CN111979174A - Method for separating hirudo nipponica salivary gland cells - Google Patents

Abstract

The invention discloses a method for separating Japanese leech salivary gland cells, which relates to the technical field of cell separation and includes the following steps: S1: placing the extracted salivary gland tissue in a pre-cooled buffer; S2: using the buffer to wash off the salivary gland tissue Blood stains; S3: transfer the salivary gland tissue treated by S2 to a petri dish; S4: add disinfectant for disinfection; S5: wash again with buffer to remove residual disinfectant; S6: transfer the salivary gland tissue after S5 treatment To the centrifuge tube, add an appropriate amount of enzyme solution for enzymolysis in a water bath; S7: Take the supernatant after S6 treatment, and add culture medium at the same time; S8: Centrifuge the salivary gland tissue after S7 treatment, discard the supernatant, add After resuspending an appropriate amount of culture medium, it was inoculated onto a culture plate. The invention provides a method for separating salivary gland cells of Japanese medicinal leech, further culturing the salivary gland cells, extracting active substances such as hirudin, and can be used for research on the secretion mechanism of salivary gland cells.

Description

A method for separating salivary gland cells of Japanese medicinal leech

technical field

The present invention relates to the technical field of cell separation, more particularly, to a method for separating salivary gland cells of Japanese leech.

Background technique

With the aggravation of population aging and changes in living environment, the incidence of cardiovascular and cerebrovascular diseases is also increasing year by year, such as hypertension, cerebral thrombosis, cerebral hemorrhage, etc., more and more patients, the corresponding prevention and treatment measures to solve the disease urgently important. Studies have shown that hirudin is the strongest anticoagulant active substance discovered in the world so far, and it is the most effective pharmaceutical raw material for the treatment of cardiovascular and cerebrovascular diseases. Japanese leech is widely distributed in China and is a traditional Chinese medicinal material in my country. With its strong anti-platelet aggregation effect and antithrombin activity, the hirudin secreted by it is a good biologically active substance, and there is a clinical demand for natural hirudin. larger.

Japanese leech (Hirudo nipponia) is the only species of leech that sucks animal blood in the Chinese Pharmacopoeia. Its salivary glands and the hirudin secreted by them have strong antithrombin activity and anti-platelet aggregation. medicinal value. The main medicinal parts of Japanese leeches lie in their salivary glands. However, due to the scarcity of research on Japanese leeches at this stage, and the main focus is on the artificial breeding of Japanese leeches and the research on bioactive components, the salivary glands of Japanese leeches are very scarce. There are few reports on separation and culture related research, which is not conducive to the development and utilization of Japanese leech resources.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a method for separating Japanese leech salivary gland cells, which is to separate the Japanese leech salivary gland cells from the tissue block, and then further cultivate the salivary gland cells to extract biological active substances such as hirudin, and can be used to extract biologically active substances such as hirudin. It is used to study the secretion mechanism of salivary gland cells; through screening experiments on the type, concentration, and digestion time of enzymes, an enzymatic hydrolysis method for separating salivary gland cells of Japanese leech was created, and the salivary gland cells of leech were separated from tissue blocks. Then, primary culture and subculture are performed to obtain hirudin. This method will further culture salivary gland cells, extract hirudin and other biologically active substances, and can be used to study the secretion mechanism of salivary gland cells.

The above-mentioned technical purpose of the present invention is achieved through the following technical solutions:

A method for separating Japanese leech salivary gland cells, comprising the following steps:

S1: Place the extracted salivary gland tissue in pre-cooled buffer;

S2: Use buffer to wash away blood stains from salivary gland tissue;

S3: transfer the salivary gland tissue treated by S2 to a petri dish;

S4: adding disinfectant for disinfection;

S5: Use the buffer to wash again, and wash away the residual disinfectant;

S6: transfer the salivary gland tissue treated in S5 to a centrifuge tube, add an appropriate amount of enzyme solution, and perform enzymatic hydrolysis in a water bath;

S7: take the supernatant after S6 treatment, and add the culture medium at the same time;

S8: centrifuge the salivary gland tissue treated in S7, discard the supernatant, add an appropriate amount of culture medium to resuspend, and then inoculate it on a culture plate.

As a preferred solution, in the process of S4, the disinfectant needs to be replaced three times, and the disinfection time for each time is 4-6 minutes, and the blowing treatment is performed during the disinfection process.

As a preferred solution, in the process of S7, 1-3 ml of the culture medium is added, and in the process of S8, the time of centrifugation is 4-6 minutes.

As a preferred solution, the enzyme solution in the S6 process needs to be screened, the salivary gland tissues obtained in S5 are grouped, and different types of enzymes are selected and treated in a water bath to obtain the best enzyme types.

As a preferred solution, the salivary gland tissues obtained by S5 are grouped, and the best enzyme species with different concentrations are selected, and subjected to water bath treatment to obtain the best concentration of the best enzyme species.

As a preferred solution, the salivary gland tissues obtained by S5 are grouped, and the optimal enzyme species with the optimal concentration is subjected to water bath treatment for different times to obtain the optimal treatment time.

In the above preferred scheme, the most suitable collagenase type II can be selected from the three enzymes: trypsin, collagenase type II and TryPLE; enzymatic hydrolysis experiments with different concentrations of collagenase type II are carried out.

As a preferred solution, in the S1 process, the method for extracting salivary gland tissue includes the following steps:

The Japanese leech was placed in clean water, then anesthetized, and the mucus on its body surface was wiped. Finally, it was fixed, and its salivary gland tissue was taken under sterile conditions.

As a preferred solution, Japanese leech was placed in purified water for 24 hours; 10% alcohol was used for anesthesia treatment.

A hirudin is obtained by applying the above-mentioned method for separating salivary gland cells of Japanese leech.

To sum up, the present invention has the following beneficial effects:

The present invention obtains leech salivary gland primary cells by enzymatic hydrolysis and digestion of Japanese leech salivary gland tissue blocks, and then conducts cell culture to extract natural hirudin. The method of engineering and synthesizing recombinant hirudin is simpler and more efficient; in addition, compared with the conventional trypsin digestion and enzymatic hydrolysis, which takes 18.5 hours, this enzymatic hydrolysis method only takes 75 minutes, which can save 93.2% of the digestion time and greatly save time. more efficient.

The invention optimizes the enzymatic hydrolysis system of Japanese leech salivary gland cells: the most suitable collagenase type II can be selected from the three enzymes of trypsin, collagenase type II and TryPLE; The conclusion is that the concentration of the enzyme has no significant effect on the enzymatic hydrolysis, so the lowest concentration is 1 mg/ml; the collagenase type II is digested in a water bath for different times, and the most suitable method is screened and then optimized. After obtaining; firstly use collagenase type Ⅱ 1mg/ml enzymolysis for 30min, and then use neutral protease 2mg/ml enzymolysis for 45min.

Enzymatic digestion of Japanese leech salivary gland cells by this method can obtain more salivary gland cells and the degree of cell damage is low. The invention is simple, time-saving and efficient, and provides an optimized method for separating leech salivary gland cells from tissue blocks.

Detailed ways

The description and claims do not use the difference in name as a way to distinguish components, but use the difference in function of the components as a criterion for distinguishing. As mentioned throughout the specification and claims, "including" is an open-ended term and should be interpreted as "including but not limited to". "Approximately" means that within an acceptable error range, those skilled in the art can solve the technical problem within a certain error range, and basically achieve the technical effect.

The enzymatic hydrolysis method for separating Japanese leech salivary gland cells is as follows:

(1) Take 15 Japanese leeches that have undergone starvation treatment and place them in purified water for 24 hours overnight. The Japanese leech was then anesthetized with 10% alcohol, and the mucus on its body surface was wiped with a sterile cotton ball dipped in 75% alcohol.

(2) Fix the head and tail on the foam board with a pin, take the salivary gland tissue under aseptic conditions, put it in the pre-cooled buffer and control the sampling time within 1h, change the buffer 3 times, wash away the blood stains .

(3) After washing away the blood stains with buffer, transfer the taken leech salivary gland tissue to a new petri dish, add 5 ml of new disinfectant, pipette, and disinfect for 5 minutes; discard the original disinfectant, remove the leech salivary gland tissue Transfer the block to a new petri dish, add 5ml of new disinfectant, pipette, and sterilize for 5 minutes; discard the original disinfectant, transfer the leech salivary gland tissue block to a new petri dish, add 5ml of new disinfectant, pipette , sterilize for 5 minutes. Discard the disinfectant, wash off the residual disinfectant with an appropriate amount of buffer, then discard the culture medium and transfer it to a new centrifuge tube.

(4) Add an appropriate amount of enzyme solution to the centrifuge tube, enzymolysis in a water bath, and shake well between intervals to fully digest the tissue block.

(5) After the water bath, the cells were resuspended by pipetting the liquid, and then the supernatant was taken into a new centrifuge tube, and 2 ml of culture medium was added to stop the enzymatic hydrolysis. Centrifuge (1200 rpm) for 5 minutes, discard the supernatant, and resuspend the cell pellet with an appropriate amount of culture medium, then inoculate it into a 24-well culture plate and observe.

(6) Screening of enzyme types by enzymolysis method: Divide the sterilized tissue blocks in step 4 into three groups, add 3ml of different types of enzyme solutions (trypsin/collagenase type II/TryPLE) and digest them in a water bath, fully digest and then Proceed to step 5, and then place it in an inverted microscope to observe the degree of cell digestion. The results showed that most of the cells in the salivary gland tissue after trypsin digestion were round, and there were many cell fragments, and the cells were damaged to a certain extent; Oval shape, with a certain degree of cell damage, but less severe than trypsin, and some cells have a long strip shape after digestion; most of the salivary gland cells after TryPLE digestion are round or irregular shapes, and some damaged cells. In conclusion, collagenase type II was selected for digestion.

(7) Screening of enzyme concentration by enzymolysis method: Divide the sterilized tissue blocks in step 4 into three groups, add different concentrations (1mg/ml/2mg/ml) and the optimal enzyme species in step 6 respectively, digest the same time Then proceed to step 5, and then place it in an inverted microscope to observe the degree of cell digestion. It was found that after the same enzyme digestion for the same time, the concentration of the enzyme had no significant effect on the enzymatic hydrolysis. In summary, 1 mg/ml collagenase type II was selected for digestion.

(8) Screening of the enzymatic digestion time of the enzymatic hydrolysis method: after adding the sterilized tissue block in step 4, after adding the optimal enzyme type in step 6 and the optimal enzyme concentration in step 7, it is divided into four groups, and they are respectively carried out for different time (( 6h/12h/18h) enzymatic digestion, then proceed to step 5, and then place it in an inverted microscope to observe the degree of cell digestion. The results showed that with the increase of digestion time, the shape of salivary gland tissue enzymolysis cells gradually became elongated, the more fully digested the tissue block, the higher the cell integrity and the less cell debris. In conclusion, 1mg/ml collagenase type II was selected for 18h digestion.

(9) Method optimization of the enzymatic hydrolysis method: To optimize the conclusion obtained in step 8, the salivary gland cells can be obtained by digesting 1 mg/ml collagenase II for 30 minutes and then 2 mg/ml neutral protease for 45 minutes. In addition, the number of salivary gland cells increased significantly, the degree of digestion was better, and the degree of cell damage was low. To sum up, the enzymatic digestion method was selected firstly with collagenase II at 1 mg/ml for 30 min, and then with neutral protease at 2 mg/ml for 45 min.

This specific embodiment is only an explanation of the present invention, and it does not limit the present invention. Those skilled in the art can make modifications without creative contribution to the present embodiment as required after reading this specification, but as long as the rights of the present invention are used All claims are protected by patent law.

Claims (9)

1. a method for separating Japanese medicine leech salivary gland cells, is characterized in that, comprises the following steps: S1: Place the extracted salivary gland tissue in pre-cooled buffer; S2: Use buffer to wash away blood stains from salivary gland tissue; S3: transfer the salivary gland tissue treated by S2 to a petri dish; S4: adding disinfectant for disinfection; S5: Use the buffer to wash again, and wash away the residual disinfectant; S6: transfer the salivary gland tissue treated in S5 to a centrifuge tube, add an appropriate amount of enzyme solution, and perform enzymatic hydrolysis in a water bath; S7: take the supernatant after S6 treatment, and add the culture medium at the same time; S8: centrifuge the salivary gland tissue treated in S7, discard the supernatant, add an appropriate amount of culture medium to resuspend, and then inoculate it on a culture plate. 2. the method for separating Japanese leech salivary gland cells according to claim 1, is characterized in that, in described S4 process, need to replace three times of disinfectant, each time of disinfection is 4-6 minutes, and carry out in the process of disinfection blowing treatment. 3. the method for separating Japanese leech salivary gland cells according to claim 2, is characterized in that, in the described S7 process, the culture solution is added 1-3ml, in the S8 process, the time of centrifugation is 4-6 minutes. 4. the method for separating Japanese medicinal leech salivary gland cells according to claim 1 and 3, is characterized in that, the enzyme solution in described S6 process needs to be screened, the salivary gland tissue that S5 obtains is grouped, chooses different kinds of enzymes And carry out water bath treatment to get the best enzyme species. 5. the method for separating Japanese medicine leech salivary gland cells according to claim 4, is characterized in that, the salivary gland tissue that S5 obtains is carried out grouping, selects the best enzyme species of different concentrations, carries out water bath treatment, obtains the best enzyme species. optimum concentration. 6. the method for separating Japanese leech salivary gland cells according to claim 5, is characterized in that, the salivary gland tissue that S5 obtains is carried out grouping, the optimum enzyme species of optimum concentration is carried out the water bath treatment of different time, obtains optimum processing time. 7. the method for separating Japanese medicine leech salivary gland cells according to claim 6, is characterized in that, in the described S1 process, the extraction method of salivary gland tissue comprises the following steps: Japanese medicine leech is placed in clear water, then carries out anesthesia treatment , and wipe the mucus on its body surface, finally fix it, and take its salivary gland tissue under sterile conditions. 8 . The method for separating Japanese leeches salivary gland cells according to claim 7 , wherein the Japanese leeches are placed in purified water for 24 hours; 10% alcohol is used for anesthesia treatment. 9 . 9 . A hirudin, characterized in that, the hirudin obtained by the method for separating the salivary gland cells of Hirudo japonica according to any one of claims 1 to 8 is applied.