 1600r/m for 2.5min, and collecting supernatant;
secondly, inoculating the supernatant collected in the first step into a complete culture medium, wherein the content of serum in the complete culture medium is 10%; culturing at 37 deg.C with 5% carbon dioxide and saturated humidity, and changing the culture solution 1 time every 30 hr; the complete culture medium used in the first step and the second step is a DMEM (DMEM) basic culture medium, and is added with double antibodies, serum and EPC (Epproduct code) factors, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 13 ng/ml;
③ after 5 days of culture, when the fibroblasts are attached to the wall and the epithelial cells are not attached to the wall, sucking the supernatant and inoculating the supernatant into another cell bottle;
fourthly, after the culture is continued for 2 days, epithelial cells start to grow adherent to the wall, the complete culture medium is replaced, and the serum content in the replaced complete culture medium is 3.5 percent; if the fibroblasts are attached to the wall, scraping fibroblast agglomerates by using an aseptic inoculating loop; the complete culture medium used in the fourth step is a basal culture medium which is DMEM and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 3.5%, and the content of the EPC factors is 13 ng/ml;
fifthly, after continuing to culture for 9 days, when the growth of the epithelial cells of the single layer is accelerated, replacing the complete culture medium, wherein the serum content in the replaced complete culture medium is 9 percent, and continuing to culture; culturing to obtain purified forest musk gland epithelial cells (as shown in figure 3); the complete culture medium used in the fifth step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 9%, and the content of the EPC factors is 13 ng/ml;
(3) transfecting the purified lentivirus of the musk forest glandular epithelial cells obtained in the step (2) to obtain transfected musk forest glandular epithelial cells;
the lentivirus transfection is SV40 virus transfection, and specifically comprises the following steps: inoculating purified forest musk glandular epithelial cells obtained in the step (2) into 6-hole plates, wherein the number of the cells in each hole is about 1 multiplied by 105A plurality of; on the next day, after the cells adhere to the wall, changing the culture solution, adding 1mL of complete culture medium, adding 20 μ L of SV40 overexpression lentivirus, mixing uniformly, continuing culturing, observing the cell state after 12h, changing the culture medium into a fresh culture medium, after the cells grow to the bottom of the plate, passing the cells into a T25 culture bottle, and culturing to obtain transfected forest musk glandular epithelial cells; the complete culture medium is DMEM as a basic culture medium, and double antibodies, serum and EPC factors are added, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 12.5 ng/ml;
(4) screening the transfected forest musk glandular epithelial cells obtained in the step (3) for successfully transfected forest musk glandular epithelial cells by puromycin (shown in a figure 4); the method comprises the following specific steps:
firstly, determining the use concentration of puromycin to be 2ug/mL, and the action time to be 2 d;
a. laying untransfected musk forest gland epithelial cells into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 3.5%;
b. after 24h, removing the old culture medium in the 24-well plate in the step a;
c. respectively adding fresh culture media containing puromycin with different concentrations into a 24-pore plate paved with cells, correspondingly replacing the fresh culture media once in 48 hours, and observing the survival ratio of the cells every day; the concentration of the puromycin is 1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL and 7ug/mL respectively; the culture medium is an epithelial cell complete culture medium, and the serum concentration is 3.5%;
d. the minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from puromycin screening; as a result, it was confirmed that puromycin was used at a concentration of 2ug/mL for an action time of 2 days.
Secondly, screening the transfected forest musk glandular epithelial cells by the puromycin use concentration and action time determined in the step I; the method comprises the following specific steps:
a. paving the transfected forest musk gland epithelial cells in the step (1) into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 3.5%;
b. after 24h, removing the culture medium in the 24-well plate in the step a,
c. adding a screening culture medium, and incubating, wherein the screening culture medium is an epithelial cell complete culture medium, the serum concentration is 3.5%, and the concentration of the added puromycin is 2 ug/mL; replacing the fresh screening culture medium once in 48 hours, and observing the survival rate of the cells every day;
d. the cells which survive at the same time point are the forest musk glandular epithelial cells which are successfully transfected;
(5) and (3) performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells (as shown in figures 5 and 6).
Example 2:
a construction method of immortalized musk forest glandular epithelial cells comprises the following steps:
(1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period; immediately transferring the collected solution to PBS containing streptomycin, wherein the streptomycin is 100U/ml;
(2) separating and purifying the forest musk gland epithelial cells and the fibroblasts of the forest musk gland tissues collected in the step (1) to obtain purified forest musk gland epithelial cells; the method specifically comprises the following steps:
firstly, cutting the musk forest gland tissue collected in the step (1) to 1-2mm3(ii) a Then digested for 5 hours at 37 ℃ with 0.3% collagenase IV; then using complete culture medium to perform resuspension washing for 2 times; centrifuging at 1300r/m for 3min, and collecting supernatant;
secondly, inoculating the supernatant collected in the first step into a complete culture medium, wherein the content of serum in the complete culture medium is 10%; culturing at 37 deg.C with 5% carbon dioxide and saturated humidity, and changing the culture solution every 24 hr for 1 time; the complete culture medium used in the first step and the second step is a DMEM (DMEM) basic culture medium, and is added with double antibodies, serum and EPC (Epproduct code) factors, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 15 ng/ml;
thirdly, after culturing for 3 days, when the fibroblasts are attached to the wall and the epithelial cells are not attached to the wall, sucking the supernatant and inoculating the supernatant into another cell bottle;
fourthly, after the culture is continued for 3 days, epithelial cells start to grow adherent to the wall, the complete culture medium is replaced, and the serum content in the replaced complete culture medium is 2 percent; if the fibroblasts are attached to the wall, scraping fibroblast agglomerates by using an aseptic inoculating loop; the complete culture medium used in the fourth step is a basal culture medium which is DMEM and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 2%, and the content of the EPC factors is 15 ng/ml;
fifthly, after continuously culturing for 8 days, when the growth of the epithelial cells of the single layer is accelerated, replacing the complete culture medium, wherein the serum content in the replaced complete culture medium is 10 percent, and continuously culturing; culturing to obtain purified musk forest glandular epithelial cells; the complete culture medium used in the fifth step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 10%, and the content of the EPC factors is 15 ng/ml;
(3) transfecting the purified lentivirus of the musk forest glandular epithelial cells obtained in the step (2) to obtain transfected musk forest glandular epithelial cells;
the lentivirus transfection is SV40 virus transfection, and specifically comprises the following steps: inoculating purified forest musk glandular epithelial cells obtained in the step (2) into 6-hole plates, wherein the number of the cells in each hole is about 1 multiplied by 105A plurality of; on the next day, after the cells adhere to the wall, changing the culture solution, adding 1mL of complete culture medium, adding 20 μ L of SV40 overexpression lentivirus, mixing uniformly, continuing culturing, observing the cell state after 12h, changing the culture medium into a fresh culture medium, after the cells grow to the bottom of the plate, passing the cells into a T25 culture bottle, and culturing to obtain transfected forest musk glandular epithelial cells; the complete culture medium is DMEM as basic culture medium, and is added with double antibody, serum and EPC factor, the content of the double antibody is 1%, the content of the serum is 10%, and the content of the EPC factor is 15ng/ml；
(4) Screening the transfected forest musk glandular epithelial cells obtained in the step (3) through puromycin to obtain successfully transfected forest musk glandular epithelial cells; the method comprises the following specific steps:
firstly, determining the use concentration of puromycin to be 2ug/mL, and the action time to be 2 d;
a. laying untransfected musk forest gland epithelial cells into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 5%;
b. after 24h, removing the culture medium from the 24-well plate in the step a;
c. respectively adding fresh culture media containing puromycin with different concentrations into a 24-pore plate paved with cells, correspondingly replacing the fresh culture media once in 48 hours, and observing the survival ratio of the cells every day; the concentration of the puromycin is 1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL and 7ug/mL respectively; the culture medium is an epithelial cell complete culture medium, and the serum concentration is 5%;
d. the minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from puromycin screening; as a result, it was confirmed that puromycin was used at a concentration of 2ug/mL for an action time of 2 days.
Secondly, screening the transfected forest musk glandular epithelial cells by the puromycin use concentration and action time determined in the step I; the method comprises the following specific steps:
a. paving the transfected forest musk gland epithelial cells in the step (1) into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 5%;
b. after 24h, the medium in the 24-well plate in step a was removed,
c. adding a screening culture medium, incubating, wherein the screening culture medium is an epithelial cell complete culture medium, the serum concentration is 5%, and the concentration of the added puromycin is 2 ug/mL; replacing the fresh screening culture medium once in 48 hours, and observing the survival rate of the cells every day;
d. the cells which survive at the same time point are the forest musk glandular epithelial cells which are successfully transfected;
(5) and performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells.
Example 3:
a construction method of immortalized musk forest glandular epithelial cells comprises the following steps:
(1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period; immediately transferring the collected solution to PBS containing streptomycin, wherein the streptomycin is 100U/ml;
(2) separating and purifying the forest musk gland epithelial cells and the fibroblasts of the forest musk gland tissues collected in the step (1) to obtain purified forest musk gland epithelial cells; the method specifically comprises the following steps:
firstly, cutting the musk forest gland tissue collected in the step (1) to 1-2mm3(ii) a Then digested for 3 hours at 38 ℃ with 0.05% collagenase IV; then using complete culture medium to perform resuspension washing for 4 times; centrifuging at 2000r/m for 2min, and collecting supernatant;
secondly, inoculating the supernatant collected in the first step into a complete culture medium, wherein the content of serum in the complete culture medium is 10%; culturing at 37 deg.C under 5% carbon dioxide and saturated humidity, and changing the culture solution every 36 hr for 1 time; the complete culture medium used in the first step and the second step is a DMEM (DMEM) basic culture medium, and is added with double antibodies, serum and EPC (Epproduct code) factors, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 10 ng/ml;
③ after culturing for 4 days, when the fibroblasts are attached to the wall and the epithelial cells are not attached to the wall, sucking the supernatant and inoculating the supernatant into another cell bottle;
fourthly, after the culture is continued for 3 days, epithelial cells start to grow adherent to the wall, the complete culture medium is replaced, and the serum content in the replaced complete culture medium is 5 percent; if the fibroblasts are attached to the wall, scraping fibroblast agglomerates by using an aseptic inoculating loop; the complete culture medium used in the fourth step is a basal culture medium which is DMEM and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 5%, and the content of the EPC factors is 10 ng/ml;
fifthly, after continuously culturing for 8 days, when the growth of the epithelial cells of the single layer is accelerated, replacing the complete culture medium, wherein the serum content in the replaced complete culture medium is 8-10%, and continuously culturing; culturing to obtain purified musk forest glandular epithelial cells; the complete culture medium used in the fifth step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 8%, and the content of the EPC factors is 10 ng/ml;
(3) transfecting the purified lentivirus of the musk forest glandular epithelial cells obtained in the step (2) to obtain transfected musk forest glandular epithelial cells;
the lentivirus transfection is SV40 virus transfection, and specifically comprises the following steps: inoculating purified forest musk glandular epithelial cells obtained in the step (2) into 6-hole plates, wherein the number of the cells in each hole is about 1 multiplied by 105A plurality of; on the next day, after the cells adhere to the wall, changing the culture solution, adding 1mL of complete culture medium, adding 20 μ L of SV40 overexpression lentivirus, mixing uniformly, continuing culturing, observing the cell state after 12h, changing the culture medium into a fresh culture medium, after the cells grow to the bottom of the plate, passing the cells into a T25 culture bottle, and culturing to obtain transfected forest musk glandular epithelial cells; the complete culture medium is DMEM as a basic culture medium, and double antibodies, serum and EPC factors are added, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 10 ng/ml;
(4) screening the transfected forest musk glandular epithelial cells obtained in the step (3) through puromycin to obtain successfully transfected forest musk glandular epithelial cells; the method comprises the following specific steps:
firstly, determining the use concentration of puromycin to be 2ug/mL, and the action time to be 2 d;
a. laying untransfected musk forest gland epithelial cells into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2%;
b. after 26h, remove the medium from the 24-well plate in step a;
c. respectively adding fresh culture media containing puromycin with different concentrations into a 24-pore plate paved with cells, correspondingly replacing the fresh culture media once in 48 hours, and observing the survival ratio of the cells every day; the concentration of the puromycin is 1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL and 7ug/mL respectively; the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2%;
d. the minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from puromycin screening; as a result, it was confirmed that puromycin was used at a concentration of 2ug/mL for an action time of 2 days.
Secondly, screening the transfected forest musk glandular epithelial cells by the puromycin use concentration and action time determined in the step I; the method comprises the following specific steps:
a. paving the transfected forest musk gland epithelial cells in the step (1) into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2%;
b. after 26h, the medium was removed from the 24-well plate in step a,
c. adding, incubating, and screening the culture medium to be an epithelial cell complete culture medium, wherein the serum concentration is 2%, and the concentration of the added puromycin is 2 ug/mL; replacing the fresh screening culture medium once in 48 hours, and observing the survival rate of the cells every day;
d. the cells which survive at the same time point are the forest musk glandular epithelial cells which are successfully transfected;
(5) and performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells.
Second, experimental example:
the immortalized forest musk gland epithelial cells obtained in example 1 were subjected to cellular immunofluorescence identification:
1. instrument and reagent required by test
(1) Apparatus, as shown in table 1:
TABLE 1 apparatus
| Name of instrument | Specification and model | Manufacturer of the product |
| Biological safety cabinet | BSC-1500ⅡA2-X | JINAN BIOBASE BIOTECH Co.,Ltd. |
| CO2 cell culture box | BC-J160S | SHANGHAI BOXUN INDUSTRY & COMMERCE Co.,Ltd. |
| Fluorescent inverted microscope | DS-Ri2 | Nikon |
| High-speed refrigerated centrifuge | Multifuge X1R | Thermo Fisher |
| Electric heating constant temperature blast air drying box | DHG-9123A | Shanghai Jinghong experiment equipment Co Ltd |
(2) Reagent consumables, as shown in table 2:
TABLE 2 reagent consumables
2. The experimental steps are as follows:
(1) cell climbing sheet
3 glass plates were placed in a 24-well plate, 1mL of medium was added to each well, and 0.02 millions of cells per well were added. The incubator is kept for 2h or overnight.
(2) Fixing
After cell mounting, the medium was aspirated, washed 1 time with PBS, and fixed with 4% PFA at 4 ℃ for 30 min. Wash 3X 5 min/time with PBS. The PBS was not aspirated for the last time and left overnight at 4 ℃.
(3) Rupture membrane closure
The slide was dewatered, placed on a petri dish support,
preparing a glass sheet sealing liquid: 0.5% Trition X-100 and PBS 1:1, then 10% serum is added, 50uL of membrane rupture sealing liquid is dripped on a waterproof membrane, and the side with cells on the slide is covered for 2 h.
(4) Primary antibody incubation
Preparing a primary antibody: dilution of antibody with PBS 1:100(200)
After the membrane is broken and sealed, 50uL of first antibody is taken out of the waterproof membrane (in a wet box), the slide (the side with cells) is covered and placed at 4 ℃ (the slide can be placed for one week at most)
(5) Incubation with secondary antibody
After incubating the secondary antibody (secondary antibody: PBS 1:500) at room temperature in the dark for 2h, the cells were washed with PBS 3X 5 min/time, stained with DAPI (DAPI: PBS 1:1000) for 5min, and washed with PBS 3X 5 min/time.
(6) Embedding
On the slide, 1 drop of Fluorocount-G was added, and the side with the cells was covered.
3. The experimental results are as follows: through immunofluorescence identification, the positive rate of the CD18 of the cell can reach more than 90%, and identification results are shown in fig. 7 and fig. 8.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention.
Claims (10)
1. A construction method of immortalized forest musk glandular epithelial cells is characterized in that: the method comprises the following steps:
(1) collecting the musk gland tissue of male adult trees which are diseased and dead in the fragrance secretion period;
(2) separating and purifying the forest musk gland epithelial cells and the fibroblasts of the forest musk gland tissues collected in the step (1) to obtain purified forest musk gland epithelial cells;
(3) transfecting the purified lentivirus of the musk forest glandular epithelial cells obtained in the step (2) to obtain transfected musk forest glandular epithelial cells;
(4) screening the transfected forest musk glandular epithelial cells obtained in the step (3) through puromycin to obtain successfully transfected forest musk glandular epithelial cells;
(5) and performing amplification culture on the screened forest musk glandular epithelial cells successfully transfected, thus obtaining the immortalized forest musk glandular epithelial cells.
2. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 1, wherein: in the step (1), the forest musk gland tissue is transferred to PBS containing streptomycin immediately after being collected.
3. The method of constructing immortalized musk forest gland epithelial cells according to claim 2, wherein: the streptomycin qing is 100U/ml.
4. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 1, wherein: in the step (2), the method for separating and purifying the musk forest glandular epithelial cells and the fibroblasts comprises the following steps:
firstly, cutting the musk forest gland tissue collected in the step (1) to 1-2mm3(ii) a Then digesting the collagen IV at 36-38 ℃ for 3-5 hours by 0.05-0.3 percent of collagenase IV; then using complete culture medium to perform suspension washing for 2-4 times; centrifuging at 2000r/m for 2-3min at 1300-;
secondly, inoculating the supernatant collected in the first step into a complete culture medium, wherein the content of serum in the complete culture medium is 10%; culturing at 37 deg.C under 5% carbon dioxide and saturated humidity, and changing the culture solution every 24-36 h for 1 time;
thirdly, after culturing for 3 to 5 days, when the fibroblasts adhere to the wall and the epithelial cells do not adhere to the wall, sucking the supernatant and inoculating the supernatant into another cell bottle;
fourthly, after the culture is continued for 2 to 3 days, epithelial cells start to grow adherent to the wall, the complete culture medium is replaced, and the serum content in the replaced complete culture medium is 2 to 5 percent; if the fibroblasts are attached to the wall, scraping fibroblast agglomerates by using an aseptic inoculating loop;
fifthly, continuously culturing for 7-10 days, replacing the complete culture medium when the growth of the epithelial cells of the single layer is accelerated, and continuously culturing until the serum content in the replaced complete culture medium is 8-10%; and culturing to obtain purified musk forest glandular epithelial cells.
5. The method of constructing immortalized musk forest gland epithelial cells according to claim 4, wherein: the complete culture medium used in the first step and the second step is a DMEM basic culture medium, and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 10-15 ng/ml;
the complete culture medium used in the step (c) is a basal culture medium which is DMEM and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 2-5%, and the content of the EPC factors is 10-15 ng/ml;
the complete culture medium used in the fifth step is DMEM as a basic culture medium, and is added with double antibody, serum and EPC factors, wherein the content of the double antibody is 1%, the content of the serum is 8-10%, and the content of the EPC factors is 10-15 ng/ml; when the monolayer cells began to confluence, they were digested with 0.25-0.50mg/ml trypsin.
6. The method for constructing the epithelial cells of the musk gland in the immortalized forest according to claim 1, wherein said method comprisesIs characterized in that: in the step (3), the lentivirus transfection is SV40 virus transfection, and specifically comprises the following steps: inoculating purified forest musk glandular epithelial cells obtained in the step (2) into 6-hole plates, wherein the number of the cells in each hole is about 1 multiplied by 105A plurality of; the next day, after the cells adhere to the wall, changing the culture solution, adding 1mL of complete culture medium, adding 20 μ L of SV40 overexpression lentivirus, mixing uniformly, continuing culturing, observing the cell state after 12h, changing into fresh complete culture medium, after the cells grow to the bottom of the plate, passing to a T25 culture bottle, and culturing to obtain the immortalized forest musk epithelial cells.
7. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 6, wherein: in the step (3), the used complete culture medium is DMEM as a basic culture medium, and is added with double antibodies, serum and EPC factors, wherein the content of the double antibodies is 1%, the content of the serum is 10%, and the content of the EPC factors is 10-15 ng/ml.
8. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 1, wherein: in the step (4), screening the transfected forest musk glandular epithelial cells through puromycin to obtain successfully transfected forest musk glandular epithelial cells specifically comprises the following steps:
firstly, determining the use concentration of puromycin to be 2ug/mL, and the action time to be 2 d;
secondly, screening the transfected forest musk glandular epithelial cells by the puromycin use concentration and action time determined in the step I.
9. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 8, wherein: in the step I, the specific steps for determining the use concentration and the action time of puromycin are as follows:
a. laying untransfected musk forest gland epithelial cells into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2-5%;
b. after 24-26h, removing the culture medium in the 24-well plate in the step a;
c. respectively adding fresh culture media containing puromycin with different concentrations into a 24-pore plate paved with cells, correspondingly replacing the fresh culture media once in 48 hours, and observing the survival ratio of the cells every day; the concentration of the puromycin is 1ug/mL, 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL and 7ug/mL respectively; the culture medium is epithelial cell complete culture medium, and the serum concentration is 2-5%;
d. the minimum puromycin concentration used is the lowest screening concentration that kills all cells within 1-4 days from puromycin screening; as a result, it was confirmed that puromycin was used at a concentration of 2ug/mL for an action time of 2 days.
10. The method for constructing the epithelial cells of musk deer in the immortalized forest according to claim 8, wherein: in the second step, the screening of the successful transfected forest musk glandular epithelial cells comprises the following specific steps:
a. paving the transfected forest musk gland epithelial cells in the step (1) into a 24-pore plate according to 0.05million per pore for incubation, wherein the culture medium is an epithelial cell complete culture medium, and the serum concentration is 2-5%;
b. after 24-26h, removing the culture medium in the 24-well plate in the step a,
c. adding a screening culture medium, incubating, wherein the screening culture medium is an epithelial cell complete culture medium, the serum concentration is 2-5%, and the concentration of the added puromycin is 2 ug/mL; replacing the fresh screening culture medium once in 48 hours, and observing the survival rate of the cells every day;
d. the cells which survive at the same time point are the forest musk glandular epithelial cells which are successfully transfected.
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