Skull marrow artificial buffer solution as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a skull bone marrow artificial buffer solution and a preparation method and application thereof. The artificial buffer solution is mainly prepared from the following raw materials: LiCl, NaHCO3, MgCl2, CaCl2, NaNO2, Na2SO4, Na3PO4. 12H2O, NaCl and glucose, and the pH (Potential of Hydrogen) of the artificial buffer solution is 4 to 5. Components of the skull bone marrow artificial buffer solution are consistent with natural components of skull bone marrow fluid, are close to the pH value of normal skull bone marrow fluid, are more suitable for skull bone marrow cell and molecule related experiments, can be used as a solvent of an injection reagent in a skull, contributes to maintaining an internal environment of the skull bone marrow, and can be used for separating bone marrow cells from the skull bone marrow. The artificial buffer solution prepared by the invention is more suitable for being added into a cell culture medium as a basic buffer solution, so that a cell culture environment is close to an in-vivo environment, and the growth of skull bone marrow cells separated in vivo is facilitated. In addition, in-vitro culture of skull stem cells and stem cells in skull bone marrow injection related research is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a skull bone marrow artificial buffer and a preparation method and application thereof. Background Art

[0002] In biochemistry and neurobiology research, since the internal environment of cells and tissues in living bodies has certain osmotic pressure, pH, and ions, in vitro cell culture or injection of liquid into living experimental animals requires a buffer that can maintain the osmotic pressure and pH stability of cells or bodies. For example, distilled water does not have a salt balance effect and destroys the structure and biological properties of biological proteins; physiological saline does not have the function of adjusting pH, and cannot ensure that intact and active substances participate in biological reactions under optimal conditions, so phosphate buffered saline (PBS) is the first choice. PBS is the most widely used buffer in biochemical research. Its main components are disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride and potassium chloride. It is generally used as a solvent and plays the role of dissolving and protecting reagents. However, for cells or tissues that require higher conditions for biologically active substances, PBS cannot meet their needs. More components need to be added to the balanced buffer to ensure the most complete characteristics of biologically active substances. Therefore, Hank's balanced saline (HBSS) is often used in cell culture. In addition to containing similar components to PBS, it also contains glucose, disodium hydrogen disulfate, sodium formate, calcium chloride, magnesium chloride, etc. These additional components provide simple nutrition for cells.

[0003] Although HBSS balanced salt solution contains more ingredients such as glucose and calcium chloride than PBS buffer and is more suitable for cell culture, there has been no research on the chemical properties of rodent skull bone marrow fluid and the chemical composition of skull bone marrow fluid is unclear. Therefore, using PBS or HBSS as a buffer or cell culture fluid component in skull bone marrow cell and molecule-related research is difficult to maintain the optimal bioactive substances required by skull bone marrow cells. The skull bone marrow is a storage nest for central nervous system immune cells. If the internal environment for the survival of skull bone marrow cells is damaged, it may affect the immune regulation of the central nervous system.

[0004] In view of this, it is urgently needed in the art to develop an artificial skull bone marrow buffer suitable for maintaining a normal internal environment of the skull bone marrow and for use in skull bone marrow-related cell and molecular experiments. Summary of the invention

[0005] The present invention fills the gap in the prior art, and develops an artificial skull bone marrow buffer based on the test results of mouse skull bone marrow fluid. The skull bone marrow artificial buffer composition of the present invention is consistent with the natural composition of skull bone marrow fluid, close to the pH of normal skull bone marrow fluid, more suitable for skull bone marrow cell and molecule related experiments, can be used as a solvent for intracranial injection reagents, and helps to maintain the internal environment of the skull bone marrow. For bone marrow cells separated from skull bone marrow, the artificial buffer prepared by the present invention is more suitable as a basic buffer to be added to the cell culture medium, so that the cell culture environment is close to the in vivo environment, which is conducive to the growth of skull bone marrow cells isolated in vivo. In addition, it is helpful for the in vitro culture of stem cells in skull stem cells and skull bone marrow injection related research.

[0006] Specifically, the present invention is realized through the following technical solutions:

[0007] In a first aspect, the present invention provides a skull bone marrow artificial buffer, which is mainly prepared from the following raw materials: LiCl, NaHCO3, MgCl2, CaCl2, NaNO2, Na2SO4, Na3PO4·12H2O, NaCl and glucose.

[0008] As an optional method, in the above-mentioned skull bone marrow artificial buffer, 100 mL of the artificial buffer is mainly prepared from the following raw materials: 40-60 mg LiCl, 15-25 g NaHCO3, 200-300 mg MgCl2, 300-500 mg CaCl2, 60-85 mg NaNO2, 80-120 mg Na2SO4, 0.5-8 g Na3PO4·12H2O, 20-30 g NaCl and 25-45 mg glucose.

[0009] As an optional method, in the above-mentioned skull bone marrow artificial buffer, 100 mL of the artificial buffer is mainly prepared from the following raw materials: 50-60 mg LiCl, 18-22 g NaHCO3, 240-280 mg MgCl2, 380-420 mg CaCl2, 70-80 mg NaNO2, 95-110 mg Na2SO4, 1-5 g Na3PO4·12H2O, 22-28 g NaCl and 35-45 mg glucose.

[0010] As an optional method, in the above-mentioned skull bone marrow artificial buffer, 100 mL of the artificial buffer is mainly prepared from the following raw materials: 55.05 mg LiCl, 19.6 g NaHCO3, 262.45 mg MgCl2, 398.54 mg CaCl2, 74.99 mg NaNO2, 103.45 mg Na2SO4, 3.11 g Na3PO4·12H2O, 26.87 g NaCl and 39 mg glucose.

[0011] As an optional method, in the above-mentioned skull bone marrow artificial buffer, 100 mL of the artificial buffer is prepared from the raw materials mentioned above.

[0012] As an optional mode, in the above-mentioned skull bone marrow artificial buffer, the pH of the skull bone marrow artificial buffer is 4-5.

[0013] As an optional mode, in the above-mentioned skull bone marrow artificial buffer, the pH value of the skull bone marrow artificial buffer is 4.53.

[0014] As an optional mode, in the above-mentioned skull bone marrow artificial buffer, the skull bone marrow artificial buffer is a rodent skull bone marrow artificial buffer.

[0015] Preferably, the rodent is a mouse or a rat.

[0016] In a second aspect, the present invention provides a method for preparing the skull bone marrow artificial buffer according to the first aspect, the method comprising the following steps:

[0017] (1) Weigh LiCl and completely dissolve it in 50 mL of deionized water;

[0018] (2) Add NaHCO3, MgCl2, CaCl2, NaNO2, Na2SO4, Na3PO4·12H2O, NaCl and glucose in sequence, stirring each ingredient until completely dissolved before adding the next ingredient;

[0019] (3) Transfer the solution prepared in step (2) to a 100 mL volumetric flask and dilute to 100 mL with deionized water;

[0020] (4) Using a pH meter, measure the initial pH of the solution prepared in step (3). If the initial pH is higher than 4-5, add 0.1 mol / L HCl solution dropwise, stir continuously and measure the pH until it reaches 4-5. If the initial pH is lower than 4-5, add 0.1 mol / L NaOH solution dropwise, stir continuously and measure the pH until it reaches 4-5. Filter impurities and water-insoluble substances with filter paper, filter with a 0.22 μm bacterial filter and use for cell culture.

[0021] As an optional manner, in the above preparation method, the preparation method further comprises storing the prepared skull bone marrow artificial buffer at 4°C.

[0022] Preferably, in the above preparation method, the preparation method comprises the following steps:

[0023] (1) Weigh 55.05 mg of LiCl and completely dissolve it in 50 mL of deionized water;

[0024] (2) Add 19.6 g NaHCO3 and stir until completely dissolved;

[0025] (3) Add 262.45 mg MgCl2 and stir until completely dissolved;

[0026] (4) Add 398.54 mg CaCl2 and stir until completely dissolved;

[0027] (5) Add 74.99 mg NaNO2 and stir until completely dissolved;

[0028] (6) Add 103.45 mg Na2SO4 and stir until completely dissolved;

[0029] (7) Add 3.11 g Na3PO4·12H2O and stir until completely dissolved;

[0030] (8) Add 26.87 g NaCl and stir until completely dissolved;

[0031] (9) Add 39 mg of glucose and stir until completely dissolved;

[0032] (10) Transfer the solution to a 100 mL volumetric flask and dilute to 100 mL with deionized water;

[0033] (11) Use a pH meter to measure the initial pH of the solution. If the initial pH is higher than 4.53, add 0.1 mol / L HCl solution dropwise, stir continuously and measure the pH until it reaches 4.53. If the initial pH is lower than 4.53, add 0.1 mol / L NaOH solution dropwise, stir continuously and measure the pH until it reaches 4.53. Filter impurities and water-insoluble substances with filter paper, and filter with a 0.22 μm bacterial filter before using for cell culture.

[0034] In the third aspect, the present invention provides the artificial skull bone marrow buffer described in the first aspect or the artificial skull bone marrow buffer prepared by the preparation method described in the second aspect for use in skull bone marrow cell and molecule related experiments, as a solvent for intracranial injection reagents, and in the cultivation of bone marrow cells isolated from the skull bone marrow.

[0035] As an option, in the above use, the bone marrow cells are bone marrow stem cells.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] Since the artificial skull bone marrow buffer of the present invention is based on the test results of mouse skull bone marrow fluid, it is prepared according to different ion concentrations, and the pH is adjusted to the pH range of skull bone marrow fluid. Compared with the PBS buffer and HBSS buffer commonly used in current neurology-related animal experiments, the artificial skull bone marrow buffer of the present invention contains various ions and sugars for the growth of bone marrow cells in the in vivo environment, does not contain potassium ions that are not detected in the skull bone marrow fluid components, and the buffer of the present invention is acidic and more suitable for skull bone marrow cells. Therefore, the artificial skull bone marrow buffer of the present invention is more suitable for the skull bone marrow to maintain a normal internal environment and promote the growth and proliferation of skull bone marrow cells, and is more suitable as a buffer for skull bone marrow-related cell and molecular experimental research, and is also more suitable as a drug solvent in skull bone marrow injection drug delivery technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Apoptosis and survival of skull bone marrow cells after culture in PBS and the novel buffer of the present invention. Figure 1 a: After culturing skull bone marrow cells in PBS buffer, they were stained with 7-AAD solution and then detected by flow cytometry to measure the 7-AAD fluorescence intensity and the proportion of apoptotic cells and living cells in total cells. Figure 1 b: After culturing skull bone marrow cells with the novel buffer of the present invention, staining with 7-AAD solution was performed, and then the 7-AAD fluorescence intensity was detected by flow cytometry to detect the proportion of apoptotic cells and living cells in total cells. Figure 1 c: The percentage of live cells in the skull bone marrow extracted cells of the two groups, n=3 / group, *P<0.05. Figure 1 d: The percentage of apoptotic cells in the skull bone marrow extracted cells of the two groups, n=3 / group, ***P<0.001. DETAILED DESCRIPTION

[0039] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0042] Unless otherwise specified, all percentages and parts in the present invention are percentages and parts by weight.

[0043] The preparation of the artificial buffer solution of skull bone marrow in the present invention is based on 100 mL of solution as an example, and each solute is weighed. If a buffer solution of different volumes needs to be prepared, the solute weight can be modified according to the volume ratio of the solution to be prepared and the 100 mL of solution prepared in the method of the present invention. The mass of the solute in the prepared solution is the mass of the solute required for 100 mL of solution multiplied by the volume ratio.

[0044] Preparation Example:

[0045] The formula of the skull bone marrow artificial buffer solution of the present invention is based on the test results of mouse skull bone marrow fluid and is prepared by adding inorganic salts into sterile deionized water according to different ion concentrations.

[0046] The preliminary experiments detected the cation concentration, anion concentration, glucose concentration and pH value in the skull bone marrow of normal 8-10 week old C57BL / 6J male mice. The experimental results showed that the cations in the skull bone marrow fluid of mice include: Li + (Concentration: 0.09 mg / mL); Na + (Concentration: 165.50 mg / mL); Mg 2+ (Concentration: 0.67 mg / mL); Ca 2+ (Concentration: 1.44mg / mL), anions include: HCO3 - (Concentration: 142.30 mg / mL); Cl - (Concentration: 423.70mg / mL); NO2 - (Concentration: 0.50mg / mL); SO4 2- (Concentration: 0.70mg / mL); PO4 3- (Concentration: 7.75 mg / mL), glucose concentration: 0.39 mg / mL, pH 4.53.

[0047] The method for preparing the artificial buffer solution of skull bone marrow of the present invention is as follows:

[0048] Taking 100mL of skull bone marrow artificial buffer as an example, the preparation steps are as follows:

[0049] (1) Weigh 55.05 mg of LiCl and completely dissolve it in 50 mL of deionized water;

[0050] (2) Add 19.6 g NaHCO3 and stir until completely dissolved;

[0051] (3) Add 262.45 mg MgCl2 and stir until completely dissolved;

[0052] (4) Add 398.54 mg CaCl2 and stir until completely dissolved;

[0053] (5) Add 74.99 mg NaNO2 and stir until completely dissolved;

[0054] (6) Add 103.45 mg Na2SO4 and stir until completely dissolved;

[0055] (7) Add 3.11 g Na3PO4·12H2O and stir until completely dissolved;

[0056] (8) Add 26.87 g NaCl and stir until completely dissolved;

[0057] (9) Add 39 mg of glucose and stir until completely dissolved;

[0058] (10) Transfer the solution to a 100 mL volumetric flask and dilute to 100 mL with deionized water;

[0059] (11) Use a pH meter to measure the initial pH of the solution. If the initial pH is higher than 4.53, add 0.1 mol / L HCl solution dropwise, stir continuously and measure the pH until it reaches 4.53. If the initial pH is lower than 4.53, add 0.1 mol / L NaOH solution dropwise, stir continuously and measure the pH until it reaches 4.53. Filter impurities and water-insoluble substances with filter paper, and filter with a 0.22 μm bacterial filter before using for cell culture.

[0060] The prepared skull bone marrow artificial buffer solution was stored in a 4°C refrigerator.

[0061] Effect embodiment:

[0062] 1. Experimental purpose:

[0063] The effects of the novel skull bone marrow artificial buffer and PBS buffer in skull bone marrow cell culture were compared.

[0064] 2. Experimental methods:

[0065] The skull bone marrow cells of 8-10 week old male C57 mice were extracted using the novel skull bone marrow artificial buffer (prepared by the above preparation example) and PBS buffer, and 10% fetal bovine serum was added and cultured for 12 hours, and then 7-AAD dead cell dye was used to distinguish the percentage of apoptotic cells and live cells in the cells to be detected. The skull bone marrow cells of each sample were collected using a flow cytometer, and single cells were first circled, and then single cells were divided into apoptotic cells (weak fluorescence intensity) and live cells (medium fluorescence intensity) according to the intensity of 7-AAD fluorescence. The experimental results were recorded, and statistical analysis was performed using GraphPad Prism (8.0.2) software. The data were expressed as mean ± standard deviation, and two independent samples were tested using t test. The difference was statistically significant when P < 0.05.

[0066] 3. Experimental results:

[0067] The experimental results are as follows Figure 1 As shown in a-1d, after the skull bone marrow cells were extracted and cultured with the new skull bone marrow artificial buffer of the present invention, the proportion of live cells was 34.76±9.07%, which was higher than 13.64±1.39% of PBS buffer (P=0.0163) ( Figure 1 c); the proportion of apoptotic cells in the new buffer group was 15.82±9.34%, which was lower than 63.41±1.01% in the PBS buffer group (P=0.0009). Figure 1 d).

[0068] 4. Experimental conclusion:

[0069] Compared with PBS buffer, the use of the novel skull bone marrow artificial buffer of the present invention is more conducive to cell survival and reduces cell apoptosis in extracting and culturing mouse skull bone marrow cells.

[0070] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A skull bone marrow artificial buffer, characterized in that: The artificial buffer is mainly prepared from the following raw materials: LiCl, NaHCO3, MgCl2, CaCl2, NaNO2, Na2SO4, Na3PO4·12H2O, NaCl and glucose.

2. The skull bone marrow artificial buffer according to claim 1, characterized in that: 100 mL of the artificial buffer is mainly prepared from the following raw materials: 40-60 mg LiCl, 15-25 g NaHCO3, 200-300 mg MgCl2, 300-500 mg CaCl2, 60-85 mg NaNO2, 80-120 mg Na2SO4, 0.5-8 g Na3PO4·12H2O, 20-30 g NaCl and 25-45 mg glucose.

3. The skull bone marrow artificial buffer according to claim 2, characterized in that: 100 mL of the artificial buffer is mainly prepared from the following raw materials: 50-60 mg LiCl, 18-22 g NaHCO3, 240-280 mg MgCl2, 380-420 mg CaCl2, 70-80 mg NaNO2, 95-110 mg Na2SO4, 1-5 g Na3PO4·12H2O, 22-28 g NaCl and 35-45 mg glucose.

4. The skull bone marrow artificial buffer according to claim 1, characterized in that: The pH of the skull bone marrow artificial buffer is 4-5.

5. The skull bone marrow artificial buffer according to claim 1, characterized in that: The skull bone marrow artificial buffer is a rodent skull bone marrow artificial buffer.

6. The method for preparing the skull bone marrow artificial buffer according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) Weigh LiCl and completely dissolve it in 50 mL of deionized water; (2) Add NaHCO3, MgCl2, CaCl2, NaNO2, Na2SO4, Na3PO4·12H2O, NaCl and glucose in sequence, stirring each ingredient until completely dissolved before adding the next ingredient; (3) Transfer the solution prepared in step (2) to a 100 mL volumetric flask and dilute to 100 mL with deionized water; (4) Using a pH meter, measure the initial pH of the solution prepared in step (3). If the initial pH is higher than 4-5, add 0.1 mol / L HCl solution dropwise, stir continuously and measure the pH until it reaches 4-5. If the initial pH is lower than 4-5, add 0.1 mol / L NaOH solution dropwise, stir continuously and measure the pH until it reaches 4-5. Filter impurities and water-insoluble substances with filter paper, filter with a 0.22 μm bacterial filter and use for cell culture.

7. The preparation method according to claim 6, characterized in that: The preparation method further comprises storing the prepared skull bone marrow artificial buffer at 4°C.

8. The artificial skull bone marrow buffer according to any one of claims 1 to 5 or the artificial skull bone marrow buffer prepared by the preparation method according to claim 6 or claim 7 is used in skull bone marrow cell and molecule related experiments, as a solvent for intracranial injection reagents, and in the cultivation of bone marrow cells separated from skull bone marrow.

9. The use according to claim 8, characterized in that: The bone marrow cells are bone marrow stem cells.

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