阅读说明：本技术 一种检测人羊膜上皮细胞体外抑制淋巴细胞增殖抑制方法 (Method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro ) 是由 朱灏 于 2021-01-22 设计创作，主要内容包括：本发明涉及淋巴细胞增殖抑制领域,更具体地,本发明涉及一种检测人羊膜上皮细胞体外抑制淋巴细胞增殖抑制方法,其包括：人羊膜上皮细胞与染色后的PBMC细胞在含有游离氨基刺激剂存在的条件下在第一完全培养基中进行共培养后,记录淋巴细胞增殖能力。本申请检测人羊膜上皮细胞体外抑制淋巴细胞增殖抑制方法操作简单,数据稳定,相对标准偏差在5％以内,抑制率高达75％,可应用在免疫调节中。(The invention relates to the field of lymphocyte proliferation inhibition, in particular to a method for detecting human amniotic epithelial cell in vitro inhibition on lymphocyte proliferation, which comprises the following steps: human amniotic epithelial cells were co-cultured with stained PBMC cells in the presence of free amino stimulators in a first complete medium, and lymphocyte proliferation capacity was recorded. The method for detecting the inhibition of the human amniotic epithelial cell proliferation in vitro is simple to operate, stable in data, within 5% of relative standard deviation and high in inhibition rate of 75%, and can be applied to immune regulation.)

1. A method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro is characterized by comprising the following steps: human amniotic epithelial cells were co-cultured with stained PBMC cells in the presence of free amino stimulators in a first complete medium, and lymphocyte proliferation capacity was recorded.

2. The method for detecting inhibition of lymphocyte proliferation by human amniotic epithelial cells in vitro according to claim 1, further comprising: inoculating the human amniotic epithelial cells: taking the human amniotic epithelial cell suspension, centrifuging for n times to remove supernatant, then using a complete culture medium for resuspension, and then inoculating and culturing, wherein n is more than or equal to 1.

3. The method for detecting inhibition of lymphocyte proliferation by human amniotic epithelial cells in vitro according to claim 2, further comprising: and (4) carrying out proliferation removal on the inoculated and cultured human amniotic epithelial cells and then continuing culturing.

4. The method for detecting inhibition of lymphocyte proliferation by human amniotic epithelial cells according to any one of claims 1-3, wherein the final concentration of the stimulating agent in the first complete culture medium is 2.2-2.6 μ g/mL.

5. The method for detecting inhibition of human amniotic epithelial cell proliferation in vitro according to claim 4, wherein the free amino-stimulating agent is anti-CD3 and/or anti-CD 28.

6. The method for detecting inhibition of human amniotic epithelial cell proliferation in vitro according to claim 5, wherein the free amino-stimulating agents are anti-CD3 and anti-CD28 at a concentration ratio of 1: (0.8-1.2).

7. The method of claim 6, wherein the complete medium contains 8-12 wt% fetal bovine serum when n is 1.

8. The method for detecting inhibition of human amniotic epithelial cell proliferation in vitro according to claim 7, wherein the complete culture medium is Gibco 1640 culture medium.

9. The method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro according to claim 8, wherein in the inoculation process of the human amniotic epithelial cells, the conditions of inoculation culture are as follows: 4.8-5.2 vol% CO₂Culturing at 30-40 deg.C for 20-24 hr.

10. Use of the method according to any one of claims 1 to 9 for detecting inhibition of proliferation of lymphocytes by human amniotic epithelial cells in vitro for immunomodulation.

Technical Field

The invention relates to the field of lymphocyte proliferation inhibition, in particular to a method for detecting in-vitro inhibition of lymphocyte proliferation by human amniotic epithelial cells.

Background

Amniotic cells are commonly referred to as cells on the transparent film which wraps amniotic fluid, and one mature amniotic membrane has an area of 2 square meters and accumulates 3 hundred million amniotic epithelial cells. As a protective layer of the embryo, the amnion not only provides nutrition for the development of the fetus, but also is a wonderful barrier for preventing two variant tissues of the mother and the fetus from mutually rejecting.

The human amniotic epithelial cells are present in the amniotic tissues on the placenta after delivery, are differentiated from the blastocyst inner cell masses of fertilized eggs which have developed to the 8 th day, have differentiation potential, and can be differentiated into 3 germ layer tissues. Under appropriate induction conditions, human amniotic epithelial cells can differentiate into hepatocytes, cardiac myocytes, nerve cells, and the like. It is an epithelial cell with stem cell characteristics, expresses stem cell marker molecules and related genes, such as CD133, CD271, TRA-1-60, Oct3/4, SOX-2, SSEA4 and Klf4, but does not express telomerase genes in stem cells. Telomerase can protect the end of chromosomes, and ensure that DNA is not shortened during replication, thereby immortalizing cells. Therefore, human amniotic epithelial cells cannot be proliferated indefinitely, and cannot be regarded as strictly stem cells. The cell surface does not express HLA-A, B, C and DR antigen, and because the antigenicity is very low, no immunological rejection reaction can be caused after allogeneic transplantation.

However, the human amniotic cells enter a logarithmic growth phase after being cultured for three days, the proliferation is slow, the inhibition effect on lymphocyte proliferation is slow, meanwhile, due to the difference of methods such as cell culture proliferation and de-proliferation, the quality of the human amniotic cells is greatly different, and the data fluctuation is large and unstable when the inhibition on lymphocyte proliferation is detected in vitro.

Disclosure of Invention

In view of the problems in the prior art, the first aspect of the present invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which comprises: human amniotic epithelial cells were co-cultured with stained PBMC cells in the presence of free amino stimulators in a first complete medium, and lymphocyte proliferation capacity was recorded.

As a preferred embodiment of the present invention, the method for detecting inhibition of proliferation of lymphocytes by human amniotic epithelial cells in vitro further comprises: inoculating the human amniotic epithelial cells: taking the human amniotic epithelial cell suspension, centrifuging for n times to remove supernatant, then using a complete culture medium for resuspension, and then inoculating and culturing, wherein n is more than or equal to 1.

As a preferred embodiment of the present invention, the method for detecting inhibition of proliferation of lymphocytes by human amniotic epithelial cells in vitro further comprises: and (4) carrying out proliferation removal on the inoculated and cultured human amniotic epithelial cells and then continuing culturing.

As a preferred embodiment of the present invention, the final concentration of the stimulating agent in the first complete medium is 2.2 to 2.6. mu.g/mL.

In a preferred embodiment of the present invention, the free amino group-stimulating agent is anti-CD3 and/or anti-CD 28.

As a preferred technical scheme of the invention, the free amino stimulant is anti-CD3 and anti-CD28, and the concentration ratio of the free amino stimulant to the free amino stimulant is 1: (0.8-1.2).

In a preferred embodiment of the present invention, when n is 1, the complete medium contains 8 to 12 wt% fetal bovine serum.

In a preferred embodiment of the present invention, the complete medium is Gibco 1640 medium.

In a preferred embodiment of the present invention, in the inoculation process of the human amniotic epithelial cells, the conditions of inoculation and culture are as follows: 4.8-5.2 vol% CO₂Culturing at 30-40 deg.C for 20-24 hr.

The invention also provides an application of the method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro in the immunoregulation.

Compared with the prior art, the invention has the following beneficial effects:

(1) the concentration of the stimulating agent is strictly controlled, so that the contact probability of the human amniotic epithelial cells and the PBMC cells is increased when the human amniotic epithelial cells are inoculated and cultured for 20-24h and are co-cultured with the stained PBMC cells;

(2) in the process of inoculating the human amniotic epithelial cells, the cell concentration is adjusted to 2 x 10⁵After the culture is carried out by the volume fraction of the cells per milliliter, the proliferation is better, and higher cell activity is kept;

(3) the inoculation process of the amniotic epithelial cells of the applicant is simple, the supernatant is removed by one-time centrifugation, insoluble floccules do not exist after the amniotic epithelial cells are resuspended by using the Gibco 1640 culture medium in the application, and the inhibition rate of the obtained human amniotic epithelial cells on lymphocyte proliferation is improved;

(4) the method for detecting the inhibition of the human amniotic epithelial cell proliferation in vitro is simple to operate, stable in data, within 5% of relative standard deviation and high in inhibition rate of 75%, and can be applied to immune regulation.

Detailed Description

The present invention is illustrated by the following specific embodiments, but is not limited to the specific examples given below.

The invention provides a method for detecting human amniotic epithelial cell in vitro inhibition of lymphocyte proliferation, which comprises the following steps: human amniotic epithelial cells were co-cultured with stained PBMC cells in the presence of free amino stimulators in a first complete medium, and lymphocyte proliferation capacity was recorded.

In one embodiment, the final concentration of the stimulating agent in the first complete medium is 2.2-2.6. mu.g/mL.

Preferably, the final concentration of the stimulant in the first complete medium is 2.4. mu.g/mL.

In one embodiment, the free amino stimulant is anti-CD3 and/or anti-CD 28.

Preferably, the free amino stimulant is anti-CD3 and anti-CD 28; further preferably, the concentration ratio of anti-CD3 to anti-CD28 is 1: (0.8-1.2); more preferably, the concentration ratio of anti-CD3 to anti-CD28 is 1: 1.

when human amniotic epithelial cells are inoculated and cultured for 20-24h, most of the human amniotic epithelial cells are grown adherently and do not reach a logarithmic growth phase, when the viable human amniotic epithelial cells are cultured together with the stained PBMC cells in the presence of a free amino stimulant in a first complete culture medium, the contact probability of the viable human amniotic epithelial cells on the PBMC cells is reduced, and the applicant unexpectedly finds that when the final concentration of the stimulant in the first complete culture medium is 2.2-2.6 mug/mL, and the stimulants are anti-CD3 and anti-CD28, the concentration ratio of the stimulants is 1: (0.8-1.2), the probability of contacting the human amniotic cells with the stained PBMC cells is significantly increased, and the applicant believes that the probable reason is that under the condition, the human amniotic epithelial cells can be stimulated to secrete a cytokine which may cause contact attraction during the process of inhibiting the proliferation of the lymphocytes.

In one embodiment, the first complete medium contains 8-12 wt% fetal bovine serum.

Preferably, the first complete medium contains 10 wt% fetal bovine serum.

Preferably, the first complete medium is Gibco 1640 medium.

The procedure for staining PBMCs according to the present invention is not particularly limited and may be routinely selected by those skilled in the art.

In one embodiment, PBMC staining using 2 u mol/L CFSE staining solution staining for 5 min.

The method for thawing the cryopreserved PBMC cells before staining the PBMC can also comprise a process of thawing the cryopreserved PBMC cells, and the specific operation is not particularly limited, and the routine selection can be performed by a person skilled in the art.

In one embodiment, the number ratio of the human amniotic epithelial cells to the stained PBMC cells is 1: (1-10).

Preferably, the number ratio of the human amniotic epithelial cells to the stained PBMC cells is 1: 5.

in one embodiment, the method for detecting inhibition of lymphocyte proliferation in vitro by human amniotic epithelial cells further comprises: inoculating the human amniotic epithelial cells: taking the human amniotic epithelial cell suspension, centrifuging for n times to remove supernatant, then using a complete culture medium for resuspension, and then inoculating and culturing, wherein n is more than or equal to 1.

In one embodiment, n is 1.

In one embodiment, the complete medium contains 8-12 wt% fetal bovine serum.

Preferably, the complete medium contains 10 wt% fetal bovine serum.

Preferably, the complete medium is Gibco 1640 medium.

In one embodiment, the conditions of the seed culture are: 4.8-5.2 vol% CO₂Culturing at 30-40 deg.C for 20-24 hr.

Preferably, the conditions of the inoculation culture are as follows: 5 vol% CO₂The culture was carried out at 37 ℃ for 22 h.

The applicant unexpectedly finds in experiments that insoluble floccules are generated when a complete culture medium is added for resuspension in the process of inoculating human amniotic epithelial cells, and the floccules still exist after being blown, and also unexpectedly finds that after cell suspension is centrifuged for multiple times to remove supernatant, the insoluble floccules are reduced when the complete culture medium is added for resuspension, but the method is complex in operation and increases time cost on one hand, and on the other hand, a small amount of insoluble floccules have great influence on lymphocyte proliferation inhibition on the other hand. After a series of researches and thinking, the applicant finds that when the complete culture medium is a Gibco 1640 culture medium, after supernatant is removed by centrifuging human amniotic epithelial cell suspension for one time, insoluble floccules cannot be generated, and the inhibition rate of lymphocyte proliferation is remarkably improved.

In one embodiment, the human amniotic epithelial cells are seeded with: taking 400g of cell suspension, centrifuging for 5min, removing supernatant, adding complete culture medium for resuspension, sampling and counting; according to the counting results, the cell concentration was adjusted to 2X 10⁵Per mL; the adjusted cell suspension was inoculated into 3 24-well culture plates, each of which was inoculated into 4 wells at 500. mu.L per well, to thereby inoculate 12 wells in total. The inoculated cell plates were placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and cultured for 22 h.

In this application, the cell concentration is adjusted to 2X 10⁵After being cultured after the culture medium is/mL, the culture medium has better proliferation and keeps higher cell activity.

In one embodiment, the method for detecting inhibition of lymphocyte proliferation in vitro by human amniotic epithelial cells further comprises: and (4) carrying out proliferation removal on the inoculated and cultured human amniotic epithelial cells and then continuing culturing.

In one embodiment, the treatment for de-proliferation includes one or more of radiation de-proliferation, repeated freezing and thawing, and addition of a de-proliferation treatment agent.

Preferably, the de-proliferation treatment agent is a complete culture medium of mitomycin C; further preferably, the concentration of the mitomycin C is 8-12 mug/mL; more preferably, the concentration of mitomycin C is 10. mu.g/mL.

In one embodiment, the time of the de-proliferation treatment is 1-3 h.

Preferably, the time of the de-proliferation treatment is 2 h.

The applicant has surprisingly found that when the antiproliferative treatment agent is a complete medium of 8-12. mu.g/ml mitomycin C, and after culturing for 1-3 hours and then culturing again, the human amniotic epithelial cells obtained at this time have a large amount of secreted factors, and can limitedly inhibit lymphocyte proliferation.

In one embodiment, the continuously culturing the human amniotic epithelial cells after the de-proliferation of the seeded culture specifically comprises: the medium was discarded from each well of the contact culture, 500. mu.l of complete medium containing 10. mu.g/mL mitomycin C was added to each well, and the mixture was placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and incubating for 2 h; after incubation, cells were removed, the medium was gently aspirated from each well, washed 2 times with 1ml of pbs per well. After washing, 1mL of complete medium was added to the culture well and the cell culture plate was placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 deg.C).

The complete medium of the present invention is not particularly limited, and is Gibco 1640 medium.

In one embodiment, the method for detecting inhibition of lymphocyte proliferation in vitro by human amniotic epithelial cells comprises the following steps:

(1) inoculating the human amniotic epithelial cells: taking the human amniotic epithelial cell suspension, centrifuging for n times to remove supernatant, then using a complete culture medium for resuspension, and then carrying out inoculation culture, wherein n is more than or equal to 1;

(2) the inoculated and cultured human amniotic epithelial cells are subjected to proliferation removal and then are continuously cultured;

(3) co-culturing the human amniotic epithelial cells obtained in the step (2) and the stained PBMC cells in a first complete culture medium in the presence of a free amino stimulant, and recording the proliferation capacity of the lymphocytes.

The invention also provides an application of the method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro in the immunoregulation.

Examples

Hereinafter, the present invention will be described in more detail by way of examples, but it should be understood that these examples are merely illustrative and not restrictive. The starting materials used in the examples which follow are all commercially available unless otherwise stated.

Example 1

The embodiment 1 of the invention provides a method for detecting the inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which comprises the following specific steps:

(1) inoculating the human amniotic epithelial cells: taking 400g of cell suspension, centrifuging for 5min, removing supernatant, adding Gibco 1640 culture medium containing 10 wt% fetal bovine serum for resuspension, sampling and counting; according to the counting results, the cell concentration was adjusted to 2X 10⁵Per mL; the adjusted cell suspension was inoculated into 3 24-well culture plates, each of which was inoculated into 4 wells at 500. mu.L per well, to thereby inoculate 12 wells in total. The inoculated cell plates were placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and culturing for 20 h;

(2) discarding the medium from each well in step (1), adding 500. mu.l of 10 wt% fetal bovine serum-containing Gibco 1640 medium containing 8. mu.g/mL mitomycin C per well, and placing in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and incubating for 3 h; after incubation, cells were removed, the medium was gently aspirated from each well, washed 2 times with 1ml of pbs per well. After washing, 1mL of Gibco 1640 medium containing 10 wt% fetal bovine serum was added to the culture well, and the cell culture plate was placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃);

(3) and (3) the human amniotic epithelial cells finally obtained in the step (2) and PBMC cells stained for 5min by using 2 mu mol/L CFSE staining solution are mixed according to the ratio of the number of 1: ratio 5 lymphocyte proliferative capacity was recorded after co-culture with 1.2. mu.g/mL anti-CD3 and 1. mu.g/mL anti-CD28 in Gibco 1640 medium containing 10 wt% fetal bovine serum.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 2

The embodiment 2 of the invention provides a method for detecting human amniotic epithelial cell in-vitro inhibition of lymphocyte proliferation, which comprises the following specific steps:

(1) inoculating the human amniotic epithelial cells: taking 400g of cell suspension, centrifuging for 5min, removing supernatant, adding Gibco 1640 culture medium containing 10 wt% fetal bovine serum for resuspension, sampling and counting; according to the counting results, the cell concentration was adjusted to 2X 10⁵Per mL; the adjusted cell suspension was inoculated into 3 24-well culture plates, each of which was inoculated into 4 wells at 500. mu.L per well, to thereby inoculate 12 wells in total. The inoculated cell plates were placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and culturing for 20 h;

(2) discarding the medium from each well in step (1), adding 500. mu.l of Gibco 1640 medium containing 12. mu.g/mL mitomycin C and 10 wt% fetal bovine serum to each well, and placing in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and incubating for 1 h; after incubation, cells were removed, the medium was gently aspirated from each well, washed 2 times with 1ml of pbs per well. After washing, 1mL of Gibco 1640 medium containing 10 wt% fetal bovine serum was added to the culture well, and the cell culture plate was placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃);

(3) and (3) the human amniotic epithelial cells finally obtained in the step (2) and PBMC cells stained for 5min by using 2 mu mol/L CFSE staining solution are mixed according to the ratio of the number of 1: ratio of 5 lymphocyte proliferation capacity was recorded after co-culture with 1.2. mu.g/mL anti-CD3 and 1.4. mu.g/mL anti-CD28 in Gibco 1640 medium containing 10 wt% fetal bovine serum.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 3

The embodiment 3 of the invention provides a method for detecting the inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which comprises the following specific steps:

(1) inoculating the human amniotic epithelial cells: taking 400g of cell suspension, centrifuging for 5min, removing supernatant, adding Gibco 1640 culture medium containing 10 wt% fetal bovine serum for resuspension, sampling and counting; according to the counting results, the cell concentration was adjusted to 2X 10⁵Per mL; the adjusted cell suspension was inoculated into 3 24-well culture plates, each of which was inoculated into 4 wells at 500. mu.L per well, to thereby inoculate 12 wells in total. The inoculated cell plates were placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and culturing for 20 h;

(2) discarding the medium from each well in step (1), adding 500. mu.l of 10 wt% fetal bovine serum-containing Gibco 1640 medium containing 10. mu.g/mL mitomycin C per well, and placing in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃), and incubating for 2 h; after incubation, cells were removed, the medium was gently aspirated from each well, washed 2 times with 1ml of pbs per well. After washing, 1mL of Gibco 1640 medium containing 10 wt% fetal bovine serum was added to the culture well, and the cell culture plate was placed in a carbon dioxide incubator (CO)₂The concentration is set as follows: 5.0 vol%, temperature settings were: 37.0 ℃);

(3) and (3) the human amniotic epithelial cells finally obtained in the step (2) and PBMC cells stained for 5min by using 2 mu mol/L CFSE staining solution are mixed according to the ratio of the number of 1: ratio of 5 lymphocyte proliferation capacity was recorded after co-culture with 1.2. mu.g/mL anti-CD3 and 1.2. mu.g/mL anti-CD28 in Gibco 1640 medium containing 10 wt% fetal bovine serum.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 4

The embodiment 4 of the invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is implemented in the same manner as the embodiment 3, except that the Gibco 1640 medium containing 10 wt% fetal bovine serum in the step (1) is replaced by DMEM/F12 medium.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 5

The embodiment 5 of the invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is implemented in the same manner as the embodiment 3, except that in the step (1), the centrifugation is repeated for 5min again to remove the supernatant for 2 times, and a Gibco 1640 culture medium containing 10 wt% fetal bovine serum is replaced by a DMEM/F12 culture medium.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 6

Example 6 of the present invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is the same as example 3 in the specific embodiment, except that 1.2 μ g/mL anti-CD3 and 1.2 μ g/mL anti-CD28 are replaced with 1 μ g/mL anti-CD3 and 1 μ g/mL anti-CD28 in step (3), respectively.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 7

Example 7 of the present invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is the same as example 3 in the specific embodiment, except that 1.2 μ g/mL anti-CD3 and 1.2 μ g/mL anti-CD28 are replaced with 1.5 μ g/mL anti-CD3 and 1.5 μ g/mL anti-CD28 in the step (3), respectively.

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 8

Example 8 of the present invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is the same as example 3 in the specific embodiment, except that 1.2 μ g/mL anti-CD3 and 1.2 μ g/mL anti-CD28 are replaced with 2.4 μ g/mL anti-CD3 in the step (3).

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Example 9

Example 9 of the present invention provides a method for detecting inhibition of human amniotic epithelial cells on lymphocyte proliferation in vitro, which is the same as example 3 in the specific embodiment, except that 1.2 μ g/mL anti-CD3 and 1.2 μ g/mL anti-CD28 are replaced with 2.4 μ g/mL anti-CD28 in the step (3).

The method for detecting the inhibition of the human amniotic epithelial cells on the proliferation of the lymphocytes in vitro is applied to immune regulation.

Performance evaluation

1. Resuspension effect: the presence of insoluble flocs in the cell fluid after resuspension in step (1) of examples 1-9 was recorded separately.

2. Human amniotic epithelial cells and PBMC cell contact probability test: in each of examples 1 to 9, the contact state between the human amniotic cells and the PBMC was observed after the co-culture for half a day in step (3), and the contact state between the human amniotic cells and the PBMC was numbered in the order of 1-9, wherein the higher the number of the PBMC in contact with the human amniotic cells, the earlier the order is, and the smaller the number is, and the number is 1-9, wherein the number is 1-2, and the number is excellent, the number is 3-6, and the number is good, and the number is 7-9, and the number is poor.

TABLE 1

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.