CN108265021A - A kind of leech cell injuring model base and its cultural method - Google Patents

Abstract

The invention discloses a culture medium for leech cells in vitro and a culture method thereof. The shredded leech tissue pieces are inoculated in a cell-enclosed culture bottle, and Insect-C Culture insect cell culture medium is added, and cultured in a cell culture box, and then cultured in a cell culture box. Cooperate with tissue block recovery method for isolation and culture, and finally obtain purified leech body cells by trypsin digestion method for further culture. Compared with other insect cell culture media, the purified somatic cells of leech were obtained more quickly and successfully, which prepared for further subculture of leech cells in vitro, and established a set of in vitro primary culture leech cell system.

Description

A kind of leech cell in vitro culture medium and culture method thereof

technical field

The invention relates to a method for culturing leech cells in vitro, belonging to the field of bioengineering.

Background technique

With the rapid development of life sciences, cell culture is an essential process for both the entire bioengineering technology and one of the biological cloning technologies, and promotes the development of cell biology, molecular biology, biochemistry development in other fields.

Leeches (Whitmania pigra Whitman) belong to the family Annelidae, Hirudoidae. According to statistics, there are more than 600 species of leeches distributed all over the world, and nearly 100 species are distributed in my country. Leeches are cold-blooded annelids that can grow and reproduce in both the north and the south of China. They mainly live in freshwater reservoirs, ditches, paddy fields, lakes and swamps, and most often in ponds rich in organic matter or in small rivers without pollution. The optimum temperature for growth is 10-40°C. When the temperature is lower than 3°C in the northern region, it enters the dormancy and hibernation period in the soil, and stinging activities will occur when the temperature is higher than 8°C in March-April of the following year. Leech is an omnivorous animal. Its main way of life is to suck the blood or body fluid of animals. It often uses plankton, insects, and mollusks in the water as its main bait. Under artificial conditions, it uses various animal offal, cooked egg yolk, compound feed, and plant residues. Freshwater snails, miscellaneous fish, earthworms, etc. are used as bait.

Studies have found that the secretion of leech salivary glands contains hirudin and other biologically active substances and has broad application prospects in medicine. Hirudin, leech hyaluronidase, antistacin, anesthetics, vasodilators, prostaglandins, etc., have the effects of lowering blood fat, protecting cardiovascular and cerebrovascular, anticoagulant and antitumor, and have important research significance. Therefore, there is a great demand for natural hirudin clinically. The existing hirudin extraction is mainly to recombine and synthesize hirudin by means of bioengineering and stimulate hirudin salivary glands to secrete hirudin. These two methods are not only costly but also yield very low hirudin. With the development of in vitro cell culture technology, it has become a hot research topic to obtain hirudin by culturing leech cells in vitro. It has been found that the hirudin obtained from this method not only has a high yield, but also has the same effect as that obtained by stimulating the secretion of leech salivary glands. comparable to hirudin. However, at present, the in vitro culture technology of leech cells is still in its infancy, and the selection of its culture medium, the determination of culture conditions, the selection of antibiotics, etc. are all problems that need to be overcome urgently.

Contents of the invention

In view of the above problems, the present invention provides a method for culturing leech cells in vitro.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A kind of leech cell culture medium in vitro, it is characterized in that, described culture medium formula is as follows:

Inorganic salt:

NaCl1420mg/L, ZnCL2876.54mg _/ L, _MgSO4 · _{7H2O1743.48mg} /L, KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L; pH adjusted to 7.4 with 1.0N KOH.

A method for culturing leech cells in vitro, inoculating shredded leech tissue pieces into a cell-enclosed culture bottle, adding the above-mentioned Insect-C Culture insect cell culture medium, cultivating them statically in a cell incubator, and then separating them with the tissue piece recovery method Cultured, and finally purified leech body cells were obtained by trypsinization for further culture. Specific steps are as follows

1) Soak the living leeches in normal saline for 1 hour to initially remove impurities in the body and ensure the vitality of the somatic cells to the maximum extent, then wash the surface mucous membranes with normal saline until clean, and finally wash them three times with distilled water and place them in 70% alcohol Soak for 5 minutes to remove bacteria on the body surface, and rinse with distilled water several times to remove alcohol;

2) Transfer the leech after skin disinfection in step 1) to the ultra-clean workbench, put it into a sterile petri dish filled with sterile PBS, and cut off the leeches with sterile scissors and tweezers Take the head tissue at both ends;

3) Transfer the head tissue in step 2) to another sterile petri dish containing PBS buffer, soak it in the verruca; cut the head tissue with a sterile scalpel, and take the epidermis The middle tissue was rinsed with sterile PBS buffer;

4) Soak the tissue in step 3) in 75% alcohol for 1 min, take it out and rinse it with sterile PBS buffer, repeat three times;

5) Place the tissue in step 4) in a sterile petri dish filled with sterile PBS, cut it into small tissue pieces with sterile scissors and tweezers, and soak for 5 minutes;

6) Discard the tissue fragments floating on the upper part of the small tissue pieces in step 5), use a 5mL pipette tip to suck up the tissue piece paste, and spread the sucked paste tissue on the bottom of the bottle with uniform size and evenly distributed on the Add 5mL of Insect-C Culture insect cell culture medium to a 25mL closed cell culture flask, and place in a cell culture incubator at 25°C for 60min.

7) Put the culture bottle after step 6) to stand still for 60 minutes to adhere to the wall, use a pipette to suck off the excess liquid at the bottom of the bottle, and use a pipette gun to slowly add 5mL of Insect-C Culture insect cell culture medium to the bottle mouth to cover the tissue block, and continue the original procedure. For subculture, replace the fresh culture medium in time when the color of the culture medium becomes darker; after 6-12 hours of primary culture, when the cells are about to cover the bottom of the bottle, remove the tissue pieces and re-inoculate the removed tissue pieces in a new culture medium cultured in an incubator and replaced with fresh culture medium in time.

8) Discard the culture medium for the cells obtained in step 7), wash with sterile PBS, add 2mL trypsin digestion solution, digest for 50s, shake the culture bottle gently, observe under the microscope, wait until the fibroblasts shrink, become round and float , when the leech body cells have shrunk but not floated, immediately add 5mL Insect-C Culture insect cell culture medium to stop digestion, shake gently, discard the culture medium, add 4mL PBS solution, and gently wash away the residual fibroblasts , add 2mL of digestion solution, after 3 minutes of digestion, discard 1mL of digestion solution, beat the bottom of the bottle from the bottom to make the cells fall off completely, then add 5mL of Insect-CCulture insect cell culture medium to stop digestion, and repeatedly blow and beat the cells to disperse the cell clusters into single cells.

9) Filter the cells obtained in step 8 with eight layers of gauze and inoculate them in a 25mL culture flask containing 5mL of Insect-C Culture insect cell culture medium. The culture temperature is 25°C and the culture time is 3 days.

The medium Insect-CCulture insect cell culture medium formula used in the present invention is:

Inorganic salt:

NaCl1420mg/L, ZnCL2876.54mg _/ L, _MgSO4 · _{7H2O1743.48mg} /L, KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L;

The pH was adjusted to 7.4 with 1.0N KOH, and 15% inactivated special-grade fetal bovine serum for insects was added before use.

The content of penicillin added to the sterile PBS buffer used in the present invention is 600 U/mL, the content of streptomycin is 600 U/mL, and the content of amphotericin B is 15 μg/mL; the size of the shredded tissue block is 1 mm ³ .

The antibiotic Insect-C Culture insect cell medium used in the present invention is added with a content of 600 U/mL of penicillin, 600 U/mL of streptomycin, 15 μg/mL of amphotericin B, and 15% of inactivated Special grade fetal bovine serum for insects.

The beneficial effects of the present invention are: the in vitro culture method for the primary culture of leech cells provided by the present invention provides an optimized insect cell culture medium, which is more rapid and successful compared with other insect cell culture medium. Purified somatic cells of leech were obtained to prepare for further in vitro subculture of leech cells, and a set of in vitro primary culture leech cell system was established. The invention provides accurate and reliable experimental basis for the subsequent in vitro culture of leech cells.

Detailed ways

The present invention can be better understood from the following examples. Then, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention.

The leech adopted in the following examples comes from: Japanese medical leech.

Example 1

A kind of leech living body growth cycle selection method provided by the present invention, concrete steps are as follows

Step 1: Soak 1-year-old, 2-year-old, and 3-year-old living leeches in normal saline for 1 hour to initially remove impurities in the body and ensure the vitality of the somatic cells to the maximum extent, then wash the surface mucous membrane with normal saline until it is clean, and finally wash it three times with distilled water Then soak in 70% alcohol for 5 minutes to remove bacteria on the body surface, and rinse with distilled water several times to remove alcohol;

Step 2: Transfer the leeches after the skin disinfection treatment in step 1 to the ultra-clean workbench, put them into a sterile petri dish filled with sterile PBS, and cut off the leeches with sterile scissors and tweezers Take the head tissue from both ends of the body;

Step 3: transfer the head tissue in step 2 to another sterile petri dish equipped with PBS buffer solution, and soak the verruca;

Step 4: The head tissue in step 3 is cut open with a sterile scalpel, and the tissue in the middle of the epidermis is washed with sterile PBS buffer;

Step 5: Soak the tissues in step 4 in 75% alcohol for 1 min, take them out and rinse with sterile PBS buffer, repeat three times;

Step 6: Place the tissue in step 5 in a sterile petri dish filled with sterile PBS, cut it into small tissue pieces with sterile scissors and tweezers, and soak for 5 minutes;

Step 7: Discard the tissue fragments floating on the upper part of the small tissue pieces in step 6, use 5mL pipette tips to absorb the tissue piece paste, and spread the absorbed paste tissue on the bottom of the bottle with uniform size and even distribution Add 5mL of Insect-C Culture antibiotic-containing insect cell culture medium to a 25mL closed cell culture flask, and culture in a cell culture incubator at 25°C for 60min.

Step 8: Leave the culture bottle after step 7 to adhere to the wall for 60 minutes, use a pipette to suck off the excess liquid at the bottom of the bottle, and use a pipette gun to slowly add 5mL Insect-C Culture insect cell culture medium containing antibiotics to cover the tissue block. Replace the fresh culture medium in time when the color of the culture medium becomes darker; after 6-12 hours of primary culture, when the cells are about to cover the bottom of the bottle, remove the tissue pieces and re-inoculate the removed tissue pieces in a new culture bottle. Culture in an incubator and replace with fresh culture medium in time.

Step 9: Discard the culture medium for the cells obtained in Step 8. After washing with sterile PBS, add 2 mL of trypsin digestion solution, digest for 50 seconds, shake the culture bottle gently, and observe under a microscope. The leech body cells have shrunk but not When it floats, immediately add 5mL insect cell culture medium containing antibiotic Insect-C Culture to stop the digestion, shake gently, discard the culture medium, add 4mL PBS solution, gently wash away the residual fibroblasts, add 2mL digestion solution, and digest After 3 minutes, discard 1mL of the digestion solution, beat the bottom of the bottle from the bottom to completely detach the cells, then add 6mL of Insect-C Culture insect cell culture medium containing antibiotics to stop the digestion, and repeatedly blow the cells to disperse the cell clusters into single cells.

Step 10: Filter the cells obtained in step 9 with eight layers of gauze and inoculate them in a 25mL culture bottle containing 5mL of Insect-C Culture insect cell culture medium. The culture temperature is 25°C and the culture time is 3 days. After that, continue to transfer to another In a 25mL culture flask containing 5mL Insect-C Culture insect cell culture medium, repeat the previous operation.

The medium Insect-CCulture insect cell culture medium formula used in the present invention is:

Inorganic salt:

NaCl1420mg/L _{, ZnCL2876.54mg} /L, _MgSO4 ·7H2O1743.48mg/L,

KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L;

The pH was adjusted to 7.4 with 1.0N KOH, and 15% inactivated special-grade fetal bovine serum for insects was added before use.

The content of penicillin added to the sterile PBS buffer used in the present invention is 600 U/mL, the content of streptomycin is 600 U/mL, and the content of amphotericin B is 15 μg/mL; the size of the shredded tissue block is 1 mm ³ .

The antibiotic Insect-C Culture insect cell medium used in the present invention is added with a content of 600 U/mL of penicillin, 600 U/mL of streptomycin, 15 μg/mL of amphotericin B, and 15% of inactivated Special grade fetal bovine serum for insects.

This example shows that 2-year-old samples can be selected to obtain free cells after 10 hours of primary culture, and the subsequent subculture cells grow adherently, and no free cells appear in 1- and 3-year-old samples after 15 hours of primary culture. The invention can be used as an accurate basis for selecting a living leech.

Example 2

A kind of leech cell primary culture optimized insect cell culture medium provided by the present invention, the specific steps are as follows

Step 1: Soak the living leeches in normal saline for 1 hour to initially remove impurities in the body and ensure the vitality of the somatic cells to the maximum extent, then wash the surface mucous membranes with normal saline until clean, and finally wash them three times with distilled water and place them in 70% alcohol Soak in water for 5 minutes to remove bacteria on the body surface, and rinse with distilled water several times to remove alcohol;

Step 2: Transfer the leech after the skin disinfection treatment in step 1 to the ultra-clean workbench, and put it into a sterile petri dish filled with 600U/mL of PBS of different antibiotics, use sterile scissors and The tweezers cut off both ends of the leech body to get the head tissue;

Step 3: transfer the head tissue in step 2 to another sterile petri dish equipped with PBS buffer solution, and soak the verruca;

Step 4: Cut the head tissue in step 3 with a sterile scalpel, and rinse with sterile PBS buffer after taking the tissue in the middle of the epidermis;

Step 5: Soak the tissue in step 4 in 75% alcohol for 1 min, take it out and rinse it with PBS buffer containing 600U/mL of different antibiotics, repeat three times;

Step 6: Place the tissue in step 5 into a sterile petri dish containing 600U/mL of different antibiotics in PBS, cut it into small pieces with sterile scissors and tweezers, and soak for 5 minutes;

Step 7: Discard the tissue fragments floating on the upper part of the small tissue pieces in step 6, use a 5mL pipette tip to absorb the paste of the tissue pieces, and spread the absorbed paste tissue on the bottom of the bottle with uniform size and evenly distributed on the Add 5mL of Insect-C Culture insect cell culture medium containing 600U/mL of different antibiotics to a 25mL closed cell culture flask, and culture in a cell incubator at 25°C for 60min.

Step 8: Leave the culture bottle after step 7 to adhere to the wall for 60 minutes, use a pipette to suck off the excess liquid at the bottom of the bottle, and use a pipette gun to slowly add 5 mL of Insect-C Culture and SFX containing 600 U/mL of different antibiotics to the bottle mouth -Insect, MGM-450 insect cell culture medium, cover the tissue block, and replace the fresh culture medium in time when the color of the culture medium becomes dark; after 6-12 hours of culture, when the cells are about to cover the bottom of the bottle, remove the tissue block, and transfer The removed tissue pieces were re-inoculated into new culture flasks, cultured in the incubator and replaced with fresh culture medium in time, and the growth of cells was observed.

Step 9: Discard the culture medium for the cells obtained in Step 8. After washing with sterile PBS, add 2 mL of trypsin digestion solution, digest for 50 seconds, shake the culture bottle gently, and observe under a microscope. The leech body cells have shrunk but not floated. Immediately add 5 mL of Insect-C Culture, SFX-Insect, MGM-450 insect cell culture medium containing 600 U/mL of different antibiotics to stop the digestion, discard the culture medium after shaking gently, add 4 mL of PBS solution, and wash gently After removing the remaining fibroblasts, add 2mL of digestive solution, and after 3 minutes of digestion, discard 1mL of the digestive solution, tap the bottom of the bottle from the bottom to make the cells fall off completely, then add 5mL of Insect-Culture and SFX containing 600U/mL of different antibiotics -Insect, MGM-450 insect cell medium to stop digestion, repeatedly pipetting cells to disperse cell clusters into single cells.

Step 10: Filter the cells obtained in step 9 with eight layers of gauze and inoculate them in a 25mL culture bottle containing 5mL of Insect-C Culture insect cell culture medium. The culture temperature is 25°C and the culture time is 3 days. After that, continue to transfer to another In a 25mL culture flask containing 5mL Insect-C Culture insect cell culture medium, repeat the previous operation.

The medium Insect-CCulture insect cell culture medium formula used in the present invention is:

Inorganic salt:

NaCl1420mg/L _{, ZnCL2876.54mg} /L, _MgSO4 ·7H2O1743.48mg/L,

KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L;

The pH was adjusted to 7.4 with 1.0N KOH, and 15% inactivated special-grade fetal bovine serum for insects was added before use.

This example shows that in the 11th hour of primary culture of Insect-Culture insect cell culture medium containing 600U/mL of the three-antibody mixture, a small amount of cells were freed from the periphery of the tissue block, and the shape was round and transparent; the culture medium was not polluted and the cells survived It took a long time, and a large number of cells adhered to the wall in the subsequent subculture experiments; after 15 hours of primary culture of SFX-Insect insect cell culture medium, the culture medium was still clear under the microscope, and the cells showed no signs of growth; MGM-450 insect cell culture After the 15th hour of primary culture, the contamination of the culture medium was light yellow and black spots appeared under the microscope, no free cells appeared around the tissue, and the cells showed signs of temporary growth. Therefore, the optimized and improved Insect-C Culture insect cell culture medium of the present invention can be used as the culture medium required for leech cell culture.

Example 3

Antibiotic class selection provided by the present invention, concrete steps are as follows

Step 1: Soak the living leeches in normal saline for 1 hour to initially remove impurities in the body and ensure the vitality of the somatic cells to the maximum extent, then wash the surface mucous membranes with normal saline until clean, and finally wash them three times with distilled water and place them in 70% alcohol Soak in water for 5 minutes to remove bacteria on the body surface, and rinse with distilled water several times to remove alcohol;

Step 2: Transfer the leech after the skin disinfection treatment in step 1 to the ultra-clean workbench, and put it into the aseptic culture of PBS containing different kinds of antibiotics (penicillin, streptomycin and amphotericin B) In the dish, use sterile scissors and tweezers to cut off the two ends of the verruca to take the head tissue;

Step 3: Transfer the head tissue from step 2 to another sterile petri dish containing different kinds of antibiotics (penicillin, streptomycin and amphotericin B) in PBS buffer, and soak the leech body;

Step 4: The head tissue in step 3 is cut open with a sterile scalpel, and the tissue in the middle of the epidermis is washed with PBS buffer containing different types of antibiotics (penicillin, streptomycin and amphotericin B) ;

Step 5: Soak the tissue in step 4 in 75% alcohol for 1 min, take it out and rinse it with PBS buffer with different concentrations of three antibody mixtures (penicillin, streptomycin and amphotericin B), repeat three times;

Step 6: Place the tissue in step 5 in a sterile petri dish filled with different types of antibiotics (penicillin, streptomycin and amphotericin B) in PBS, and use sterile scissors and tweezers to cut into small tissue pieces, And soak for 5min;

Step 7: Discard the tissue fragments floating on the upper part of the small tissue pieces in step 6, use a 5mL pipette tip to absorb the paste of the tissue pieces, and spread the absorbed paste tissue on the bottom of the bottle with uniform size and evenly distributed on the Add 5mL of Insect-C Culture insect cell culture medium containing different antibiotics (penicillin, streptomycin and amphotericin B) to a 25mL cell-closed culture flask, and culture in a cell culture incubator at 25°C for 60min.

Step 8: Leave the culture bottle after step 7 to adhere to the wall for 60 minutes, suck off the excess liquid at the bottom of the bottle with a pipette, and slowly add 5 mL of different antibiotics (penicillin, streptomycin and amphotericin) to the mouth of the bottle with a pipette gun. B) Insect-CCulture insect cell culture medium, cover the tissue block, and replace the fresh culture medium in time when the color of the culture medium becomes dark; after 6-12 hours of primary culture, when the cells are about to cover the bottom of the bottle, remove the tissue block, and put The removed tissue pieces were re-inoculated into new culture bottles, cultured in the incubator and replaced with fresh culture medium in time.

Step 9: Discard the culture medium from the cells obtained in step 8, wash with PBS containing different types of antibiotics (penicillin, streptomycin and amphotericin B), add 2mL trypsin digestion solution, digest for 50s, shake the culture bottle gently , observed under a microscope, when the leech body cells have shrunk but not floated, immediately add 5 mL of Insect-C Culture insect cell culture medium containing different types of antibiotics (penicillin, streptomycin and amphotericin B) to stop digestion, and shake gently Discard the culture medium, add 4mL PBS solution containing different kinds of antibiotics (penicillin, streptomycin and amphotericin B), gently wash away the residual fibroblasts, add 2mL digestion solution, digest for 2-3.5min, discard Remove 1mL of the digestion solution, beat the bottom of the bottle from the bottom to make the cells fall off completely, then add 5mL of Insect-C Culture insect cell culture medium containing different types of antibiotics (penicillin, streptomycin and amphotericin B) to stop the digestion, and repeatedly blow and beat the cells to make the cells clumps dispersed into individual cells.

Step 10: Filter the cells obtained in Step 9 with eight layers of gauze and inoculate them in 25 mL culture flasks containing 5 mL of Insect-C Culture insect cell culture medium containing different types of antibiotics (penicillin, streptomycin and amphotericin B). After the culture temperature was 25°C and the culture time was 3 days, they were transferred to another 25 mL culture flask containing 5 mL of Insect-C Culture insect cell culture medium, and the previous operation was repeated.

The medium Insect-CCulture insect cell culture medium formula used in the present invention is:

Inorganic salt:

NaCl1420mg/L _{, ZnCL2876.54mg} /L, _MgSO4 ·7H2O1743.48mg/L,

KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L;

The pH was adjusted to 7.4 with 1.0N KOH, and 15% inactivated special-grade fetal bovine serum for insects was added before use.

This example shows that when the antibiotic concentration in the same type of medium is 600U/mL, only in the presence of penicillin, streptomycin and amphotericin B at the same time, the cells around the tissue grow and free cells appear during primary culture. The culture medium is non-polluted, and a large number of cells adhere to the wall during the subculture, and the growth condition is good; Penicillin, streptomycin and amphotericin B alone or in combination of two will all appear pollution.

Embodiment 4 The antibiotic concentration selection provided by the present invention, the specific steps are as follows

Step 1: Soak the living leeches in normal saline for 1 hour to initially remove impurities in the body and ensure the vitality of the somatic cells to the maximum extent, then wash the surface mucous membranes with normal saline until clean, and finally wash them three times with distilled water and place them in 70% alcohol Soak in water for 5 minutes to remove bacteria on the body surface, and rinse with distilled water several times to remove alcohol;

Step 2: Transfer the leech after the skin disinfection treatment in step 1 to the ultra-clean workbench, and put it into a container filled with different concentrations of three-antibody mixture (penicillin, streptomycin and amphotericin B) in PBS. In a sterile petri dish, use sterile scissors and tweezers to cut off both ends of the verruca to take the head tissue;

Step 3: Transfer the head tissue in step 2 to another sterile petri dish filled with different concentrations of three antibody mixtures (penicillin, streptomycin and amphotericin B) in PBS buffer, and soak the verruca;

Step 4: Cut the head tissue in step 3 with a sterile scalpel, and after taking the tissue in the middle of the epidermis, use different concentrations of three antibody mixtures (penicillin, streptomycin and amphotericin B) in PBS buffer liquid flushing;

Step 5: Soak the tissue in step 4 in 75% alcohol for 1 min, take it out and rinse it with PBS buffer with different concentrations of three antibody mixtures (penicillin, streptomycin and amphotericin B), repeat three times;

Step 6: Place the tissue in step 5 in a sterile petri dish containing different concentrations of three-antibody mixture (penicillin, streptomycin and amphotericin B) in PBS, and cut it into small pieces with sterile scissors and tweezers. Tissue blocks, soaked for 5 minutes;

Step 7: Discard the tissue fragments floating on the upper part of the small tissue pieces in step 6, use a 5mL pipette tip to absorb the paste of the tissue pieces, and spread the absorbed paste tissue on the bottom of the bottle with uniform size and evenly distributed on the Add 5mL of three antibody mixtures (penicillin, streptomycin and amphotericin B) of different concentrations to a 25mL cell-closed culture flask, and insect-culture insect cell culture medium at 25°C for 60min.

Step 8: Leave the culture bottle after step 7 to adhere to the wall for 60 minutes, use a pipette to suck off the excess liquid at the bottom of the bottle, and use a pipette gun to slowly add 5 mL of the three-antibody mixture containing different concentrations (penicillin, streptomycin and Amphotericin B) Insect-C Culture insect cell culture medium, cover the tissue pieces, and replace the fresh medium in time when the color of the culture medium becomes dark; after 6-12 hours of primary culture, remove the tissue pieces when the cells are about to cover the bottom of the bottle , and the removed tissue pieces were re-inoculated into new culture flasks, cultured in the incubator and replaced with fresh culture medium in time.

Step 9: Discard the culture medium for the cells obtained in step 8, wash with different concentrations of three-antibody mixture (penicillin, streptomycin and amphotericin B) in PBS, add 2 mL of trypsin digestion solution, digest for 50 seconds, and shake gently In the culture flask, observe under the microscope, when the leech body cells have shrunk but not floated, immediately add 5 mL of the three-antibody mixture (penicillin, streptomycin and amphotericin B) containing different concentrations of Insect-C Culture insect cell culture medium to stop digestion After gently shaking, discard the culture medium, add 4mL of three-antibody mixture (penicillin, streptomycin and amphotericin B) of different concentrations in PBS, wash gently to remove residual fibroblasts, add 2mL of digestion solution, and digest After 2-3.5 minutes, discard 1mL of the digestion solution, tap the bottom of the bottle from the bottom to make the cells fall off completely, and then add 5mL of the three-antibody mixture (penicillin, streptomycin and amphotericin B) containing different concentrations. Insect-C Culture Insect Cell Culture The base terminates the digestion, and the cells are repeatedly pipetted to disperse the cell clusters into single cells.

Step 10: Filter the cells obtained in step 9 with eight layers of gauze and inoculate them in 25 mL of Insect-C Culture insect cell culture medium containing different concentrations of three antibody mixtures (penicillin, streptomycin and amphotericin B) 5 mL In the bottle, the culture temperature is 25°C, and after 3 days of culture time, continue to transfer to another 25mL culture bottle containing 5mL Insect-C Culture insect cell culture medium, and repeat the previous operation.

The medium Insect-CCulture insect cell culture medium formula used in the present invention is:

Inorganic salt:

NaCl1420mg/L _{, ZnCL2876.54mg} /L, _MgSO4 ·7H2O1743.48mg/L,

KCl2080mg/L, _KH2PO4 · _2H2O1000mg /L;

Amino acids:

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine di Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L;

Vitamins:

D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L;

other:

Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L;

The pH was adjusted to 7.4 with 1.0N KOH, and 15% inactivated special-grade fetal bovine serum for insects was added before use.

This example shows that in the same type of medium, when the content of penicillin is 600U/mL, the content of streptomycin is 600U/mL, and the content of amphotericin B is 15μg/mL, the cell growth condition is the best, and there will be no pollution At the same time, the cell survival time is long; when the penicillin content is 200U/mL, the streptomycin content is 200U/mL, and the amphotericin B content is 5μg/mL, the culture medium becomes turbid after primary culture for 10 hours, and the surrounding tissues The cells are free, and the cells do not grow; when the content of penicillin is 400U/mL, the content of streptomycin is 400U/mL, and the content of amphotericin B is 5μg/mL, the culture solution is light yellow after primary culture for 15 hours, and There are black granular substances around the tissue, no cells are free, and the cells do not grow; when the content of penicillin is 800U/mL, the content of streptomycin is 800U/mL, and the content of amphotericin B is 20μg/mL, the primary culture is 15h After 20 hours of culture, the culture medium was clear, and there were no free cells around the tissue, and the cells did not grow.

Claims (10)

1. a kind of leech cell injuring model base, which is characterized in that the culture medium prescription is as follows：

Inorganic salts：

NaCl1420mg/L, ZnCL₂ 876.54mg/L, MgSO₄·7H₂O1743.48mg/L, KCl2080mg/L, KH2PO₄· 2H₂O 1000mg/L；

Amino acid：

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, altheine 400mg/L, L-Aspartic acid 350mg/L, L-cysteine dihydrochloride 160mg/L, Pidolidone 3500mg/L, L-Histidine hydrochloride 2000mg/L, l-Isoleucine 200mg/L, L-Leu 150mg/L, L lysine HCL 850mg/L, l-methionine 100mg/L, L-phenylalanine 100mg/L, L-PROLINE 500mg/L, L-threonine 200mg/L, L-Trp 150mg/L, L- Tyrosine phosphatase disodium hydrogen 580mg/L, Valine 200mg/L ,-alanine 400mg/L, l-Alanine 225mg/L；

Vitamin：

D-Biotin 0.15mg/L, D-VB5 calcium 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, hydrochloric acid pyridoxamine 0.15mg/L, riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/ L；

It is other：

Sucrose 2000mg/L, D-Glucose 1500mg/L, D-Fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L MALIC ACID 90mg/L；

PH is adjusted with 1.0N KOH to 7.4.

2. a kind of leech cell injuring model method, is cultivated using culture medium described in claim 1, which is characterized in that packet Include following steps：

1）Leech salivary organization block after shredding is inoculated in cell closing culture bottle, adds in Insect-CCulture elder brothers Worm cell culture medium, quiescent culture 50-70min in 25-28 DEG C of cell incubator；

2）Cooperation tissue block absorption method is separately cultured；

3）The leech body cell for obtaining purifying is digested by pancreatin.

A kind of 3. leech cell injuring model method according to claim 2, which is characterized in that step 1）Middle leech saliva Glandular tissue block is obtained by following processing method：

A, water intaking leech live body impregnates 1h in physiological saline, impurity and ensures the work of its body cell to greatest extent in preliminary removing body Power, then its surface mucosa is cleaned to clean with physiological saline, it is finally cleaned three times with distilled water and is placed in 70% alcohol and impregnated 5min removes body surface bacterium, is repeated as many times with distilled water and washes alcohol；

B, the leech after epidermis is disinfected will be carried out in step a to be transferred in super clear workbench, and put it into and be contained with nothing In the sterile petri dish of bacterium PBS, cut off leech body both ends with sterile scissors and tweezers and take head tissue；

C, the head tissue in step b is transferred in another sterile petri dish equipped with PBS buffer solution, dipped leech body；It will Head tissue splits head tissue with aseptic operation knife, and is rinsed after the tissue among epidermis is taken with sterile PBS buffer；

D, the tissue in step c is placed in 75% alcohol after impregnating 1min, is rinsed, repeated with sterile PBS buffer after taking-up Three times；

E, the tissue in step d is placed in the sterile petri dish for being contained with sterile PBS, is shredded into sterile scissors with tweezers small Tissue block, and impregnate 5min；The tissue debris for swimming in top is discarded, the leech salivary organization block after being shredded shreds The size of tissue block is 1mm afterwards³。

A kind of 4. leech cell injuring model method according to claim 2, which is characterized in that step 1）Middle cultivation temperature It is 25 DEG C, incubation time 60min.

A kind of 5. leech cell injuring model method according to claim 2, which is characterized in that Insect- used CCulture insect cell medium based formulas is as follows：

Inorganic salts：

NaCl1420mg/L, ZnCL₂ 876.54mg/L, MgSO₄·7H₂O1743.48mg/L, KCl2080mg/L, KH2PO₄· 2H₂O 1000mg/L；

Amino acid：

DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, altheine 400mg/L, L-Aspartic acid 350mg/L, L-cysteine dihydrochloride 160mg/L, Pidolidone 3500mg/L, L-Histidine hydrochloride 2000mg/L, l-Isoleucine 200mg/L, L-Leu 150mg/L, L lysine HCL 850mg/L, l-methionine 100mg/L, L-phenylalanine 100mg/L, L-PROLINE 500mg/L, L-threonine 200mg/L, L-Trp 150mg/L, L- Tyrosine phosphatase disodium hydrogen 580mg/L, Valine 200mg/L ,-alanine 400mg/L, l-Alanine 225mg/L；

Vitamin：

D-Biotin 0.15mg/L, D-VB5 calcium 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, hydrochloric acid pyridoxamine 0.15mg/L, riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/ L；

It is other：

Sucrose 2000mg/L, D-Glucose 1500mg/L, D-Fructose 500mg/L, maltose 600mg/L, α-ketoglutaric acid 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L MALIC ACID 90mg/L；

PH is adjusted with 1.0N KOH to 7.4, uses the special superfine fetal calf serum of preceding 15% insect for adding in inactivation.

A kind of 6. leech cell injuring model method according to claim 2, which is characterized in that step 2）Described in organize The operating process of block absorption method is：Take out step 1）Culture bottle, with suction pipe absorb bottom of bottle surplus liquid, using liquid-transfering gun in bottle Mouth is slowly added to 4-6mL Insect-CCulture insect cell mediums, and be annihilated tissue block, continues original cuiture, culture solution Fresh medium is replaced when darkening in time；After cultivating 6-12h, when cell will be paved with bottom of bottle, tissue block is removed, and will The tissue block of removal is reinoculated in new culture bottle, is cultivated in incubator and is replaced fresh medium in time.

A kind of 7. leech cell injuring model method according to claim 3, which is characterized in that the sterile PBS bufferings The content that penicillin is added in liquid is 600U/mL, and the content of streptomysin is 600U/mL, and the content of amphotericin B is 15 μ g/mL.

A kind of 8. leech cell injuring model method according to claim 6, which is characterized in that step 2）It was separately cultured Insect-CCulture insect cell mediums in journey separately add in the content of penicillin as 600U/mL, the content of streptomysin For 600U/mL, the content of amphotericin B is 15 μ g/mL, the special superfine fetal calf serum of 15% insect of inactivation.

A kind of 9. leech cell injuring model method according to claim 2, which is characterized in that step 3）The pancreatin disappears Change method is：By step 2）Obtained cell discards culture solution, after sterile PBS cleanings, adds in 2mL pancreatin digestive juices, digestion 40 ~60s, gently shakes culture bottle, micro- Microscopic observation, the existing contraction of leech body cell but when not floating, adds in 3-6mL immediately Insect-CCulture insect cell mediums terminate digestion, and culture solution is discarded after gently shaking, and add in 4mL PBS liquid, gently It rinses except after remaining fibroblast, 2mL pancreatin digestive juices are added in, after digesting 2~4min, 1mL digestive juices is discarded, from bottom Patting bottom of bottle completely falls off cell, adds 3-6mLInsect-CCulture insect cell mediums and terminates digestion, repeatedly Cell is blown and beaten, cell mass is made to be dispersed into individual cells, conducive to culture；It is inoculated in after eight layers of filtered through gauze of obtained cell containing 3- In the 25mL culture bottles of 6mL Insect-CCulture insect cell mediums, 25-28 DEG C of cultivation temperature, incubation time 2- 4d。

10. a kind of leech cell injuring model method according to claim 9, which is characterized in that first time pancreatin digests Optimal time is 50s, and second is 3min, and Insect-CCulture insect cell mediums optimal addn is 5mL, is trained 25 DEG C of temperature is supported, after incubation time 3d, continues to transfer in another insect cell mediums of Insect-CCulture containing 5mL In 25mL culture bottles, last action is repeated.