CN114190362B - A kind of placenta preservation liquid and the preservation method of placenta - Google Patents

Abstract

The invention relates to the technical field of medicine, and particularly discloses a placenta preservation solution and a placenta preservation method. The placenta preservation solution comprises the following components in parts by weight: 100-150 parts of PBS buffer solution; 3-5 parts of glucose; 1-2 parts of reduced glutathione; 2-3 parts of adenosine; 1-2 parts of streptomycin; 0.5 to 1 portion of gentamicin; 1-2 parts of traditional Chinese medicine extract. The research shows that: the placenta preservation solution is added with a traditional Chinese medicine extract which is extracted from traditional Chinese medicines of ephedra and bupleurum, so that the survival rate of the placenta mesenchymal stem cells can be effectively improved.

Description

A kind of placenta preservation liquid and the preservation method of placenta

technical field

The invention relates to the field of medical technology, in particular to a placenta preservation solution and a placenta preservation method.

Background technique

The placenta is an important organ for material exchange between the fetus and the mother. It is a tissue-combining organ between the mother and the child formed by the joint growth of the embryonic embryonic membrane and the maternal endometrium during human pregnancy. After a pregnant woman gives birth, the placenta is released from the body. However, the isolated placenta contains a large number of mesenchymal stem cells; placental mesenchymal stem cells (mesenchymal stem cells, MSCs) are a kind of pluripotent stem cells, which are pluripotent cells with self-replication ability; such as placental mesenchymal stem cells Differentiate under suitable conditions: epithelial stem cells, neural stem cells, liver stem cells, muscle cells, osteoblasts, chondrocytes, stromal cells and other cells. It can be used to repair damaged or diseased tissues and organs, and treat cardiovascular and cerebrovascular diseases, nervous system diseases, liver diseases, bone tissue diseases, corneal injuries, burns, myopathy and other diseases.

However, the placental mesenchymal stem cells in the isolated placenta will soon lose their activity; it is not conducive to the subsequent isolation and culture of placental mesenchymal stem cells. Therefore, it is of great application value to provide a placenta preservation solution that can maintain a high activity of placental mesenchymal stem cells in an isolated placenta.

Contents of the invention

In order to overcome at least one of the technical problems in the prior art, the present invention provides a placenta preservation solution.

The above-mentioned technical problems to be solved by the present invention are realized through the following technical solutions:

A placenta preservation solution, which comprises the following components in parts by weight: 100-150 parts of PBS buffer solution; 3-5 parts of glucose; 1-2 parts of reduced glutathione; 2-3 parts of adenosine; streptomycin 1 to 2 parts; 0.5 to 1 part of Daqingmycin; 1 to 2 parts of traditional Chinese medicine extract.

The inventors have shown through a large number of experimental studies that the placental mesenchymal stem cells in the placenta can have a higher survival rate by preserving the isolated placenta in the above-mentioned placenta preservation solution.

Preferably, the placenta preservation solution comprises the following components in parts by weight: 100-120 parts of PBS buffer solution; 4-5 parts of glucose; 1-1.5 parts of reduced glutathione; 2-2.5 parts of adenosine ; Streptomycin 1 to 1.5 parts; Daqingmycin 0.5 to 1 part; Chinese medicine extract 1 to 2 parts.

Preferably, the Chinese medicine extract is prepared by the following steps:

The traditional Chinese medicine ephedra and bupleurum are mixed, and then heated and refluxed with ethanol for extraction. After the extraction, the extract is concentrated and dried to obtain an alcohol extract, which is the traditional Chinese medicine extract.

What the inventor needs to explain here is that the raw materials for the preparation of Chinese medicine extracts play a decisive role in whether the prepared Chinese medicine extracts can effectively improve the survival rate of placental mesenchymal stem cells; All drugs can improve the survival rate of placental mesenchymal stem cells. The inventors have shown through a large number of experiments that adding traditional Chinese medicine extracts extracted from traditional Chinese medicines ephedra and Bupleurum chinensis into the placental preservation solution can effectively improve the survival rate of placental mesenchymal stem cells. Further studies have shown that adding traditional Chinese medicine extracts extracted from Chinese medicines Ephedra and Bupleurum to the placental preservation solution can improve the survival rate of placental mesenchymal stem cells much more than adding Chinese medicines such as ephedra or Bupleurum alone. Other traditional Chinese medicines are Chinese medicine extracts extracted from raw materials.

Preferably, the ethanol is an aqueous ethanol solution with a volume fraction of 30% to 50%.

Most preferably, the ethanol is an aqueous ethanol solution with a volume fraction of 40%.

Preferably, the weight ratio of the traditional Chinese medicine ephedra and Bupleuri is 3-5:1.

Most preferably, the weight ratio of the traditional Chinese medicine Ephedra and Bupleurum is 4:1.

Preferably, the ratio of the total weight of the traditional Chinese medicine ephedra and Bupleuri to ethanol is 1g:10-20mL.

Most preferably, the ratio of the total weight of the traditional Chinese medicine Ephedra and Bupleurum to the ethanol is 1g:15mL.

Preferably, the heating to reflux extraction refers to reflux extraction under the condition of heating to ethanol boiling for 1-2 hours.

Further, put the ethanol extract on a macroporous resin column; first elute with an aqueous ethanol solution with a volume fraction of 3% to 5% to remove impurities; then elute with an aqueous ethanol solution with a volume fraction of 16% to 20%, and collect the volume fraction The fractions eluted with 16%-20% ethanol aqueous solution are concentrated and dried to obtain the traditional Chinese medicine extract.

Preferably, first use 2 to 4 times column volume of ethanol water solution with a volume fraction of 3% to 5% to remove impurities; , collect the fractions eluted with ethanol aqueous solution with a volume fraction of 16% to 20%, concentrate and dry to obtain the Chinese medicine extract.

Further studies by the inventors have shown that adding the traditional Chinese medicine extract prepared by the macroporous resin column chromatography method to the preservation solution can further greatly improve the survival rate of placental mesenchymal stem cells.

The present invention also provides a method for preserving the placenta, which comprises the following steps: soaking the freshly collected placenta in the above-mentioned placenta preserving solution, and preserving at 0-5°C.

Beneficial effects: the present invention provides a brand-new placental preservation solution, which can effectively improve the survival of placental mesenchymal stem cells by adding the traditional Chinese medicine extract extracted from the traditional Chinese medicine ephedra and Bupleuri. Rate.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples, but the examples do not limit the present invention in any form.

The PBS buffer solution in the following examples was prepared by the following method: Weigh 8.0g NaCl, 0.2gKCl, 1.44g Na ₂ HPO ₄ , 0.24g KH ₂ PO ₄ and dissolve them in 800mL distilled water, adjust the solution to 7.4 with HCl, and finally Add distilled water to make up to 1L to get 0.01M PBS buffer.

The preparation of embodiment 1 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the traditional Chinese medicine Extract;

Wherein, the weight ratio of the traditional Chinese medicine ephedra and Bupleurum is 4:1; the dosage ratio of the total weight of the traditional Chinese medicine ephedra and Bupleurum to the ethanol aqueous solution is 1g:15mL.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The preparation of embodiment 2 placenta preservation solution

Composition of raw materials by weight: 150 parts of PBS buffer solution; 5 parts of glucose; 1 part of reduced glutathione; 2.5 parts of adenosine; 1 part of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 30%, and carry out reflux extraction for 1 hour under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the Chinese medicine extract thing;

Wherein, the weight ratio of the traditional Chinese medicine ephedra and bupleurum is 3:1; the dosage ratio of the total weight of the traditional Chinese medicine ephedra and bupleurum to the ethanol aqueous solution is 1g:10mL.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The preparation of embodiment 3 placenta preservation solution

Composition of raw materials by weight: 100 parts of PBS buffer solution; 3 parts of glucose; 1 part of reduced glutathione; 2 parts of adenosine; 1 part of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 50%, and carry out reflux extraction for 2 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the Chinese medicine extract thing;

Wherein, the weight ratio of the traditional Chinese medicine ephedra and Bupleurum is 5:1; the dosage ratio of the total weight of the traditional Chinese medicine ephedra and Bupleurum to the ethanol aqueous solution is 1g:20mL.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The preparation of embodiment 4 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the alcohol extract; Wherein, the weight ratio of Chinese medicine Ephedra and Bupleurum is 4:1; the consumption ratio of the total weight of Chinese medicine Ephedra and Bupleurum to ethanol aqueous solution is 1g:15mL;

Further macroporous resin column (wherein packing is equipped with the macroporous resin that model is HP-20 with alcohol extract; The weight consumption of macroporous resin is 30 times of alcohol extract weight); 4% ethanol aqueous solution elution removes impurity; Then use 5 times column volume volume fraction to be 18% ethanol aqueous solution elution, collect the flow part that the volume fraction is 18% ethanol aqueous solution elution, get final product after concentrating and drying Chinese herbal extracts.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Example 4 and Example 1 is that the Chinese medicine extract is further prepared through a macroporous resin column.

Preparation of comparative example 1 placenta preservation solution

Composition in parts by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin and dakinycin into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Comparative Example 1 and Example 1 is that no traditional Chinese medicine extract is added to the placenta preservation solution described in Comparative Example 1.

Preparation of comparative example 2 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

Take the traditional Chinese medicine ephedra, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the traditional Chinese medicine extract;

Wherein, the consumption ratio of traditional Chinese medicine ephedra and ethanol aqueous solution is 1g:15mL.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Comparative Example 2 and Example 1 is that the raw materials for the preparation of Chinese medicine extracts are different; Comparative Example 2 only uses the Chinese medicine Ephedra as raw material to prepare the Chinese medicine extract; while Example 1 uses the Chinese medicine Mahuang and Bupleurum as raw materials to prepare Chinese medicine Extract.

Preparation of comparative example 3 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

Take the traditional Chinese medicine Bupleurum bupleuri, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the traditional Chinese medicine extract;

Wherein, the dosage ratio of the traditional Chinese medicine Bupleurum bupleuri and the ethanol aqueous solution is 1g:15mL.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Comparative Example 3 and Example 1 is that the raw materials for the preparation of Chinese medicine extracts are different; Comparative Example 2 only uses the traditional Chinese medicine Bupleuri as raw material to prepare the Chinese medicine extract; while Example 1 uses the Chinese medicine Ephedra and Bupleurum as raw materials. Chinese herbal extracts.

Preparation of comparative example 4 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the alcohol extract; Wherein, the weight ratio of Chinese medicine Ephedra and Bupleurum is 4:1; the consumption ratio of the total weight of Chinese medicine Ephedra and Bupleurum to ethanol aqueous solution is 1g:15mL;

Further the ethanol extract is put on the macroporous resin column (which is filled with the macroporous resin of HP-20; the weight consumption of the macroporous resin is 30 times of the ethanol extract weight); Miscellaneous; Then use 5 times column volume volume fraction to be 8% ethanol aqueous solution elution, collect the flow part that the volume fraction is 8% ethanol aqueous solution elution, promptly obtain described Chinese medicine extract after concentrating and drying.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Comparative Example 4 and Example 4 is that the elution conditions of the macroporous resin column are different; Comparative Example 4 is first eluted with water to remove impurities, and then eluted with an aqueous ethanol solution with a volume fraction of 8%; while Example 4 is First use 4% ethanol aqueous solution to remove impurities, and then use 18% ethanol aqueous solution to elute.

Preparation of comparative example 5 placenta preservation solution

Composition by weight of raw materials: 120 parts of PBS buffer solution; 4 parts of glucose; 1.5 parts of reduced glutathione; 2 parts of adenosine; 1.5 parts of streptomycin;

The Chinese medicine extract is prepared by the following method:

After mixing the traditional Chinese medicine ephedra and bupleurum, add ethanol aqueous solution with a volume fraction of 40%, and carry out reflux extraction for 1.5 hours under the condition of heating the ethanol aqueous solution to boiling; after the extraction is completed, the extract is concentrated and dried to obtain the alcohol extract; Wherein, the weight ratio of Chinese medicine Ephedra and Bupleurum is 4:1; the consumption ratio of the total weight of Chinese medicine Ephedra and Bupleurum to ethanol aqueous solution is 1g:15mL;

Further macroporous resin column (wherein packing is equipped with the macroporous resin that model is HP-20 with alcohol extract; The weight consumption of macroporous resin is 30 times of alcohol extract weight); 20% ethanol aqueous solution for elution and impurity removal; then use 5 times of the column volume for eluting with 40% ethanol aqueous solution, collect the fractions eluted with 40% ethanol aqueous solution, concentrate and dry to obtain the obtained Chinese herbal extracts.

Preparation method: Add glucose, reduced glutathione, adenosine, streptomycin, dakinmycin and traditional Chinese medicine extracts into PBS buffer solution, stir evenly to obtain the placenta preservation solution.

The difference between Comparative Example 5 and Example 4 is that the elution conditions of the macroporous resin column are different; Comparative Example 5 is eluted with 20% ethanol aqueous solution to remove impurities, and then washed with 40% ethanol aqueous solution. and the embodiment 4 is firstly eluted with 4% ethanol aqueous solution to remove impurities, and then eluted with 18% ethanol aqueous solution.

Experimental example

Put the freshly collected placenta into the placenta collection boxes equipped with the placenta preservation solutions prepared in Examples 1-3 and Comparative Examples 1-5, respectively, and soak them in the amount of the placenta preservation solution to submerge the placenta; and store them at 4°C 7 days; then isolated placental mesenchymal stem cells.

Adjust the freshly isolated placental stem cells to a cell suspension with a density of 1×10 ⁶ cells/mL; then add 0.4% trypan blue staining solution and mix well; then take the cell suspension with trypan blue staining solution 20 μL was added to a cell counting plate, and the cell viability was detected with a Countstar cell counter; the results of the cell viability detection are shown in Table 1.

Table 1. Cell viability detection results

cell viability Embodiment 1 placenta preservation solution 80.32% Embodiment 2 placenta preservation solution 76.44% Embodiment 3 placenta preservation solution 72.18% Embodiment 4 placenta preservation solution 94.45% Comparative example 1 placenta preservation solution 46.18% Comparative example 2 placenta preservation solution 58.49% Comparative example 3 placenta preservation solution 55.37% Comparative example 4 placenta preservation solution 84.18% Comparative example 5 placenta preservation solution 77.27%

It can be seen from the test results of cell viability in Table 1 that after 7 days after the isolated placenta was stored in the placenta preservation solution prepared in Examples 1-4, the cell viability of the placental mesenchymal stem cells was above 72%. ; Much higher than the cell viability preserved in the placental preservation solution in Comparative Example 1 without adding the Chinese medicine extract. This shows that the addition of traditional Chinese medicine extracts extracted from the traditional Chinese medicine ephedra and Bupleurum chinensis as raw materials in the placental preservation solution can greatly increase the cell viability of placental mesenchymal stem cells.

It can also be seen from the cell viability detection results in Table 1 that after the isolated placenta was stored in the placenta preservation solution prepared in Example 4 for 7 days, the cell viability of the placental mesenchymal stem cells in it all reached 94.45%, which was the same as Compared with the placental preservation solution prepared in Example 1, there is a further significant improvement; this illustrates: the Chinese medicine extract is further processed by the above-mentioned macroporous resin column chromatography method of the present invention, and can further enrich a large amount of The active ingredients further significantly increased the cell viability of placental mesenchymal stem cells.

It can also be seen from the cell viability detection results in Table 1 that after the isolated placenta was stored in the placental preservation solution prepared in Comparative Examples 2 and 3 for 7 days, the cell viability of the placental mesenchymal stem cells therein was the same as that in Comparative Example 1. Although there is improvement compared, the magnitude is not large; far less than the magnitude of promotion of Example 1 relative to Comparative Example 1. This shows that the raw materials for the preparation of Chinese medicine extracts play a decisive role in whether the prepared Chinese medicine extracts can effectively improve the cell viability of placental mesenchymal stem cells; The cell viability of placental mesenchymal stem cells must be added to the placental preservation solution to add Chinese medicine extracts extracted from the traditional Chinese medicine ephedra and Bupleuri as raw materials, in order to greatly increase the cell viability of placental mesenchymal stem cells.

It can also be seen from the cell viability detection results in Table 1 that after 7 days after the isolated placenta was stored in the placenta preservation solution prepared in Comparative Example 4, the cell viability of the placental mesenchymal stem cells therein was compared with that of Example 1 , the promotion was not obvious, and its promotion range was far less than that of Example 4; in addition, after the isolated placenta was stored in the placenta preservation solution prepared in Comparative Example 5 for 7 days, the cell viability of the placental mesenchymal stem cells therein Compared with Example 1, it has also been reduced; this illustrates: the elution condition of macroporous resin plays a decisive role in whether the Chinese medicine extract with better cell viability of improving placental mesenchymal stem cells can be prepared ; Only under the macroporous resin elution conditions described in the present invention (i.e. first use volume fraction is 3% ~ 5% ethanol aqueous solution elution to remove impurities; then use volume fraction is 16% ~ 20% ethanol aqueous solution elution , collecting the fractions eluted with ethanol aqueous solution with a volume fraction of 16% to 20%), the prepared Chinese medicine extract can further greatly improve the cell viability of placental mesenchymal stem cells; while under other macroporous resin elution conditions The prepared traditional Chinese medicine extract can not further significantly increase the cell viability of the placental mesenchymal stem cells, and even reduces the cell viability of the placental mesenchymal stem cells.

Claims (4)

1. A placenta preservation solution, characterized in that it comprises the following components by weight: 100 to 150 parts of PBS buffer solution; 3 to 5 parts of glucose; 1 to 2 parts of reduced glutathione; 2 to 3 parts of adenosine 1-2 parts of streptomycin; 0.5-1 part of daqingmycin; 1-2 parts of traditional Chinese medicine extract; The Chinese medicine extract is prepared by the following steps: Take the traditional Chinese medicine Ephedra and Bupleurum and mix them with ethanol for heating and reflux extraction. After the extraction is completed, the extract is concentrated and dried to obtain an alcoholic extract; the ethanol used is an ethanol aqueous solution with a volume fraction of 40%; the Chinese medicine Ephedra and Bupleurum The weight ratio is 4:1; the total weight of Chinese medicine ephedra and Bupleurum and the consumption ratio of ethanol are 1g:15mL; Further, put the ethanol extract on a macroporous resin column; first use 3% to 5% ethanol aqueous solution with a volume fraction of 2 to 4 times the column volume to elute and remove impurities; then use a volume fraction of 3 to 6 times the column volume to 16%-20% ethanol aqueous solution is eluted, and the fractions eluted with the volume fraction of 16%-20% ethanol aqueous solution are collected, concentrated and dried to obtain the traditional Chinese medicine extract. 2. The placenta preservation solution according to claim 1, characterized in that it comprises the following components in parts by weight: 100-120 parts of PBS buffer solution; 4-5 parts of glucose; 1-1.5 parts of reduced glutathione; 2-2.5 parts of adenosine; 1-1.5 parts of streptomycin; 0.5-1 part of daqingmycin; 1-2 parts of traditional Chinese medicine extract. 3 . The placenta preservation solution according to claim 1 , wherein the heating and reflux extraction refers to reflux extraction under the condition of heating to ethanol boiling for 1 to 2 hours. 4 . 4. A method for preserving the placenta, characterized in that it comprises the following steps: soaking the freshly collected placenta in the placenta preservation solution according to any one of claims 1-3, and storing it at 0-5°C.