阅读说明：本技术 一种抗消化系统肿瘤的浆水无细胞培养液 (Serous acellular culture solution for resisting digestive system tumors ) 是由 陈朋 刘宇恒 张仁涛 马万通 周忠坤 胡树坚 马云浩 杜康嘉 于 2020-07-03 设计创作，主要内容包括：本发明涉及生物制药技术领域,尤其涉及一种抗消化系统肿瘤的浆水无细胞培养液及其制备方法和应用,所述的浆水无细胞培养液通过以下方法制备：取浆水加入RPMI培养基或DMEM培养基重悬沉淀,恒温培养,离心获得的培养液即为浆水无细胞培养液。所述的浆水中含有植物乳杆菌、副干酪乳杆菌、木糖葡萄球菌和发酵乳杆菌中的一种或者几种,所述的浆水无细胞培养液对结直肠癌、胃癌、肝癌和胰腺癌均具有较强抑制作用,且所述的浆水无细胞培养液来源于食品,是浆水的主要成分,安全,副作用小。(The invention relates to the technical field of biological pharmacy, in particular to a serous acellular culture solution for resisting digestive system tumors, a preparation method and application thereof, wherein the serous acellular culture solution is prepared by the following method: adding the plasma into RPMI culture medium or DMEM culture medium to perform basic suspension precipitation, performing constant temperature culture, and centrifuging to obtain a culture solution, namely the plasma-water cell-free culture solution. The serous fluid contains one or more of lactobacillus plantarum, lactobacillus paracasei, staphylococcus xylosus and lactobacillus fermentum, the serous fluid cell-free culture solution has a strong inhibition effect on colorectal cancer, gastric cancer, liver cancer and pancreatic cancer, and the serous fluid cell-free culture solution is derived from food, is a main component of the serous fluid, and is safe and small in side effect.)

1. A serous fluid cell-free culture solution is characterized by being prepared by the following method:

adding the plasma into RPMI culture medium or DMEM culture medium to perform basic suspension precipitation, performing constant temperature culture, and centrifuging to obtain a culture solution, namely the plasma-water cell-free culture solution.

2. The aqueous slurry cell-free culture solution according to claim 1, wherein the aqueous slurry contains one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

3. The aqueous slurry cell-free culture solution according to claim 2, wherein the aqueous slurry contains Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus), and Lactobacillus fermentum (Lactobacillus fermentum).

4. The aqueous slurry cell-free culture solution according to claim 1, which is prepared by the following method:

(1) centrifuging the slurry, discarding the supernatant, resuspending the lower precipitate with MRS culture medium, culturing at constant temperature to obtain slurry primary culture, centrifuging, discarding the supernatant, and precipitating the slurry primary culture for use;

(2) suspending the serous fluid primary culture precipitate obtained in the step (1) by using an RPMI culture medium or a DMEM culture medium, adjusting the pH value to the optimum pH value of the cells, and culturing at constant temperature to obtain a serous fluid secondary culture;

(3) centrifuging the serous fluid secondary culture obtained in the step (2) and taking supernatant fluid;

(4) and (4) filtering the supernatant obtained in the step (3) by using a sterile filter membrane to obtain the serous acellular culture solution.

5. The aqueous slurry cell-free culture solution according to claim 4, wherein the aqueous slurry contains one or more probiotics selected from Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

6. The aqueous slurry cell-free culture solution according to claim 5, wherein the aqueous slurry contains Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus), and Lactobacillus fermentum (Lactobacillus fermentum).

7. The serous fluid cell-free culture solution according to claims 1 to 6, wherein the preparation method of serous fluid in the step (1) comprises the following steps:

mixing 1-3 parts of freeze-dried vegetable powder and 5-15 parts of auxiliary materials according to parts by weight, adding 100 parts of boiling water, cooling, adding 1-3 parts of fermentation seeds, wherein the fermentation seeds contain one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum, and standing for 24-48 h at 30 ℃ to obtain the slurry.

8. The slurry-water cell-free culture solution according to claim 7, wherein the fermentation seed contains Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus), and Lactobacillus fermentum (Lactobacillus fermentum).

9. The aqueous acellular culture solution according to claim 1, wherein in step (3), the RPMI medium or the DMEM medium contains 10% fetal bovine serum.

10. Use of the serous acellular culture fluid according to claims 1-6 for the preparation of a medicament for the treatment of tumors of the digestive system.

11. The use according to claim 10, wherein the tumor of the digestive system is one or more of colorectal cancer, gastric cancer, liver cancer and pancreatic cancer.

12. A method for preparing a serous fluid cell-free culture solution is characterized in that serous fluid is taken and added into an RPMI culture medium or DMEM culture medium to carry out basic suspension precipitation, the pH is adjusted to be the optimum pH of cells, constant temperature culture is carried out, and a culture solution obtained by centrifugation is the serous fluid cell-free culture solution.

13. The method of claim 12, wherein the slurry contains one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

14. The method of preparing a slurry-water cell-free culture solution according to claim 12, wherein the slurry contains Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

15. The method for preparing the slurry-water cell-free culture solution according to claim 12, comprising the steps of:

(1) centrifuging the slurry, discarding the supernatant, resuspending the lower precipitate with MRS culture medium, culturing at constant temperature to obtain slurry primary culture, centrifuging, discarding the supernatant, and precipitating the slurry primary culture for use;

(2) suspending the serous fluid primary culture precipitate obtained in the step (1) by using an RPMI culture medium or a DMEM culture medium, adjusting the pH value to the optimum pH value of the cells, and culturing at constant temperature to obtain a serous fluid secondary culture;

(3) centrifuging the serous fluid secondary culture obtained in the step (2) and taking supernatant fluid;

(4) and (4) filtering the supernatant obtained in the step (3) by using a sterile filter membrane to obtain the serous acellular culture solution.

16. The method for preparing the cell-free culture solution in slurry water according to claim 15, wherein the slurry water contains one or more probiotics selected from Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

17. The method of preparing a slurry-water cell-free culture solution according to claim 16, wherein the slurry contains Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

18. The method for preparing a serum-free cell culture solution according to claims 12 to 17, wherein the method for preparing the serum in the step (1) specifically comprises the following steps:

mixing 1-3 parts of freeze-dried vegetable powder and 5-15 parts of auxiliary materials according to parts by weight, adding 100 parts of boiling water, cooling, adding 1-3 parts of fermentation seeds, wherein the fermentation seeds contain one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum, and standing for 24-48 h at 30 ℃ to obtain the slurry.

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a serous acellular culture solution for resisting digestive system tumors and a preparation method and application thereof.

Background

WHO statistics in 2017 showed a total of 5600 million population deaths in 2015, with 4000 million non-infectious disease (NCD) deaths accounting for 70% of the total population. Cancer accounts for 22% as the second cause of death by NCD. The global cancer statistics of 2012 show that worldwide colorectal cancer (CRC) ranks the top three in mortality and morbidity in developed countries, and ranks the top three in mortality and mortality of both men and women in developing countries. In recent years, the CRC morbidity and mortality in China are 14.2/10 ten thousand and 7.4/10 ten thousand, which are the fifth place. At the present stage, the incidence rate of colorectal cancer in China is obviously increased, the incidence rate of colorectal cancer is the highest among the population ages of 41-65, and the colorectal cancer shows a stable increasing trend. At present, the colon cancer is mainly treated by surgical resection and postoperative administration of therapeutic drugs such as 5-Fu and the like to prevent tumor recurrence. However, at present, first-line chemotherapy drugs such as 5-Fu generally act on the DNA structure of cells to influence the proliferation thereof, and lack selectivity and targeting.

The serous fluid is a fermented food with long history, and is particularly popular with people in northwest China. Research finds that the serofluid has a plurality of probiotic functions such as: promoting digestion, regulating blood fat, maintaining intestinal homeostasis and the like, and has potential application value in the prevention of diabetes, hypertension, digestive system cancer and the like. Serous fluid has been regarded as a good medicine for treating gastrointestinal and respiratory diseases in the traditional Chinese medicine book. The serous fluid contains rich probiotic resources, and microorganisms in the serous fluid are naturally domesticated and selected for thousands of years, so that some microorganisms with probiotic functional characteristics are stored, and the resources suitable for oriental intestinal probiotics provide wide resources for research and development. Researchers carry out systematic analysis on microbial communities of various types of pulp water prepared by adopting the traditional process, and find that one or more of probiotics such as lactobacillus plantarum, lactobacillus paracasei, staphylococcus xylosus, lactobacillus fermentum and the like are high-abundance flora of the pulp water, and even if the sources of the pulp water are different, the types of the dominant flora are basically the same.

The inventor unexpectedly finds that the serous water cell-free culture solution obtained by adding serous water into an RPMI culture medium or a DMEM culture medium for fermentation has strong inhibition effect on colorectal cancer, gastric cancer, liver cancer and pancreatic cancer cells, and has the characteristics of low toxicity and high efficiency.

Disclosure of Invention

In view of the above technical problems, a first object of the present invention is to provide a cell-free culture solution in slurry, which is prepared by the following method:

adding the plasma into RPMI culture medium or DMEM culture medium to perform basic suspension precipitation, performing constant temperature culture, and centrifuging to obtain a culture solution, namely the plasma-water cell-free culture solution.

Further, the slurry water contains one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

Further, the water slurry contains Lactobacillus plantarum, Lactobacillus paracasei, staphylococcus xylosus and Lactobacillus fermentum.

Further, the serous water cell-free culture solution is prepared by the following method:

(1) centrifuging the slurry, discarding the supernatant, resuspending the lower precipitate with MRS culture medium, culturing at constant temperature to obtain slurry primary culture, centrifuging, discarding the supernatant, and precipitating the slurry primary culture for use;

(2) suspending the serous fluid primary culture precipitate obtained in the step (1) by using an RPMI culture medium or a DMEM culture medium, adjusting the pH value to the optimum pH value of the cells, and culturing at constant temperature to obtain a serous fluid secondary culture;

(3) centrifuging the serous fluid secondary culture obtained in the step (2) and taking supernatant fluid;

(4) and (4) filtering the supernatant obtained in the step (3) by using a sterile filter membrane to obtain the serous acellular culture solution.

Further, the water slurry contains one or more probiotics of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

Further, the water slurry contains Lactobacillus plantarum, Lactobacillus paracasei, staphylococcus xylosus and Lactobacillus fermentum.

Further, the preparation method of the slurry in the step (1) specifically comprises the following steps:

mixing 1-3 parts of freeze-dried vegetable powder and 5-15 parts of auxiliary materials according to parts by weight, adding 100 parts of boiling water, cooling, adding 1-3 parts of fermentation seeds, wherein the fermentation seeds contain one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum, and standing at 30 ℃ for 24-48 hours to obtain the slurry.

Further, the fermentation seeds contain Lactobacillus plantarum, Lactobacillus paracasei, staphylococcus xylosus and Lactobacillus fermentum.

Further, in the step (3), the RPMI medium or the DMEM medium contains 10% fetal bovine serum.

The second purpose of the invention is to provide the application of the serous water cell-free culture solution in preparing the medicine for treating the tumor of the digestive system.

Furthermore, the digestive system tumor is one or more of colorectal cancer, gastric cancer, liver cancer and pancreatic cancer.

The third purpose of the invention is to provide a preparation method of the serous water acellular culture solution, which comprises the steps of adding serous water into RPMI culture medium or DMEM culture medium for basic suspension precipitation, adjusting the pH value to the optimum pH value of cells, carrying out constant temperature culture, and centrifuging to obtain the culture solution, namely the serous water acellular culture solution.

Further, the slurry water contains one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

Further, the water slurry contains Lactobacillus plantarum, Lactobacillus paracasei, staphylococcus xylosus and Lactobacillus fermentum.

Further, the preparation method comprises the following steps:

(1) centrifuging the slurry, discarding the supernatant, resuspending the lower precipitate with MRS culture medium, culturing at constant temperature to obtain slurry primary culture, centrifuging, discarding the supernatant, and precipitating the slurry primary culture for use;

(2) suspending the serous fluid primary culture precipitate obtained in the step (1) by using an RPMI culture medium or a DMEM culture medium, adjusting the pH value to the optimum pH value of the cells, and culturing at constant temperature to obtain a serous fluid secondary culture;

(3) centrifuging the serous fluid secondary culture obtained in the step (2) and taking supernatant fluid;

(4) and (4) filtering the supernatant obtained in the step (3) by using a sterile filter membrane to obtain the serous acellular culture solution.

Further, the water slurry contains one or more probiotics of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), staphylococcus xylosus (staphylococcus xylosus) and Lactobacillus fermentum (Lactobacillus fermentum).

Further, the water slurry contains Lactobacillus plantarum, Lactobacillus paracasei, staphylococcus xylosus and Lactobacillus fermentum.

Further, the preparation method of the slurry in the step (1) specifically comprises the following steps:

mixing 1-3 parts of freeze-dried vegetable powder and 5-15 parts of auxiliary materials according to parts by weight, adding 100 parts of boiling water, cooling, adding 1-3 parts of fermentation seeds, wherein the fermentation seeds contain one or more of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei subsp. paracasei), Staphylococcus xylosus (Staphylococcus xylosus) and Lactobacillus fermentum, and standing at 30 ℃ for 24-48 hours to obtain the slurry.

Compared with the prior art, the invention has the following advantages:

a. the serous fluid cell-free culture solution has strong inhibition effect on colorectal cancer, gastric cancer, liver cancer and pancreatic cancer.

b. The serous fluid cell-free culture solution is derived from food, is a main component of serous fluid, and is safe and small in side effect.

Drawings

FIG. 1 cell experiment of plasma water cell-free culture solution for anti-colorectal cancer

FIG. 2 experiment of anti-gastric cancer cells with aqueous slurry cell-free culture solution

FIG. 3 experiment of anti-hepatoma cells with serum-free cell culture medium

FIG. 4 plasma Water cell free culture solution experiment for pancreatic cancer cell resistance

Detailed Description

The scope of the present invention will be described below with reference to specific embodiments, but the scope of the present invention is not limited to the following examples.