CN107129962A - A kind of primary culture method of Hirudo japonica salivary gland cell - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a primary culture method of Japanese hirudo salivary gland cells, inoculating the shredded tissue pieces in a 25cm closed cell culture bottle, adding 5mL SFX- ^INSCET insect cell culture fluid , placed in a biochemical incubator at 29°C for static culture. The present invention will greatly increase the output of natural hirudin; a large amount of salivary gland cells can be obtained after a very small amount of wild Japanese leech salivary gland cells are cultured in vitro, and a large amount of natural hirudin can be separated and extracted, which is beneficial to the protection of wild Japanese leech populations , to achieve the goal of both in-depth development and utilization and vigorous protection of animal germplasm resources. The invention is fast, effective and highly reproducible, perfects the culture of leech cells, and provides reliable test data and basis for the research on the primary culture of leech cells.

Description

A kind of primary culture method of Japanese medical hirudo salivary gland cells

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a method for primary culture of Japanese hirudo salivary gland cells.

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. Japanese medical leeches (Hirudo nipponia) belong to the phylum Annelids, which include the subphylum Annulus, class Hirudo, order Hirudo nipponia, family Hirudo, and genus Hirudo. 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. They live in rivers, wet land, and fields. Some live by feeding on the blood of mammals such as fish, frogs, water birds, cattle, horses, and humans, and some feed on earthworms, shrimps, snails, and mussels. The body fluids, tissues and even the whole body of invertebrates can survive. The leech animals that suck the blood of mammals for a living, the salivary gland secretions contain hirudin and other biologically active substances, and have broad application prospects in medicine are collectively called medical leeches in the world. Studies have found that medical leech salivary gland secretions contain a variety of active substances: hirudin, leech hyaluronidase, antistacin, anesthetics, vasodilators, prostaglandins, etc. As one of the important sources of natural hirudin, Japanese medicine leech has the effects of lowering blood fat, protecting cardiovascular and cerebrovascular, anticoagulant and antitumor, and is known as "soft gold" and has important research significance. At present, there are two main ways to obtain hirudin at home and abroad. One is to directly separate and extract natural hirudin from the salivary glands of leeches, or stimulate the leeches to secrete salivary glands with arginine, and then use special equipment to collect them. Saliva, the natural hirudin obtained by this method has a very small yield and high cost; the other is to synthesize recombinant hirudin by genetic engineering. Although it is clinically different from natural hirudin, it is resistant to The coagulation activity is low, the specificity of anticoagulant effect will be affected by the concentration of recombinant hirudin, and the anticoagulant effect is correspondingly high with high purity. Therefore, there is a great demand for natural hirudin clinically.

To sum up, the problems existing in the prior art are: the current method for obtaining hirudin has the disadvantages of extremely small hirudin yield, high cost and low anticoagulant activity.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a method for primary culture of Japanese leech salivary gland cells.

The present invention is achieved in this way, a primary culture method of Japanese Hirudo salivary gland cells, the primary culture method of the Japanese Hirudo salivary gland cells comprises inoculating the shredded tissue pieces in a ^25cm closed cell culture flask , add 5 mL of SFX-INSCET insect cell culture medium, and place in a biochemical incubator for static culture at 29°C.

Further, before the inoculation:

Step 1, use water to remove the sticky secretions on the body surface of Japanese medical leeches. After cleaning up, soak in 0.3% potassium permanganate solution first, and then soak in alcohol solution to disinfect Japanese medical leeches;

Step 2, in the ultra-clean workbench, transfer the Japanese medical leech sterilized on the body surface to a disposable sterile petri dish filled with PBS buffer, immerse the Japanese medical leech in the PBS buffer, and use high temperature and high pressure sterilized Handle the surgical scissors and tweezers to remove the front head of the Japanese medical leech;

Step 3, transfer the head to another disposable sterile petri dish filled with PBS buffer, submerge it in PBS buffer, cut the front head of Japanese medical leeches along the middle of the oropharynx, and remove the pharyngeal wall Inner epidermis and outer skin, rinsed with PBS;

Step 4, after soaking the salivary glands in the alcohol solution, quickly transfer them to three vessels each equipped with PBS buffer solution (containing 800 U/mL of penicillin/streptomycin/amphotericin mixed solution) for soaking, And in the third petri dish filled with PBS buffer solution (containing 800 U/mL penicillin/streptomycin/amphotericin mixed solution), cut it into tissue pieces.

Further, in the first step, soak in 0.3% potassium permanganate solution for 10 minutes, and then soak in 10% alcohol solution for 10 minutes to disinfect the leeches.

Further, after the salivary gland is placed in 75% ethanol solution and soaked for 5-7S in the described step 4, it is transferred to three each containing 1mL PBS buffer (containing 800U/mL of penicillin/streptomycin/amphotericin) Soak in each vessel for 5 minutes.

Further, in step 4, cut into tissue pieces with a size of 0.5 mm ³ -1 mm ³ .

Another object of the present invention is to provide a Japanese leech cultured by using the primary culture method of the Japanese leech salivary gland cells.

The advantages and positive effects of the present invention are: to carry out the selection and screening test of the primary culture method of Japanese medical leech salivary gland cells, the type of medium, the type and concentration of antibiotics, etc., to establish the Japanese medical leech primary cell culture system, and to realize the medical leech salivary gland cells Primary culture for further subculture. If the in vitro culture of Hirudo salivary gland cells can be realized, and can be subcultured, the way to obtain natural hirudin - directly extracting and separating from Hirudo salivary gland cells cultured in vitro, will always maintain a high level of anticoagulant activity, and greatly Improve the output of natural hirudin, which is significantly higher than the existing methods of directly separating and extracting natural hirudin from the salivary glands of leeches and stimulating the leeches with arginine to secrete their salivary glands and then collecting saliva. A very small amount of wild Japanese leech salivary gland cells can be cultured in vitro to obtain a large number of salivary gland cells, and a large amount of natural hirudin can be isolated and extracted, which is conducive to the protection of wild Japanese leech populations, achieving both deep development and utilization and vigorous protection of animal species quality resource goals. The primary culture of Japanese hirudo salivary gland cells is carried out by adopting the invention, and better culture effect is obtained.

The invention is fast, effective and highly reproducible, perfects the culture of leech cells, and provides reliable test data and basis for the research on the primary culture of leech cells.

Description of drawings

Fig. 1 is a flow chart of the primary culture method of Japanese hirudo salivary gland cells provided by the embodiment of the present invention.

detailed description

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The application principle of the present invention will be described in detail below in conjunction with the accompanying drawings.

The present invention selects the most suitable culture medium SFX-INSCET insect cell culture medium from RPMI1640 culture medium, SFX-INSCET insect cell culture medium and M199 culture medium; Ampicillin, gentamicin sulfate, penicillin/streptomycin/amphotericin B mixed solution, and ampicillin were selected to select the most suitable antibiotic penicillin/streptomycin/amphotericin B mixed solution; Streptomycin/amphotericin B concentration gradient test was carried out, and the most suitable concentration of 800U/mL was screened out; thus the culture system most suitable for the primary culture of Japanese medical leech cells was preliminarily screened out.

As shown in Figure 1, the primary culture method of Japanese medicine leech salivary gland cells provided by the embodiment of the present invention comprises the following steps:

S101: Remove the viscous secretion on the body surface of Japanese medical leech with water. After cleaning up, soak in 0.3% potassium permanganate solution for 10 minutes, and then soak in 10% alcohol solution for 10 minutes to disinfect Japanese medical leeches;

S102: In the ultra-clean workbench, transfer the Japanese leeches that have been sterilized on the body surface to a disposable sterile petri dish filled with PBS buffer. Use surgical scissors and tweezers to remove the front head of the Japanese medical leech;

S103: Transfer the head to another disposable sterile petri dish filled with PBS buffer, submerge it in PBS buffer, cut the front head of Japanese medical leeches along the middle of the oropharynx, and remove the pharyngeal wall Epidermis and outer skin, rinsed with PBS;

S104: After soaking the salivary glands in 75% alcohol solution for 5-7S, quickly transfer them to three vessels each containing 1mL of PBS buffer solution (containing 800U/mL penicillin/streptomycin/amphotericin mixed solution) Soak in each of them for 5 minutes, and cut them into about 0.5mm ³ -1mm ³ size in the third Petri dish filled with PBS buffer solution (containing 800U/mL penicillin/streptomycin/amphotericin mixed solution) organization block;

S105: Inoculate the shredded tissue pieces into a 25 cm ² closed cell culture flask, add SFX-INSCET insect cell culture medium, and place in a biochemical incubator for static culture at 29°C.

The application effects of the present invention will be described in detail below in conjunction with experiments.

The specific steps of the test are as follows:

Step 1, use water to remove the sticky secretions on the body surface of Japanese medical leeches. After cleaning up, soak in 0.3% potassium permanganate solution for 10 minutes, and then soak in 10% alcohol solution for 10 minutes to disinfect Japanese medical leeches;

Step 2, in the ultra-clean workbench, transfer the Japanese medical leech sterilized on the body surface to a disposable sterile petri dish filled with PBS buffer, immerse the Japanese medical leech in the PBS buffer, and use high temperature and high pressure sterilized Handle the surgical scissors and tweezers to remove the front head of the Japanese medical leech;

Step 3, transfer the head to another disposable sterile petri dish filled with PBS buffer, submerge it in PBS buffer, cut the front head of Japanese medical leeches along the middle of the oropharynx, and remove the pharyngeal wall Inner epidermis and outer skin, rinsed with PBS;

Step 4, after soaking the salivary glands in 75% ethanol solution for 5-7S, quickly transfer them to three chambers each equipped with 1mL PBS buffer solution (containing 800U/mL penicillin/streptomycin/amphotericin mixed solution) Soak in each vessel for 5 minutes, and cut it into 0.5mm ³ -1mm ³ size in the third Petri dish filled with PBS buffer solution (containing 800U/mL penicillin/streptomycin/amphotericin mixed solution) left and right tissue blocks;

Step 5, screening of the type of primary culture medium: inoculate the shredded tissue pieces into 25 cm ² closed cell culture flasks, divide them into three groups, add different types of culture medium (medium volume 5 mL), place in Static culture in a biochemical incubator at 29°C, replace half of the culture medium after 7 days, and add 20% inactivated standard fetal bovine serum; results Primary cell culture in RPMI1640 medium, on the second day of inoculation, visual observation Most of the tissue blocks are attached to the bottom of the wall. Under the microscope, it can be seen that the shredded tissue blocks are of different sizes and shapes, and the edges appear as brighter bright bands. The culture medium is clear and free of impurities; the 3-4 days of inoculation and culture , it was observed with the naked eye that the tissue block was attached to the bottom of the wall, the culture medium was clear, and a small amount of black impurities were observed in the culture medium under a microscope; on the 5th day of inoculation culture, the culture medium was observed to be turbid with the naked eye, the white transparent medium turned yellow slightly, and pollutants Distributed in dots, a large amount of black fine sand-like impurities were observed under an inverted microscope, and the cells showed no signs of growth. The primary cell culture was carried out in M199 medium, on the 2nd to 3rd day of inoculation culture, half of the tissue block was not adhered to the wall, and the culture medium was clear. Under the inverted microscope, it was observed that the tissue size was different, the shape was various, and the edge appeared relatively Bright bright band, no impurities in the culture medium; 4-5 days after inoculation and culture, most of the tissue pieces are still not attached to the wall, the culture medium is clear under an inverted microscope, and the cells have no signs of growth; about 6-7 days after inoculation and culture, the color of the culture medium It became light and turbid, and a layer of white gauze-like pollutants floated in the culture solution with the naked eye. The primary cell culture was carried out in SFX-INSCET insect cell culture medium. On the 2nd to 3rd day of inoculation culture, it was observed with the naked eye that most of the tissue pieces were attached to the bottom of the wall. Under an inverted microscope, it was observed that the tissue size was different, the shape was various, and the edges appeared. It is a relatively bright bright band. On the 3rd to 4th day of inoculation and culture, the culture medium is still clear under an inverted microscope, and the cells have no signs of growth; on the 5th to 6th day of inoculation and culture, a small amount of cells are free from the periphery of the tissue block, and the cell shape is Round and transparent.

Step 6, Screening of primary cultured antibiotics: Inoculate the shredded tissue pieces in a 25cm ² closed cell culture flask, add the optimal medium in step S105, divide into six groups, add different kinds of the same concentration respectively (200U/mL) mixed solution of antibiotic/PBS buffer, placed in a biochemical incubator at 29°C for static culture; the results were compared in the same medium (ie SFX-INSCET insect cell culture medium) after the same concentration of 200U/mL The growth, survival and contamination status of primary cells treated with different types of antibiotics. It was found that the survival time of the tissue pieces soaked in the penicillin/streptomycin/amphotericin B mixed solution was the longest during the primary culture, and the culture medium was kept free of contamination. It also takes the longest time to be polluted.

Step 7, screening of antibiotic concentration in primary culture ^: Inoculate the shredded tissue pieces in a 25cm closed cell culture flask, add the optimal medium in step S105, divide into six groups, add different concentrations, step The best antibiotic in S106 was cultured statically in a biochemical incubator at 29°C; the results were compared to the growth of primary cells soaked in the same antibiotic solution (ie "three-antibody" solution) but with different concentrations of antibiotics in the same medium Survival and pollution state, it was found that after the antibiotic concentration was 800U/mL (ie antibiotic: PBS buffer = 800U: 1mL), the cell contamination state was the smallest, the cell survival time was the longest, and new cell growth was observed .

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (6)

1. a primary culture method of Japanese medicine hirudo salivary gland cells, it is characterized in that, the primary culture method of described Japanese medicine hirudo salivary gland cells comprises the tissue piece after shredding is inoculated in 25cm ^In the cell sealing culture bottle, Add 5 mL of SFX-INSCET insect cell culture medium, and place it in a biochemical incubator at 29°C for static culture. 2. the primary culture method of Japanese medicine leech salivary gland cells as claimed in claim 1, is characterized in that, before described inoculation needs: Step 1, remove the viscous secretions on the body surface of Japanese leech with water, after cleaning, first soak in 0.3% potassium permanganate solution, then soak in alcohol solution, and disinfect the Japanese leech; Step 2, in the ultra-clean workbench, transfer the Japanese medical leech sterilized on the body surface to a disposable sterile petri dish filled with PBS buffer, immerse the Japanese medical leech in the PBS buffer, and use high temperature and high pressure sterilized Handle the surgical scissors and tweezers to remove the front head of the Japanese medical leech; Step 3, transfer the head to another disposable sterile petri dish filled with PBS buffer, submerge it in PBS buffer, cut the front head of Japanese medical leeches along the middle of the oropharynx, and remove the pharyngeal wall Inner epidermis and outer skin, rinsed with PBS; Step 4, after soaking the salivary glands in the alcohol solution, quickly transfer them to three vessels each containing PBS buffer solution for soaking, and in the third Petri dish containing PBS buffer solution, cut it into tissue pieces; the PBS buffer contains 800 U/mL of penicillin/streptomycin/amphotericin mixed solution. 3. the primary culture method of Japanese medicine hirudo salivary gland cells as claimed in claim 2, is characterized in that, in described step 1, be put in 0.3% potassium permanganate solution and soak 10min, then put into 10% alcoholic solution Soak in water for 10 minutes to disinfect Japanese medical leeches. 4. the primary culture method of Japanese medicine hirudo salivary gland cells as claimed in claim 2, is characterized in that, in described step 4, after salivary gland is placed in 75% alcohol solution and soaks 5-7S, it is transferred to three rapidly. Soak in each vessel filled with 1mL PBS buffer solution (containing 800U/mL penicillin/streptomycin/amphotericin mixed solution) for 5min each. 5. The method for primary culture of Japanese medical hirudo salivary gland cells as claimed in claim 2, characterized in that, in said step 4, the cells are cut into 0.5mm ³ -1mm ³ sized tissue pieces. 6. A Japanese leech cultivated by the method for primary culture of salivary gland cells of any one of claims 1 to 5.