Preparation method of low endotoxin silkworm blood lymph fluid inhibiting blackening reaction

Abstract

The application discloses a preparation method of silkworm blood lymph fluid with inhibited melanization reaction and low endotoxin, which comprises the following steps: low endotoxin silkworm variety selection, silkworm body disinfection and impurity removal, silkworm body low-temperature anesthesia, blood lymph fluid collection, anti-melanization treatment, phenol oxidase inactivation treatment, sediment removal and anaerobic preservation, so as to finally obtain the silkworm blood lymph fluid with inhibited melanization reaction and low endotoxin. The silkworm blood lymph fluid extracted by the prior art contains a large amount of endotoxin, which is extremely harmful to in-vitro cell culture and is one of the bottlenecks for replacing fetal bovine serum with insect blood lymph fluid. The application can reduce the endotoxin concentration in the silkworm lymph fluid. In view of the problems caused by quinone derivatives generated by melanization reaction, the application can reduce the phenol oxidase proenzyme activity in the silkworm blood lymph fluid, long-term inhibit the melanization reaction of the blood lymph fluid, reduce the accumulation of toxic quinones, and make the prepared silkworm blood lymph fluid capable of being used for long-term in-vitro cell culture.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically, to a method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content. Background Technology

[0002] Cell culture is an experimental technique that simulates the conditions of cell-derived and tissue-based environments by culturing cells in an in vitro container. It is an indispensable tool in various research fields such as biology, biochemistry, and medicine. Maintaining in vitro cell culture requires not only providing suitable culture medium type, air composition, and temperature, but also adding serum to the cell growth medium to supplement the comprehensive nutrients needed for cell growth, including hormones, growth factors, binding proteins, attachment and diffusion factors, etc. Currently, the main source of serum on the market is fetal bovine serum (FBS). Obtaining FBS requires removing the fetus from the womb of a pregnant cow awaiting slaughter and extracting blood from the fetus through cardiac puncture under sterile conditions. To increase the blood volume, special instruments are usually used to stimulate the heartbeat. It is reported that the annual global production of FBS exceeds 500,000 liters, which means that more than 1,000,000 fetuses are slaughtered every year, raising concerns about animal welfare.

[0003] During the metamorphosis of the silkworm pupal stage, the intensity of cellular life activities is more vigorous than in the larval stage, involving the secretion of various hormones and the synthesis of growth factors. Numerous studies have shown that the abundant active proteins in silkworm pupal hemolymph can prolong cell lifespan and inhibit apoptosis, meaning that silkworm pupal hemolymph could be a good substitute for fetal bovine serum. However, due to the presence of numerous Gram-negative bacteria in the silkworm's environment, when bacteria invade the silkworm's body, they activate the silkworm's phenoloxidase proenzyme system, causing a melanization reaction and catalyzing the formation of large amounts of cytotoxic quinone derivatives. The lysed bacteria then release endotoxins into the hemolymph. Studies have shown that hemolymph without heat-induced phenoloxidase inactivation cannot induce cell growth and proliferation; while adding vitamin C to the culture medium has a poor inhibitory effect on phenoloxidase activity. As the culture time increases, the vitamin C concentration decreases, and toxic quinones accumulate rapidly, making silkworm hemolymph extracted using current techniques unsuitable for direct long-term in vitro cell culture. Therefore, there is an urgent need to find a silkworm hemolymph extraction method that can inhibit the melanization reaction and has low endotoxin content. Relevant research has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing silkworm hemolymph with low endotoxin content and inhibiting melanization reaction. Existing methods for extracting silkworm hemolymph contain high levels of endotoxins, which are extremely detrimental to in vitro cell culture and are a bottleneck restricting the use of insect hemolymph to replace fetal bovine serum. This invention can reduce the endotoxin concentration in silkworm hemolymph. Furthermore, addressing the problems caused by quinone derivatives produced during the melanization reaction, this invention can reduce the activity of prophenoloxidase in silkworm hemolymph, thereby inhibiting the melanization reaction in the hemolymph for a long period, reducing the accumulation of toxic quinones, and enabling the prepared silkworm hemolymph to be used for long-term in vitro cell culture.

[0005] This invention is achieved through the following technical solution: a method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content, comprising the following steps:

[0006] 1) Selection of low endotoxin silkworm varieties: Use sterile silkworms and cultivate them in a sterile environment until the pupal stage;

[0007] 2) Disinfection and removal of impurities from silkworms: On a sterile operating table, take sterile silkworms that are 2-3 days old in the pupal stage, disinfect the pupae with alcohol, and then rinse them.

[0008] 3) Low-temperature anesthesia of silkworms: After step 2), remove the moisture from the surface of the silkworm pupae, and then place the silkworm pupae on ice for low-temperature anesthesia for 1 to 5 minutes, preferably 3 minutes;

[0009] 4) Collection of hemolymph: Use a sterile scalpel to cut the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph.

[0010] 5) Anti-melanization treatment: Add glutamine to the obtained hemolymph at a concentration of 30-60 mmol / L, preferably at a concentration of 40 mmol / L;

[0011] 6) Phenol oxidase inactivation treatment: After being fully dissolved in step 5), incubate in a water bath at 60°C for 30-45 minutes;

[0012] 7) Removal of precipitate: After step 6), cool to 2-4℃ (preferably 4℃), centrifuge at 1000×g for 10min, take the supernatant and filter it 1-2 times with a filter membrane with a pore size of 0.1-0.2μm, preferably take the supernatant and filter it 2 times with a filter membrane with a pore size of 0.2μm.

[0013] 8) Anaerobic preservation treatment: After step 7), silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin is obtained, and then vacuum sealed packaging is used.

[0014] The obtained silkworm hemolymph fluid (vacuum-sealed packaging) needs to be stored and transported frozen, with a shelf life of 12 months. It should be thawed gradually before use and should not be repeatedly frozen and thawed.

[0015] To further improve the preparation method of silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content as described in this invention, the following setup is adopted: the sterile silkworms are selected from those fed with sterile artificial feed.

[0016] To further improve the preparation method of silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content as described in this invention, the following setting is specifically adopted: the disinfection of the pupa with alcohol is specifically: wiping the pupa with a cotton ball containing 75% alcohol by volume.

[0017] To further improve the preparation method of silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content as described in this invention, the following settings are adopted: In step 2), PBS buffer or sterile water is used for rinsing.

[0018] To further improve the preparation method of silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content as described in this invention, the following setting is adopted: the removal of moisture from the surface of the silkworm pupa specifically involves wiping the silkworm pupa dry with sterile absorbent paper.

[0019] To further improve the preparation method of silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content as described in this invention, the following settings are adopted: the water bath time in step 6) is 30 minutes or 45 minutes.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. During the silkworm pupal stage, the cells in the tissues exhibit vigorous life activities, and their hemolymph contains abundant lipids, proteins, hormones, and growth factors. Using this invention, the cell culture medium prepared from sterile silkworms can not only provide sufficient nutrients for cells in vitro, but also reduce endotoxins in the hemolymph, inhibit melanization, and increase the growth rate of cells in vitro.

[0022] 2. The presence of a large number of Gram-negative bacteria in the silkworm's living environment leads to a high level of endotoxins in the silkworm hemolymph extracted by existing methods. This is extremely detrimental to in vitro cell culture and is one of the bottlenecks restricting the use of insect hemolymph to replace fetal bovine serum. This invention can reduce the concentration of endotoxins in silkworm hemolymph.

[0023] 3. The prophenoloxidase system in silkworm hemolymph activates a melanization reaction upon contact with antigens, catalyzing the release of large amounts of cytotoxic quinone derivatives. Existing methods use the addition of vitamin C to inhibit the melanization reaction, but with the degradation and metabolism of vitamin C, toxic quinones accumulate rapidly, leading to cell failure and even death. This invention can reduce the prophenoloxidase activity in silkworm hemolymph, inhibit the melanization reaction in hemolymph in the long term, reduce the accumulation of toxic quinones, and enable the prepared silkworm hemolymph to be used for long-term in vitro cell culture.

[0024] 4. This invention aims to reduce the main cytotoxic substances in the hemolymph of silkworms. Based on the endotoxin production pattern, sterile silkworms raised with sterile artificial feed are selected, which reduces bacterial contamination of the hemolymph from the source. Therefore, the content of endotoxins produced by bacteria in the hemolymph prepared by this invention is extremely low. This method is simple to operate, reduces the complex endotoxin removal process, and is low in cost.

[0025] 5. This invention aims to reduce the main cytotoxic substances in the hemolymph of silkworms. Based on the principle of insect hemolymph melanization reaction, it completely inactivates phenol oxidase by water bath heating, and then uses the addition of glutamine to inhibit the hemolymph melanization reaction of silkworms for a long time, so that the quinone derivatives in the hemolymph of silkworms are kept at a low concentration for a long time, which is more beneficial to the long-term culture of cells.

[0026] 6. To improve the in vitro cell growth rate of hemolymph culture, this invention selects silkworm pupae in their 2nd-3rd day pupal stage as hemolymph donors, taking advantage of the vigorous life activities during the silkworm pupal stage. Hemolymph at this stage is rich in nutrients and growth factors, resulting in better cell growth promotion. Compared with other hemolymph extraction methods, the pupal stage of the silkworms used in this invention is longer than that of the 5th instar silkworm, and the pupae do not need to feed, allowing for low-temperature storage and enabling hemolymph extraction much faster than with 5th instar silkworms. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0029] The purpose of this invention is to provide a method for preparing silkworm hemolymph fluid that can replace fetal bovine serum, inhibit melanization, and has low endotoxin levels. During the silkworm pupal stage, cellular life activities are vigorous, and its hemolymph fluid is rich in lipids, proteins, hormones, and growth factors. Cell culture medium prepared from sterile silkworms can not only provide sufficient nutrients for in vitro cells but also reduce endotoxin levels in the hemolymph, inhibit melanization, and increase the growth rate of in vitro cells.

[0030] Example 1:

[0031] A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content, comprising the following steps:

[0032] (1) Selection of low endotoxin silkworm varieties

[0033] Sterile silkworms fed with sterile artificial feed were selected and cultured in a sterile environment until the pupal stage.

[0034] (2) Disinfection of silkworms and removal of impurities

[0035] On a sterile operating table, take 400 sterile silkworms that are 2-3 days old in the pupal stage, wipe the pupae with cotton balls containing 75% alcohol by volume, and then rinse with PBS buffer.

[0036] (3) Low-temperature anesthesia of silkworms

[0037] Use sterile absorbent paper to dry the silkworm pupae, then place them on ice for 3 minutes to achieve low-temperature anesthesia.

[0038] (4) Collection of hemolymph

[0039] Use a sterile scalpel to cut open the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph.

[0040] (5) Anti-blackening treatment

[0041] Add glutamine to the obtained hemolymph at a concentration of 40 mmol / L;

[0042] (6) Phenol oxidase inactivation treatment

[0043] After fully dissolving, bathe in a 60°C water bath for 30 minutes;

[0044] (7) Deprecipitation treatment

[0045] After cooling to 4℃, centrifuge at 1000×g for 10min, and filter the supernatant twice through a filter membrane with a pore size of 0.2μm.

[0046] (8) Anaerobic preservation treatment

[0047] After vacuum sealing 100 mL of the obtained filtrate, a vacuum-packed silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content is obtained.

[0048] The vacuum-packed silkworm hemolymph fluid obtained needs to be stored and transported frozen, with a shelf life of 12 months. It should be thawed gradually before use and should not be repeatedly frozen and thawed.

[0049] Example 2:

[0050] A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content, comprising the following steps:

[0051] (1) Selection of low endotoxin silkworm varieties

[0052] Sterile silkworms fed with sterile artificial feed were selected and cultured in a sterile environment until the pupal stage.

[0053] (2) Disinfection of silkworms and removal of impurities

[0054] On a sterile operating table, take 400 sterile silkworms that are 2-3 days old in the pupal stage, wipe the pupae with cotton balls containing 75% alcohol by volume, and then rinse with sterile water.

[0055] (3) Low-temperature anesthesia of silkworms

[0056] Use sterile absorbent paper to dry the silkworm pupae, then place them on ice for 3 minutes to achieve low-temperature anesthesia.

[0057] (4) Collection of hemolymph

[0058] Use a sterile scalpel to cut open the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph.

[0059] (5) Anti-blackening treatment

[0060] Add glutamine to the obtained hemolymph at a concentration of 40 mmol / L;

[0061] (6) Phenol oxidase inactivation treatment

[0062] After fully dissolving, bathe in a 60°C water bath for 30 minutes;

[0063] (7) Deprecipitation treatment

[0064] After cooling to 4℃, centrifuge at 1000×g for 10min, and filter the supernatant twice through a filter membrane with a pore size of 0.2μm.

[0065] (8) Anaerobic preservation treatment

[0066] After vacuum sealing 100 mL of the obtained filtrate, a vacuum-packed silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content is obtained.

[0067] The vacuum-packed silkworm hemolymph fluid obtained needs to be stored and transported frozen, with a shelf life of 12 months. It should be thawed gradually before use and should not be repeatedly frozen and thawed.

[0068] Example 3:

[0069] A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content, comprising the following steps:

[0070] (1) Selection of low endotoxin silkworm varieties

[0071] Sterile silkworms fed with sterile artificial feed were selected and cultured in a sterile environment until the pupal stage.

[0072] (2) Disinfection of silkworms and removal of impurities

[0073] On a sterile operating table, take 400 sterile silkworms that are 2-3 days old in the pupal stage, wipe the pupae with cotton balls containing 75% alcohol by volume, and then rinse with PBS buffer.

[0074] (3) Low-temperature anesthesia of silkworms

[0075] Use sterile absorbent paper to dry the silkworm pupae, then place them on ice for 3 minutes to achieve low-temperature anesthesia.

[0076] (4) Collection of hemolymph

[0077] Use a sterile scalpel to cut open the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph.

[0078] (5) Anti-blackening treatment

[0079] Add glutamine to the obtained hemolymph at a concentration of 40 mmol / L;

[0080] (6) Phenol oxidase inactivation treatment

[0081] After fully dissolving, bathe in a 60°C water bath for 45 minutes;

[0082] (7) Deprecipitation treatment

[0083] After cooling to 4℃, centrifuge at 1000×g for 10min, and filter the supernatant twice through a filter membrane with a pore size of 0.2μm.

[0084] (8) Anaerobic preservation treatment

[0085] After vacuum sealing 100 mL of the obtained filtrate, a vacuum-packed silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content is obtained.

[0086] The vacuum-packed silkworm hemolymph fluid obtained needs to be stored and transported frozen, with a shelf life of 12 months. It should be thawed gradually before use and should not be repeatedly frozen and thawed.

[0087] Example 4:

[0088] A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content, comprising the following steps:

[0089] (1) Selection of low endotoxin silkworm varieties

[0090] Sterile silkworms fed with sterile artificial feed were selected and cultured in a sterile environment until the pupal stage.

[0091] (2) Disinfection of silkworms and removal of impurities

[0092] On a sterile operating table, take 400 sterile silkworms that are 2-3 days old in the pupal stage, wipe the pupae with cotton balls containing 75% alcohol by volume, and then rinse with PBS buffer or sterile water.

[0093] (3) Low-temperature anesthesia of silkworms

[0094] Use sterile absorbent paper to dry the silkworm pupae, then place them on ice for low-temperature anesthesia for 1 to 5 minutes, preferably 3 minutes.

[0095] (4) Collection of hemolymph

[0096] Use a sterile scalpel to cut open the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph.

[0097] (5) Anti-blackening treatment

[0098] Glutamine was added to the obtained hemolymph at a concentration of 30–60 mmol / L, preferably at a concentration of 40 mmol / L.

[0099] (6) Phenol oxidase inactivation treatment

[0100] After fully dissolving, bathe in a 60°C water bath for 30–45 minutes;

[0101] (7) Deprecipitation treatment

[0102] After cooling to 2-4℃ (preferably 4℃), centrifuge at 1000×g for 10min, and filter the supernatant 1-2 times with a filter membrane with a pore size of 0.1-0.2μm, preferably filter the supernatant 2 times with a filter membrane with a pore size of 0.2μm.

[0103] (8) Anaerobic preservation treatment

[0104] After vacuum sealing 100 mL of the obtained filtrate, a vacuum-packed silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content is obtained.

[0105] The vacuum-packed silkworm hemolymph fluid obtained needs to be stored and transported frozen, with a shelf life of 12 months. It should be thawed gradually before use and should not be repeatedly frozen and thawed.

[0106] Table 1 shows the experimental data on the treatment conditions and melanization time of sterile silkworm pupa hemolymph.

[0107]

[0108] Note: Blackening time of hemolymph: The optical density of the sample was measured at 460 nm using a UV120 spectrophotometer. The reaction time recorded when the absorbance was equal to that of the control group was the blackening time.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a low endotoxin silkworm (Bombyx mori) hemolymph solution that inhibits melanization, characterized by comprising the steps of: Includes the following steps: ​ 1) Selection of low endotoxin silkworm varieties: Use sterile silkworms and cultivate them in a sterile environment until the pupal stage; 2) Disinfection and impurity removal of silkworms: On a sterile operating table, take sterile silkworms that are 2-3 days old in the pupal stage, disinfect the pupae with alcohol, and then rinse them; 3) Low-temperature anesthesia of silkworms: After step 2), remove the moisture from the surface of the silkworm pupae, and then place the silkworm pupae on ice for low-temperature anesthesia for 1 to 5 minutes; 4) Collection of hemolymph: Use a sterile scalpel to cut the membrane connecting the thoracic and abdominal segments of the silkworm pupa, creating an open wound on the back of the pupa. While gently squeezing the head and tail of the pupa with your fingers, use a Pasteur tube to collect the overflowing hemolymph. 5) Anti-melanization treatment: Add glutamine to the obtained hemolymph at a concentration of 30-60 mmol / L; 6) Phenol oxidase inactivation treatment: After complete dissolution in step 5), incubate in a 60°C water bath for 30-45 minutes; 7) Removal of precipitate: After step 6), cool to 2~4℃, centrifuge at 1000×g for 5~15min, and take the supernatant and filter it 1~2 times with a filter membrane with a pore size of 0.1~0.2µm; 8) Anaerobic preservation treatment: After step 7), silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin is obtained, and then vacuum sealed packaging is used.

2. The method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 1, characterized in that: The sterile silkworms are selected from those fed with sterile artificial feed.

3. A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 1 or 2, characterized in that: The specific method of disinfecting the pupae with alcohol is as follows: wipe the pupae with a cotton ball containing 75% alcohol by volume.

4. A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 1 or 2, characterized in that: In step 2), PBS buffer or sterile water is used for rinsing.

5. A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 1 or 2, characterized in that: The specific method for removing moisture from the surface of silkworm pupae is to wipe the silkworm pupae dry with sterile absorbent paper.

6. The method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 3, characterized in that: The water bath time in step 6) is 30 to 45 minutes.

7. The method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 4, characterized in that: The water bath time in step 6) is 30 minutes or 45 minutes.

8. The method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 5, characterized in that: The water bath time in step 6) is 30 minutes or 45 minutes.

9. A method for preparing silkworm hemolymph fluid with inhibited melanization reaction and low endotoxin content according to claim 1 or 2, characterized in that: The water bath time in step 6) is 30 minutes or 45 minutes.

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