Method for measuring neurotrophic factor content in cell lysate and culture supernatant of hAMCs cultured in vitro

Abstract

The application discloses a cell lysate of hAMCs cultured in vitro and a determination method of neurotrophic factor content of culture supernatant, wherein the hAMCs are cultured in vitro, when the cells grow to 80%-90% confluence, subculture is carried out, until the cells reach 80% confluence, then centrifugation is carried out to obtain the culture supernatant of hAMCs, PBS buffer is added to the hAMCs from which the supernatant is removed, centrifugal cleaning is carried out, the cells are broken in an ultrasonic cell disrupter to obtain the cell lysate of hAMCs, and finally, the neurotrophic factor content of the culture supernatant and the cell lysate of hAMCs is determined by using an ELISA method.

Description

Technical Field

[0001] This invention relates to a method for determining the content of neurotrophic factors in cell lysates and culture supernatants of cultured hAMCs in vitro. Background Technology

[0002] The human amnion is a semi-transparent membrane that surrounds the fetus by the placenta. Its surface lacks nerves, blood vessels, muscles, and lymphatic tissues. Amniotic epithelial cells (hAEC) and amniotic mesenchymal stem cells (hAMSCs) can be isolated from it. Amniotic mesenchymal stem cells not only have significant self-renewal capacity and multi-lineage differentiation potential across germ layers, but also, compared to embryonic stem cells and other adult stem cells, are abundant, readily available, free from ethical controversies, have low immunogenicity, and pose no tumorigenic risk. Furthermore, they possess strong paracrine or autocrine functions by secreting various bioactive factors such as immunosuppressive factors and neurotrophic factors. These characteristics endow them with immeasurable clinical application value in tissue engineering, cell therapy, gene therapy, and other related regenerative medicine fields.

[0003] The expression and secretion of neurotrophic factors by cultured amniotic mesenchymal stem cells (hAMSCs) are crucial for neuronal growth and survival. Neurotrophic factors are protein molecules produced by nerve-innervated tissues (such as muscles) and astrocytes. They typically enter nerve endings via receptor-mediated endocytosis and then reach the cell body via retrograde axonal transport, promoting the synthesis of related proteins and thus supporting neuronal growth, development, and functional integrity. Currently, there is no reliable method for measuring the neurotrophic factor content in hAMSCs. Therefore, accurately measuring the neurotrophic factor content secreted by amniotic mesenchymal stem cells is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Methods for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs in vitro.

[0007] Includes the following steps:

[0008] Step (A): Collect human placental amniotic membrane tissue, digest it with trypsin at least twice, then stop the trypsin digestion with DMEM / F12 culture medium with a VDMEM:VF12 ratio of 1:1, and finally digest it again with DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I to obtain a cell suspension. Filter the cell suspension to obtain a single-cell suspension.

[0009] Step (B): Add the single-cell suspension to a centrifuge tube containing culture medium and centrifuge to remove the supernatant. Then, inoculate the cells into a new culture medium and culture them. Change the culture medium within 24-48 hours. When the cells grow to 80%-90% confluence, passage them until the cells reach 80% confluence to obtain in vitro cultured hAMCs. Then, continue to passage and expand the hAMCs.

[0010] Step (C): Take the culture medium containing hAMCs from step (B) and add it to a centrifuge tube for centrifugation. Then, take the supernatant from the centrifuge tube into a dish for later use to obtain the culture supernatant of hAMCs.

[0011] Step (D): Take the centrifuge tube from step (C) after removing the supernatant, add PBS buffer and centrifuge to wash 1-3 times. After washing, place it in an ultrasonic cell disruptor to disrupt the cells and obtain the cell lysate of hAMCs. Then, aspirate the cell lysate of hAMCs into a dish for later use.

[0012] Step (E) involves using ELISA to determine the neurotrophic factor content in the culture supernatant and cell lysate of hAMCs.

[0013] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs, in step (A), the final concentration of trypsin is 0.1-2.5 g / L, and trypsin digests human placental amniotic tissue at room temperature for 30-60 min.

[0014] The final concentration of collagenase IV was 1.0-2.0 g / L, and the final concentration of deoxyribonuclease I was 0.1-0.2 g / L. The human placental amniotic tissue was re-digested in DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I at 37°C for 30-60 min.

[0015] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs, in step (B), the cell culture medium is a mixture of L-DMEM with 10% FBS, 0.25% trypsin, 5.5% NaHCO3, and penicillin-streptomycin.

[0016] Centrifuge the single-cell suspension in centrifuge tubes at 1500-2000 rpm for 15-30 min;

[0017] Single-cell suspensions were inoculated into new culture medium and cultured in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 (volume fraction).

[0018] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs, in step (C), the culture medium containing hAMCs is centrifuged in a centrifuge tube at 1200-1800 rpm for 8-10 min.

[0019] The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs in vitro includes the following steps in step (D): taking the centrifuge tube from which the supernatant was removed in step (C), adding PBS buffer, and centrifuging to wash 1-3 times, including the following process:

[0020] (D1) Take the centrifuge tube after removing the supernatant, add 8-12 ml of PBS buffer for the first time, and wash at 3000-4000 rpm for 5-8 min to remove the PBS buffer.

[0021] (D2) Add 6-8 ml of PBS buffer for the second time, and wash at 2000-3000 rpm for 2-4 min to remove the PBS buffer;

[0022] (D3) Add 6-8 ml of PBS buffer for the third time, and wash at 1200-2000 rpm for 2-4 min to remove the PBS buffer.

[0023] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs, in step (D), after cleaning the centrifuge tubes after removing the supernatant, the conditions for cell disruption in an ultrasonic cell disruptor are: power 50W-150W, working time 1s, interval 2s, temperature below 30℃ (ice bath), and total ultrasonic duration 10-30min.

[0024] The aforementioned method for determining the neurotrophic factor content in the cell lysate and culture supernatant of cultured hAMCs, step (E), involves using ELISA to determine the neurotrophic factor content in the culture supernatant and cell lysate of hAMCs, respectively, and includes the following steps:

[0025] (E1) Take the culture supernatant and cell lysis buffer of hAMCs, and use serum-free cell culture medium as a control. Dilute with distilled water to obtain dilution a, dilution b and dilution c. Dilution a is the culture supernatant of hAMCs, dilution b is the cell lysis buffer of hAMCs and dilution c is the serum-free cell culture medium.

[0026] (E2) Take three sets of ELISA plates, with at least 3 plates in each set. Add diluent a, diluent b and diluent c to the wells of the three sets of ELISA plates respectively. After mixing the ELISA plates thoroughly, let them stand at 37°C for 120-140 minutes.

[0027] (E3) Wash the microplate thoroughly with washing solution 4-6 times, then filter dry;

[0028] (E4) Add the first antibody working solution to each well of the microplate, mix the microplate thoroughly, and let it stand at 37°C for 60-80 minutes. Repeat (E3).

[0029] (E5) Add enzyme-labeled antibody working solution to each well of the ELISA plate, let the ELISA plate stand at 37℃ for 30-40 min, and then repeat (E3).

[0030] (E6) Add substrate working solution to each well of the microplate, let the microplate stand in the dark at 37°C for 15-20 minutes, and then add stop solution and mix well.

[0031] (E7) Within 30 minutes, use an ELISA reader to detect the absorbance values ​​of the three ELISA plates at 450 nm. Plot the absorbance values ​​of the standards 2000, 1000, 500, 250, 125, 62.5, 31.2, and 0 pg / ml on graph paper to draw a standard curve.

[0032] (E8) Based on the absorbance value detected by the microplate reader in (E7), find the corresponding neurotrophic factor content on the standard curve, and then multiply it by the dilution factor to obtain the neurotrophic factor content of hAMCs culture supernatant and cell lysate.

[0033] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs (E1), the culture supernatant and cell lysate of hAMCs, as well as the serum-free cell culture medium, are diluted 10-15 times with distilled water.

[0034] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs (E3), the washing buffer is prepared by diluting concentrated washing buffer with redistilled water at a ratio of 1:20.

[0035] In the aforementioned method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs, in steps (E2)-(E6), the amount of diluent a, diluent b, diluent c, first antibody working solution, enzyme-labeled antibody working solution, substrate working solution, and stop solution added to the wells of the ELISA plate is 100-200 μL.

[0036] The beneficial effects of this invention are as follows: The method for determining the neurotrophic factor content in the cell lysate and culture supernatant of cultured hAMCs in vitro involves culturing hAMCs in vitro until the cells reach 80%-90% confluence, then passaged until the cells reach 80% confluence. The culture supernatant of hAMCs is obtained by centrifugation, and the hAMCs after removing the supernatant are washed with PBS buffer by centrifugation. The cells are then disrupted in an ultrasonic cell disruptor to obtain the cell lysate of hAMCs. Finally, the neurotrophic factor content in the culture supernatant and cell lysate of hAMCs is determined by ELISA. This method is easy to operate, has a small detection error, and can effectively and accurately determine the neurotrophic factor content of hAMCs. Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments.

[0038] The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to the present invention includes the following steps:

[0039] Step (A): Collect human placental amniotic membrane tissue, digest it with trypsin at least twice, then stop the trypsin digestion with DMEM / F12 culture medium with a VDMEM:VF12 ratio of 1:1, and finally digest it again with DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I to obtain a cell suspension. Filter the cell suspension to obtain a single-cell suspension.

[0040] In step (A), the final concentration of trypsin is 0.1-2.5 g / L, and trypsin digests human placental amniotic tissue at room temperature for 30-60 min;

[0041] The final concentration of collagenase IV was 1.0-2.0 g / L, and the final concentration of deoxyribonuclease I was 0.1-0.2 g / L. The human placental amniotic tissue was re-digested in DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I at 37°C for 30-60 min.

[0042] Step (B): Add the single-cell suspension to a centrifuge tube containing culture medium and centrifuge to remove the supernatant. Then, inoculate the cells into a new culture medium and culture them. Change the culture medium within 24-48 hours. When the cells grow to 80%-90% confluence, passage them until the cells reach 80% confluence to obtain in vitro cultured hAMCs. Then, continue to passage and expand the hAMCs.

[0043] In step (B), the cell culture medium is a mixture of L-DMEM, 10% FBS, 0.25% trypsin, 5.5% NaHCO3, and penicillin-streptomycin.

[0044] Centrifuge the single-cell suspension in centrifuge tubes at 1500-2000 rpm for 15-30 min;

[0045] Single-cell suspensions were inoculated into new culture medium and cultured in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 (volume fraction).

[0046] Step (C): Take the culture medium containing hAMCs from step (B) and add it to a centrifuge tube for centrifugation. Then, take the supernatant from the centrifuge tube into a dish for later use to obtain the culture supernatant of hAMCs.

[0047] In step (C), the culture medium containing hAMCs is centrifuged in centrifuge tubes at 1200-1800 rpm for 8-10 min;

[0048] Step (D): Take the centrifuge tube from step (C) after removing the supernatant, add PBS buffer and centrifuge to wash 1-3 times. After washing, place it in an ultrasonic cell disruptor to disrupt the cells and obtain the cell lysate of hAMCs. Then, aspirate the cell lysate of hAMCs into a dish for later use.

[0049] In step (D), take the centrifuge tube from which the supernatant was removed in step (C), add PBS buffer, and centrifuge to wash 1-3 times, including the following process:

[0050] (D1) Take the centrifuge tube after removing the supernatant, add 8-12 ml of PBS buffer for the first time, and wash at 3000-4000 rpm for 5-8 min to remove the PBS buffer.

[0051] (D2) Add 6-8 ml of PBS buffer for the second time, and wash at 2000-3000 rpm for 2-4 min to remove the PBS buffer;

[0052] (D3) Add 6-8 ml of PBS buffer for the third time, and wash at 1200-2000 rpm for 2-4 min to remove the PBS buffer;

[0053] In step (D), after cleaning, the centrifuge tubes after removing the supernatant are placed in an ultrasonic cell disruptor under the following conditions: power 50W-150W, working time 1s, interval 2s, temperature below 30℃, ice bath, and total ultrasonic time 10-30min.

[0054] Step (E): The content of neurotrophic factors in the culture supernatant and cell lysate of hAMCs was determined by ELISA.

[0055] Step (E) involves using ELISA to determine the neurotrophic factor content in the culture supernatant and cell lysate of hAMCs, including the following procedures:

[0056] (E1) Take the culture supernatant and cell lysis buffer of hAMCs, and use serum-free cell culture medium as a control. Dilute with distilled water to obtain dilution a, dilution b and dilution c. Dilution a is the culture supernatant of hAMCs, dilution b is the cell lysis buffer of hAMCs and dilution c is the serum-free cell culture medium.

[0057] In (E1), the culture supernatant and cell lysate of hAMCs and serum-free cell culture medium were diluted 10-15 times with distilled water;

[0058] (E2) Take three sets of ELISA plates, with at least 3 plates in each set. Add diluent a, diluent b and diluent c to the wells of the three sets of ELISA plates respectively. After mixing the ELISA plates thoroughly, let them stand at 37°C for 120-140 minutes.

[0059] (E3) Wash the microplate thoroughly with washing solution 4-6 times, then filter dry;

[0060] In (E3), the washing solution is prepared by diluting concentrated washing solution with redistilled water at a ratio of 1:20;

[0061] (E4) Add the first antibody working solution to each well of the microplate, mix the microplate thoroughly, and let it stand at 37°C for 60-80 minutes. Repeat (E3).

[0062] (E5) Add enzyme-labeled antibody working solution to each well of the ELISA plate, let the ELISA plate stand at 37℃ for 30-40 min, and then repeat (E3).

[0063] (E6) Add substrate working solution to each well of the microplate, let the microplate stand in the dark at 37°C for 15-20 minutes, and then add stop solution and mix well.

[0064] (E7) Within 30 minutes, use an ELISA reader to detect the absorbance values ​​of the three ELISA plates at 450 nm. Plot the absorbance values ​​of the standards 2000, 1000, 500, 250, 125, 62.5, 31.2, and 0 pg / ml on graph paper to draw a standard curve.

[0065] (E8) Based on the absorbance value detected by the microplate reader in (E7), find the corresponding neurotrophic factor content on the standard curve, and then multiply it by the dilution factor to obtain the neurotrophic factor content of hAMCs culture supernatant and cell lysate.

[0066] In (E2)-(E6), the amount of diluent a, diluent b, diluent c, first antibody working solution, enzyme-labeled antibody working solution, substrate working solution and stop solution added to the wells of the ELISA plate is 100-200ul.

[0067] An example of specifically determining the neurotrophic factor content based on the method for measuring the neurotrophic factor content in cell lysates and culture supernatants of cultured hAMCs in vitro:

[0068] 1. Human placental amniotic membrane tissue was collected and digested with trypsin at a final concentration of 2.5 g / L for 40 min at room temperature. This was repeated 3 times. Trypsin was then stopped with DMEM / F12 culture medium with a VDMEM:VF12 ratio of 1:1. Finally, the tissue was digested again with DMEM / F12 culture medium containing collagenase IV at a final concentration of 2.0 g / L and deoxyribonuclease I at a final concentration of 0.2 g / L to obtain a cell suspension. The cell suspension was filtered to obtain a single-cell suspension.

[0069] 2. Prepare the culture medium. The cell culture medium is a mixture of L-DMEM, 10% FBS, 0.25% trypsin, 5.5% NaHCO3, and penicillin-streptomycin. Add the single-cell suspension to a centrifuge tube containing the culture medium and centrifuge at 2000 rpm for 30 min. Remove the supernatant, inoculate the single-cell suspension into a new culture medium, and incubate at 37°C, saturated humidity, and 5% CO2 for 3 days. Change the culture medium within 24 hours. When the cells grow to 85% confluence, passage them until the cells reach 80% confluence to obtain in vitro cultured hAMCs. Then, continue to passage and expand the hAMCs.

[0070] 3. Add the culture medium containing hAMCs to a centrifuge tube and centrifuge at 1800 rpm for 10 min. Then, transfer the supernatant from the centrifuge tube to a dish for later use to obtain the culture supernatant of hAMCs.

[0071] 4. Take the centrifuge tube after removing the supernatant, add PBS buffer and centrifuge to wash 3 times. After washing, place it in an ultrasonic cell disruptor to disrupt the cells. Set the cell disruption conditions as follows: power 50W-150W, working time 1s, interval 2s, temperature below 30℃, ice bath, and total sonication time 30min. Obtain the cell lysate of hAMCs and aspirate the cell lysate of hAMCs into a dish for later use.

[0072] The specific cleaning process is as follows:

[0073] 4-1. Take the centrifuge tube after removing the supernatant, add 12 ml of PBS buffer for the first time, wash at 4000 rpm for 8 min, and remove the PBS buffer.

[0074] 4-2. Add 6 ml of PBS buffer for the second time, and wash at 2500 rpm for 4 min to remove the PBS buffer;

[0075] 4-3. Add 6 ml of PBS buffer for the third time, wash at 1200 rpm for 2 min to remove the PBS buffer;

[0076] 5. The content of neurotrophic factors in the culture supernatant and cell lysate of hAMCs was determined by ELISA.

[0077] The specific process is as follows:

[0078] 5-1. Take the culture supernatant and cell lysis buffer of hAMCs, and use serum-free cell culture medium as a control. Dilute with distilled water 10 times to obtain dilution a, dilution b and dilution c. Dilution a is the culture supernatant of hAMCs, dilution b is the cell lysis buffer of hAMCs and dilution c is the serum-free cell culture medium.

[0079] 5-2. Take three sets of microplates, three in each set. Add diluent a, diluent b and diluent c to the wells of the three sets of microplates respectively, and add 100 μL to each well. Mix the microplates thoroughly and let them stand at 37°C for 120 min.

[0080] 5-3. Wash the microplate thoroughly 5 times with washing solution, which is a concentrated washing solution diluted 1:20 with redistilled water, and then filter dry.

[0081] 5-4. Add 100 μL of the first antibody working solution to each well of the microplate, mix the microplate thoroughly, let it stand at 37°C for 60 min, and then wash the plate again.

[0082] 5-5. Add 100 μL of enzyme-labeled antibody working solution to each well of the ELISA plate, incubate the ELISA plate at 37°C for 30 min, and then repeat the washing process.

[0083] 5-6. Add 100 μL of substrate working solution to each well of the microplate, incubate the microplate in the dark at 37°C for 15 min, and then add 100 μL of stop solution and mix well.

[0084] 5-7. Within 30 minutes, use an ELISA reader to detect the absorbance values ​​of the three ELISA plates at 450 nm. Plot the absorbance values ​​of the standards (2000, 1000, 500, 250, 125, 62.5, 31.2, 0 pg / ml) on graph paper to create a standard curve.

[0085] 5-8. Based on the absorbance values ​​detected by the ELISA reader, find the corresponding neurotrophic factor content on the standard curve, and then multiply by the dilution factor to obtain the neurotrophic factor content of hAMCs culture supernatant and cell lysate. Specifically, the neurotrophic factors are NT-3 neurotrophic factor, NGF nerve growth factor, BDNF brain-derived neurotrophic factor, and bFGF fibroblast growth factor.

[0086] The absorbance values ​​of neurotrophic factors in hAMC culture supernatant and cell lysate obtained according to the above detection process are shown in the table below at 450 nm:

[0087] Serum-free cell culture medium hAMCs cell lysate hAMCs culture supernatant NT-3 three absorbance values 1.105 1.133 1.307 1.55 1.559 1.642 1.422 1.394 1.211 NGF three groups of absorbance values 0.302 0.307 0.358 0.991 0.875 0.854 1.164 1.106 1.216 BDNF three groups of absorbance values 0.195 0.211 0.203 0.278 0.308 0.247 0.306 0.292 0.257 bFGF three groups of absorbance values 0.141 0.185 0.125 0.415 0.421 0.411 0.432 0.448 0435

[0088] Based on the absorbance values ​​of NT-3, NGF, BDNF, and bFGF detected at 450 nm, a standard curve was plotted on graph paper with the absorbance values ​​of standards 2000, 1000, 500, 250, 125, 62.5, 31.2, and 0 pg / ml as the x-axis and the absorbance values ​​as the y-axis. The corresponding contents were then found on the standard curve graph and multiplied by the dilution factor to obtain the specific NT-3, NGF, BDNF, and bFGF contents of the hAMC culture supernatant and cell lysate.

[0089] In summary, the method for determining the neurotrophic factor content in the cell lysate and culture supernatant of cultured hAMCs according to the present invention involves culturing hAMCs in vitro until the cells reach 80%-90% confluence, then passaged until the cells reach 80% confluence. The culture supernatant of hAMCs is then obtained by centrifugation, and the hAMCs after removing the supernatant are washed with PBS buffer by centrifugation. The cells are then disrupted using an ultrasonic cell disruptor to obtain the cell lysate. Finally, the neurotrophic factor content in the culture supernatant and cell lysate of hAMCs is determined using ELISA. This method is easy to operate, has small detection errors, and can effectively and accurately determine the neurotrophic factor content of hAMCs.

[0090] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the content of neurotrophic factors in cell lysate and culture supernatant of cultured hAMCs in vitro, characterized in that, Includes the following steps: Step (A): Collect human placental amniotic membrane tissue, digest it with trypsin at least twice, then stop the trypsin digestion with DMEM / F12 culture medium with a VDMEM:VF12 ratio of 1:1, and finally digest it again with DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I to obtain a cell suspension. After filtration, obtain a single cell suspension. Step (B): Add the single-cell suspension to a centrifuge tube containing culture medium and centrifuge to remove the supernatant. Then, inoculate the cells into a new culture medium and culture them. When the cells grow to 80%-90% confluence, passage them until the cells reach 80% confluence to obtain in vitro cultured hAMCs. Continue passage culture to expand the cells. Step (C): Take the culture medium containing hAMCs from step (B) and add it to a centrifuge tube for centrifugation. Then, take the supernatant from the centrifuge tube into a dish for later use to obtain the culture supernatant of hAMCs. Step (D): Take the centrifuge tube from which the supernatant was removed in step (C), add PBS buffer and centrifuge to wash, then place it in an ultrasonic cell disruptor to disrupt the cells and obtain the cell lysate of hAMCs. Pipette the cell lysate of hAMCs into a dish for later use. Step (E): The content of neurotrophic factors in the culture supernatant and cell lysate of hAMCs was determined by ELISA. Specifically: (E1) Take the culture supernatant and cell lysis buffer of hAMCs, and use serum-free cell culture medium as a control. Dilute with distilled water to obtain dilutions a, b, and c. Dilution a is the culture supernatant of hAMCs, dilution b is the cell lysis buffer of hAMCs, and dilution c is the serum-free cell culture medium. (E2) Take three sets of ELISA plates, with at least 3 plates in each set. Add diluents a, b, and c to the wells of the three sets of ELISA plates respectively. Mix the ELISA plates thoroughly and let them stand at 37°C for 120-140 minutes. (E3) Wash the microplate thoroughly with washing solution 4-6 times, then filter dry; (E4) Add the first antibody working solution to each well of the microplate, mix the microplate thoroughly, and let it stand at 37°C for 60-80 min. Repeat (E3). (E5) Add enzyme-labeled antibody working solution to each well of the ELISA plate, and let the ELISA plate stand at 37°C for 30-40 min. Repeat (E3). (E6) Add substrate working solution to each well of the microplate, incubate the microplate in the dark at 37°C for 15-20 minutes, and then add stop solution and mix well. (E7) Within 30 minutes, use an ELISA reader to detect the absorbance of the three ELISA plates at 450 nm. Plot the standard curve on graph paper with the standard as the x-axis and the absorbance as the y-axis. (E8) Based on the absorbance value detected by the microplate reader in (E7), find the corresponding neurotrophic factor content on the standard curve, and then multiply it by the dilution factor to obtain the neurotrophic factor content of hAMCs culture supernatant and cell lysate.

2. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to claim 1, characterized in that, In step (A), the final concentration of trypsin is 0.1-2.5 g / L, and trypsin digests human placental amniotic tissue at room temperature for 30-60 min; The final concentration of collagenase IV was 1.0-2.0 g / L, and the final concentration of deoxyribonuclease I was 0.1-0.2 g / L. The human placental amniotic tissue was re-digested in DMEM / F12 culture medium containing collagenase IV and deoxyribonuclease I at 37°C for 30-60 min.

3. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to claim 1, characterized in that, In step (B), the cell culture medium is a mixture of L-DMEM, 10% FBS, 0.25% trypsin, 5.5% NaHCO3, and penicillin-streptomycin. Centrifuge the single-cell suspension in centrifuge tubes at 1500-2000 rpm for 15-30 min; Single-cell suspensions were inoculated into new culture medium and cultured in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 (volume fraction).

4. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs according to claim 1, characterized in that, In step (C), the culture medium containing hAMCs is centrifuged in centrifuge tubes at 1200-1800 rpm for 8-10 min.

5. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs according to claim 1, characterized in that, In step (D), take the centrifuge tube from step (C) after removing the supernatant, add PBS buffer, and centrifuge to wash 1-3 times, including the following steps: (D1) Take the centrifuge tube after removing the supernatant, add 8-12 ml of PBS buffer for the first time, and wash at 3000-4000 rpm for 5-8 min to remove the PBS buffer. (D2) Add 6-8 ml of PBS buffer for the second time, and wash at 2000-3000 rpm for 2-4 min to remove the PBS buffer; (D3) Add 6-8 ml of PBS buffer for the third time, and wash at 1200-2000 rpm for 2-4 min to remove the PBS buffer.

6. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to claim 1, characterized in that, In step (D), after cleaning, the centrifuge tubes after removing the supernatant are placed in an ultrasonic cell disruptor under the following conditions: power 50W-150W, working time 1s, interval 2s, temperature below 30℃ (ice bath), and total ultrasonic time 10-30min.

7. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to claim 1, characterized in that, In (E1), the culture supernatant and cell lysate of hAMCs, as well as the serum-free cell culture medium, were diluted 10-15 times with distilled water.

8. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of cultured hAMCs according to claim 1, characterized in that, In (E3), the washing solution is prepared by diluting concentrated washing solution with redistilled water at a ratio of 1:

20.

9. The method for determining the neurotrophic factor content in cell lysate and culture supernatant of in vitro cultured hAMCs according to claim 1, characterized in that, In (E2)-(E6), the amount of diluent a, diluent b, diluent c, first antibody working solution, enzyme-labeled antibody working solution, substrate working solution and stop solution added to the well of the ELISA plate is 100-200ul.